Marcie Kritzik

Pancreatic Expression of Keratinocyte Growth Factor Leads to Differentiation of Islet Hepatocytes and Proliferation of Duct Cells

Keratinocyte growth factor, (KGF), a member of the fibroblast growth factor (FGF) family, is involved in wound healing. It also promotes the differentiation of many epithelial tissues and proliferation of epithelial cells as well as pancreatic duct cells. Additionally, many members of the highly homologous FGF family (including KGF), influence both growth and cellular morphology in the developing embryo. We have previously observed elevated levels of KGF in our interferon-gamma transgenic mouse model of pancreatic regeneration. To understand the role of KGF in pancreatic differentiation, we generated insulin promoter-regulated KGF transgenic mice. Remarkably, we have found that ectopic KGF expression resulted in the emergence of hepatocytes within the islets of Langerhans in the pancreas. Additionally, significant intra-islet duct cell proliferation in the pancreata of transgenic KGF mice was observed. The unexpected appearance of hepatocytes and proliferation of intra-islet duct cells in the pancreata of these mice evidently stemmed directly from local exposure to KGF.

The stromal cell–derived factor-1α/CXCR4 ligand–receptor axis is critical for progenitor survival and migration in the pancreas

The SDF-1α/CXCR4 ligand/chemokine receptor pair is required for appropriate patterning during ontogeny and stimulates the growth and differentiation of critical cell types. Here, we demonstrate SDF-1α and CXCR4 expression in fetal pancreas. We have found that SDF-1α and its receptor CXCR4 are expressed in islets, also CXCR4 is expressed in and around the proliferating duct epithelium of the regenerating pancreas of the interferon (IFN) γ–nonobese diabetic mouse. We show that SDF-1α stimulates the phosphorylation of Akt, mitogen-activated protein kinase, and Src in pancreatic duct cells. Furthermore, migration assays indicate a stimulatory effect of SDF-1α on ductal cell migration. Importantly, blocking the SDF-1α/CXCR4 axis in IFNγ-nonobese diabetic mice resulted in diminished proliferation and increased apoptosis in the pancreatic ductal cells. Together, these data indicate that the SDF-1α–CXCR4 ligand receptor axis is an obligatory component in the maintenance of duct cell survival, proliferation, and migration during pancreatic regeneration.

BMP4 Regulates Pancreatic Progenitor Cell Expansion through Id2

Inhibitor of DNA binding (Id) proteins bind to and inhibit the function of basic helix-loop-helix (bHLH) transcription factors including those that regulate pancreatic development. Moreover, bone morphogenetic proteins (BMPs) regulate the expression of Ids. We hypothesized that BMP4 and Id proteins play a role in the expansion and differentiation of epithelial progenitor cells. We demonstrate that BMP4 induces the expression of Id2 along with the expansion of AR42J pancreatic epithelial cells. Furthermore, neutralization of BMP4 significantly reduced duct epithelial cell expansion in a mouse model of islet regeneration. BMP4 stimulation promotes Id2 binding to the bHLH transcription factor NeuroD, which is required for the differentiation of pancreatic islet cells. Therefore, our results indicate that BMP4 stimulation blocks the differentiation of endocrine progenitor cells and instead promotes their expansion thereby revealing a novel paradigm of signaling explaining the balance between expansion and differentiation of pancreatic duct epithelial progenitors. Understanding the mechanisms of BMP and Id function elucidates a key step during pancreas embryogenesis, which is important knowledge for expanding pancreatic progenitors in vitro.

Transcription factor expression during pancreatic islet regeneration.

Recent studies by a number of laboratories have identified transcription factors that are involved in pancreatic development. Indeed, marked abnormalities in pancreatic development result from deficiencies in these molecules, which include, among others, PDX-1, islet-1 (Isl-1), and Pax-6. These studies have prompted us to evaluate the expression of Isl-1 and Pax-6 in the pancreas of the interferon-gamma (IFNgamma) transgenic mouse, which exhibits new islet growth and expansion of ducts throughout the life of the animal. We have previously demonstrated that PDX-1 is strikingly expressed in the ducts of the IFNgamma transgenic mouse. This latter observation compelled us to examine expression of hepatocyte nuclear factor-3beta (HNF3beta), which mediates PDX-1 gene transcription, in the IFNgamma transgenic pancreas as well. As a result of these studies, we now demonstrate marked expression of these transcription factors in the pancreatic ducts of IFNgamma transgenic mice. These data suggest a role for these transcription factors during pancreatic regeneration in the IFNgamma transgenic mouse.

Identification and expansion of pancreatic stem/progenitor cells

Pancreatic islet transplantation represents an attrative approach for the treatment of diabetes. However, the limited availability of donor islets has largely hampered this approach. In this respect, the use of alternative sources of islets such as the ex vivo expansion and differentiation of functional endocrine cells for treating diabetes has become the major focus of diabetes research. Adult pancreatic stem cells/progenitor cells have yet to be recognized because limited markers exist for their identification. While the pancreas has the capacity to regenerate under certain circumstance, questions where adult pancreatic stem/progenitor cells are localized, how they are regulated, and even if the pancreas harbors a stem cell population need to be resolved. In this article, we review the recent achievements both in the identification as well as in the expansion of pancreatic stem/progenitor cells.

FGFR3 Is a Negative Regulator of the Expansion of Pancreatic Epithelial Cells

Fibroblast growth factors (FGFs) and their receptors (FGFRs) are key signaling molecules for pancreas development. Although FGFR3 is a crucial developmental gene, acting as a negative regulator of bone formation, its participation remains unexplored in pancreatic organogenesis. We found that FGFR3 was expressed in the epithelia in both mouse embryonic and adult regenerating pancreata but was absent in normal adult islets. In FGFR3 knockout mice, we observed an increase in the proliferation of epithelial cells in neonates, leading to a marked increase in islet areas in adults. In vitro studies showed that FGF9 is a very potent ligand for FGFR3 and activates extracellular signal–related kinases (ERKs) in pancreatic cell lines. Moreover, FGFR3 blockade or FGFR3 deficiency led to increased proliferation of pancreatic epithelial cells in vivo. This was accompanied by an increase in the proportion of potential islet progenitor cells. Thus, our results show that FGFR3 signaling inhibits the expansion of the immature pancreatic epithelium. Consequently, this study suggests that FGFR3 participates in regulating pancreatic growth during the emergence of mature islet cells.

Nodal and lefty signaling regulates the growth of pancreatic cells

Nodal and its antagonist, Lefty, are important mediators specifying the laterality of the organs during embryogenesis. Nodal signals through activin receptors in the presence of its co‐receptor, Cripto. In the present study, we investigated the possible roles of Nodal and Lefty signaling during islet development and regeneration. We found that both Nodal and Lefty are expressed in the pancreas during embryogenesis and islet regeneration. In vitro studies demonstrated that Nodal inhibits, whereas Lefty enhances, the proliferation of a pancreatic cell line. In addition, we showed that Lefty‐1 activates MAPK and Akt phosphorylation in these cells. In vivo blockade of endogenous Lefty using neutralizing Lefty‐1 monoclonal antibody results in a significantly decreased proliferation of duct epithelial cells during islet regeneration. This is the first study to decipher the expression and function of Nodal and Lefty in pancreatic growth. Importantly, our results highlight a novel function of Nodal‐Lefty signaling in the regulation of expansion of pancreatic cells. Developmental Dynamics 237:1255‐1267, 2008.

PYY in the expanding pancreatic epithelium

Gut peptide YY (PYY) plays an important role in regulating metabolism and is expressed during the ontogeny of the pancreas. However, its biological role during endocrine cell formation is not fully understood, and its role, if any, during pancreatic regeneration in the adult has not yet been explored. The knowledge of factors involved in beta cell renewal in adult animals is clearly relevant for the design of treatment strategies for type 1 diabetes. We therefore sought to determine if observations during fetal pancreas formation also apply to pancreatic growth in adult animals. Indeed, we have found marked expansion of the PYY-expressing population during pancreatic regeneration. In addition, we demonstrate the presence of cells co-expressing PYY and the critical pancreatic transcription factor pancreatic duodenal homeobox 1 (PDX-1). Interestingly, these cells also co-expressed specific islet hormones during pancreatic development and re-growth, suggesting a developmental relationship. Furthermore, we have found that PYY can act in concert with IGF-1 to stimulate cellular responsiveness in pancreatic epithelial cells in vitro. Our data suggest that PYY may be a mediator of islet cell development, as well as a cofactor for growth factor responses, not only during fetal pancreas formation but also during regeneration in adult animals.

21 Pancreatic Stem Cells

β-CELL LOSS: A TERMINAL PROCESS Insulin-dependent, or type I, diabetes mellitus (IDDM) is a devastating disease in which affected individuals depend on daily injections of exogenous insulin for regulation of glucose homeostasis and survival. The lifetime dependency on insulin invariably leads to a variety of debilitating complications that threaten quality of life and significantly shorten life expectancy of IDDM patients. Insulin-producing cells in the pancreatic islets of Langerhans are the targets for selective inflammatory destruction in IDDM. This loss of the pancreatic islets is a terminal process, since the pancreas lacks any significant ability to regenerate insulin-producing cells. Indeed, pancreatic islet cells have a very low rate of growth in adults, and knowledge of the factors that regulate islet growth is lacking. Increased knowledge of islet stem cells and potential islet regeneration could allow critical advances in the development of new therapies for IDDM. Knowledge regarding pancreatic stem cells has grown dramatically in recent years, but continued progress is still hampered by the lack of in vitro and in vivo assay systems for stem cell activity and function. In this review, we focus on pancreatic stem cells during morphogenesis and regeneration, as well as on the molecular markers associated with these cells. ONTOGENY OF PANCREATIC ENDOCRINE CELLS During embryogenesis, the pancreas arises from the foregut, initially as two distinct bulges on either side of the duodenum, termed the dorsal and ventral buds (for review, see Slack 1995). The two buds grow independently, with both endocrine and exocrine tissue components present...