AP-1 function in mesenchymal cell fate decision: The role of ribosomal S6 kinase and Fra1

S26

Abstracts / Bone 44 (2009) S18–S55

c Department of Molecular Genetics, The University of Texas MD Anderson Cancer Center, Houston, Texas, United States d Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, Texas, United States

Bone mass is maintained by the balanced action of osteoblastic bone formation and osteoclastic bone resorption. This remodeling process is regulated by many systemic and local factors. Identification of molecules that affect bone mass is important for understanding and potential treatment of bone disease. We identified Collagen triple helix repeat containing 1 (Cthrc1) as a downstream target of bone morphogenetic protein-2 in ATDC5 cells by PCR-based suppression subtractive hybridization, followed by differential hybridization. Cthrc1, a glycosylated protein with a signal sequence, was highly expressed in osteogenic MC3T3-E1 cells by northern blot analyses and in bone tissues by in situ hybridization analyses. To investigate the role of Cthrc1 in bone, we generated Cthrc1-null mice and transgenic mice which overexpress Cthrc1 in osteoblasts (Cthrc1 transgenic mice). Micro-CT and bone histomorphometric analyses revealed that Cthrc1-null mice displayed low bone mass by suppressed bone formation, whereas Cthrc1 transgenic mice showed high bone mass by accelerated bone formation. Osteoblast proliferation, assessed by BrdU incorporation, was suppressed in Cthrc1-null mice and stimulated in Cthrc1 transgenic mice, respectively. Furthermore, mRNA expression levels of alkaline phosphatase, Col1a1, and Osteocalcin in primary osteoblasts harvested from calvaria were decreased in Cthrc1-null mice, and increased in Cthrc1 transgenic mice, respectively. Our results indicate that Cthrc1 positively regulates bone formation by stimulating osteoblast proliferation and differentiation.

levels of Smad1. Similar reduction by PPM1A was observed in wildtype Smad5 and Smad8. PPM1A decreased protein levels of another mutant Smad1, in which a Smurf consensus sequence in the linker region has been destroyed, suggesting that Smurf E3 ubiquitin ligases may not be involved in this process. Treatment cells with a proteasome inhibitor blocked the inhibitory effects of PPM1A on Smad1. In contrast to wild-type, a mutant PPM1A lacking a phosphatase activity did not inhibit BMP signaling and failed to decrease the Smad protein levels. Moreover, knockdown of endogenous PPM1A by siRNA stimulated BMP activity in C2C12 myoblasts. Taken together, these findings suggested that PPM1A suppresses BMP signaling by stimulating Smad degradation independent of dephosphorylation at the carboxyl termini and this inhibitory regulation may play an important role in physiological BMP activities. doi:10.1016/j.bone.2009.01.060

023 AP-1 function in mesenchymal cell fate decision: The role of ribosomal S6 kinase and Fra1 F. Driesslera,c, J. Lutherb, M. Meggesc, A. Reichardtc, A. Schillingd, M. Amlingd, J. Davidb,c a Bone and Mineral Research Program, Garvan Institute of Medical Research, Sydney, NSW, Australia b Department of Internal Medicine 3, Rheumatology and Immunology, University of Erlangen-Nuremberg, Erlangen, Germany c Bone Cell Differentiation Group, Deutsches Rheuma- Forschungszentrum, Berlin, Germany d Center for Biomechanics, Experimental Trauma Surgery and Skeletal Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

doi:10.1016/j.bone.2009.01.059

022 Protein phosphatase magnesium-dependent 1A inhibits BMP signaling by stimulating Smad degradation independent of dephosphorylation at the carboxyl termini S. Kokabua,b, J. Nojimaa,b, T. Fukudaa, K. Kanomataa, T. Yodab, T. Katagiria a Division of Pathophysiology, Research Center for Genomic Medicine, Saitama Medical University, Hidaka-shi, Saitama, Japan b Department of Oral and Maxillofacial Surgery, Saitama Medical University, Moroyama-machi, Saitama, Japan BMPs induce bone formation through binding to specific receptors. Type I BMP receptors phosphorylate the carboxyl terminal serine residues of Smad1/5/8 to activate downstream signaling of BMPs, such as an Id1 gene expression. Recently, we found that osteoblastic differentiation of myoblasts was induced by an over-expression of a constitutively activated Smad1, in which the carboxyl terminal serine residues have been substituted by aspartic acid residues, suggesting that the Smad signaling pathway is critical for the bone formation induced by BMPs. Protein phosphatase magnesium-dependent 1A (PPM1A) has been shown to suppress BMP activities by dephosphorylating the carboxyl terminal phospho-serine residues as substrates. We report here that PPM1A suppresses BMP signaling via a novel mechanism. In co-transfection experiments, PPM1A inhibited both Id1 promoter activity and ALP activity induced by not only BMP4-treatment but also an over-expression of the constitutively active Smad1. This finding indicated that the inhibitory activity of PPM1A on BMP signaling was independent of dephosphorylation of Smads at the carboxyl termini. Western blotting and immunohistochemical analysis indicated that PPM1A reduced protein

While mice over-expressing the Fos-related protein Fra-1 develop progressive osteosclerosis due to increased osteoblastic differentiation, mice lacking the Fos kinase Rsk2 develop progressive osteopenia with impaired mineralization. These observations suggested a role for Fra-1 phosphorylation by Rsk2 in controlling osteoblast activity. To test this hypothesis, fra-1 transgenic mice lacking Rsk2 were generated and phenotyped. The mice die prematurely, were severely growth retarded and developed both a severe osteosclerosis and a bone mineralization defect suggesting that Fra-1 and Rsk2 function independently in bone. However, a total lipodystrophy was observed in the double mutated mice. When analysing the parental strains, a progressive lipodystrophy of the white adipose tissue (WAT) was also observed in fra1 transgenic but not in Rsk2-deficient mice. The phenotype was more pronounced in females than in males. Brown adipose tissue (BAT) was unaffected. An increased number of immature adipocytes were found in histological sections of the WAT isolated of fra-1 transgenic mice. In agreement, the expression of markers for fat maturation, Glut4 and aP2 were decreased. The phenotype was cell autonomous, since adipogenic potential of primary osteoblasts (POBs) isolated from the calvaria of fra-1 transgenic mice was impaired. The effect was Fra1-dependent as shown by the constitutive or inducible over-expression of Fra-1 in adipogenic cell line strongly inhibiting adipocyte differentiation. Osteoblasts and adipocytes differentiate from pluripotent bone marrow mesenchymal stromal cells (MSCs) that can also differentiate into other mesenchymal lineages: myotubes (muscle), chondrocytes (cartilage) or fibroblasts. However, the expression of key regulators of mesemchymal cell fate (i.e. Runx2, Sox9, MyoD, C/EBP (beta) and C/EBP (delta) was unchanged, indicative of a normal commitment of the cells. However, the adipogenic transcription factors controlling adipocyte maturation, C/EBP (alpha) and PPAR (gamma2), were down-regulated in fra1–tg POBs and in adipogenic cell lines over-expressing Fra-1.

Abstracts / Bone 44 (2009) S18–S55

Thus, our data demonstrate that increased bone mass can be observed in lean mice. Indeed, in addition to promoting osteoblastogenesis, Fra-1 inhibits adipocyte maturation, this latter being caused by C/EBP (alpha) and PPAR (gamma2) down-regulation. Moreover, we show that Rsk2 is regulating Fra-1 function in fat tissue but not in the bone. Understanding AP-1 function in mesenchymal cell fate decision may provide valuable insights into mesenchymal tissue regeneration and metabolic diseases. doi:10.1016/j.bone.2009.01.061

024 CREB induces BMP2 transcription in osteoblasts and CREB knockout reduces bone mass in mice M. Zhaoa, J. Edwardsa, S. Koa, R. Parlatoc, S. Harrisb, G. Mundya a Medicine/Clinical Pharmacology, Vanderbilt University, Nashville, Tennessee, United States b Periodontics, University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States c Molecular Biology of the Cell I, German Cancer Research Center, Heidelberg, Germany Transcription factor CREB (cAMP-response element binding protein) plays an essential role in osteoblasts to mediate the anabolic signaling of intermittent dosage of PTH in bone. However, the downstream mechanisms of CREB in osteoblasts have to be demonstrated. Here, we have characterized the skeletal phenotype of CREB knockout mice and identified the potential transcriptional target of CREB in osteoblasts. The results of m CT measurement and alizarin red/alcian blue, ALP and von Kossa staining, have shown that global knockout of CREB in mice caused a dwarfism phenotype and a significant reduction of bone volume and mineralization in the embryonic skeleton. Since the global CREB KO mice do not survive after birth, we have generated the osteoblast-specific CREB KO mice (CREBfl;2.3ColCre) and examined their skeletal phenotype. m CT results have shown that bone volume of two-month-old CREBfl;2.3ColCre KO mice substantially reduced, along with decreased trabecular number and thickness and increased trabecular separation, compared with that of control mice. These results suggest that CREB is critical for both normal skeletal development and postnatal bone mass. Interestingly, we found that expression of BMP2, an important factor for osteoblast differentiation and bone formation, was reduced in the bones of CREB KO mice. Since there exists a similar osteopenic phenotype between CREB KO CREBfl;2.3ColCre) and BMP2 KO (BMP2fl;3.6ColCre) mice, and the BMP2 promoter contains multiple cAMP response elements (CRE), we hypothesized that the function of CREB in bone is mediated at least in part through BMP2. In cell culture, we found that both CREB and PTH stimulated BMP2 gene expression in osteoblasts. We also found that pharmacological manipulation of CREB phosphorylation by cAMP/PKA activator IBMX or inhibitor KT5720 affected CREB transactivation of the BMP2 expression. Furthermore, through promoter mutation studies, we demonstrated that CREB transactivated BMP2 gene by directly interacting with a specific CRE in the BMP2 promoter. Lastly, we have shown that overexpression of CREB promoted osteoblast differentiation and this action was blocked by addition of noggin in the cultures. Together, these results suggest that CREB plays an important role in osteogenesis embryonically and postnatally, and this function is mediated by up-regulation of BMP2 transcription in osteoblasts. doi:10.1016/j.bone.2009.01.062

S27

025 Osteoblast IL-33 mRNA expression is regulated by PTH, and IL-33 treatment causes both increased osteoblastic matrix mineralisation and reduced osteoclast formation in vitro H. Saleha,b,c, J.M.W. Quinna,b,c, T. Martinc, M.T. Gillespiea a Prince Henry's Institute, Clayton, VIC, Australia b Department of Medicine, University of Melbourne, Fitzroy, VIC, Australia c St. Vincent's Institute, Fitzroy, VIC, Australia IL-33 is a Th2 stimulating pro-inflammatory cytokine related to IL-1 and IL-18, its actions mediated by receptor ST2L. We have previously found that IL-33, like IL-18, indirectly inhibits osteoclast formation via T lymphocytes but does not directly affect osteoclast formation from RANKL-stimulated bone marrow macrophages (BMM). In mouse bone sections, anti-IL-33 antibody clearly immunostained osteoblasts and chondrocytes and some osteoclasts but not osteocytes or most bone marrow cells. DNA microarray array studies also showed ST2L mRNA expression in matured osteoblastic Kusa 4b10 cells is increased by PTH treatment, observations confirmed by further RT-PCR analysis. PTH treatment also increased IL-33 mRNA levels in osteoblasts. Long term osteoblastic cultures treated with ascorbate (which increases osteoblastic differentiation) also increased IL-33 mRNA levels. With long term pro-adipogenic treatment (dexamethasone, insulin and IBMX) of immature Kusa 4b10 cells IL33 mRNA levels increased but ST2L mRNA levels decreased. Since osteoblasts express ST2L we investigated IL-33 action on osteoblasts to identify possible autocrine actions. IL-33 promoted matrix mineralisation by primary osteoblasts. Furthermore, in long term ascorbate stimulated primary osteoblasts in which expression of osteocytic features are apparent (e.g. sclerostin and DMP-1 expression), IL-33 reduced sclerostin mRNA levels after 6 and 24 h of treatment, although other PTH regulated genes in these cells, such as ephrin B2 were not affected. We also investigated IL-33 effects on osteoblastic support of osteoclastogenesis. Osteoclast formation from BMM stimulated by 1,25 dihydroxyvitamin D3-treated Kusa O pre-osteoblastic cells was blocked in the presence of IL-33 (20 ng/ml), an action ablated by anti-GM-CSF antibody. GM-CSF mRNA was strongly upregulated by IL-33 treatment, as indeed was RANKL mRNA. However, Kusa O/BMM co-cultures treated with IL-33 and anti-GM-CSF antibody (without other stimulus) induced osteoclast formation only weakly. Thus we have found evidence that IL-33 stimulates osteoblastic function while indirectly, through two separate mechanisms, inhibiting osteoclast formation. IL-33 thus may play a role in maintaining bone mass, perhaps participating in the anabolic actions of PTH. doi:10.1016/j.bone.2009.01.063

026 A dairy-based protein, calcium and vitamin D supplement preserves trabecular bone and reduces falls in aged care residents S. Iuliano-Burnsa, K. Kinga, J. Woodsc, A. Evansb, A. Ghasem-Zadehb, E. Seemana a Endocrinology, Medicine, University of Melbourne/Austin Health, West Heidelberg, VIC, Australia b Bone and Mineral Research Unit, Austin Health, West Heidelberg, VIC, Australia c Nutrition and Dietetics, Monash University, Clayton, VIC, Australia Aged care residents are at higher risk of falls and fractures than elderly living in the community. Nutrient deficiencies may increase falls and fracture risk by contributing to bone loss and body sway and reducing muscle mass and strength. We aimed to determine if a dairybased protein, calcium and vitamin D supplement incorporated into