Bone morphogenetic proteins in inflammation, glucose homeostasis and adipose tissue energy metabolism

Accepted Manuscript Title: Bone morphogenetic proteins in inflammation, glucose homeostasis and adipose tissue energy metabolism Author: Lovorka Grgurevic Gitte Lund Christensen Tim Schulz Slobodan Vukicevic PII: DOI: Reference:

S1359-6101(15)00093-3 http://dx.doi.org/doi:10.1016/j.cytogfr.2015.12.009 CGFR 912

To appear in:

Cytokine & Growth Factor Reviews

Received date: Revised date: Accepted date:

12-10-2015 10-12-2015 23-12-2015

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BMP review for CGFR

Bone morphogenetic proteins in inflammation, glucose homeostasis and adipose tissue energy metabolism Lovorka Grgurevic1#, Gitte Lund Christensen2#, Tim Schulz3,4*, Slobodan Vukicevic1* 1

University of Zagreb School of Medicine, Center for Translational and Clinical Research,

Laboratory for Mineralized Tissues, Zagreb, Croatia 2

Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark

3

German Institute of Human Nutrition Potsdam-Rehbrücke, Nuthetal, Germany

4

German Center for Diabetes Research (DZD), München-Neuherberg, Germany

#

These authors equally contributed to this manuscript

*Correspondence should be addressed to: [email protected] and [email protected]

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BMP review for CGFR

Graphical abstract

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BMP review for CGFR

Highlights 1. BMPs mediate inflammatory processes in the gastrointestinal system, skeletal and cardiovascular diseases 2. BMPs affect systemic energy balance by targeting the pancreas as well as brown and white adipose tissues 3. BMPs regulate pancreatic development and insulin secretion 4. BMPs act as inducers of brown adipocyte formation and thermogenic function

. Abstract Bore morphogenetic proteins (BMPs) are members of the transforming growth factor (TGF)-β superfamily, a group of secreted proteins that regulate embryonic development. This review summarizes the effects of BMPs on physiological processes not exclusively linked to the musculoskeletal system. Specifically, we focus on the involvement of BMPs in inflammatory disorders, e.g. fibrosis, inflammatory bowel disease, anchylosing spondylitis, rheumatoid arthritis. Moreover, we discuss the role of BMPs in the context of vascular disorders, and explore the role of these signalling proteins in iron homeostasis (anaemia, hemochromatosis) and oxidative damage. The second and third parts of this review focus on BMPs in the development of metabolic pathologies such as type-2 diabetes mellitus and obesity. The pancreatic beta cells are the sole source of the hormone insulin and BMPs have recently been implicated in pancreas development as well as control of adult glucose homeostasis. Lastly, we review the recently recognized role of BMPs in brown adipose tissue formation and their consequences for energy expenditure and adiposity. In summary, BMPs play a pivotal role in metabolism beyond their role in skeletal homeostasis. However, increased understanding of these pleiotropic functions also highlights the necessity of tissue-specific strategies when harnessing BMP action as a therapeutic target.

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BMP review for CGFR BMPs are large dimeric proteins synthesized and folded in the cytoplasm and cleaved by proteases during secretion. They are highly conserved molecules whose biologically active C-terminal peptide is released into the extracellular compartment to bind membrane receptors on target cells. Ligand binding induces constitutively-active BMP type II receptors to trans phosphorylate the BMP type I receptor which in turn phosphorylates the intracellular BMP effector proteins SMAD1/5/8 whereas TGFβ1 typically activates Smad2 and Smad3 [1-5]. The activated Smads translocate from the cytosol to the nucleus and form complexes with other transcription factors to bind and activate the expression of target genes. “Canonical” signalling appears to mediate the principal effects of BMPs, although activation of other signalling pathways could be crucial in their role in several biological responses including inflammation, glucose regulation and energy metabolism, which will be covered in this review.

BONE MORPHOGENETIC PROTEINS IN INFLAMMATION The role of BMPs in inflammatory disorders includes chronic liver disease, inflammatory bowel disease, iron deficiency anemia, vascular disease, atherosclerosis, rheumatoid arthritis, ankylosing spondylitis, rare bone disorders and implantation of commercially available bone devices containing BMP2 and BMP7 [6]. BMPs role in inflammatory disorders of the gastrointestinal system The liver provides a generic model of inflammation and repair, demonstrating an intensive interplay between the epithelial, inflammatory cells and myofibroblasts [7], following intoxication and hepatocyte damage. Fibrogenesis is one of the most prominent pathophysiological processes in a number of chronic diffuse liver diseases including viral hepatitis, fatty liver, autoimmune diseases and alcoholic liver disease [8]. Numerous animal models and human studies showed a correlation between inflammation and fibrosis leading to cirrhosis that is characterized by an excessive extracellular matrix deposition that leads to progressive liver dysfunction. In response to various factors that may cause liver damage, inflammation promotes fibrosis through a number of mechanisms and cell mediators (inflammatory cytokines and chemical neurotransmitters) (Figure 1). A characteristic feature of inflammation is the activation of hepatic stellate cells (HSCs) to a myofibroblast-like phenotype that is proliferative, fibrogenic and contractile, synthesizing a large amount of ECM components.

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BMP review for CGFR

Figure 1. The role of BMPs in liver fibrogenesis. After persistent and chronic liver injury which is associated with inflammatory response, liver regeneration fails and large number of cytokines are released from damaged tissue. The most important fibrogenic mediators such as transforming growth factor-beta (TGFβ), platelet-derived growth factor (PDGF), tumor necrosis factor α (TNFα) and connective tissue growth factor (CTGF) induce the recruitment of inflammatory cells. Apoptosis of damaged hepatocytes and inflammatory cells stimulates activation of QHSC and their transdifferentiation to myofibroblasts through activated hepatic stellate cells which secrete large number of ECM components. BMP7 inhibits TGFβ activity and also has an antiapoptotic and antiinflammatory effect. There is a close topographical relationship between the site of inflammation and the development of fibrosis in vivo. Although there are substantial numbers of leukocytes in the resting liver, injury results in a massive accumulation and contemporaneous activation of resident and recruited inflammatory cells. A variety of signal transduction pathways and cytokines has effects in the development of liver fibrosis such as TGFβ1, BMP7 and Smads [9]. TGFβ1 has been identified as the profibrogenic master cytokine, promoting transition of HSC to a myofibroblast-like phenotype, and inhibition of ECM degradation by HSCs through the expression of tissue inhibitor of metalloproteinases (TIMPs). Platelet-derived growth factor (PDGF), connective tissue growth factor (CTGF) and tumor necrosis factor (TNFα) have emerged as most potent proliferative HSC cytokines [10], while, BMPs are important in hepatic development and liver regeneration. BMP9 is specifically expressed in the liver tissue and may induce liver fibrosis through ALK1, endoglin, Id1, hepcidin and Snail [11]. BMP7 has been shown to inhibit TGFβ dependent epithelial to mesenchymal transition (EMT) of hepatocytes and also has an antiapoptotic and anti-inflammatory effect [10]. For example, it reduces the progression of fibrosis in mice intoxicated with CCl4 and prevents EMT of hepatocytes accelerating liver regeneration after partial hepatectomy which is reduced after administration of an 5

BMP review for CGFR anti-BMP7 antibody [12]. The expression of BMP7 in the liver increases with increased hepatic inflammatory grade and fibrosis. Anti-inflammatory and anti-fibrogenic effects of BMP7 have also been shown in patients with chronic hepatitis B [13]. Various cytokines contribute to the control of hepatitis C virus (HCV) replication. BMP7 inhibits the intracellular replication of the HCV subgenome in a dose-dependent manner via a cell-cycle mediated arrest in host Huh7/Rep-Feo cells. Recombinant adenovirus carrying BMP7 (AAV-BMP7) in a rat experimental model of induced hepatic fibrosis suggests that BMP7 inhibits the hepatic fibrosis and promotes liver regeneration [14]. Expression of BMP7 was also confirmed in mesenchymal stem cells (MSCs) which have therapeutic effects in various diseases. MSC mitigate cirrhosis through their production of BMP7 against the fibrogenic effect of TGFβ1 in the injured liver [15]. In contrast, BMP2 inhibits the division of hepatocytes, thereby reducing the liver regeneration after hepatectomy [16]. BMPs are also involved in the process of pancreatic fibrosis which is a common pathological characteristic of chronic pancreatitis (CP), a major cause of pancreatic cancer. During early progression of pancreatic fibrosis, BMP2 and phosphorylated Smad1 (pSmad1) levels increase, followed by a decreased expression level suggesting dynamic changes of BMP expression in correlation with fibrosis. The BMP2 inhibitory effect on TGFβ and ECM formation was confirmed in the primary mouse and human pancreatic stellate cells [17]. The anti-inflammatory effect of BMPs was also tested in a model of gastric inflammation after infection of transgenic mice (that express either BMP inhibitor noggin or the β galactosidase gene under the control of a BMP responsive element and BMP4 mice) with Helicobacter pylori or Helicobacter feolis [18]. BMP signalling reduces the inflammation and the presence of dysplastic changes in the gastric mucosa after infection. The BMP pathway is also critical for epithelial morphogenesis and inflammation of the oesophagus. In the model of eosinophilic esophagitis (EoE), in which basal progenitor cells become hyperplastic after inflammation and stimulation of BMP/NRF2 pathways were disrupted [19]. The key feature of the inflammatory bowel disease (IBD) is an impaired epithelial repair. The specific expression pattern of BMPs and components of the BMP signalling pathway were observed in all three germ layers suggesting an essential role in the gastrointestinal (GI) tract development [20]. Although the GI is characterized by a remarkable ability to resist the physiological inflammation, when the intestinal homeostasis is corrupted the disease occurs. IBD, describes a set of chronic gastrointestinal illnesses, including Crohn's disease and ulcerative colitis (UC), of multifactorial etiology with complex interactions between genetic and environmental factors culminating in a sustained activation of the mucosal immune and non-immune response, facilitated by defects in the intestinal epithelial barrier and mucosal immune system leading to an active inflammation and tissue 6

BMP review for CGFR destruction. In mucosal transcriptomic expression profiles of biopsies from patients with UC expression of BMP/Retinoic acid inducible Neural-specific 3 (BRINP3) was significantly downregulated. Low levels of expression could predispose the characteristics of a mucosal inflammation [21]. The trinitrobenzene sulfonic acid (TNBS) experimental model of colitis allows a comparison between the acute and chronic stage of colitis and the “late” stage of human IBD regarding the tissue damage and clinical signs of the disease, but missing the “early” stage in humans, which occurs mostly without symptoms [22]. BMP7, when applied prophylactically or therapeutically in rats with TNBS induced colitis, reduced the intestinal inflammation due to a decrement of proinflammatory cytokine Interleukin (IL)-6 and increased expression of synergistic BMPs, receptor type IB and BMPspecific receptor regulated Smads (BR-Smads) and downregulation of BMP antagonists [23]. BMPs and BMP pathway components are however preserved in the colon of diseased animals and alternations in their expression in IBD are recovered by the BMP7 therapy [24]. The disruption of TGFβ/Smad signalling pathway in IBD is characterized by a high expression of Smad7 and reduced Smad3 phosphorylation, leading to an inadequate inflammatory cell response to TGFβ1, and a persisting inflammation, whereas Smad7 inhibition leads to its restoration. In Smad3 knockout mice there is an increased expression of TGFβ1 and Smad7 in the intestine with an absence of macroscopic lesions and a reduction of fibrosis [25]. On the contrary, BMP7 downregulates the expression level of inhibitory Smads, supporting the observation that blocking Smad7 is a key event in the regulation of inflammation in IBD. In another study after intracolonical administration of adeno-associated virus delivering BMP7, oxidative damage and attenuated complementary mucosal cell proliferation was prevented [26]. Recently, targeting SMAD7 with mongersen (formulation containing a 21-base single-strand phosphorothioate oligonucleotide) that hybridizes to the human SMAD7 messenger RNA and facilitates RNase H-mediated RNA degradation through a classic antisense mechanism. Mongersen has previously been shown to down-regulate Smad7 and prevent and alleviate Crohn’s disease–like colitis in mice [27]. In a multicenter, randomized, placebo-controlled, double-blind, phase 2 clinical trial, patients with moderate-to-severe Crohn’s disease were randomly assigned to receive one of three doses of mongersen which had clinical benefit for study participants which was supported also by normalization of C-reactive peptide. The data from this phase 2 study provide evidence of the efficacy and adverse-effect profile of mongersen in active Crohn’s disease and also influence of SMAD7 in inflammatory reaction of Crohn’s disease [28]. IBD can be complicated by the development of an iron deficiency anemia that is partly explained by the intestinal blood loss. The recent discovery of the mechanism that regulates iron metabolism has revealed an important role for hepcidin, an iron regulating hormone secreted by hepatocytes [29,30]. Hepcidin binds to and down-regulates the iron exporter ferroportin, which is expressed on macrophages and duodenal 7

BMP review for CGFR enterocytes, thereby inhibiting, respectively, the recycling of iron from erythrocytes and the absorption of iron from the diet. Thus, elevated circulating hepcidin leads to decreased serum iron, iron-restricted impairment of erythropoiesis and, ultimately, anemia. A key molecule involved in sensing serum iron and regulating hepcidin expression appropriately is the human hemochromatosis protein (HFE), an atypical class I major histocompatibility protein expressed on the hepatocyte surface [31]. Hepcidin expression in the liver can be induced through two major pathways: the inflammatory pathway via IL-6, and iron-sensing pathway by BMP6. Also GATA 6 protein is upregulated during inflammation in concert with hepcidin [32]. The binding of BMPs to their receptor and the co-receptor hemojuvelin (HJV) expressed on hepatocytes initiates a cascade of events, including the phosphorylation of receptor SMAD1/5/8, that culminates in the transcriptional upregulation of the hepcidin gene. Of the various BMPs, BMP6 appears to be the most functionally relevant regulator of hepcidin in vivo: its expression is modulated by circulating iron levels and its absence in mice leads to a marked iron overload [33,34]. BMP6 plays an essential role in colitis since neutralization of BMP6 significantly reduces the hepcidin serum levels. Interestingly, intestinal inflammation was not associated with an increased liver BMP6 mRNA or BMP6-dependent signalling activity. BMP6 is required for normal expression of hepcidin, but the actual stimulus for colitis-induced hepcidin up-regulation involved another factor such as IL-6 which is produced by the liver and other tissues during colitis. IL-6 has been shown to increase the hepcidin expression in vitro and in vivo, and IL-6-induced hepcidin up-regulation has been proposed to play an important role in the pathogenesis of the anemia of chronic disease (ACD). These results suggest a mechanism in which signals activated by increased IL-6 expression interact with constitutive signals provided by basal levels of BMP6 to promote the hepcidin transcription. The combination of both sets of signals determines the amount of hepcidin expressed and blocking either one leads to lowering of the hepcidin production. This explanation is consistent with earlier observation indicating that an intact BMP-SMAD pathway is required for IL-6-induced hepcidin up-regulation and that signals activated by BMPs and IL-6 probably interact at the level of the hepcidin promoter [30,35]. Lowering of elevated levels of hepcidin has a beneficial effect on serum iron in multiple models of colitis, probably as a result of increased ferroportin-mediated release of iron into the circulation. Thus, inhibiting hepcidin expression or function in inflammatory conditions such as IBD may help to correct the anemia of IBD and may also attenuate the intestinal inflammation [30].

BMPs in inflammatory skeletal disease Clinical studies have suggested an association between the BMP pathway and the inflammatory process in skeletal diseases. In both ankylosing spondylitis (AS) and rheumatoid arthritis (RA) 8

BMP review for CGFR patients serum concentrations of BMP2 and BMP7 were higher when compared to healthy individuals [36,37]. In the peripheral blood cells, expression of BMP4 and BMP6 was reduced in patients with RA. BMP4 correlated negatively in RA and positively in psoriatic arthritis (PsA) with the disease activity [38]. RA is characterized by a synovial inflammation and hyperplasia, leading to cartilage and bone damage. The pro-inflammatory cytokines such as tumor necrosis factor-α (TNFα), interleukin- 1β (IL-1β), and IL-6 associate with up regulated BMP2 and BMP6 level and Smad pathway activation in the arthritis synovium [39-41] while expression of BMP4 and BMP5 mRNA was found to be significantly decreased [42]. Neutrophil granulocyte activation in the synovial fluid of patients with RA may be due to cell exposure to different stimuli [43]. In proteomic analysis of plasma from patients with RA, PsA and non-inflammatory arthritis, some difference was found in protein expression pattern between inflammatory and non-inflammatory rheumatic conditions. Regarding distinctions between RA and PsA, four proteins were down-regulated in PsA and upregulated in RA as compared to non-inflammation arthritis (NIA) (SERPIN A11 – Q86U17, complement factor H-related protein 5 – Q9BXR6, cartilage acidic protein 1 – Q9NQ79 and coagulation factor IX – P00749). A larger study is needed to refine those findings regarding the level of inflammation associated with the expression of specific proteins [44]. It has been reported that lactoferrin (LF) may represent a marker of neutrophil granulocyte activation and also could activate BMP7 gene expression through the mitogen-activated protein kinase ERK pathway in joint chondrocytes. Antirheumatic treatments had no effect on BMP activation in the synovium [45]. Structural damage in AS is characterized by extensive new cartilage and bone formation leading to a progressive ankylosis of the spine and sacroiliac joints. Recent data clearly demonstrate that both persistent inflammation and progression towards ankylosis contribute to the loss of function and disability [46]. In such rheumatoid conditions bone loss is related to inflammation-associated osteoporosis and new bone formation, as has been often seen in patients with AS. That can be explained by different biological mechanisms steering on the one hand at the bone remodeling cycle and, on the other, to new bone formation originating from the enthesis [47]. These first steps result in a local production of different BMPs by various cell types including inflammatory cells like in peripheral blood mononuclear cells [48] where upregulation of gene expression for BMP2, -4 and -7 is present after stimulation with TNFα and IL-1β. Recent studies have demonstrated that weekly local administration of BMP7 inhibits the progression of osteoarthritis in vivo. BMP7 has a potential of inhibiting inflammation in the joint and the degeneration of cartilage in inflammatory arthritis in a zymosan-induced arthritis (ZIA) model. The mechanism by which BMP7 counteracts IL-1β signalling suggests that BMP7 reverses the mitogen-activated protein kinase signalling via inhibition of IL-1β-induced P38 phosphorylation [49,50]. These findings support an anti-inflammatory effect 9

BMP review for CGFR of BMP7 in several inflammatory models of the bone. BMP signalling is dynamically activated in collagen-induced arthritis, where blocking of TNFα increased expression of BMP7 [48,51]. Among the potential biomarkers of bone remodeling in AS, BMP7 beside DKK-1 (dickkopf-1) displayed significant time alterations and correlative interactions during an anti-TNF treatment [52]. In AS patients IgG autoantibodies against noggin were reported at a higher level than in healthy individuals which could contribute to neo-ossification in those patients [53]. Inhibition of BMP signalling by noggin results in the protection against arthritis and ankylosis, not only in a preventive setting in which gene transfer was performed before the onset of the disease but also in a therapeutic setup where gene transfer was performed at the onset of clinical symptoms. BMPs are also related to the rare inherited connective tissue disorder fibrodysplasia ossificans progressiva (FOP) that causes formation of a secondary skeleton of heterotopic bone throughout the body. The histopathology of FOP lesions in destructive inflammatory stage is characterized by accumulation of mononuclear cells and perivascular infiltrates. The FOP phenotype and progressive heterotopic ossification suggested that the primary molecular pathology involves BMPs. Mutant receptor (ACVR1/ALK2) causes increased BMP pathway signalling, triggered by tissue injury which initiates a cascade of events that lead to the secondary skeleton formation. Latest results suggest that those mutations causing FOP by gaining responsiveness to the normally antagonistic ligand activin A, demonstrating that this ligand is necessary and sufficient for driving heterotopic ossification [54,55]. BMP2 and BMP7 are clinically used to promote bone formation in cases of bone fracture delayed unions and spinal fusions [56,57]. The FDA has approved the use of INFUSE bone graft BMP2 to enhance the growth of bone in anterior lumbar interbody spine fusion and open tibial fractures. Diffuse soft-tissue swelling, presumably inflammatory in nature, was noted in the neck structures and esophagus when used off label in cervical spine fusion patients [58]. High doses of BMP2 induce structurally abnormal bone and inflammation in vivo [59]. Also incidence of postoperative wound complication like prolonged serous drainage associated with the use of rhBMP2 in a large series of patients with acute trauma or posttraumatic reconstruction was documented [60]. Comparison of BMP7 and BMP2 was also performed and indicated that BMP7 was associated with smaller soft tissue oedema than BMP2 [61]. Complications observed with the clinical use of BMP2 (Infuse) and BMP7 (Osigraft) bone devices are a consequence of a limited understanding of the molecular mechanisms of BMP mediated function in bone remodelling during the formulation of the bone devices. The amount of BMP2 and -7 in current commercial BMP devices significantly exceeds their biological need, containing a supraphysiologic dose, which upon use is lost from the injury site, leading to increased bioavailability elsewhere, and eventually to uncontrolled immunological responses, including formation of antibodies, exuberant ectopic bone formation and other undesirable 10

BMP review for CGFR inflammation mediated side effects [58,62]. There is an obvious medical need for the development of a new osteogenic device that will offer a safe and cost-effective healing. To improve patient safety and efficacy there is a need for strategies to suppress non-osteogenic BMP2 signalling targets [59]. A novel osteogenic device OSTEOGROW aimed to accelerate bone regeneration has been developed [58]. It contains biologically compatible autologous carrier made from the peripheral blood (whole blood containing device, WBCD), that significantly limits the inflammation processes common with commercial bone devices. Small amounts of BMP6 are added to the carrier WBCD to accelerate and enhance bone formation, as evidenced in preclinical models of bone repair [58,63].

BMPs in inflammatory vascular diseases Atherosclerosis is a chronic inflammatory disease of the arterial wall preferentially occurring in lesion-prone areas and leading to regional ischemia and ischemic complications such as a stroke and myocardial infarction [64,65]. The earliest measurable markers of atherogenesis include the expression of inflammatory adhesion molecules, the loss of bioavailable nitric oxide (NO) production, and an increase in reactive oxygen species (ROS) levels from NADPH oxidases. BMPs modulate the endothelial cell (EC) inflammation and differentiation in response to proatherogenic oscillatory shear stress, oxidative stress and proinflammatory cytokines [66]. Once up-regulated, BMP2 and BMP4 induce a proinflammatory endothelial phenotype resulting in an enhanced leukocyte adhesion to the endothelial surface in vitro [67]. BMP2 expression is, for example, increased by exposure of endothelial cells to proinflammatory stimuli such as TNFα and production of endothelial microparticles (EMPs) which release BMP2 and calcium after damage of endothelial cells causing osteogenic differentiation of vascular smooth muscle cells (VSMCS) [68]. Accordingly, chronic infusion of BMP4 in mice leads to endothelial dysfunction and arterial hypertension [69]. It has been shown that BMP4 is increased in response to a high fat diet which then leads to upregulated BMP2 and -4 expression and an proinflammatory effect on the endothelium surface [70,71]. Initially ECs in lesion-prone areas experience unstable flow conditions, such as low shear stress and oscillatory shear stress (OS), which induces BMP4 expression and stimulates the production of ROS in a nox1- and p47phox-dependent manner. ROS then stimulates ICAM-1 expression and monocyte binding, leading to the foam cell (FC) formation and atherosclerosis. The discovery of BMP4 as a mechanosensitive, proinflammatory cytokine stimulating ROS production provides not only a better understanding of the role of shear stress in vascular biology and pathophysiology, but also an opportunity for the development of novel diagnostic and therapeutic approaches. BMP4 exerts prooxidant, prohypertensive, and proinflammatory effects only in the systemic circulation, whereas pulmonary arteries are protected from such BMP4-related adverse effects. The vascular bed-specific 11

BMP review for CGFR endothelial effects of BMP4 are likely to contribute to its differential pathophysiological role in the systemic vs pulmonary circulation. Mutation of the BMP type II receptor (BMPRII) underlies most cases of heritable pulmonary arterial hypertension (PAH). Pulmonary artery smooth muscle cells from patients with PAH exhibit attenuated growth suppression by BMP and abnormal mitogen response to TGFβ1 which is associated with inappropriately altered NF-κβ signalling and enhanced induction of IL-6 and IL-8 [72]. Knockdown of BMPRII in endothelial cells induced monocyte adhesion through expression ICAM-1 and VCAM-1 (Figure 2). Loss of BMPRII induced endothelial inflammation and atherosclerosis, while BMPRII expression was gradually lost in the progression of human coronary artery atherosclerosis [73].

Figure 2. The role of BMPs in inflammatory vascular disease. Oxidative and oscillatory shear stress (OS) together with proinflammatory cytokines induce BMP2 and -4 up regulation resulting in an increased reactive oxygen species (ROS) production, intercellular adhesion molecule-1 (ICAM-1) expression, monocyte (Mo) binding and adhesion, leading to the foam cell (FC) formation and atherosclerosis. EC- endothelial cell, M- tunica media, APF- atherosclerotic plaque formation. BMP signalling is modulated by extracellular ligand antagonists and a large number of regulatory proteins such as BMP endothelial precursor cell– derived regulator (BMPER) and Matrix GLA (MGP). BMPER is a secreted glycoprotein that binds directly to BMPs and modulates their function in a dose dependent manner. BMPER has anti-inflammatory properties and protective effects against BMP signalling, that controls inflammatory cell response by NFκB [67]. MGP antagonizes BMP activity through a direct protein-protein interaction. MGP binds and inhibits BMP2 and -4 effects. Overexpression of MGP limits the BMP activity, endothelial inflammation, atherosclerotic lesion formation and lesion calcification in a mouse model of atherosclerosis. In addition, increased MGP 12

BMP review for CGFR limits the expression of ALK1 and VEGF in atherosclerotic lesions. Loss of MGP results in an increased BMP activity. MGP deficiency leads to chondrogenic transdifferentiation and calcification of vascular smooth muscle cells (SMC) [66,70,71]. Recently, it has been shown that inhibition of BMP signalling in ApoE−/− mice by transgene expression of MGP resulted in a diminished Smad1/5/8 signalling, as well as a reduced inflammation, lesion formation, and calcification after fat feeding [66]. On the other hand, MGP deficient ApoE−/− mice displayed enhanced Smad1/5/8 signalling and extensive calcification of the media [70]. TGFβ1, but not BMPs, activates Smad1/5 in macrophages, where Smad1/5 is responsible for proatherogenic effects. The balance between Smad1/5 and Smad2/3 signalling defines the outcome of the effect of TGFβ on atherosclerosis [74]. It has also been demonstrated that treatment with BMP7 caused a significant decrease in the levels of pro inflammatory cytokines IL-6, TNFα and monocyte chemotactic protein-1(MCP-1) [75]. BMP7 has a potential to polarize monocytes into M2 macrophages which are required for tissue response and potential treatment of atherosclerosis. Effect of angiotensin receptor blockade by valsartan and BMP inhibition by noggin was evaluated to demonstrate that noggin significantly reduces blood glucose levels, reactive oxygen species production in the aorta, and gene transcription and proteins expression of inflammatory molecules in the vascular wall [76]. Thus, the BMP pathway may be a potentially therapeutic target in diabetic inflammatory vascular disease.

THE ROLE OF BMP SIGNALLING IN PANCREAS DEVELOPMENT, BETA-CELL FUNCTION AND GLUCOSE HOMEOSTASIS The sedentary western lifestyle has lead to an explosion of the number of obese individuals and the prevalence of type 2 diabetes [77]. In obese and inactive individuals, the increased amounts of circulating free fatty acids and inflammatory cytokines lead to decreased insulin sensitivity in periferal tissues (reviewed in [78,79]). This puts pressure on the pancreatic beta-cells to secrete more insulin to maintain glucose homeostasis. In healthy individuals, the beta cells will adopt by increased proliferation and insulin secretion. As long as the beta-cells are able to keep up with the increasing needs for insulin, the blood glucose levels remain normal. Only when the beta-cells are no longer capable of producing sufficient amounts of insulin, hyperglycemia and diabetes develop [80]. Concordantly, a significant reduction in both beta-cell mass and function is observed in individuals with type 2 diabetes compared to normoglycemic individuals with the same insulin sensitivity [81]. There has been significant focus on understanding the nature of the beta-cell defects that lead to the development of diabetes. In this part we will focus on the role of BMPs in pancreatic beta-cells during pancreas development and in the progression towards type 2 diabetes. 13

BMP review for CGFR

BMP signalling in pancreas development Emerging data point to essential roles of BMPs in pancreatic beta-cell dysfunction associated with type 2 diabetes. Important lessons about the effect of BMP signalling on pancreatic endocrine cells can be learned from the characterization of the role of BMPs in pancreas development. Along with a plethora of growth factors, including Notch, Hedgehog, FGF and TGF-β family proteins, correctly timed BMP signalling is very central in early pancreas organogenesis and endocrine cell differentiation [82-85]. BMP2, 4, 5 and 7 are expressed in and secreted from the mesenchyme, which surrounds the developing pancreas [82,83,85]. Central transcription factors directing pancreas development are homeodomain transcription factors and members of the basic helix-loop-helix (bHLH) protein family. All pancreatic tissues arise from progenitors characterized by their expression of the homeodomain transcription factor Pdx1 [86]. After the origin of the early gut tube from the endoderm, the pancreas originates at first as a dorsal bud and later a ventral bud, which after rotation of the gut fuse to one organ [87,88]. Inhibition of BMP signalling by mutant receptors or Smad4 deletion affects ventral pancreas outgrowth but has less effect on the development of the dorsal pancreas [83,84]. Later Pdx1-Cre driven deletion of Smad4 has no effect on pancreas development [89]. Likewise, early inhibition of BMP signalling by noggin only in the adjacent mesenchyme severely restricts pancreatic epithelial branching and generation of pancreatic exocrine tissue [82,83]. Thus, it appears that the timing of BMP inhibition determines the outcome. Generally, inhibition of BMP signalling promotes endocrine cell development, whereas induction of Bmp signalling at a later stage inhibits both ventral and dorsal endocrine cell development [82,83]. In a study of chicken pancreas development, Smad1/5/8 phosphorylation is observed primarily in the mesenchyme surrounding the developing pancreas, indicating that BMP signalling in the mesenchyme may direct pancreas development through an indirect effect [82]. It appears that a short wave of BMP signalling is essential in early pancreas development, to secure proper pancreatic development, specifically of the epithelium and exocrine tissue. Bmp4 and BMPRIA expression is decreased at embryonic day 13-14 in mice, just when endocrine cells start to differentiate, and remains low throughout gestation [85]. In combination with secretion of Hedgehog ligands and Bmp inhibitors from the dorsal organizer and adjacent cells this properly allows the induction of Pdx1 and endocrine cell development [83]. In addition to the role as a regulator of Pdx1, BMP signalling induces expression of the four Inhibitor of DNA binding proteins (Id1-4) [90], which function as inhibitors of the central beta-cell factors Neurogenin3 (Ngn3) and NeuroD [91]. Timed and restricted Ngn3 expression is essential for 14

BMP review for CGFR directing cells into the endocrine linage [86], whereas NeuroD expression is essential for late stage beta-cell maturation [92]. Thus, BMP signalling is decreased when endocrine cell maturation progresses and underscores the need for spatial and strictly timed expression of BMPs and their inhibitiors during the pancreatic outgrowth and cell type specification. Interestingly, this has effectively been mimicked in the recently published protocols of embryonic stem cell to beta-cell differentiation [93-95], where inhibition of BMP signalling at late stage differentiation is essential to promote beta-cell maturation. In several studies designed to investigate the role of BMP signalling under pancreatic development, inhibition of Bmp signalling resulted in disturbed pancreatic development [82,85]. In contrast, Insulin-cre driven deletion of BMPRIA in beta-cells or transgene expression of Pdx1-cre driving a dominant negative BMPRIA lead to near normal macroscopic development of the pancreas. These mice exhibited a less differentiated islet cell gene expression profile and less glucose responsive betacells [96]. As in other studies, investigating pancreatic development, Insulin- or Pdx1 driven inhibition of Bmp signalling may not adversely affect pancreas development [89],, probably due to the late induction of the inhibition. On the other hand, Pdx1 driven overexpression of Bmp6 resulted in severe pancreas agenesis [85]. In future genetic animal models it is essential to be able to separate effects on beta-cell development and adult beta-cell function. Thus, it would be informative if future animal models are designed to allow undisturbed pancreas development followed by conditional knockout of Bmps and or their receptors in adulthood.

Expression and regulation of BMP signalling in adulthood/diabetes development As learned from the developmental studies, the late stage endocrine cell development specifies the transcription factor network needed to maintain functional maturity of pancreatic endocrine cells. Since the central factors Pdx1 and NeuroD are inhibited by BMP signalling one may consider BMP expression/signalling to be disallowed in the late stage differentiation and terminally differentiated endocrine cells. In adult mouse islets the BMPRIA as well as BMPRII are expressed in both alpha and beta-cells [96,97]. In addition, BMPRIB and the ligands BMP2, 3, 4, 6 and 7 are expressed, albeit at low levels, suggesting that external sources of BMPs are relevant activators of BMP signalling in the pancreas. Likewise, the SMAD proteins and targets like the Id-proteins are expressed [97-99]. Thus, the entire BMP signalling machinery is present in pancreatic islets. Interestingly, it appears that all the above mentioned proteins are expressed at similar or higher levels in pancreatic alpha-cells compared to beta-cells [97]. Yet nothing is known about the effect of BMP signalling and functional effects in pancreatic alpha-cells. 15

BMP review for CGFR BMP2 expression has been found to be upregulated by inflammatory cytokines in several tissues [40,100,101]. During the development of both type 1 and type 2 diabetes, the pancreatic islets are exposed to high or low grade inflammation [102-104]. Pancreatic beta-cells are very sensitive to inflammatory cytokines due to high expression of the IL-1R [105]. Exposure of both human and rodent pancreatic islets to inflammatory cytokines induces the expression of BMP2 [90,106,107]. In vitro, cytokine exposure of primary islets induced upregulation and secretion of BMP2 leads to upregulation of Id1-4 expression, indicating autocrine effects of the released BMP2 [107]. Moreover Bmp2 and Id1 expression is also increased by free fatty acids [107,108]. These observations are also reflected in increased expression of BMP2/4 and ID1 in islets from type 2 diabetic mice [90,99,109]. Moreover BMP2 expression in islets correlates positively with Hba1c levels in humans [110]. In clonal beta-cells expressing a HNF1α frameshift mutation known to cause mature onset diabetes of the young (MODY), BMP3 expression is decreased. This is associated with decreased Insulin expression [111]. Likewise, prolonged treatment of primary islets of Langerhans with Bmp4 decreases the expression of Bmp3, which is associated with beta-cell dysfunction but without effect on Insulin gene or protein expression [112].

Effect of BMPs on glucose homeostasis and pancreatic islet function Systemically, BMP expression and secretion may arise from several tissues, which respond to the inflammatory diabetogenic environment. BMP2/4 expression has been reported to be upregulated in arteries, kidneys, retina, and islets in the diabetic environment [76,90,113-115]. Systemic inhibition of BMP2/4 by noggin improves glucose homeostasis in type 2 diabetic mice. Whether this is caused by a direct effect on beta-cell function remains elusive until further investigation [76]. Insulin driven beta-cell specific deletion of BMPRIA, BMPRI, as well as a whole body, heterozygous BMPRIA null allele attenuated glucose homeostasis, indicating a positive role for BMP signalling in the regulation of glucose homeostasis [96,116]. In addition, transgenic expression of BMP4 in beta-cells, and systemic administration of BMP4 protein to adult mice was shown to stimulate insulin secretion [96]. A study of mice lacking the BMP inhibitor Sosdc1 similarly reported improved beta-cell function in vivo [117]. However, this was associated with decreased expression of BMP responsive genes [117]. Thus, although deletion of Sostdc1 should increase BMP signalling it appear that the increased beta-cell function was associated with reduced BMP signalling. A high throughput screen has identified BMP9 as a factor with the potential to regulate blood glucose homeostasis via inhibition of hepatic glucose production and activation of key enzymes of lipid metabolism in normal and diabetic mice. Moreover, BMP9 injections stimulated insulin secretion in vivo but had no effect on insulin secretion from beta-cell lines [118]. 16

BMP review for CGFR The conflicting conclusions from these studies may arise from developmental differences or from indirect effects of BMPs mediated through other tissues. The contribution of BMPs secreted locally in the pancreas versus circulating BMPs arising from other tissues is difficult to determine in vivo and therefore calls for ex vivo investigation of primary tissue. The functional role of BMP signalling in pancreatic beta-cells has recently been investigated in vitro [90,112]. Stimulation of primary pancreatic islets and a rodent insulinoma cell line with BMP4 inhibits glucose-stimulated insulin secretion and BMP2 and 4 concomitantly inhibit rodent beta-cell proliferation [90]. In addition, inhibition of endogenous release of BMP2 and 4 by the soluble BMPRIA-Fc improves both insulin secretion and basal and growth factor induced proliferation [90]. In mature pancreatic islets, the expression of BMP2 and 4 is low, but increases in response to inflammatory cytokines, free fatty acids as well as in the diabetic environment [90,107]. Inflammatory cytokines and BMP2/4 stimulation of primary pancreatic islets induce the expression of Id1-4 [90,107]. Id1 expression is also increased in islets from type 2 diabetic mice [99]. The Id proteins have been shown to bind to the bHLH protein NeuroD, which is essential for maintaining beta-cell maturity and function [92]. Interestingly, deletion of Id1 renders mice resistant to diet induced hyperglycemia [119], indicating that BMP signalling leading to Id1 expression is increased in the diabetic setting and promotes the development of diabetes. These mice also show increased energy expenditure and reduced insulin resistance in response to high fat feeding [120]. This may point to an effect of BMP signalling and Id1 expression in the regulation of insulin resistance. Conversely, in a mouse model heterozygous for the BMPRIA, the mice showed disturbed glucose homeostasis and signs of insulin resistance [116]. Thus in vivo, it is difficult to determine if the effects on glucose homeostasis are related to effects on insulin resistance or direct effects on beta-cell proliferation and function. Given that systemic noggin treatment improves glucose homeostasis in type 2 diabetic mice, there may be several targets adding to this effect [114]. In addition, it has recently been shown that transient stimulation of human pancreatic exocrine tissue with BMP4 or 7 results in differentiation of exocrine cells into insulin producing cells following removal of the BMP stimulus [121]. If this holds true it may explain some of the divergences between in vitro and in vivo findings. Future in vivo experiments allowing conditional deletion of BMP signalling in animal models will reveal the role of BMPs in specific tissues under diabetes development. Moreover, evaluation of systemic BMP inhibition in a diabetic animal model should allow more specific evaluation of effects on beta-cell function versus insulin resistance.

THE ROLE OF BONE MORPHOGENETIC PROTEINS IN BROWN ADIPOCYTE BIOLOGY AND ENERGY METABOLISM 17

BMP review for CGFR The worldwide increase of adiposity and its co-morbidities, particularly diabetes and cardiovascular complications, has made the development of therapeutic approaches an urgent necessity. Obesity develops as a consequence of excessive nutrient intake and lipid storage in white adipose tissue (WAT) [122]. Brown adipose tissue (BAT) is a specialized type of fat that controls systemic energy homeostasis through body temperature regulation [123]. This process, also known as thermogenesis, has been proposed as a target to treat metabolic disease. The purpose of this section is to summarize recent discoveries on the role of BMP signalling in brown adipocyte formation and function and to discuss its potential for the development of anti-obesity therapies. Brown adipocytes, unlike their white counterpart, are uniquely equipped with uncoupling protein (UCP)-1, a protein that dissociates the mitochondrial proton gradient from ATP production, generating heat instead [124,125]. Since the recent re-discovery of metabolically active BAT in adult humans [126-131], it has been suggested that brown fat is also involved in control of human adiposity [132,133]. Due to its remarkable capacity for nutrient uptake and combustion, BAT also appears to regulate glucose metabolism and insulin sensitivity [134-136], as well as lipid homeostasis [137]. Among the most intriguing recent developments in brown fat biology has been the recognition that two distinct populations of brown adipocytes exist: The classical brown adipocytes that are morphologically and functionally characterized by their abundance of mitochondria, multilocular lipid droplets, and expression of UCP1 at all times. Classical BAT is found in the scapular and subscapular as well as in the deeper neck regions of rodents and humans [123,138,139]. The second type of BAT, alternatively termed brite (brown-in-white) or beige fat, emerges within white adipose tissue in response to cold-induced stimulation of β-adrenergic signalling and potentially endocrine stimulation by other secreted factors [140-142]. This process has also been termed browning of WAT. It is currently assumed that brite/beige adipocytes can either transdifferentiate from pre-existing white adipocytes [143] and/or arise from de novo differentiation of WAT-resident progenitors after cold exposure [144,145]. Little is known about the heterogeneity of the adipogenic progenitor cells in white adipose tissue, but recent studies suggest that only a subset of progenitors may possess brown adipogenic potential. In response to cold, this population of cells could either differentiate directly into brite/beige cells or give rise to a sub-population of white adipocytes with the potential to convert into brite/beige adipocytes [142,146,147]. In line with their functional distinction, developmental lineage tracing studies have established that classical BAT of the interscapular location arises from progenitors that also give rise to skeletal muscle and dermis [148,149]. Their lineage is thus developmentally distinct from the progenitor cells that give rise to many, but not all depots of WAT [150].

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BMP review for CGFR Lastly, and despite the difference of their development origins, brown and white adipocyte biology can be divided into three distinct stages: (1) Commitment of a multipotent mesenchymal progenitor towards the adipogenic lineage; (2) clonal expansion/ differentiation of committed progenitors (the pre-adipocytes); and (3) the metabolic function of terminally differentiated, i.e. mature, adipocytes [122,151]. Based on this sequential progress, this overview will address the role of BMPs in the stages of adipogenic lineage commitment, differentiation, and mature adipocyte function specializing primarily on the classical brown as well as the brite/beige adipocytes (summarized in Figure 3).

19

BMP review for CGFR

Figure 3. Brown adipogenesis and BMPs. Brown adipogenesis can be subdivided into three distinct stages: Lineage committment, adipogenic differentiation and function, i.e. thermogenesis. Distinctr members of the BMP-ligand family can promote (red) or inhibit (blue) these individual sub-stages of brown adipogenesis. The underlying mechanisms include canonical signalling processes and interaction with important transcription factors. (Abbreviations: EBF2: Early B-cell factor 2; PPARγ: Peroxisome proliferator-activated receptor gamma; PRDM16: PR domain containing 16; ROCK: Rho-associated protein kinase; ZFP423: Zinc finger protein 423) 20

BMP review for CGFR The role of BMPs in embryonic lineage commitment of brown adipogenic progenitors All major developmental pathways, e.g. BMP-, FGF-, Hedgehog-, Notch- and Wnt-signalling have been implicated in the development of brown adipocytes over the past years (reviewed in [152]). The role of BMPs in brown adipocyte development was initially recognized following the observation that BMP7-deficient mice display a marked impairment of BAT formation during embryogenesis [153]. It should be noted that loss of BMP7 results in perinatal lethality due to a variety of defects unrelated to BAT, somewhat complicating the interpretation of these observations. However, it was also determined that pre-treatment of a multipotent progenitor cell line, C3H10T1/2, with BMP7 prior to adipogenic differentiation committed these cells towards a brown adipogenic fate [153]. Similar results were obtained in primary adipogenic progenitor cells isolated from BAT, WAT and skeletal muscle [154]. These data indicate that BMP7 plays a critical role in the initial commitment steps towards brown adipogenesis. Of note, several studies have so far confirmed that BMP7, and also BMP4, promote brown adipogenesis in human pre-adipocytes, altogether suggesting that BMP signalling generally may act through different BMP ligands to direct this process [155,156]. Analysis of the downstream signalling cascade revealed that two type 1 BMP receptors, Activin A receptor type 1 (ACVR1), and BMPRIA), but not BMPRIB are required for normal embryonic development of BAT [157]. In this study, both receptors were separately deleted in the developing skeletal muscle and BAT of mice by intercrossing animals carrying floxed alleles for the respective receptors with the Cre-driver under control of the myf5 gene promoter (Myf5-Cre). It was previously demonstrated that Cre-drivers that are active during early embryonic myogenesis, such as Myf5-Cre and Pax7-Cre, concomitantly cause recombination in the common myo-adipogenic stem cells localized in the dermomyotome. These cells give rise to the myogenic and classical brown adipogenic lineages during embryogenesis [148,149]. Animals displayed a marked reduction of BAT size at birth and throughout life. Reduced BAT size was confirmed during embryonic development for mice with Myf5-Cre driven deletion of BMPRIA. Moreover, it could be determined that loss of BMPRIA leads to reduced proliferation of the developing brown fat pad at embryonic day 16.5 (E16.5). Apoptosis, on the other hand, remained unchanged in the dermomyotomal areas from where BAT derives throughout the second half of embryogenesis, i.e. from E9.5 to postnatal day 1 (P1). These findings taken together indicate that BMP signalling is required for the expansion steps of the developing BAT during embryogenesis [157]. Interestingly, ablation of BMPRIA or ACVR1 in cultured brown preadipocytes reduced the differentiation capacity alone and following pre-treatment with BMP7, and differentiation was completely abolished in cells lacking both receptors further supporting that intact BMP signalling is required for brown pre-adipocyte differentiation [157].

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BMP review for CGFR Likewise, pre-treatment of embryoid bodies (EBs) generated from mouse embryonic stem cells with BMP4 promotes to formation of adipocytes when EBs were seeded onto gelatin-coated tissue culture plates [158]. These findings indicate that BMPs are involved in the early steps of pre-adipocyte determination. Given that in many cases brown adipogenic potential was not directly determined, the underlying mechanisms and the identity of the different BMP ligands which play a role under in vivo settings warrant further investigation. Two transcription factors, PR domain containing (Prdm)-16 and early B-cell factor (Ebf)-2, have recently been implicated in the lineage determination processes of the developing BAT [146,149]. Interestingly, some experimental evidence provides support for the notion that these transcription factors may interact with BMP signalling [153,159,160]. This in turn would suggest that these signals may act in concert with BMPs to direct the initial commitment steps of the myo-adipogenic progenitors towards the embryonic BAT lineage. In this line, the transcription factor zinc finger protein (Zfp)-423 is involved in the commitment steps of early adipose progenitors in BAT and WAT [161,162]. Moreover, the ZFP423 protein possesses an interaction domain for the protein SMAD4, a canonical BMP target, and its expression can additionally be induced by treatment with BMP4. The authors go on to show that while the involvement in adipogenic lineage commitment of Zfp423 appears to be independent of the SMAD4-interaction, its capacity to promote adipogenic differentiation absolutely depends on this interaction [161].

The role of BMPs in the molecular regulation of (brown) pre-adipocyte differentiation The role of BMPs and other members of the transforming growth factor (TGF)-β superfamily in the molecular regulation of (white) adipocyte differentiation has been a focus of recent reviews [163,164]. With regard to white adipocyte formation, it has been demonstrated repeatedly that BMPs, BMP-2 and -4, and -7 among them, are critical regulators of the developmental fate decision between osteogenic and adipogenic lineages [151,165-167]. The signalling biology underlying these processes is very complex and the dual role of some BMPs in promoting adipogenesis and/or osteogenesis relies on differences in the local concentrations of BMPs and differential expression of individual BMP receptors [151,166,168]. In relation to possible regulation of brown adipogenesis, the interpretation of many early studies is somewhat impeded due to the use of the 3T3L1-cell line which possesses very little or no brown adipogenic potential. Secondly, and with the exception of more recent studies, the expression of brown adipocyte markers such as UCP1 was simply not assessed when other cell models, such as C3H10T1/2 cells, were used. Here, BMP4 was initially described as a factor that drives adipogenic commitment and differentiation into white adipocytes [151,169,170], a conclusion that is also 22

BMP review for CGFR supported by the demonstration that BMP4 suppresses UCP1-expression in brown pre-adipocytes [153]. While these studies were all conducted in murine cells, the situation appears to be more complicated in human pre-adipocytes where both BMP4 and -7 can promote brown adipogenesis [155,156]. The differentiation of pre-adipocytes into mature brown or white adipocytes requires induction of two major pro-adipogenic transcription factors, Peroxisome proliferator-activated receptor (PPAR)γ and CCAAT/enhancer-binding protein (C/EBP)-α [151]. As discussed above, the physical interaction of the ZFP423 with BMP4-induced SMAD4 proteins amplifies induction of PPARγ, thus providing a mechanism by which BMPs could directly affect adipogenic differentiation [161]. While many aspects of the transcriptional control of these early adipogenic events are similar in brown and white adipogenesis, several factors have been identified that specifically promote a brown adipogenic phenotype. Among these, Prdm16, and additionally PPARγ-coactivator (PGC)-1α, have been shown to be master regulators of the brown adipogenic differentiation program: these factors activate mitochondrial biogenesis and expression of brown adipocyte specific proteins, such as UCP1[122,149,171]. Interestingly, BMP7 can induce expression of all above mentioned transcription factors in pre-adipocytes [153,154]. Canonical BMP signalling is relayed through the Smad and p38 mitogen activated protein kinase (p38 MAPK) signalling cascades [172]. Since p38 MAPK is an upstream regulator of PGC1α, the direct link between BMPs and this cascade is straightforward [173,174]. Accordingly, inhibition of p38 MAPK blocks the brown adipogenic effects of BMP7 [153]. The situation is more intricate with Smad signalling: R-SMAD proteins can be sub-divided into SMAD1/5/8 which relay the anti-proliferative, pro-adipogenic effects of BMPs, and SMAD2/3, which mediate the pro-proliferative, anti-adipogenic effects of Activins and transforming growth factor (TGF)-β, respectively (reviewed in [164,172]). While the majority of these observations have been made in white adipocytes, emerging evidence indicates that this is also true for BAT. For instance, the brown adipogenic effects of BMP7 are, at least in part, mediated by SMAD1/5/8 [153,157,175]. Interestingly, the neuropeptide orexin, presumably through activation of the G-protein α-subunit Gq, appears to stimulate brown adipogenesis through a BMPRIA-dependent activation of SMAD1/5 signalling [176]. The negative effects of TGFβ on brown adipogenesis are mediated by SMAD3: Loss of this transcription factor promotes brown adipogenesis by de-repressing PGC1α, as did antibody-mediated inactivation of TGFβ1 [177]. Pharmacological inhibition of Activin receptor IIB (ActRIIB), the main receptor for growth and differentiation factor-8 (GDF8; also known as myostatin), also increased BAT mass and energy expenditure via inhibition of SMAD3, a process that was not observed in WAT [178]. Although this approach also resulted in increased muscle mass, further cell culture analyses 23

BMP review for CGFR established a cell-autonomous mechanism where inhibition of ActRIIB induced expression of UCP1 in brown adipocytes. Accordingly, exposure of progenitors to GDF8 inhibited brown adipocyte marker expression. Intriguingly, BMP7 (also: BMPs -2 and -4) and GDF8 countered each other’s activating effects on reporters for SMAD1/5/8 and SMAD2/3, respectively, suggesting that these to pathways closely interact to control brown adipocyte maturation [178]. Likewise, treatment with TGFβ1 and Activin A markedly inhibited brown adipogenic gene expression [179]. It should be noted that no effects of BMP7 on brown adipogenesis were observed in this study. Meanwhile, since a cell line deficient for the tumor suppressor p53 was used, a factor that appears to be required for brown adipogenesis [180], the mechanisms by which BMP7 induces brown adipogenesis could also be perturbed.

The role of BMPs in browning of WAT The mechanisms discussed above specifically refer to the role of BMP signalling in classical brown adipocytes. Despite the developmental distinctions of BAT and brite/beige fat, these processes are often mirrored by similar effects on the browning mechanisms of WAT. Accordingly, it was recently demonstrated that BMP7 acts through a pathway that involves inhibition of Rho-associated protein kinase (ROCK)-activity and changed G- over F-actin dynamics [181]. This in turn led to inhibition of the downstream target of this process, myocardin-related transcription factor A (MRTFA). Interestingly, loss of MRTFA also resulted in a highly significant induction of browning of WAT and increased energy expenditure and resistance to diet-induced obesity [181]. Other BMPs have also implicated in browning. Surprisingly, over-expression of BMP4 under control of the adipocyte protein-2 (aP2)-promoter markedly increased browning of WAT by targeting PGC1α. On the other hand, adipose tissue-specific deletion of BMP4 decreased brown adipogenesis in WAT and BAT [182]. These findings demonstrate impressively that the composition of the local microenvironment plays a critical role in the specific effects of individual BMPs and it remains to be determined whether BMP4 acts directly on the adipogenic progenitor cells or converts mature white into brite/beige adipocytes. Other members of the TGFβ superfamily have also been implicated in this process. For instance, it was recently shown that a recombinant BMP9-derivative, MB109, promotes brown adipocyte gene expression in human pre-adipocytes, enhances browning of WAT and improves glucose homeostasis following systemic administration [183]. GDF5 not only induced thermogenesis in classical BAT but also promoted browning of subcutaneous WAT. This in turn increased systemic energy expenditure and led to a lean phenotype during high fat diet feeding of mice [184,185]. Similarly, and as discussed above for classical BAT, loss of GDF8 resulted in browning of WAT [186]. The negative regulator 24

BMP review for CGFR of BAT, the TGFβ/SMAD3 axis, also appears to inhibit browning. Hence, genetic ablation of SMAD3 also conferred beneficial metabolic features to WAT, such as increased expression of UCP1, higher rates of fatty acid oxidation and increased resistance to cold [177]. These findings taken together suggest that modulation of BMP/TGFβ signalling might be a feasible approach to convert white into energy expending brite/beige adipocytes, thereby improving the metabolic pathology of obesity and its associated diseases. A number of BMPs and other members of the TGFβ-superfamily have been linked to browning. However, it was recently pointed out that many of the browning factors identified to date warrant further detailed evaluation [127]. Specifically, all factors require analysis of their browning capacity under different temperature regimes, i.e. cold and thermoneutrality, to determine whether they can act in concert with or independent of the main driver of brown adipocyte thermogenesis, the sympathetic nervous system and activation of the β-adrenergic receptors [127].

Role of BMPs in thermogenesis and control of energy homeostasis Finally, a subject deserving special attention is the role of centrally administered BMPs in energy balance. An elegant study by Whittle et al. established that BMP8b acts locally in BAT and centrally to induce thermogenesis [187]. In the periphery, expression of BMP8b is induced by cold and other thermogenic regimens in the classical BAT. BMP8b null mice displayed a marked propensity for diet-induced adiposity and their BAT showed defective lipolysis. The authors go on to demonstrate that BMP8b sensitized brown adipocytes to sympathetic input via the adrenergic receptors. If injected into the hypothalamus, BMP8b inhibits AMP-activated protein kinase (AMPK) signalling in the ventromedial hypothalamus which results in increased sympathetic tone and BAT-mediated energy expenditure [187]. These findings taken together demonstrate the role of BMPs for regulation of thermogenesis locally in BAT as well as centrally via hypothalamic mechanisms. Of note, intracerebroventricular injections of BMP7 markedly inhibited food intake via mammalian target of rapamycin (mTOR) signalling and independent of leptin-action, highlighting yet another role of BMPs in energy homeostasis [188]. In mature adipocytes, BMP7 seems to play a similar role as BM8b: Treatment of mature brown adipocytes with this BMP increased mitochondrial activity and fatty acid uptake [189]. Systemic administration of BMP7 resulted in increased expression of UCP1 in WAT depots. Importantly, this effect was only observed when BMP7 was injected in combination with the β3-adrenergic receptor agonist, CL316,243, [154]. This was further corroborated by an interesting study showing that BMP7’s positive effects on thermogenesis, BAT activity, lipolysis, and browning of WAT were only evident at sub-thermoneutrality [190]. Thermoneutrality is defined as the temperature at which homeotherms are no longer required to expend energy to maintain their body temperature. The results on BMP8b and BMP7 taken together suggest that BMP signalling 25

BMP review for CGFR could act as a sensitizer of adrenergic signalling in mature brown and brite/beige adipocytes, thereby increasing the response to sympathetic stimulation. This principle may in the future hold potential for the development of treatment strategies that target the sympathetic input locally, i.e. in the level of the adipocyte.

Perspectives Involvement of BMPs in various metabolic functions raises the question of the necessity for organspecific strategies for therapeutic modulation of BMP signalling. For instance, inhibition of BMP action in several organ systems may help to reduce pro-fibrotic and pro-inflammatory processes. Moreover, individual BMP signalling components display highly distinct effects on pancreatic development, function and adult glucose homeostasis. This altogether suggests that BMPs act on a cell type-specific rather than tissue-specific level in the pancreas and likely also in other organs. Additionally, BMPs appear to be required for the formation of brown adipocytes and browning of WAT, and the related benefits for metabolic health, adding yet another layer of physiological complexity. Future studies are required to further address the role of BMPs outside the classical musculoskeletal system to attain (i) a cell type-specific resolution of the activation/ inhibition mechanisms for this signalling cascade; and (ii) a better understanding of potential by-effects in nontarget tissues during therapeutic interventions involving BMPs.

Acknowledgements L.G. is supported by the Croatian Science Foundation (grant No. O-1259-2015). T.J.S. is supported by the German Research Foundation (grant No. SCHU 2445/2-1), and the European Research Council (ERC StG No. 311082). T.J.S is also supported by the German Center for Diabetes Research (DZD). G.L.C. holds a post-doctoral grant from the Danish Diabetes Academy supported by the Novo Nordisk Foundation. S. V. is supported by the FP7 HEALTH cooperative project (grant agreement No. 279239) and the Croatian Scientific Center of Excellence for Reproductive and Regenerative Medicine.

Conflicts of interest: The authors declare no conflicts of interest.

References 1. Nakashima M, Reddi AH. The application of bone morphogenetic proteins to dental tissue engineering. Nat Biotechnol 2003; 21:1025-1032. 26

BMP review for CGFR 2. Sanchez-Duffhues G, Hiepen C, Knaus P, Ten DP. Bone morphogenetic protein signaling in bone homeostasis. Bone 2015; 80:43-59. 3. Vukicevic S, Stavljenic A, Pecina M. Discovery and clinical applications of bone morphogenetic proteins. Eur J Clin Chem Clin Biochem 1995; 33:661-671. 4. Sieber C, Kopf J, Hiepen C, Knaus P. Recent advances in BMP receptor signaling. Cytokine Growth Factor Rev 2009; 20:343-355. 5. Korchynskyi O, van Bezooijen RL, Lowik CWGM, ten Dijke P. Bone morphogenetic protein receptors and their nuclear effectors in bone formation. In: Vukicevic S, Sampath TK (editors). Bone morphogenetic proteins: regeneration of bone and beyond. Basel-Boston-Berlin:Birkauser Verlag; 2004, p.9-114. 6. Vukicevic S, Grgurevic L. Bone Morphogenetic Proteins in Inflammation. In: Parnham M (editors). Encyclopedia of Inflammatory Diseases. Basel:Springer Verlag; 2015, p.1-15. 7. Bataller R, Brenner DA. Liver fibrosis. J Clin Invest 2005; 115:209-218. 8. Ramachandran P, Iredale JP. Liver fibrosis: a bidirectional model of fibrogenesis and resolution. QJM 2012; 105:813-817. 9. Povero D, Busletta C, Novo E, di Bonzo LV, Cannito S, Paternostro C, et al. Liver fibrosis: a dynamic and potentially reversible process. Histol Histopathol 2010; 25:1075-1091. 10. Zeisberg M, Hanai J, Sugimoto H, Mammoto T, Charytan D, Strutz F, et al. BMP-7 counteracts TGF-beta1-induced epithelial-to-mesenchymal transition and reverses chronic renal injury. Nat Med 2003; 9:964-968. 11. Bi J, Ge S. Potential roles of BMP9 in liver fibrosis. Int J Mol Sci 2014; 15:20656-20667. 12. Sugimoto H, Yang C, LeBleu VS, Soubasakos MA, Giraldo M, Zeisberg M, et al. BMP-7 functions as a novel hormone to facilitate liver regeneration. FASEB J 2007; 21:256-264. 13. Cao H, Shu X, Chen LB, Zhang K, Xu QH, Li G. [The relationship of expression of BMP-7 in the liver and hepatic inflammation and fibrosis in patients with chronic HBV infection]. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi 2010; 24:101-103. 14. Hao ZM, Cai M, Lv YF, Huang YH, Li HH. Oral administration of recombinant adeno-associated virus-mediated bone morphogenetic protein-7 suppresses CCl(4)-induced hepatic fibrosis in mice. Mol Ther 2012; 20:2043-2051. 15. Li B, Shao Q, Ji D, Li F, Chen G. Mesenchymal stem cells mitigate cirrhosis through BMP7. Cell Physiol Biochem 2015; 35:433-440. 16. Xu CP, Ji WM, van den Brink GR, Peppelenbosch MP. Bone morphogenetic protein-2 is a negative regulator of hepatocyte proliferation downregulated in the regenerating liver. World J Gastroenterol 2006; 12:7621-7625. 27

BMP review for CGFR 17. Gao X, Cao Y, Yang W, Duan C, Aronson JF, Rastellini C, et al. BMP2 inhibits TGF-betainduced pancreatic stellate cell activation and extracellular matrix formation. Am J Physiol Gastrointest Liver Physiol 2013; 304:G804-G813. 18. Takabayashi H, Shinohara M, Mao M, Phaosawasdi P, El-Zaatari M, Zhang M, et al. Antiinflammatory activity of bone morphogenetic protein signaling pathways in stomachs of mice. Gastroenterology 2014; 147:396-406. 19. Jiang M, Ku WY, Zhou Z, Dellon ES, Falk GW, Nakagawa H, et al. BMP-driven NRF2 activation in esophageal basal cell differentiation and eosinophilic esophagitis. J Clin Invest 2015; 125:1557-1568. 20. Batts LE, Polk DB, Dubois RN, Kulessa H. Bmp signaling is required for intestinal growth and morphogenesis. Dev Dyn 2006; 235:1563-1570. 21. Smith PJ, Levine AP, Dunne J, Guilhamon P, Turmaine M, Sewell GW, et al. Mucosal transcriptomics implicates under expression of BRINP3 in the pathogenesis of ulcerative colitis. Inflamm Bowel Dis 2014; 20:1802-1812. 22. Fiocchi C. Early and late inflammatory bowel disease: why and how are they different? Curr Opin Gastroenterol 2011; 27:317-320. 23. Maric I, Poljak L, Zoricic S, Bobinac D, Bosukonda D, Sampath KT, et al. Bone morphogenetic protein-7 reduces the severity of colon tissue damage and accelerates the healing of inflammatory bowel disease in rats. J Cell Physiol 2003; 196:258-264. 24. Monteleone G, Pallone F, MacDonald TT. Smad7 in TGF-beta-mediated negative regulation of gut inflammation. Trends Immunol 2004; 25:513-517. 25. Latella G, Vetuschi A, Sferra R, Zanninelli G, D'Angelo A, Catitti V, et al. Smad3 loss confers resistance to the development of trinitrobenzene sulfonic acid-induced colorectal fibrosis. Eur J Clin Invest 2009; 39:145-156. 26. Hao Z, Yang X, Lv Y, Li S, Purbey BK, Su H. Intracolonically administered adeno-associated virus-bone morphogenetic protein-7 ameliorates dextran sulphate sodium-induced acute colitis in rats. J Gene Med 2012; 14:482-490. 27. Hadad A. SMAD7: un oligonucletido antisentido oral para el tratamiento de la enfermedad de Crohn. Enfermedad Inflamatoria Intestinal al D+şa 2015; 14:39-40. 28. Monteleone G, Neurath MF, Ardizzone S, Di SA, Fantini MC, Castiglione F, et al. Mongersen, an oral SMAD7 antisense oligonucleotide, and Crohn's disease. N Engl J Med 2015; 372:1104-1113. 29. Hentze MW, Muckenthaler MU, Galy B, Camaschella C. Two to tango: regulation of Mammalian iron metabolism. Cell 2010; 142:24-38.

28

BMP review for CGFR 30. Wang L, Trebicka E, Fu Y, Ellenbogen S, Hong CC, Babitt JL, et al. The bone morphogenetic protein-hepcidin axis as a therapeutic target in inflammatory bowel disease. Inflamm Bowel Dis 2012; 18:112-119. 31. Gao J, Chen J, Kramer M, Tsukamoto H, Zhang AS, Enns CA. Interaction of the hereditary hemochromatosis protein HFE with transferrin receptor 2 is required for transferrin-induced hepcidin expression. Cell Metab 2009; 9:217-227. 32. Bagu ET, Layoun A, Calve A, Santos MM. Friend of GATA and GATA-6 modulate the transcriptional up-regulation of hepcidin in hepatocytes during inflammation. Biometals 2013; 26:1051-1065. 33. Pauk M, Grgurevic L, Brkljacic J, Kufner V, Bordukalo-Niksic T, Grabusic K, et al. Exogenous BMP7 corrects the plasma iron overload and bone loss in Bmp6-/- mice. Int Orthop 2014; 39:161172. 34.

Andriopoulos B, Jr., Corradini E, Xia Y, Faasse SA, Chen S, Grgurevic L, et al. BMP6 is a key endogenous regulator of hepcidin expression and iron metabolism. Nat Genet 2009; 41:482-487.

35. Shanmugam NK, Ellenbogen S, Trebicka E, Wang L, Mukhopadhyay S, Lacy-Hulbert A, et al. Tumor necrosis factor alpha inhibits expression of the iron regulating hormone hepcidin in murine models of innate colitis. PLoS One 2012; 7:e3813636. Park MC, Chung SJ, Park YB, Lee SK. Relationship of angiogenic factors to disease activity and radiographic damage in rheumatoid arthritis. Clin Exp Rheumatol 2008; 26:881-886. 37. Biver E, Hardouin P, Caverzasio J. The "bone morphogenic proteins" pathways in bone and joint diseases: translational perspectives from physiopathology to therapeutic targets. Cytokine Growth Factor Rev 2013; 24:69-81. 38. Grcevic D, Jajic Z, Kovacic N, Lukic IK, Velagic V, Grubisic F, et al. Peripheral blood expression profiles of bone morphogenetic proteins, tumor necrosis factor-superfamily molecules, and transcription factor Runx2 could be used as markers of the form of arthritis, disease activity, and therapeutic responsiveness. J Rheumatol 2010; 37:246-256. 39. Drexler SK, Kong PL, Wales J, Foxwell BM. Cell signalling in macrophages, the principal innate immune effector cells of rheumatoid arthritis. Arthritis Res Ther 2008; 10:21640. Lories RJ, Derese I, Ceuppens JL, Luyten FP. Bone morphogenetic proteins 2 and 6, expressed in arthritic synovium, are regulated by proinflammatory cytokines and differentially modulate fibroblast-like synoviocyte apoptosis. Arthritis Rheum 2003; 48:2807-2818. 41. Takahashi T, Muneta T, Tsuji K, Sekiya I. BMP-7 inhibits cartilage degeneration through suppression of inflammation in rat zymosan-induced arthritis. Cell Tissue Res 2011; 344:321-332. 29

BMP review for CGFR 42. Bramlage CP, Haupl T, Kaps C, Ungethum U, Krenn V, Pruss A, et al. Decrease in expression of bone morphogenetic proteins 4 and 5 in synovial tissue of patients with osteoarthritis and rheumatoid arthritis. Arthritis Res Ther 2006; 8:R5843. Guo X, Wang XF. Signaling cross-talk between TGF-beta/BMP and other pathways. Cell Res 2009; 19:71-88. 44. Grazio S, Razdorov G, Erjavec I, Grubisic F, Kusic Z, Punda M, et al. Differential expression of proteins with heparin affinity in patients with rheumatoid and psoriatic arthritis: a preliminary study. Clin Exp Rheumatol 2013; 31:665-671. 45. Verschueren PC, Lories RJ, Daans M, Theate I, Durez P, Westhovens R, et al. Detection, identification and in vivo treatment responsiveness of bone morphogenetic protein (BMP)-activated cell populations in the synovium of patients with rheumatoid arthritis. Ann Rheum Dis 2009; 68:117123. 46. Machado P, Landewe R, Braun J, Hermann KG, Baker D, van der Heijde D. Both structural damage and inflammation of the spine contribute to impairment of spinal mobility in patients with ankylosing spondylitis. Ann Rheum Dis 2010; 69:1465-1470. 47. Carter S, Lories RJ. Osteoporosis: a paradox in ankylosing spondylitis. Curr Osteoporos Rep 2011; 9:112-115. 48. Chen MH, Chen HA, Chen WS, Chen MH, Tsai CY, Chou CT. Upregulation of BMP-2 expression in peripheral blood mononuclear cells by proinflammatory cytokines and radiographic progression in ankylosing spondylitis. Mod Rheumatol 2015; 1-6. 49. Chubinskaya S, Hurtig M, Rueger DC. OP-1/BMP-7 in cartilage repair. Int Orthop 2007; 31:773781. 50. Chubinskaya S, Segalite D, Pikovsky D, Hakimiyan AA, Rueger DC. Effects induced by BMPS in cultures of human articular chondrocytes: comparative studies. Growth Factors 2008; 26:275-283. 51. Daans M, Lories RJ, Luyten FP. Dynamic activation of bone morphogenetic protein signaling in collagen-induced arthritis supports their role in joint homeostasis and disease. Arthritis Res Ther 2008; 10:R11552. Korkosz M, Gasowski J, Leszczynski P, Pawlak-Bus K, Jeka S, Siedlar M, et al. Effect of tumour necrosis factor-alpha inhibitor on serum level of dickkopf-1 protein and bone morphogenetic protein7 in ankylosing spondylitis patients with high disease activity. Scand J Rheumatol 2014; 43:43-48. 53. Tsui FW, Tsui HW, Las HF, Pritzker KP, Inman RD. Serum levels of novel noggin and sclerostin-immune complexes are elevated in ankylosing spondylitis. Ann Rheum Dis 2014; 73:18731879.

30

BMP review for CGFR 54. Hatsell SJ, Idone V, Wolken DM, Huang L, Kim HJ, Wang L, et al. ACVR1R206H receptor mutation causes fibrodysplasia ossificans progressiva by imparting responsiveness to activin A. Sci Transl Med 2015; 7:303ra13755. Kaplan FS, Pignolo RJ, Shore EM. From mysteries to medicines: drug development for fibrodysplasia ossificans progressive. Expert Opin Orphan Drugs 2013; 1:637-649. 56. White AP, Vaccaro AR, Hall JA, Whang PG, Friel BC, McKee MD. Clinical applications of BMP-7/OP-1 in fractures, nonunions and spinal fusion. Int Orthop 2007; 31:735-741. 57. Moghaddam A, Elleser C, Biglari B, Wentzensen A, Zimmermann G. Clinical application of BMP 7 in long bone non-unions. Arch Orthop Trauma Surg 2010; 130:71-76. 58. Vukicevic S, Oppermann H, Verbanac D, Jankolija M, Popek I, Curak J, et al. The clinical use of bone morphogenetic proteins revisited: a novel biocompatible carrier device OSTEOGROW for bone healing. Int Orthop 2014; 38:635-647. 59. Zara JN, Siu RK, Zhang X, Shen J, Ngo R, Lee M, et al. High doses of bone morphogenetic protein 2 induce structurally abnormal bone and inflammation in vivo. Tissue Eng Part A 2011; 17:1389-1399. 60. Sagi HC, Jordan CJ, Barei DP, Serrano-Riera R, Steverson B. Indomethacin prophylaxis for heterotopic ossification after acetabular fracture surgery increases the risk for nonunion of the posterior wall. J Orthop Trauma 2014; 28:377-383. 61. Lee KB, Taghavi CE, Murray SS, Song KJ, Keorochana G, Wang JC. BMP induced inflammation: a comparison of rhBMP-7 and rhBMP-2. J Orthop Res 2012; 30:1985-1994. 62. Dumic-Cule I, Brkljacic J, Rogic D, Bordukalo-Niksic T, Tikvica Luetic A, Draca N, et al. Systemically available bone morphogenetic protein 2 and 7 affect bone metabolism. Int Orthop 2014; 63. Peric M, Dumic-Cule I, Grcevic D, Matijasic M, Verbanac D, Grgurevic L, et al. The rational use of animal models in the evaluation of novel bone regenerative therapies. Bone 2015; 70:70-86. 64. Ross R. Atherosclerosis--an inflammatory disease. N Engl J Med 1999; 340:115-126. 65. Libby P, Ridker PM, Maseri A. Inflammation and atherosclerosis. Circulation 2002; 105:11351143. 66. Yao Y, Bennett BJ, Wang X, Rosenfeld ME, Giachelli C, Lusis AJ, et al. Inhibition of bone morphogenetic proteins protects against atherosclerosis and vascular calcification. Circ Res 2010; 107:485-494. 67. Helbing T, Rothweiler R, Ketterer E, Goetz L, Heinke J, Grundmann S, et al. BMP activity controlled by BMPER regulates the proinflammatory phenotype of endothelium. Blood 2011; 118:5040-5049.

31

BMP review for CGFR 68. Buendia P, Montes de OA, Madueno JA, Merino A, Martin-Malo A, Aljama P, et al. Endothelial microparticles mediate inflammation-induced vascular calcification. FASEB J 2015; 29:173-181. 69. Miriyala S, Gongora Nieto MC, Mingone C, Smith D, Dikalov S, Harrison DG, et al. Bone morphogenic protein-4 induces hypertension in mice: role of noggin, vascular NADPH oxidases, and impaired vasorelaxation. Circulation 2006; 113:2818-2825. 70. Pardali E, ten Dijke P. TGFbeta signaling and cardiovascular diseases. Int J Biol Sci 2012; 8:195213. 71. Nakagawa Y, Ikeda K, Akakabe Y, Koide M, Uraoka M, Yutaka KT, et al. Paracrine osteogenic signals via bone morphogenetic protein-2 accelerate the atherosclerotic intimal calcification in vivo. Arterioscler Thromb Vasc Biol 2010; 30:1908-1915. 72. Davies RJ, Holmes AM, Deighton J, Long L, Yang X, Barker L, et al. BMP type II receptor deficiency confers resistance to growth inhibition by TGF-beta in pulmonary artery smooth muscle cells: role of proinflammatory cytokines. Am J Physiol Lung Cell Mol Physiol 2012; 302:L604-L615. 73. Simic T. Anti-inflammatory and anti-atherogenic role of BMP receptor II in atherosclerosis. Future Cardiol 2013; 9:619-622. 74. Nurgazieva D, Mickley A, Moganti K, Ming W, Ovsyi I, Popova A, et al. TGF-beta1, but not bone morphogenetic proteins, activates Smad1/5 pathway in primary human macrophages and induces expression of proatherogenic genes. J Immunol 2015; 194:709-718. 75. Rocher C, Singla R, Singal PK, Parthasarathy S, Singla DK. Bone morphogenetic protein 7 polarizes THP-1 cells into M2 macrophages. Can J Physiol Pharmacol 2012; 90:947-951. 76. Koga M, Engberding N, Dikalova AE, Chang KH, Seidel-Rogol B, Long JS, et al. The bone morphogenic protein inhibitor, noggin, reduces glycemia and vascular inflammation in db/db mice. Am J Physiol Heart Circ Physiol 2013; 305:H747-H755. 77. Danaei G, Finucane MM, Lu Y, Singh GM, Cowan MJ, Paciorek CJ, et al. National, regional, and global trends in fasting plasma glucose and diabetes prevalence since 1980: systematic analysis of health examination surveys and epidemiological studies with 370 country-years and 2.7 million participants. Lancet 2011; 378:31-40. 78. Lionetti L, Mollica MP, Lombardi A, Cavaliere G, Gifuni G, Barletta A. From chronic overnutrition to insulin resistance: the role of fat-storing capacity and inflammation. Nutr Metab Cardiovasc Dis 2009; 19:146-152. 79. Donath MY, Shoelson SE. Type 2 diabetes as an inflammatory disease. Nat Rev Immunol 2011; 11:98-107. 80. Weir GC, Bonner-Weir S. Five stages of evolving beta-cell dysfunction during progression to diabetes. Diabetes 2004; 53 Suppl 3:S16-S21. 32

BMP review for CGFR 81. Butler AE, Janson J, Bonner-Weir S, Ritzel R, Rizza RA, Butler PC. Beta-cell deficit and increased beta-cell apoptosis in humans with type 2 diabetes. Diabetes 2003; 52:102-110. 82. Ahnfelt-Ronne J, Ravassard P, Pardanaud-Glavieux C, Scharfmann R, Serup P. Mesenchymal bone morphogenetic protein signaling is required for normal pancreas development. Diabetes 2010; 59:1948-1956. 83. Chung WS, Andersson O, Row R, Kimelman D, Stainier DY. Suppression of Alk8-mediated Bmp signaling cell-autonomously induces pancreatic beta-cells in zebrafish. Proc Natl Acad Sci U S A 2010; 107:1142-1147. 84. Wandzioch E, Zaret KS. Dynamic signaling network for the specification of embryonic pancreas and liver progenitors. Science 2009; 324:1707-1710. 85. Dichmann DS, Miller CP, Jensen J, Scott Heller R, Serup P. Expression and misexpression of members of the FGF and TGFbeta families of growth factors in the developing mouse pancreas. Developmental Dynamics 2003; 226:663-674. 86. Gu G, Dubauskaite J, Melton DA. Direct evidence for the pancreatic lineage: NGN3+ cells are islet progenitors and are distinct from duct progenitors. Development 2002; 129:2447-2457. 87. Collombat P, Hecksher-Sorensen J, Serup P, Mansouri A. Specifying pancreatic endocrine cell fates. Mech Dev 2006; 123:501-512. 88. Gittes GK. Developmental biology of the pancreas: a comprehensive review. Dev Biol 2009; 326:4-35. 89. Bardeesy N, Cheng KH, Berger JH, Chu GC, Pahler J, Olson P, et al. Smad4 is dispensable for normal pancreas development yet critical in progression and tumor biology of pancreas cancer. Genes Dev 2006; 20:3130-3146. 90. Bruun C, Christensen GL, Jacobsen ML, Kanstrup MB, Jensen PR, Fjordvang H, et al. Inhibition of beta cell growth and function by bone morphogenetic proteins. Diabetologia 2014; 57:2546-2554. 91. Ghil SH, Jeon YJ, Suh-Kim H. Inhibition of BETA2/NeuroD by Id2. Exp Mol Med 2002; 34:367-373. 92. Gu C, Stein GH, Pan N, Goebbels S, Hornberg H, Nave KA, et al. Pancreatic beta cells require NeuroD to achieve and maintain functional maturity. Cell Metab 2010; 11:298-310. 93. Russ HA, Parent AV, Ringler JJ, Hennings TG, Nair GG, Shveygert M, et al. Controlled induction of human pancreatic progenitors produces functional beta-like cells in vitro. EMBO J 2015; 34:1759-1772. 94. Pagliuca FW, Millman JR, Gurtler M, Segel M, Van DA, Ryu JH, et al. Generation of functional human pancreatic beta cells in vitro. Cell 2014; 159:428-439.

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BMP review for CGFR 95. Rezania A, Bruin JE, Arora P, Rubin A, Batushansky I, Asadi A, et al. Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells. Nat Biotechnol 2014; 32:1121-1133. 96. Goulley J, Dahl U, Baeza N, Mishina Y, Edlund H. BMP4-BMPR1A signaling in beta cells is required for and augments glucose-stimulated insulin secretion. Cell Metab 2007; 5:207-219. 97. Benner C, van der Meulen T, Caceres E, Tigyi K, Donaldson CJ, Huising MO. The transcriptional landscape of mouse beta cells compared to human beta cells reveals notable species differences in long non-coding RNA and protein-coding gene expression. BMC Genomics 2014; 15:62098. Kjorholt C, Akerfeldt MC, Biden TJ, Laybutt DR. Chronic hyperglycemia, independent of plasma lipid levels, is sufficient for the loss of beta-cell differentiation and secretory function in the db/db mouse model of diabetes. Diabetes 2005; 54:2755-2763. 99. Bensellam M, Montgomery MK, Luzuriaga J, Chan JY, Laybutt DR. Inhibitor of differentiation proteins protect against oxidative stress by regulating the antioxidant-mitochondrial response in mouse beta cells. Diabetologia 2015; 58:758-770. 100. Fukui N, Zhu Y, Maloney WJ, Clohisy J, Sandell LJ. Stimulation of BMP-2 expression by proinflammatory cytokines IL-1 and TNF-alpha in normal and osteoarthritic chondrocytes. J Bone Joint Surg Am 2003; 85-A Suppl 3:59-66. 101. Fowler MJ, Jr., Neff MS, Borghaei RC, Pease EA, Mochan E, Thornton RD. Induction of bone morphogenetic protein-2 by interleukin-1 in human fibroblasts. Biochem Biophys Res Commun 1998; 248:450-453. 102. Richardson SJ, Willcox A, Bone AJ, Foulis AK, Morgan NG. Islet-associated macrophages in type 2 diabetes. Diabetologia 2009; 52:1686-1688. 103. Willcox A, Richardson SJ, Bone AJ, Foulis AK, Morgan NG. Analysis of islet inflammation in human type 1 diabetes. Clin Exp Immunol 2009; 155:173-181. 104. Boni-Schnetzler M, Thorne J, Parnaud G, Marselli L, Ehses JA, Kerr-Conte J, et al. Increased interleukin (IL)-1beta messenger ribonucleic acid expression in beta -cells of individuals with type 2 diabetes and regulation of IL-1beta in human islets by glucose and autostimulation. J Clin Endocrinol Metab 2008; 93:4065-4074. 105. Boni-Schnetzler M, Boller S, Debray S, Bouzakri K, Meier DT, Prazak R, et al. Free fatty acids induce a proinflammatory response in islets via the abundantly expressed interleukin-1 receptor I. Endocrinology 2009; 150:5218-5229.

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BMP review for CGFR 106. Eizirik DL, Sammeth M, Bouckenooghe T, Bottu G, Sisino G, Igoillo-Esteve M, et al. The human pancreatic islet transcriptome: expression of candidate genes for type 1 diabetes and the impact of pro-inflammatory cytokines. PLoS Genet 2012; 8:e1002552107. Ibarra UA, Friberg J, Christensen DP, Lund CG, Billestrup N. Inflammatory Cytokines Stimulate Bone Morphogenetic Protein-2 Expression and Release from Pancreatic Beta Cells. J Interferon Cytokine Res 2015; 108. Busch AK, Cordery D, Denyer GS, Biden TJ. Expression profiling of palmitate- and oleateregulated genes provides novel insights into the effects of chronic lipid exposure on pancreatic betacell function. Diabetes 2002; 51:977-987. 109. Keller MP, Choi Y, Wang P, Davis DB, Rabaglia ME, Oler AT, et al. A gene expression network model of type 2 diabetes links cell cycle regulation in islets with diabetes susceptibility. Genome Res 2008; 18:706-716. 110. Fadista J, Vikman P, Laakso EO, Mollet IG, Esguerra JL, Taneera J, et al. Global genomic and transcriptomic analysis of human pancreatic islets reveals novel genes influencing glucose metabolism. Proc Natl Acad Sci U S A 2014; 111:13924-13929. 111. Bonner C, Farrelly AM, Concannon CG, Dussmann H, Baquie M, Virard I, et al. Bone morphogenetic protein 3 controls insulin gene expression and is down-regulated in INS-1 cells inducibly expressing a hepatocyte nuclear factor 1A-maturity-onset diabetes of the young mutation. J Biol Chem 2011; 286:25719-25728. 112. Christensen GL, Jacobsen ML, Wendt A, Mollet IG, Friberg J, Frederiksen KS, et al. Bone morphogenetic protein 4 inhibits insulin secretion from rodent beta cells through regulation of calbindin1 expression and reduced voltage-dependent calcium currents. Diabetologia 2015; 58:12821290. 113. Hussein KA, Choksi K, Akeel S, Ahmad S, Megyerdi S, El-Sherbiny M, et al. Bone morphogenetic protein 2: a potential new player in the pathogenesis of diabetic retinopathy. Exp Eye Res 2014; 125:79-88. 114. Koga M, Yamauchi A, Kanaoka Y, Jige R, Tsukamoto A, Teshima N, et al. BMP4 is increased in the aortas of diabetic ApoE knockout mice and enhances uptake of oxidized low density lipoprotein into peritoneal macrophages. J Inflamm (Lond) 2013; 10:32115. Tominaga T, Abe H, Ueda O, Goto C, Nakahara K, Murakami T, et al. Activation of bone morphogenetic protein 4 signaling leads to glomerulosclerosis that mimics diabetic nephropathy. J Biol Chem 2011; 286:20109-20116.

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BMP review for CGFR 116. Scott GJ, Ray MK, Ward T, McCann K, Peddada S, Jiang FX, et al. Abnormal glucose metabolism in heterozygous mutant mice for a type I receptor required for BMP signaling. Genesis 2009; 47:385-391. 117. Henley KD, Gooding KA, Economides AN, Gannon M. Inactivation of the dual Bmp/Wnt inhibitor Sostdc1 enhances pancreatic islet function. Am J Physiol Endocrinol Metab 2012; 303:E752-E761. 118. Chen C, Grzegorzewski KJ, Barash S, Zhao Q, Schneider H, Wang Q, et al. An integrated functional genomics screening program reveals a role for BMP-9 in glucose homeostasis. Nat Biotechnol 2003; 21:294-301. 119. Akerfeldt MC, Laybutt DR. Inhibition of Id1 augments insulin secretion and protects against high-fat diet-induced glucose intolerance. Diabetes 2011; 60:2506-2514. 120. Satyanarayana A, Klarmann KD, Gavrilova O, Keller JR. Ablation of the transcriptional regulator Id1 enhances energy expenditure, increases insulin sensitivity, and protects against age and diet induced insulin resistance, and hepatosteatosis. FASEB J 2012; 26:309-323. 121. Klein D, Alvarez-Cubela S, Lanzoni G, Vargas N, Prabakar KR, Boulina M, et al. BMP-7 induces adult human pancreatic exocrine-to-endocrine conversion. Diabetes 2015; 122. Rosen ED, Spiegelman BM. What we talk about when we talk about fat. Cell 2014; 156:20-44. 123. Cannon B, Nedergaard J. Brown adipose tissue: function and physiological significance. Physiol Rev 2004; 84:277-359. 124. Klingenberg M, Huang SG. Structure and function of the uncoupling protein from brown adipose tissue. Biochim Biophys Acta 1999; 1415:271-296. 125. Ricquier D, Mory G. Factors affecting brown adipose tissue activity in animals and man. Clin Endocrinol Metab 1984; 13:501-520. 126. Cypess AM, Lehman S, Williams G, Tal I, Rodman D, Goldfine AB, et al. Identification and importance of brown adipose tissue in adult humans. N Engl J Med 2009; 360:1509-1517. 127. Nedergaard J, Bengtsson T, Cannon B. Unexpected evidence for active brown adipose tissue in adult humans. Am J Physiol Endocrinol Metab 2007; 293:E444-E452. 128. Saito M, Okamatsu-Ogura Y, Matsushita M, Watanabe K, Yoneshiro T, Nio-Kobayashi J, et al. High incidence of metabolically active brown adipose tissue in healthy adult humans: effects of cold exposure and adiposity. Diabetes 2009; 58:1526-1531. 129. van Marken Lichtenbelt WD, Vanhommerig JW, Smulders NM, Drossaerts JM, Kemerink GJ, Bouvy ND, et al. Cold-activated brown adipose tissue in healthy men. N Engl J Med 2009; 360:15001508.

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BMP review for CGFR 130. Virtanen KA, Lidell ME, Orava J, Heglind M, Westergren R, Niemi T, et al. Functional brown adipose tissue in healthy adults. N Engl J Med 2009; 360:1518-1525. 131. Zingaretti MC, Crosta F, Vitali A, Guerrieri M, Frontini A, Cannon B, et al. The presence of UCP1 demonstrates that metabolically active adipose tissue in the neck of adult humans truly represents brown adipose tissue. FASEB J 2009; 23:3113-3120. 132. Yoneshiro T, Aita S, Matsushita M, Kayahara T, Kameya T, Kawai Y, et al. Recruited brown adipose tissue as an antiobesity agent in humans. J Clin Invest 2013; 123:3404-3408. 133. Yoneshiro T, Ogawa T, Okamoto N, Matsushita M, Aita S, Kameya T, et al. Impact of UCP1 and beta3AR gene polymorphisms on age-related changes in brown adipose tissue and adiposity in humans. Int J Obes (Lond) 2013; 37:993-998. 134. Bakker LE, Boon MR, van der Linden RA, Arias-Bouda LP, van Klinken JB, Smit F, et al. Brown adipose tissue volume in healthy lean south Asian adults compared with white Caucasians: a prospective, case-controlled observational study. Lancet Diabetes Endocrinol 2014; 2:210-217. 135. Chondronikola M, Volpi E, Borsheim E, Porter C, Annamalai P, Enerback S, et al. Brown adipose tissue improves whole-body glucose homeostasis and insulin sensitivity in humans. Diabetes 2014; 63:4089-4099. 136. Stanford KI, Middelbeek RJ, Townsend KL, An D, Nygaard EB, Hitchcox KM, et al. Brown adipose tissue regulates glucose homeostasis and insulin sensitivity. J Clin Invest 2013; 123:215-223. 137. Bartelt A, Bruns OT, Reimer R, Hohenberg H, Ittrich H, Peldschus K, et al. Brown adipose tissue activity controls triglyceride clearance. Nat Med 2011; 17:200-205. 138. Cypess AM, White AP, Vernochet C, Schulz TJ, Xue R, Sass CA, et al. Anatomical localization, gene expression profiling and functional characterization of adult human neck brown fat. Nat Med 2013; 19:635-639. 139. Lidell ME, Betz MJ, Dahlqvist LO, Heglind M, Elander L, Slawik M, et al. Evidence for two types of brown adipose tissue in humans. Nat Med 2013; 19:631-634. 140. Nedergaard J, Cannon B. The browning of white adipose tissue: some burning issues. Cell Metab 2014; 20:396-407. 141. Petrovic N, Walden TB, Shabalina IG, Timmons JA, Cannon B, Nedergaard J. Chronic peroxisome proliferator-activated receptor gamma (PPARgamma) activation of epididymally derived white adipocyte cultures reveals a population of thermogenically competent, UCP1-containing adipocytes molecularly distinct from classic brown adipocytes. J Biol Chem 2010; 285:7153-7164. 142. Wu J, Bostrom P, Sparks LM, Ye L, Choi JH, Giang AH, et al. Beige adipocytes are a distinct type of thermogenic fat cell in mouse and human. Cell 2012; 150:366-376.

37

BMP review for CGFR 143. Himms-Hagen J, Melnyk A, Zingaretti MC, Ceresi E, Barbatelli G, Cinti S. Multilocular fat cells in WAT of CL-316243-treated rats derive directly from white adipocytes. Am J Physiol Cell Physiol 2000; 279:C670-C681. 144. Lee YH, Petkova AP, Mottillo EP, Granneman JG. In vivo identification of bipotential adipocyte progenitors recruited by beta3-adrenoceptor activation and high-fat feeding. Cell Metab 2012; 15:480-491. 145. Wang QA, Tao C, Gupta RK, Scherer PE. Tracking adipogenesis during white adipose tissue development, expansion and regeneration. Nat Med 2013; 19:1338-1344. 146. Wang W, Kissig M, Rajakumari S, Huang L, Lim HW, Won KJ, et al. Ebf2 is a selective marker of brown and beige adipogenic precursor cells. Proc Natl Acad Sci U S A 2014; 111:14466-14471. 147. Xue R, Lynes MD, Dreyfuss JM, Shamsi F, Schulz TJ, Zhang H, et al. Clonal analyses and gene profiling identify genetic biomarkers of the thermogenic potential of human brown and white preadipocytes. Nat Med 2015; 21:760-768. 148. Lepper C, Fan CM. Inducible lineage tracing of Pax7-descendant cells reveals embryonic origin of adult satellite cells. Genesis 2010; 48:424-436. 149. Seale P, Bjork B, Yang W, Kajimura S, Chin S, Kuang S, et al. PRDM16 controls a brown fat/skeletal muscle switch. Nature 2008; 454:961-967. 150. Sanchez-Gurmaches J, Guertin DA. Adipocytes arise from multiple lineages that are heterogeneously and dynamically distributed. Nat Commun 2014; 5:4099151. Otto TC, Lane MD. Adipose development: from stem cell to adipocyte. Crit Rev Biochem Mol Biol 2005; 40:229-242. 152. Schulz TJ, Tseng YH. Brown adipose tissue: development, metabolism and beyond. Biochem J 2013; 453:167-178. 153. Tseng YH, Kokkotou E, Schulz TJ, Huang TL, Winnay JN, Taniguchi CM, et al. New role of bone morphogenetic protein 7 in brown adipogenesis and energy expenditure. Nature 2008; 454:1000-1004. 154. Schulz TJ, Huang TL, Tran TT, Zhang H, Townsend KL, Shadrach JL, et al. Identification of inducible brown adipocyte progenitors residing in skeletal muscle and white fat. Proc Natl Acad Sci U S A 2011; 108:143-148. 155. Elsen M, Raschke S, Tennagels N, Schwahn U, Jelenik T, Roden M, et al. BMP4 and BMP7 induce the white-to-brown transition of primary human adipose stem cells. Am J Physiol Cell Physiol 2014; 306:C431-C440.

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BMP review for CGFR 156. Gustafson B, Hammarstedt A, Hedjazifar S, Hoffmann JM, Svensson PA, Grimsby J, et al. BMP4 and BMP Antagonists Regulate Human White and Beige Adipogenesis. Diabetes 2015; 64:1670-1681. 157. Schulz TJ, Huang P, Huang TL, Xue R, McDougall LE, Townsend KL, et al. Brown-fat paucity due to impaired BMP signalling induces compensatory browning of white fat. Nature 2013; 495:379383. 158. Taha MF, Valojerdi MR, Mowla SJ. Effect of bone morphogenetic protein-4 (BMP-4) on adipocyte differentiation from mouse embryonic stem cells. Anat Histol Embryol 2006; 35:271-278. 159. El-Magd MA, Allen S, McGonnell I, Otto A, Patel K. Bmp4 regulates chick Ebf2 and Ebf3 gene expression in somite development. Dev Growth Differ 2013; 55:710-722. 160. Warner DR, Horn KH, Mudd L, Webb CL, Greene RM, Pisano MM. PRDM16/MEL1: a novel Smad binding protein expressed in murine embryonic orofacial tissue. Biochim Biophys Acta 2007; 1773:814-820. 161. Gupta RK, Arany Z, Seale P, Mepani RJ, Ye L, Conroe HM, et al. Transcriptional control of preadipocyte determination by Zfp423. Nature 2010; 464:619-623. 162. Gupta RK, Mepani RJ, Kleiner S, Lo JC, Khandekar MJ, Cohen P, et al. Zfp423 expression identifies committed preadipocytes and localizes to adipose endothelial and perivascular cells. Cell Metab 2012; 15:230-239. 163. Schulz TJ, Tseng YH. Emerging role of bone morphogenetic proteins in adipogenesis and energy metabolism. Cytokine Growth Factor Rev 2009; 20:523-531. 164. Zamani N, Brown CW. Emerging roles for the transforming growth factor-{beta} superfamily in regulating adiposity and energy expenditure. Endocr Rev 2011; 32:387-403. 165. Ahrens M, Ankenbauer T, Schroder D, Hollnagel A, Mayer H, Gross G. Expression of human bone morphogenetic proteins-2 or -4 in murine mesenchymal progenitor C3H10T1/2 cells induces differentiation into distinct mesenchymal cell lineages. DNA Cell Biol 1993; 12:871-880. 166. Chen TL, Shen WJ, Kraemer FB. Human BMP-7/OP-1 induces the growth and differentiation of adipocytes and osteoblasts in bone marrow stromal cell cultures. J Cell Biochem 2001; 82:187199. 167. Kang Q, Song WX, Luo Q, Tang N, Luo J, Luo X, et al. A comprehensive analysis of the dual roles of BMPs in regulating adipogenic and osteogenic differentiation of mesenchymal progenitor cells. Stem Cells Dev 2009; 18:545-559. 168. Chen D, Ji X, Harris MA, Feng JQ, Karsenty G, Celeste AJ, et al. Differential roles for bone morphogenetic protein (BMP) receptor type IB and IA in differentiation and specification of mesenchymal precursor cells to osteoblast and adipocyte lineages. J Cell Biol 1998; 142:295-305. 39

BMP review for CGFR 169. Bowers RR, Kim JW, Otto TC, Lane MD. Stable stem cell commitment to the adipocyte lineage by inhibition of DNA methylation: role of the BMP-4 gene. Proc Natl Acad Sci U S A 2006; 103:13022-13027. 170. Tang QQ, Otto TC, Lane MD. Commitment of C3H10T1/2 pluripotent stem cells to the adipocyte lineage. Proc Natl Acad Sci U S A 2004; 101:9607-9611. 171. Puigserver P, Wu Z, Park CW, Graves R, Wright M, Spiegelman BM. A cold-inducible coactivator of nuclear receptors linked to adaptive thermogenesis. Cell 1998; 92:829-839. 172. Nohe A, Keating E, Knaus P, Petersen NO. Signal transduction of bone morphogenetic protein receptors. Cell Signal 2004; 16:291-299. 173. Akimoto T, Pohnert SC, Li P, Zhang M, Gumbs C, Rosenberg PB, et al. Exercise stimulates Pgc-1alpha transcription in skeletal muscle through activation of the p38 MAPK pathway. J Biol Chem 2005; 280:19587-19593. 174. Hata K, Nishimura R, Ikeda F, Yamashita K, Matsubara T, Nokubi T, et al. Differential roles of Smad1 and p38 kinase in regulation of peroxisome proliferator-activating receptor gamma during bone morphogenetic protein 2-induced adipogenesis. Mol Biol Cell 2003; 14:545-555. 175. Zhang H, Schulz TJ, Espinoza DO, Huang TL, Emanuelli B, Kristiansen K, et al. Cross talk between insulin and bone morphogenetic protein signaling systems in brown adipogenesis. Mol Cell Biol 2010; 30:4224-4233. 176. Sellayah D, Bharaj P, Sikder D. Orexin is required for brown adipose tissue development, differentiation, and function. Cell Metab 2011; 14:478-490. 177. Yadav H, Quijano C, Kamaraju AK, Gavrilova O, Malek R, Chen W, et al. Protection from obesity and diabetes by blockade of TGF-beta/Smad3 signaling. Cell Metab 2011; 14:67-79. 178. Fournier B, Murray B, Gutzwiller S, Marcaletti S, Marcellin D, Bergling S, et al. Blockade of the activin receptor IIb activates functional brown adipogenesis and thermogenesis by inducing mitochondrial oxidative metabolism. Mol Cell Biol 2012; 32:2871-2879. 179. Yoshida H, Kanamori Y, Asano H, Hashimoto O, Murakami M, Kawada T, et al. Regulation of brown adipogenesis by the Tgf-beta family: involvement of Srebp1c in Tgf-beta- and Activin-induced inhibition of adipogenesis. Biochim Biophys Acta 2013; 1830:5027-5035. 180. Molchadsky A, Ezra O, Amendola PG, Krantz D, Kogan-Sakin I, Buganim Y, et al. p53 is required for brown adipogenic differentiation and has a protective role against diet-induced obesity. Cell Death Differ 2013; 20:774-783. 181. McDonald ME, Li C, Bian H, Smith BD, Layne MD, Farmer SR. Myocardin-related transcription factor A regulates conversion of progenitors to beige adipocytes. Cell 2015; 160:105118. 40

BMP review for CGFR 182. Qian SW, Tang Y, Li X, Liu Y, Zhang YY, Huang HY, et al. BMP4-mediated brown fat-like changes in white adipose tissue alter glucose and energy homeostasis. Proc Natl Acad Sci U S A 2013; 110:E798-E807. 183. Kuo MM, Kim S, Tseng CY, Jeon YH, Choe S, Lee DK. BMP-9 as a potent brown adipogenic inducer with anti-obesity capacity. Biomaterials 2014; 35:3172-3179. 184. Hinoi E, Iezaki T, Fujita H, Watanabe T, Odaka Y, Ozaki K, et al. PI3K/Akt is involved in brown adipogenesis mediated by growth differentiation factor-5 in association with activation of the Smad pathway. Biochem Biophys Res Commun 2014; 450:255-260. 185. Hinoi E, Nakamura Y, Takada S, Fujita H, Iezaki T, Hashizume S, et al. Growth differentiation factor-5 promotes brown adipogenesis in systemic energy expenditure. Diabetes 2014; 63:162-175. 186. Zhang C, McFarlane C, Lokireddy S, Masuda S, Ge X, Gluckman PD, et al. Inhibition of myostatin protects against diet-induced obesity by enhancing fatty acid oxidation and promoting a brown adipose phenotype in mice. Diabetologia 2012; 55:183-193. 187. Whittle AJ, Carobbio S, Martins L, Slawik M, Hondares E, Vazquez MJ, et al. BMP8B increases brown adipose tissue thermogenesis through both central and peripheral actions. Cell 2012; 149:871-885. 188. Townsend KL, Suzuki R, Huang TL, Jing E, Schulz TJ, Lee K, et al. Bone morphogenetic protein 7 (BMP7) reverses obesity and regulates appetite through a central mTOR pathway. FASEB J 2012; 26:2187-2196. 189. Townsend KL, An D, Lynes MD, Huang TL, Zhang H, Goodyear LJ, et al. Increased mitochondrial activity in BMP7-treated brown adipocytes, due to increased. Antioxid Redox Signal 2013; 19:243-257. 190. Boon MR, van den Berg SA, Wang Y, van den Bossche J, Karkampouna S, Bauwens M, et al. BMP7 activates brown adipose tissue and reduces diet-induced obesity only at subthermoneutrality. PLoS One 2013; 8:e74083-

Lovorka Grgurevic conducts research in the Center for Translational and Clinical Research (CETRI), University of Zagreb School of Medicine, on formulation development and toxicology testing. She explores the structure and function of bone morphogenetic proteins (BMPs) in biological fluids, has discovered circulating osteogenic proteins and associated molecules in the plasma, and then investigated their effectiveness in bone healing and in models of acute and chronic renal failure. She contributed significantly to the discovery of a new carrier for BMPs and tested its efficacy in animal 41

BMP review for CGFR models of bone defects. Dr Grgurevic discovered novel biomarkers for bone repair, breast and prostate cancer prognosis. She received a new laboratory installation grant from the Croatian Science Foundation and was awarded by the Croatian Academy of Sciences and Arts for scientific achievements.

Gitte Lund Christensen completed her Msc degree in Biology from University of Copenhagen in 2003. She did a PhD in the cardio vascular field studying signal transduction from the Angiotensin II type 1 receptor. Current she is a Post doc at the University of Copenhagen at the laboratory of pancreatic islet biology. In collaboration with Professor Nils Billestrup, she works on elucidating the role of BMP2 and 4 in pancreatic beta-cells dysfunction associated with the development of diabetes. Gitte is supported by a post doc grant from The Danish Diabetes Academy.

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BMP review for CGFR Tim J. Schulz heads a junior research group at the German Institute of Human Nutrition in PotsdamRehbrücke, Germany. He received his doctorate in biochemistry and nutritional sciences from the Friedrich-Schilller-University of Jena, Germany. He subsequently completed his post-doctoral training on BMPs and brown adipose tissue biology in the laboratory of Dr. Yu-Hua Tseng at Joslin Diabetes Center and Harvard Medical School. His present research interests include the role of aging in regenerative decline and adipocyte formation. Dr. Schulz has received stipends from the German Research Foundation (DFG) and the Mary K. Iacocca family foundation. He is a recipient of a starting grant from the European Research Council and an Emmy Noether-fellowship from the DFG.

Slobodan Vukicevic is a full professor and head of Laboratory of Mineralized Tissues and Proteomic Center at the Center for Translational and Clinical Research, University of Zagreb School of Medicine. Scientific interests comprise bone and cartilage morphogenetic proteins and development of drugs for regeneration of bone, kidney, pancreas and heart muscle. Invited speaker at international conferences and universities. Received international awards for achievements in science. Chairman and organizer of several international conferences on calcified tissues. Member of EMBO, World Academy of Arts and Sciences (WAAS) and Croatian Academy of Sciences and Arts (CASA). Author of more than 170 manuscripts, editor of 4 books on BMPs and inventor on 33 patents. Founder of Genera Research, innovation-based biotechnology company developing a novel regenerative therapy for bone defects via coordinating the collaborative FP7 program OSTEOGROW.

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BMP review for CGFR

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