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Pancreatic stellate cells – Multi-functional cells in the pancreas
Pancreatology 13 (2013) 102e105
Contents lists available at SciVerse ScienceDirect
Pancreatology journal homepage: www.elsevier.com/locate/pan
Review article
Pancreatic stellate cells e Multi-functional cells in the pancreas Atsushi Masamune*, Tooru Shimosegawa Division of Gastroenterology, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 November 2012 Received in revised form 10 December 2012 Accepted 10 December 2012
There is accumulating evidence that activated pancreatic stellate cells (PSCs) play a pivotal role in pancreatic fibrosis in chronic pancreatitis and pancreatic cancer. In addition, we have seen great progress in our understanding of the cell biology of PSCs and the interactions between PSCs and other cell types in the pancreas. In response to pancreatic injury or inflammation, quiescent PSCs are activated to myofibroblast-like cells. Recent studies have shown that the activation of intracellular signaling pathways such as mitogen-activated protein kinases plays a role in the activation of PSCs. microRNAs might also play a role, because the microRNA expression profiles are dramatically altered in the process of activation. In addition to producing extracellular matrix components such as type I collagen, PSCs have a wide variety of cell functions related to local immunity, inflammation, angiogenesis, and exocrine and endocrine functions in the pancreas. From this point of view, the interactions between PSCs and other cell types such as pancreatic exocrine cells, endocrine cells, and cancer cells have attracted increasing attention of researchers. PSCs might regulate exocrine functions in the pancreas through the cholecystokinin-induced release of acetylcholine. PSCs induce apoptosis and decrease insulin expression in b-cells, suggesting a novel mechanism of diabetes in diseased pancreas. PSCs promote the progression of pancreatic cancer by multiple mechanisms. Recent studies have shown that PSCs induce epithelial emesenchymal transition and enhance the stem-cell like features of pancreatic cancer cells. In conclusion, PSCs should now be recognized as not only profibrogenic cells but as multi-functional cells in the pancreas. Copyright Ó 2012, IAP and EPC. Published by Elsevier India, a division of Reed Elsevier India Pvt. Ltd. All rights reserved.
Keywords: Pancreatitis Pancreatic cancer Desmoplastic reaction Fibrosis MicroRNA Signal transduction
1. Introduction Pancreatic fibrosis is a characteristic feature of chronic pancreatitis (CP) and desmoplastic reaction associated with pancreatic cancer. However, for a long time, the molecular mechanisms remained largely unknown, mainly due to the lack of appropriate in vitro models and the unavailability of histological samples. In 1998, two groups independently reported the identification, isolation and characterization of the star-shaped cells in the pancreas, namely pancreatic stellate cells (PSCs) [1,2]. Since then, evidence has been accumulating to indicate that activated PSCs play a pivotal role in the development of pancreatic fibrosis
Abbreviations used: CP, chronic pancreatitis; ECM, extracellular matrix; EMT, epithelialemesenchymal transition; GLUT, glucose transporter; HSC, hepatic stellate cell; IL, interleukin; MAP, mitogen-activated protein; miRNA, microRNA; PSC, pancreatic stellate cell; MMP, matrix metalloproteinase; SMA, smooth muscle actin; TIMP, tissue inhibitor of metalloproteinase; TLR, Toll-like receptor; VEGF, vascular endothelial growth factor. * Corresponding author. Tel.: þ81 22 717 7172; fax: þ81 22 717 7177. E-mail address: [email protected] (A. Masamune).
in CP and pancreatic cancer [3e7]. Recently, we have seen great progress in our understanding of the cell biology of PSCs and the interactions between PSCs and other cell types in the pancreas. In this overview, we will briefly summarize our current knowledge in this field.
2. Activation of PSCs In normal pancreas, stellate cells are quiescent and can be identified by the presence of vitamin A-containing lipid droplets in the cytoplasm. In response to pancreatic injury or inflammation, quiescent PSCs undergo morphological and functional changes to become myofibroblast-like cells which express a-smooth muscle actin (a-SMA). This step is called “activation”. Activated PSCs lose lipid droplets, actively proliferate, migrate, and produce large amounts of extracellular matrix (ECM) components such as type I collagen and fibronectin [1e7]. In vitro studies have identified that cytokines and growth factors including transforming growth factor-b, tumor necrosis factor-a, interleukin (IL)-1, IL-6 and activin A as well as ethanol and its metabolites, oxidative stress and
1424-3903/$ e see front matter Copyright Ó 2012, IAP and EPC. Published by Elsevier India, a division of Reed Elsevier India Pvt. Ltd. All rights reserved. http://dx.doi.org/10.1016/j.pan.2012.12.058
A. Masamune, T. Shimosegawa / Pancreatology 13 (2013) 102e105
pressure, induce the activation of PSCs [3e7]. Cytokines and growth factors produced during pancreatic injury or inflammation by acinar cells, inflammatory cells, platelets, and endothelial cells, would activate PSCs and regulate these cell functions. Interestingly, PSCs themselves produce a variety of cytokines and growth factors [3e7]. Therefore, in addition to paracrine regulation by neighboring cells, PSCs might be activated in an autocrine manner. 3. Mechanisms of PSCs activation The mechanism responsible for the activation of PSCs is not yet completely understood, but includes the activation of intracellular signaling pathways such as mitogen-activated protein (MAP) kinases and Rho/Rho kinase (reviewed in 5). For example, during the process of activation, stress fiber formation is increased, suggesting cytoskeletal reorganization is involved in this process [8]. The small GTP-binding protein Rho has emerged as an important regulator of the actin cytoskeleton and cell morphology through the downstream effector Rho kinase. Specific inhibitors of Rhoe Rho kinase (Y-27632 and HA-1077) inhibit the transformation of quiescent PSCs to myofibroblast-like cells in vitro [8]. Similarly, inhibitors of p38 MAP kinase inhibit the activation of PSCs, supporting the role of these signaling pathways in the activation of PSCs [9]. Recently, microRNAs (miRNAs) have attracted the attention of researchers. miRNAs are small, non-coding RNAs consisting of 20e 23 nucleotides that target the 30 untranslated region sequence of messenger RNA for destabilization [10]. miRNAs regulate a variety of cell functions such as cell proliferation, apoptosis, differentiation, and carcinogenesis [10]. We compared the miRNA expression profiles between quiescent and activated rat PSCs. We found that the miRNA signature was dramatically altered during the activation of PSCs (Fig. 1) (Masamune A et al. unpublished observations). Pathway analysis (Ingenuity Systems, Redwood City, CA) showed that the altered miRNAs were associated with cellular movement, growth, and death, suggesting a role of miRNAs in the activation of PSCs.
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4. Cell functions of PSCs In addition to playing a key role in fibrogenesis, PSCs have a variety of cell functions. These cells not only produce ECM, but also synthezise matrix-degrading enzymes of the matrix metalloproteinase (MMP) family and their inhibitors [tissue inhibitors of metalloproteinases (TIMPs)] [11]. PSCs have been shown to secrete MMP-2, MMP-9, and MMP-13, and to express TIMP-1 and TIMP-2 [11]. Thus, PSCs have the ability to produce as well as to degrade ECM, suggesting that they might play a role in the maintenance of the normal tissue architecture by regulating ECM turnover. In this context, the resolution of mouse caerulein-induced pancreatitis involves transient activation of PSCs and deposition of ECM proteins, as well as the transient upregulation of MMPs and TIMPs [12]. The increased expression of the cytoskeletal protein a-SMA confers increased contractile potential, which is further increased by endothelin-1 [13]. Because PSCs are also located around ductal and vascular structures [1e7], it would be of interest to see whether the contraction of PSCs is involved in the regulation of vascular and ductal tone in the pancreas. PSCs produce a broad range of pro-inflammatory and antiinflammatory cytokines, growth factors, and adipocytokines (Table 1), suggesting pro-inflammatory and anti-inflammatory roles of PSCs. Expression of these cytokines is often associated with the activation of PSCs. For example, IL-33 is a recently identified member of the IL-1 gene family [14]. IL-33 expression was low in freshly isolated, quiescent rat PSCs but was increased upon activation, indicating that IL-33 induction was associated with the transformation to an a-SMA-positive myofibroblastic phenotype. In addition, we and others have shown that both freshly-isolated and activated PSCs express Toll-like receptors (TLRs), which are proteins involved in the activation of innate immunity [15,16]. PSCs express TLR2, which recognizes pathogen-associated molecular patterns of Gram-positive bacteria, and TLR4, which recognizes lipopoysaccharides of Gram-negative bacteria. PSCs could perform endocytosis and phagocytosis of foreign bodies, suggesting that PSCs might play a role in the local immune functions in the pancreas. Another novel function we identified in PSCs is related to angiogenesis. PSCs constitutively produce vascular endothelial growth factor (VEGF), the production of which is increased by hypoxia [17]. In addition to VEGF, PSCs expressed several angiogenesis-regulating molecules including VEGF receptors, angiopoietin-1 and its receptor Tie-2, and vasohibin-1. Conditioned media of PSCs induced angiogenesis in vitro, as shown by increased tube formation on Matrigel, and in vivo, as shown by directed vessel formation in nude mice. Very recently, we have found that PSCs express glucose transporters (GLUTs). PSCs expressed Glut1 and Glut 3, but not Glut 2 or Glut 4, suggesting a novel role of PSCs in the metabolism of glucose (Masamune A et al. unpublished observations). Therefore, in
Table 1 PSCs express a wide range of prtoinflammatory and anti-inflammatory cytokines, growth factors, and adipocytokines. IL-6 IL-8 IL-10 IL-23 IL-24 IL-32 IL-33 RANTES Fig. 1. miRNA expression profiles were different between quiescent and activated PSCs. Total RNAs including miRNAs were prepared from freshly-isolated rat quiescent PSCs (day 0) and culture-activated rat PSCs (day 14). The miRNA expression profiles in these cells were compared using the Agilent’s miRNA microarray.
MCP-1: monocyte chemoattractant protein-1. FGF: fibroblast growth factor. CTGF: connective tissue growth factor. TGF: transforming growth factor.
MCP-1 FGF CTGF TGF-b Activin/Nodal VEGF Angiopoietin Chemerin
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A. Masamune, T. Shimosegawa / Pancreatology 13 (2013) 102e105
addition to the production of ECMs, PSCs have a wide variety of cell functions. 5. The origin and source of PSCs What is the origin of PSCs? Mesodermal, endodermal, and neuro-ectodermal origins might be possible, but no studies have addressed this issue to date. In hepatic stellate cells (HSCs), several studies have attempted to clarify this issue. For example, the neuroectoderm proposal for the origins of HSC was refuted by a study by Cassiman et al., [18] who used a genetic cell lineage mapping technique with Rosa26YFPflox mice crossed with mice expressing Cre under the control of the neural crest-specific Wnt1 promoter/ enhancer. Definitive novel evidence for the mesodermal origin of HSCs has recently been presented by Asahina and colleagues [19,20] with the use of the mesoderm-specific MesP1Cre. A conditional cell lineage analysis using Wt1CreERT/Rosa26LacZflox or ROSA26mTmGflox mice, revealed Wt1-positive septum transversum giving rise to mesothelial cells, submesothelial cells, HSCs and perivascular mesenchymal cells during liver development [19]. That study also demonstrated Wt1-positive mesothelial/submesothelial cells migrating inward from the liver surface to generate liver mesenchymal cells including HSCs. Similar lineage tracing techniques need to be used to determine the exact origin of PSCs. In terms of the PSCs within normal pancreas and their increased numbers during pancreatic injury, the question often asked has been whether these cells arise wholly from resident cells or whether there is a contribution to this PSC population from other sources. Using sex-mismatched, bone marrow transplantation from male green mice to female mice, we could identify desmin-positive, bone marrow-derived cells in the pancreas [21]. Bone marrowderived cells accounted for about 8% of the total desmin-positive cells. After the induction of pancreatitis by caerulein, approximately 20% of a-SMA-positive cells contained Y chromosome in the nucleus. suggesting they originated in bone marrow. Therefore, bone marrow contributes to the populations of both quiescent and activated PSCs. Endothelial- or epithelialemesenchymal transition (EMT) may also contribute to the population of activated PSCs during pancreatic injury, but further studies are required in this area. 6. Interactions between PSCs and other cell types in the pancreas Because PSCs are morphologically and functionally very similar to HSCs, it is important to determine whether PSCs are identical to or different from HSCs. A DNA array study showed that 29 out of 23,000 genes (i.e. 0.1%) were different between PSCs and HSCs [22], suggesting that organ-specific influences may be relevant to stellate cell biology. Therefore, research on PSCs should develop in directions more relevant to the patho-physiology of the pancreas. In this context, the interactions between PSCs and other cell types in the pancreas have attracted increasing attention. For example, the expression of cholecystokinin receptors in PSCs has been reported [23]. Cholecystokinin-8 stimulated acetylcholine release in PSCs, leading to the stimulation of exocrine functions in acinar cells. These findings suggest a novel role of PSCs in the regulation of exocrine functions in the pancreas. Similarly, we examined the interaction between PSCs and b-cells. PSCs decreased insulin mRNA expression as well as secretion of insulin by b-cells. (Kikuta K et al., unpublished observations). In addition, PSCs induced apoptosis of b-cells demonstrating that PSCs induced changes in b-cells both quantitatively and qualitatively. Another important interaction is between PSCs and cancer cells. It has been reported that PSCs promote cancer
Fig. 2. Multiple interactions between PSCs and other cell types in the pancreas. Evidence is accumulating to indicate that PSCs interact with other cell types in the pancreas including acinar cells, islets, endothelial cells, inflammatory cells, cancer cells, and cancer stem cells. The interaction of PSCs with duct cells has not yet been fully elucidated.
cell progression by multiple mechanisms such as increased proliferation, migration and metastasis, and by protecting cancer cells from the induction of gemcitabine- and radiation-induced apoptosis [4,7,24e28]. We have previously reported that PSCs induced EMT in pancreatic cancer cells, as shown by fibroblast-like cell morphology, decreased expression of epithelial markers and increased expression of mesenchymal markers [29]. EMT is a developmental process that allows a polarized epithelial cell to undergo multiple biochemical changes that enable it to assume a mesenchymal phenotype [29]. Including enhanced migratory capacity, invasiveness, elevated resistance to apoptosis and greatly increased production of extracellular matrix components [30]. EMT is now considered a critical process in cancer progression, and EMT induction in cancer cells results in the acquisition of invasive and metastatic properties as well as resistance to conventional therapies. Very recently, we have shown that PSCs enhanced stem-cell like phenotypes in pancreatic cancer cells [31]. PSCs increased the spheroid formation and the expression of stem cell markers such as ATP-binding cassette sub-family G member 2 and nestin. In vivo, PSCs enhanced tumorigenicity, suggesting that PSCs might form a niche for pancreatic cancer stem cells. From the above discussion it is evident that various interactions between PSCs and other cell types play a role in healthy and diseased pancreas (Fig. 2). These interactions are mediated mainly by cytokines and growth factors such as platelet-derived growth factor and transforming growth factor-b [3e7,32]. However, we have recently shown that PSCs express a wide range of connexins, which are subunits of gap junctions [33]. The expression of a variety of connexins in PSCs suggested the presence of connexin-mediated intercellular communication between PSCs and other types of cells.
7. Conclusions In addition to producing ECM components, PSCs have a wide variety of cell functions. The interactions between PSCs and other cell types play critical roles in healthy and diseased pancreas. PSCs should be recognized as not just pro-fibrogenic cells but are multifunctional cells in the pancreas.
A. Masamune, T. Shimosegawa / Pancreatology 13 (2013) 102e105
Financial supports This work was supported in part by Grant-in-Aid from Japan Society for the Promotion of Science (23591008) and by the Research Committee of Intractable Pancreatic Diseases provided by the Ministry of Health, Labor, and Welfare of Japan. Acknowledgments We are grateful to Dr. Eriko Nakano for excellent artworks. We apologize for being unable to include all the references related to this field because of space limitation. References [1] Apte MV, Haber PS, Applegate TL, Norton ID, McCaughan GW, Korsten MA, et al. Periacinar stellate shaped cells in rat pancreas: identification, isolation and culture. Gut 1998;43:128e33. [2] Bachem MG, Schneider E, Gross H, Weidenbach H, Schmid RM, Menke A, et al. Identification, culture, and characterization of pancreas stellate cells in rats and humans. Gastroenterology 1998;115:421e32. [3] Omary MB, Lugea A, Lowe AW, Pandol SJ. The pancreatic stellate cell: a star on the rise in pancreatic diseases. J Clin Invest 2007;117:50e9. [4] Vonlaufen A, Phillips PA, Xu Z, Goldstein D, Pirola RC, Wilson JS, et al. Pancreatic stellate cells and pancreatic cancer cells: an unholy alliance. Cancer Res 2008;68:7707e10. [5] Masamune A, Shimosegawa T. Signal transduction in pancreatic stellate cells. J Gastroenterol 2009;44:249e60. [6] Masamune A, Watanabe T, Kikuta K, Shimosegawa T. Roles of pancreatic stellate cells in pancreatic inflammation and fibrosis. Clin Gastroenterol Hepatol 2009;7:S48e54. [7] Erkan M, Adler G, Apte MV, Bachem MG, Buchholz M, Detlefsen S, et al. StellaTUM: current consensus and discussion on pancreatic stellate cell research. Gut 2012;61:172e8. [8] Masamune A, Kikuta K, Satoh M, Satoh K, Shimosegawa T. Rho kinase inhibitors block activation of pancreatic stellate cells. Br J Pharmacol 2003;140: 1292e302. [9] Masamune A, Satoh M, Kikuta K, Sakai Y, Satoh A, Shimosegawa T. Inhibition of p38 mitogen-activated protein kinase blocks activation of rat pancreatic stellate cells. J Pharmacol Exp Ther 2003;304:8e14. [10] Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell 2009; 136:215e33. [11] Phillips PA, McCarroll JA, Park S, Wu MJ, Pirola R, Korsten M, et al. Rat pancreatic stellate cells secrete matrix metalloproteinases: implications for extracellular matrix turnover. Gut 2003;52:275e82. [12] Lugea A, Nan L, French SW, Bezerra JA, Gukovskaya AS, Pandol SJ. Pancreas recovery following cerulein-induced pancreatitis is impaired in plasminogendeficient mice. Gastroenterology 2006;131:885e99. [13] Masamune A, Satoh M, Kikuta K, Suzuki N, Shimosegawa T. Endothelin-1 stimulates contraction and migration of rat pancreatic stellate cells. World J Gastroenterol 2005;11:6144e51. [14] Masamune A, Watanabe T, Kikuta K, Satoh K, Kanno A, Shimosegawa T. Nuclear expression of interleukin-33 in pancreatic stellate cells. Am J Physiol Gastrointest Liver Physiol 2010;299:G821e32.
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[15] Vonlaufen A, Xu Z, Daniel B, Kumar RK, Pirola R, Wilson J, et al. Bacterial endotoxin: a trigger factor for alcoholic pancreatitis? Evidence from a novel, physiologically relevant animal model. Gastroenterology 2007;133: 1293e303. [16] Masamune A, Kikuta K, Watanabe T, Satoh K, Satoh A, Shimosegawa T. Pancreatic stellate cells express toll-like receptors. J Gastroenterol 2008;43: 352e62. [17] Masamune A, Kikuta K, Watanabe T, Satoh K, Hirota M, Shimosegawa T. Hypoxia stimulates pancreatic stellate cells to induce fibrosis and angiogenesis in pancreatic cancer. Am J Physiol Gastrointest Liver Physiol 2008;295: G709e17. [18] Cassiman D, Barlow A, Vander Borght S, Libbrecht L, Pachnis V. Hepatic stellate cells do not derive from the neural crest. J Hepatol 2006;44:1098e104. [19] Asahina K, Tsai SY, Li P, Ishii M, Maxson Jr RE, Sucov HM, et al. Mesenchymal origin of hepatic stellate cells, submesothelial cells, and perivascular mesenchymal cells during mouse liver development. Hepatology 2009;49:998e 1011. [20] Asahina K, Zhou B, Pu WT, Tsukamoto H. Septum transversum-derived mesothelium gives rise to hepatic stellate cells and perivascular mesenchymal cells in developing mouse liver. Hepatology 2011;53:983e95. [21] Watanabe T, Masamune A, Kikuta K, Hirota M, Kume K, Satoh K, et al. Bone marrow contributes to the population of pancreatic stellate cells. Am J Physiol Gastrointest Liver Physiol 2009;297:G1138e46. [22] Buchholz M, Kestler HA, Holzmann K, Ellenrieder V, Schneiderhan W, Siech M, et al. Transcriptome analysis of human hepatic and pancreatic stellate cells: organ-specific variations of a common transcriptional phenotype. J Mol Med (Berl) 2005;83:795e805. [23] Phillips PA, Yang L, Shulkes A, Vonlaufen A, Poljak A, Bustamante S, et al. Pancreatic stellate cells produce acetylcholine and may play a role in pancreatic exocrine secretion. Proc Natl Acad Sci U S A 2010;107:17397e402. [24] Bachem MG, Schunemann M, Ramadani M, Siech M, Beger H, Buck A, et al. Pancreatic carcinoma cells induce fibrosis by stimulating proliferation and matrix synthesis of stellate cells. Gastroenterology 2005;128:907e21. [25] Hwang RF, Moore T, Arumugam T, Ramachandran V, Amos KD, Rivera A, et al. Cancer-associated stromal fibroblasts promote pancreatic tumor progression. Cancer Res 2008;68:918e26. [26] Xu Z, Vonlaufen A, Phillips PA, Fiala-Beer E, Zhang X, Yang L, et al. Role of pancreatic stellate cells in pancreatic cancer metastasis. Am J Pathol 2010; 177:2585e96. [27] Mantoni TS, Lunardi S, Al-Assar O, Masamune A, Brunner TB. Pancreatic stellate cells radioprotect pancreatic cancer cells through b1-integrin signaling. Cancer Res 2011;71:3453e8. [28] Erkan M, Hausmann S, Michalski CW, Fingerle AA, Dobritz M, Kleeff J, et al. The role of stroma in pancreatic cancer: diagnostic and therapeutic implications. Nat Rev Gastroenterol Hepatol 2012;9:454e67. [29] Kikuta K, Masamune A, Watanabe T, Ariga H, Itoh H, Hamada S, et al. Pancreatic stellate cells promote epithelial-mesenchymal transition in pancreatic cancer cells. Biochem Biophys Res Commun 2010;403:380e4. [30] Thiery JP, Acloque H, Huang RY, Nieto MA. Epithelial-mesenchymal transitions in development and disease. Cell 2009;139:871e90. [31] Hamada S, Masamune A, Takikawa T, Suzuki N, Kikuta K, Hirota M, et al. Pancreatic stellate cells enhance stem cell-like phenotypes in pancreatic cancer cells. Biochem Biophys Res Commun 2012;421:349e54. [32] Masamune A, Kikuta K, Satoh M, Kume K, Shimosegawa T. Differential roles of signaling pathways for proliferation and migration of rat pancreatic stellate cells. Tohoku J Exp Med 2003;199:69e84. [33] Masamune A, Suzuki N, Kikuta K, Ariga H, Hayashi S, Takikawa T, et al. Connexins regulate cell functions in pancreatic stellate cells. Pancreas 2012 Aug 10 [Epub ahead of print].
Contents lists available at SciVerse ScienceDirect
Pancreatology journal homepage: www.elsevier.com/locate/pan
Review article
Pancreatic stellate cells e Multi-functional cells in the pancreas Atsushi Masamune*, Tooru Shimosegawa Division of Gastroenterology, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 November 2012 Received in revised form 10 December 2012 Accepted 10 December 2012
There is accumulating evidence that activated pancreatic stellate cells (PSCs) play a pivotal role in pancreatic fibrosis in chronic pancreatitis and pancreatic cancer. In addition, we have seen great progress in our understanding of the cell biology of PSCs and the interactions between PSCs and other cell types in the pancreas. In response to pancreatic injury or inflammation, quiescent PSCs are activated to myofibroblast-like cells. Recent studies have shown that the activation of intracellular signaling pathways such as mitogen-activated protein kinases plays a role in the activation of PSCs. microRNAs might also play a role, because the microRNA expression profiles are dramatically altered in the process of activation. In addition to producing extracellular matrix components such as type I collagen, PSCs have a wide variety of cell functions related to local immunity, inflammation, angiogenesis, and exocrine and endocrine functions in the pancreas. From this point of view, the interactions between PSCs and other cell types such as pancreatic exocrine cells, endocrine cells, and cancer cells have attracted increasing attention of researchers. PSCs might regulate exocrine functions in the pancreas through the cholecystokinin-induced release of acetylcholine. PSCs induce apoptosis and decrease insulin expression in b-cells, suggesting a novel mechanism of diabetes in diseased pancreas. PSCs promote the progression of pancreatic cancer by multiple mechanisms. Recent studies have shown that PSCs induce epithelial emesenchymal transition and enhance the stem-cell like features of pancreatic cancer cells. In conclusion, PSCs should now be recognized as not only profibrogenic cells but as multi-functional cells in the pancreas. Copyright Ó 2012, IAP and EPC. Published by Elsevier India, a division of Reed Elsevier India Pvt. Ltd. All rights reserved.
Keywords: Pancreatitis Pancreatic cancer Desmoplastic reaction Fibrosis MicroRNA Signal transduction
1. Introduction Pancreatic fibrosis is a characteristic feature of chronic pancreatitis (CP) and desmoplastic reaction associated with pancreatic cancer. However, for a long time, the molecular mechanisms remained largely unknown, mainly due to the lack of appropriate in vitro models and the unavailability of histological samples. In 1998, two groups independently reported the identification, isolation and characterization of the star-shaped cells in the pancreas, namely pancreatic stellate cells (PSCs) [1,2]. Since then, evidence has been accumulating to indicate that activated PSCs play a pivotal role in the development of pancreatic fibrosis
Abbreviations used: CP, chronic pancreatitis; ECM, extracellular matrix; EMT, epithelialemesenchymal transition; GLUT, glucose transporter; HSC, hepatic stellate cell; IL, interleukin; MAP, mitogen-activated protein; miRNA, microRNA; PSC, pancreatic stellate cell; MMP, matrix metalloproteinase; SMA, smooth muscle actin; TIMP, tissue inhibitor of metalloproteinase; TLR, Toll-like receptor; VEGF, vascular endothelial growth factor. * Corresponding author. Tel.: þ81 22 717 7172; fax: þ81 22 717 7177. E-mail address: [email protected] (A. Masamune).
in CP and pancreatic cancer [3e7]. Recently, we have seen great progress in our understanding of the cell biology of PSCs and the interactions between PSCs and other cell types in the pancreas. In this overview, we will briefly summarize our current knowledge in this field.
2. Activation of PSCs In normal pancreas, stellate cells are quiescent and can be identified by the presence of vitamin A-containing lipid droplets in the cytoplasm. In response to pancreatic injury or inflammation, quiescent PSCs undergo morphological and functional changes to become myofibroblast-like cells which express a-smooth muscle actin (a-SMA). This step is called “activation”. Activated PSCs lose lipid droplets, actively proliferate, migrate, and produce large amounts of extracellular matrix (ECM) components such as type I collagen and fibronectin [1e7]. In vitro studies have identified that cytokines and growth factors including transforming growth factor-b, tumor necrosis factor-a, interleukin (IL)-1, IL-6 and activin A as well as ethanol and its metabolites, oxidative stress and
1424-3903/$ e see front matter Copyright Ó 2012, IAP and EPC. Published by Elsevier India, a division of Reed Elsevier India Pvt. Ltd. All rights reserved. http://dx.doi.org/10.1016/j.pan.2012.12.058
A. Masamune, T. Shimosegawa / Pancreatology 13 (2013) 102e105
pressure, induce the activation of PSCs [3e7]. Cytokines and growth factors produced during pancreatic injury or inflammation by acinar cells, inflammatory cells, platelets, and endothelial cells, would activate PSCs and regulate these cell functions. Interestingly, PSCs themselves produce a variety of cytokines and growth factors [3e7]. Therefore, in addition to paracrine regulation by neighboring cells, PSCs might be activated in an autocrine manner. 3. Mechanisms of PSCs activation The mechanism responsible for the activation of PSCs is not yet completely understood, but includes the activation of intracellular signaling pathways such as mitogen-activated protein (MAP) kinases and Rho/Rho kinase (reviewed in 5). For example, during the process of activation, stress fiber formation is increased, suggesting cytoskeletal reorganization is involved in this process [8]. The small GTP-binding protein Rho has emerged as an important regulator of the actin cytoskeleton and cell morphology through the downstream effector Rho kinase. Specific inhibitors of Rhoe Rho kinase (Y-27632 and HA-1077) inhibit the transformation of quiescent PSCs to myofibroblast-like cells in vitro [8]. Similarly, inhibitors of p38 MAP kinase inhibit the activation of PSCs, supporting the role of these signaling pathways in the activation of PSCs [9]. Recently, microRNAs (miRNAs) have attracted the attention of researchers. miRNAs are small, non-coding RNAs consisting of 20e 23 nucleotides that target the 30 untranslated region sequence of messenger RNA for destabilization [10]. miRNAs regulate a variety of cell functions such as cell proliferation, apoptosis, differentiation, and carcinogenesis [10]. We compared the miRNA expression profiles between quiescent and activated rat PSCs. We found that the miRNA signature was dramatically altered during the activation of PSCs (Fig. 1) (Masamune A et al. unpublished observations). Pathway analysis (Ingenuity Systems, Redwood City, CA) showed that the altered miRNAs were associated with cellular movement, growth, and death, suggesting a role of miRNAs in the activation of PSCs.
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4. Cell functions of PSCs In addition to playing a key role in fibrogenesis, PSCs have a variety of cell functions. These cells not only produce ECM, but also synthezise matrix-degrading enzymes of the matrix metalloproteinase (MMP) family and their inhibitors [tissue inhibitors of metalloproteinases (TIMPs)] [11]. PSCs have been shown to secrete MMP-2, MMP-9, and MMP-13, and to express TIMP-1 and TIMP-2 [11]. Thus, PSCs have the ability to produce as well as to degrade ECM, suggesting that they might play a role in the maintenance of the normal tissue architecture by regulating ECM turnover. In this context, the resolution of mouse caerulein-induced pancreatitis involves transient activation of PSCs and deposition of ECM proteins, as well as the transient upregulation of MMPs and TIMPs [12]. The increased expression of the cytoskeletal protein a-SMA confers increased contractile potential, which is further increased by endothelin-1 [13]. Because PSCs are also located around ductal and vascular structures [1e7], it would be of interest to see whether the contraction of PSCs is involved in the regulation of vascular and ductal tone in the pancreas. PSCs produce a broad range of pro-inflammatory and antiinflammatory cytokines, growth factors, and adipocytokines (Table 1), suggesting pro-inflammatory and anti-inflammatory roles of PSCs. Expression of these cytokines is often associated with the activation of PSCs. For example, IL-33 is a recently identified member of the IL-1 gene family [14]. IL-33 expression was low in freshly isolated, quiescent rat PSCs but was increased upon activation, indicating that IL-33 induction was associated with the transformation to an a-SMA-positive myofibroblastic phenotype. In addition, we and others have shown that both freshly-isolated and activated PSCs express Toll-like receptors (TLRs), which are proteins involved in the activation of innate immunity [15,16]. PSCs express TLR2, which recognizes pathogen-associated molecular patterns of Gram-positive bacteria, and TLR4, which recognizes lipopoysaccharides of Gram-negative bacteria. PSCs could perform endocytosis and phagocytosis of foreign bodies, suggesting that PSCs might play a role in the local immune functions in the pancreas. Another novel function we identified in PSCs is related to angiogenesis. PSCs constitutively produce vascular endothelial growth factor (VEGF), the production of which is increased by hypoxia [17]. In addition to VEGF, PSCs expressed several angiogenesis-regulating molecules including VEGF receptors, angiopoietin-1 and its receptor Tie-2, and vasohibin-1. Conditioned media of PSCs induced angiogenesis in vitro, as shown by increased tube formation on Matrigel, and in vivo, as shown by directed vessel formation in nude mice. Very recently, we have found that PSCs express glucose transporters (GLUTs). PSCs expressed Glut1 and Glut 3, but not Glut 2 or Glut 4, suggesting a novel role of PSCs in the metabolism of glucose (Masamune A et al. unpublished observations). Therefore, in
Table 1 PSCs express a wide range of prtoinflammatory and anti-inflammatory cytokines, growth factors, and adipocytokines. IL-6 IL-8 IL-10 IL-23 IL-24 IL-32 IL-33 RANTES Fig. 1. miRNA expression profiles were different between quiescent and activated PSCs. Total RNAs including miRNAs were prepared from freshly-isolated rat quiescent PSCs (day 0) and culture-activated rat PSCs (day 14). The miRNA expression profiles in these cells were compared using the Agilent’s miRNA microarray.
MCP-1: monocyte chemoattractant protein-1. FGF: fibroblast growth factor. CTGF: connective tissue growth factor. TGF: transforming growth factor.
MCP-1 FGF CTGF TGF-b Activin/Nodal VEGF Angiopoietin Chemerin
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addition to the production of ECMs, PSCs have a wide variety of cell functions. 5. The origin and source of PSCs What is the origin of PSCs? Mesodermal, endodermal, and neuro-ectodermal origins might be possible, but no studies have addressed this issue to date. In hepatic stellate cells (HSCs), several studies have attempted to clarify this issue. For example, the neuroectoderm proposal for the origins of HSC was refuted by a study by Cassiman et al., [18] who used a genetic cell lineage mapping technique with Rosa26YFPflox mice crossed with mice expressing Cre under the control of the neural crest-specific Wnt1 promoter/ enhancer. Definitive novel evidence for the mesodermal origin of HSCs has recently been presented by Asahina and colleagues [19,20] with the use of the mesoderm-specific MesP1Cre. A conditional cell lineage analysis using Wt1CreERT/Rosa26LacZflox or ROSA26mTmGflox mice, revealed Wt1-positive septum transversum giving rise to mesothelial cells, submesothelial cells, HSCs and perivascular mesenchymal cells during liver development [19]. That study also demonstrated Wt1-positive mesothelial/submesothelial cells migrating inward from the liver surface to generate liver mesenchymal cells including HSCs. Similar lineage tracing techniques need to be used to determine the exact origin of PSCs. In terms of the PSCs within normal pancreas and their increased numbers during pancreatic injury, the question often asked has been whether these cells arise wholly from resident cells or whether there is a contribution to this PSC population from other sources. Using sex-mismatched, bone marrow transplantation from male green mice to female mice, we could identify desmin-positive, bone marrow-derived cells in the pancreas [21]. Bone marrowderived cells accounted for about 8% of the total desmin-positive cells. After the induction of pancreatitis by caerulein, approximately 20% of a-SMA-positive cells contained Y chromosome in the nucleus. suggesting they originated in bone marrow. Therefore, bone marrow contributes to the populations of both quiescent and activated PSCs. Endothelial- or epithelialemesenchymal transition (EMT) may also contribute to the population of activated PSCs during pancreatic injury, but further studies are required in this area. 6. Interactions between PSCs and other cell types in the pancreas Because PSCs are morphologically and functionally very similar to HSCs, it is important to determine whether PSCs are identical to or different from HSCs. A DNA array study showed that 29 out of 23,000 genes (i.e. 0.1%) were different between PSCs and HSCs [22], suggesting that organ-specific influences may be relevant to stellate cell biology. Therefore, research on PSCs should develop in directions more relevant to the patho-physiology of the pancreas. In this context, the interactions between PSCs and other cell types in the pancreas have attracted increasing attention. For example, the expression of cholecystokinin receptors in PSCs has been reported [23]. Cholecystokinin-8 stimulated acetylcholine release in PSCs, leading to the stimulation of exocrine functions in acinar cells. These findings suggest a novel role of PSCs in the regulation of exocrine functions in the pancreas. Similarly, we examined the interaction between PSCs and b-cells. PSCs decreased insulin mRNA expression as well as secretion of insulin by b-cells. (Kikuta K et al., unpublished observations). In addition, PSCs induced apoptosis of b-cells demonstrating that PSCs induced changes in b-cells both quantitatively and qualitatively. Another important interaction is between PSCs and cancer cells. It has been reported that PSCs promote cancer
Fig. 2. Multiple interactions between PSCs and other cell types in the pancreas. Evidence is accumulating to indicate that PSCs interact with other cell types in the pancreas including acinar cells, islets, endothelial cells, inflammatory cells, cancer cells, and cancer stem cells. The interaction of PSCs with duct cells has not yet been fully elucidated.
cell progression by multiple mechanisms such as increased proliferation, migration and metastasis, and by protecting cancer cells from the induction of gemcitabine- and radiation-induced apoptosis [4,7,24e28]. We have previously reported that PSCs induced EMT in pancreatic cancer cells, as shown by fibroblast-like cell morphology, decreased expression of epithelial markers and increased expression of mesenchymal markers [29]. EMT is a developmental process that allows a polarized epithelial cell to undergo multiple biochemical changes that enable it to assume a mesenchymal phenotype [29]. Including enhanced migratory capacity, invasiveness, elevated resistance to apoptosis and greatly increased production of extracellular matrix components [30]. EMT is now considered a critical process in cancer progression, and EMT induction in cancer cells results in the acquisition of invasive and metastatic properties as well as resistance to conventional therapies. Very recently, we have shown that PSCs enhanced stem-cell like phenotypes in pancreatic cancer cells [31]. PSCs increased the spheroid formation and the expression of stem cell markers such as ATP-binding cassette sub-family G member 2 and nestin. In vivo, PSCs enhanced tumorigenicity, suggesting that PSCs might form a niche for pancreatic cancer stem cells. From the above discussion it is evident that various interactions between PSCs and other cell types play a role in healthy and diseased pancreas (Fig. 2). These interactions are mediated mainly by cytokines and growth factors such as platelet-derived growth factor and transforming growth factor-b [3e7,32]. However, we have recently shown that PSCs express a wide range of connexins, which are subunits of gap junctions [33]. The expression of a variety of connexins in PSCs suggested the presence of connexin-mediated intercellular communication between PSCs and other types of cells.
7. Conclusions In addition to producing ECM components, PSCs have a wide variety of cell functions. The interactions between PSCs and other cell types play critical roles in healthy and diseased pancreas. PSCs should be recognized as not just pro-fibrogenic cells but are multifunctional cells in the pancreas.
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Financial supports This work was supported in part by Grant-in-Aid from Japan Society for the Promotion of Science (23591008) and by the Research Committee of Intractable Pancreatic Diseases provided by the Ministry of Health, Labor, and Welfare of Japan. Acknowledgments We are grateful to Dr. Eriko Nakano for excellent artworks. We apologize for being unable to include all the references related to this field because of space limitation. References [1] Apte MV, Haber PS, Applegate TL, Norton ID, McCaughan GW, Korsten MA, et al. Periacinar stellate shaped cells in rat pancreas: identification, isolation and culture. Gut 1998;43:128e33. [2] Bachem MG, Schneider E, Gross H, Weidenbach H, Schmid RM, Menke A, et al. Identification, culture, and characterization of pancreas stellate cells in rats and humans. Gastroenterology 1998;115:421e32. [3] Omary MB, Lugea A, Lowe AW, Pandol SJ. The pancreatic stellate cell: a star on the rise in pancreatic diseases. J Clin Invest 2007;117:50e9. [4] Vonlaufen A, Phillips PA, Xu Z, Goldstein D, Pirola RC, Wilson JS, et al. Pancreatic stellate cells and pancreatic cancer cells: an unholy alliance. Cancer Res 2008;68:7707e10. [5] Masamune A, Shimosegawa T. Signal transduction in pancreatic stellate cells. J Gastroenterol 2009;44:249e60. [6] Masamune A, Watanabe T, Kikuta K, Shimosegawa T. Roles of pancreatic stellate cells in pancreatic inflammation and fibrosis. Clin Gastroenterol Hepatol 2009;7:S48e54. [7] Erkan M, Adler G, Apte MV, Bachem MG, Buchholz M, Detlefsen S, et al. StellaTUM: current consensus and discussion on pancreatic stellate cell research. Gut 2012;61:172e8. [8] Masamune A, Kikuta K, Satoh M, Satoh K, Shimosegawa T. Rho kinase inhibitors block activation of pancreatic stellate cells. Br J Pharmacol 2003;140: 1292e302. [9] Masamune A, Satoh M, Kikuta K, Sakai Y, Satoh A, Shimosegawa T. Inhibition of p38 mitogen-activated protein kinase blocks activation of rat pancreatic stellate cells. J Pharmacol Exp Ther 2003;304:8e14. [10] Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell 2009; 136:215e33. [11] Phillips PA, McCarroll JA, Park S, Wu MJ, Pirola R, Korsten M, et al. Rat pancreatic stellate cells secrete matrix metalloproteinases: implications for extracellular matrix turnover. Gut 2003;52:275e82. [12] Lugea A, Nan L, French SW, Bezerra JA, Gukovskaya AS, Pandol SJ. Pancreas recovery following cerulein-induced pancreatitis is impaired in plasminogendeficient mice. Gastroenterology 2006;131:885e99. [13] Masamune A, Satoh M, Kikuta K, Suzuki N, Shimosegawa T. Endothelin-1 stimulates contraction and migration of rat pancreatic stellate cells. World J Gastroenterol 2005;11:6144e51. [14] Masamune A, Watanabe T, Kikuta K, Satoh K, Kanno A, Shimosegawa T. Nuclear expression of interleukin-33 in pancreatic stellate cells. Am J Physiol Gastrointest Liver Physiol 2010;299:G821e32.
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[15] Vonlaufen A, Xu Z, Daniel B, Kumar RK, Pirola R, Wilson J, et al. Bacterial endotoxin: a trigger factor for alcoholic pancreatitis? Evidence from a novel, physiologically relevant animal model. Gastroenterology 2007;133: 1293e303. [16] Masamune A, Kikuta K, Watanabe T, Satoh K, Satoh A, Shimosegawa T. Pancreatic stellate cells express toll-like receptors. J Gastroenterol 2008;43: 352e62. [17] Masamune A, Kikuta K, Watanabe T, Satoh K, Hirota M, Shimosegawa T. Hypoxia stimulates pancreatic stellate cells to induce fibrosis and angiogenesis in pancreatic cancer. Am J Physiol Gastrointest Liver Physiol 2008;295: G709e17. [18] Cassiman D, Barlow A, Vander Borght S, Libbrecht L, Pachnis V. Hepatic stellate cells do not derive from the neural crest. J Hepatol 2006;44:1098e104. [19] Asahina K, Tsai SY, Li P, Ishii M, Maxson Jr RE, Sucov HM, et al. Mesenchymal origin of hepatic stellate cells, submesothelial cells, and perivascular mesenchymal cells during mouse liver development. Hepatology 2009;49:998e 1011. [20] Asahina K, Zhou B, Pu WT, Tsukamoto H. Septum transversum-derived mesothelium gives rise to hepatic stellate cells and perivascular mesenchymal cells in developing mouse liver. Hepatology 2011;53:983e95. [21] Watanabe T, Masamune A, Kikuta K, Hirota M, Kume K, Satoh K, et al. Bone marrow contributes to the population of pancreatic stellate cells. Am J Physiol Gastrointest Liver Physiol 2009;297:G1138e46. [22] Buchholz M, Kestler HA, Holzmann K, Ellenrieder V, Schneiderhan W, Siech M, et al. Transcriptome analysis of human hepatic and pancreatic stellate cells: organ-specific variations of a common transcriptional phenotype. J Mol Med (Berl) 2005;83:795e805. [23] Phillips PA, Yang L, Shulkes A, Vonlaufen A, Poljak A, Bustamante S, et al. Pancreatic stellate cells produce acetylcholine and may play a role in pancreatic exocrine secretion. Proc Natl Acad Sci U S A 2010;107:17397e402. [24] Bachem MG, Schunemann M, Ramadani M, Siech M, Beger H, Buck A, et al. Pancreatic carcinoma cells induce fibrosis by stimulating proliferation and matrix synthesis of stellate cells. Gastroenterology 2005;128:907e21. [25] Hwang RF, Moore T, Arumugam T, Ramachandran V, Amos KD, Rivera A, et al. Cancer-associated stromal fibroblasts promote pancreatic tumor progression. Cancer Res 2008;68:918e26. [26] Xu Z, Vonlaufen A, Phillips PA, Fiala-Beer E, Zhang X, Yang L, et al. Role of pancreatic stellate cells in pancreatic cancer metastasis. Am J Pathol 2010; 177:2585e96. [27] Mantoni TS, Lunardi S, Al-Assar O, Masamune A, Brunner TB. Pancreatic stellate cells radioprotect pancreatic cancer cells through b1-integrin signaling. Cancer Res 2011;71:3453e8. [28] Erkan M, Hausmann S, Michalski CW, Fingerle AA, Dobritz M, Kleeff J, et al. The role of stroma in pancreatic cancer: diagnostic and therapeutic implications. Nat Rev Gastroenterol Hepatol 2012;9:454e67. [29] Kikuta K, Masamune A, Watanabe T, Ariga H, Itoh H, Hamada S, et al. Pancreatic stellate cells promote epithelial-mesenchymal transition in pancreatic cancer cells. Biochem Biophys Res Commun 2010;403:380e4. [30] Thiery JP, Acloque H, Huang RY, Nieto MA. Epithelial-mesenchymal transitions in development and disease. Cell 2009;139:871e90. [31] Hamada S, Masamune A, Takikawa T, Suzuki N, Kikuta K, Hirota M, et al. Pancreatic stellate cells enhance stem cell-like phenotypes in pancreatic cancer cells. Biochem Biophys Res Commun 2012;421:349e54. [32] Masamune A, Kikuta K, Satoh M, Kume K, Shimosegawa T. Differential roles of signaling pathways for proliferation and migration of rat pancreatic stellate cells. Tohoku J Exp Med 2003;199:69e84. [33] Masamune A, Suzuki N, Kikuta K, Ariga H, Hayashi S, Takikawa T, et al. Connexins regulate cell functions in pancreatic stellate cells. Pancreas 2012 Aug 10 [Epub ahead of print].