TGF-beta isoform signaling regulates secondary transition and mesenchymal-induced endocrine development in the embryonic mouse pancreas

Transforming growth factor-beta (TGF-beta) superfamily signaling has been implicated in many developmental processes, including pancreatic development. Previous studies are conflicting with regard to an exact role for TGF-beta signaling in various aspects of pancreatic organogenesis. Here we have investigated the role of TGF-beta isoform signaling in embryonic pancreas differentiation and lineage selection. The TGF-beta isoform receptors (RI, RII and ALK1) were localized mainly to both the pancreatic epithelium and mesenchyme at early stages of development, but then with increasing age localized to the pancreatic islets and ducts. To determine the specific role of TGF-beta isoforms, we functionally inactivated TGF-beta signaling at different points in the signaling cascade. Disruption of TGF-beta signaling at the receptor level using mice overexpressing the dominant-negative TGF-beta type II receptor showed an increase in endocrine precursors and proliferating endocrine cells, with an abnormal accumulation of endocrine cells around the developing ducts of mid-late stage embryonic pancreas. This pattern suggested that TGF-beta isoform signaling may suppress the origination of secondary transition endocrine cells from the ducts. Secondly, TGF-beta isoform ligand inhibition with neutralizing antibody in pancreatic organ culture also led to an increase in the number of endocrine-positive cells. Thirdly, hybrid mix-and-match in vitro recombinations of transgenic pancreatic mesenchyme and wild-type epithelium also led to increased endocrine cell differentiation, but with different patterns depending on the directionality of the epithelial-mesenchymal signaling. Together these results suggest that TGF-beta signaling is important for restraining the growth and differentiation of pancreatic epithelial cells, particularly away from the endocrine lineage. Inhibition of TGF-beta signaling in the embryonic period may thus allow pancreatic epithelial cells to progress towards the endocrine lineage unchecked, particularly as part of the secondary transition of pancreatic endocrine cell development. TGF-beta RII in the ducts and islets may normally serve to downregulate the production of beta cells from embryonic ducts.     

Expression of a dominant-negative mutant TGF-beta type II receptor in transgenic mice reveals essential roles for TGF-beta in regulation of growth and differentiation in the exocrine pancreas.

Using a dominant-negative mutant receptor (DNR) approach in transgenic mice, we have functionally inactivated transforming growth factor-beta (TGF-beta) signaling in select epithelial cells. The dominant-negative mutant type II TGF-beta receptor blocked signaling by all three TGF-beta isoforms in primary hepatocyte and pancreatic acinar cell cultures generated from transgenic mice, as demonstrated by the loss of growth inhibitory and gene induction responses. However, it had no effect on signaling by activin, the closest TGF-beta family member. DNR transgenic mice showed increased proliferation of pancreatic acinar cells and severely perturbed acinar differentiation. These results indicate that TGF-beta negatively controls growth of acinar cells and is essential for the maintenance of a differentiated acinar phenotype in the exocrine pancreas in vivo. In contrast, such abnormalities were not observed in the liver. Additional abnormalities in the pancreas included fibrosis, neoangiogenesis and mild macrophage infiltration, and these were associated with a marked up-regulation of TGF-beta expression in transgenic acinar cells. This transgenic model of targeted functional inactivation of TGF-beta signaling provides insights into mechanisms whereby loss of TGF-beta responsiveness might promote the carcinogenic process, both through direct effects on cell proliferation, and indirectly through up-regulation of TGF-betas with associated paracrine effects on stromal compartments.     

Conditional expression of Smad7 in pancreatic beta cells disrupts TGF-beta signaling and induces reversible diabetes mellitus

Identification of signaling pathways that maintain and promote adult pancreatic islet functions will accelerate our understanding of organogenesis and improve strategies for treating diseases like diabetes mellitus. Previous work has implicated transforming growth factor-beta (TGF-beta) signaling as an important regulator of pancreatic islet development, but has not established whether this signaling pathway is required for essential islet functions in the adult pancreas. Here we describe a conditional system for expressing Smad7, a potent inhibitor of TGF-beta signaling, to identify distinct roles for this pathway in adult and embryonic beta cells. Smad7 expression in Pdx1+ embryonic pancreas cells resulted in striking embryonic beta cell hypoplasia and neonatal lethality. Conditional expression of Smad7 in adult Pdx1+ cells reduced detectable beta cell expression of MafA, menin, and other factors that regulate beta cell function. Reduced pancreatic insulin content and hypoinsulinemia produced overt diabetes that was fully reversed upon resumption of islet TGF-beta signaling. Thus, our studies reveal that TGF-beta signaling is crucial for establishing and maintaining defining features of mature pancreatic beta cells.     

Role of TGF-beta1 in the development of pancreatic fibrosis in Otsuka Long-Evans Tokushima Fatty rats

Recently established Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model of naturally occurring obesity diabetes, exhibit progressive accumulation of connective tissue in the pancreas. The present study was designed to determine the pathogenic role of transforming growth factor-beta1 (TGF-beta1) in the development of pancreatic fibrosis in OLETF rats by investigating the serial changes in the expression of TGF-beta1 and extracellular matrix (ECM) in the pancreas. Progressive proliferation of connective tissue arose from the interstitial region surrounding islets at 20 wk of age and extended to the exocrine pancreas adjacent to the islets. TGF-beta1 mRNA levels in the pancreas increased at 20 wk of age and reached a peak value at 30 wk of age. Fibronectin (FN) and procollagen types I and III mRNAs peaked at 20 wk of age and remained at higher levels than those in the nondiabetic counterparts Long-Evans Tokushima Otsuka rats until 50 wk of age. Immunoreactivities for TGF-beta1 and FN were found in islets of OLETF rats at 20 wk of age and were seen in acinar and interstitial cells at 50 wk of age. Moreover, alpha-smooth muscle actin was located at interstitial region surrounding the islets. Proliferation of the connective tissue in the pancreas of OLETF rats closely correlated with expression of TGF-beta1 and ECM. Our results suggest that the development of pancreatic fibrosis in OLETF rats extends from endocrine to exocrine pancreas and that TGF-beta1 is involved in pancreatic fibrosis of OLETF rats.     

Modulation of endocrine pancreas development but not beta-cell carcinogenesis by Sprouty4

Sprouty (Spry) proteins modulate signal transduction pathways elicited by receptor tyrosine kinases (RTK). Depending on cell type and the particular RTK, Spry proteins exert dual functions: They can either repress RTK-mediated signaling pathways, mainly by interfering with the Ras/Raf/mitogen-activated protein kinase pathway or sustaining RTK signal transduction, for example by sequestering the E3 ubiquitin-ligase c-Cbl and thus preventing ubiquitylation, internalization, and degradation of RTKs. Here, by the inducible expression of murine Spry4 in pancreatic beta cells, we have assessed the functional role of Spry proteins in the development of pancreatic islets of Langerhans in normal mice and in the Rip1Tag2 transgenic mouse model of beta-cell carcinogenesis. beta cell-specific expression of mSpry4 provokes a significant reduction in islet size, an increased number of alpha cells per islet area, and impaired islet cell type segregation. Functional analysis of islet cell differentiation in cultured PANC-1 cells shows that mSpry4 represses adhesion and migration of differentiating pancreatic endocrine cells, most likely by affecting the subcellular localization of the protein tyrosine phosphatase PTP1B. In contrast, transgenic expression of mSpry4 during beta-cell carcinogenesis does not significantly affect tumor outgrowth and progression to tumor malignancy. Rather, tumor cells seem to escape mSpry4 transgene expression.     

Disruption of growth hormone receptor gene causes diminished pancreatic islet size and increased insulin sensitivity in mice

Growth hormone, acting through its receptor (GHR), plays an important role in carbohydrate metabolism and in promoting postnatal growth. GHR gene-deficient (GHR(-/-)) mice exhibit severe growth retardation and proportionate dwarfism. To assess the physiological relevance of growth hormone actions, GHR(-/-) mice were used to investigate their phenotype in glucose metabolism and pancreatic islet function. Adult GHR(-/-) mice exhibited significant reductions in the levels of blood glucose and insulin, as well as insulin mRNA accumulation. Immunohistochemical analysis of pancreatic sections revealed normal distribution of the islets despite a significantly smaller size. The average size of the islets found in GHR(-/-) mice was only one-third of that in wild-type littermates. Total beta-cell mass was reduced 4.5-fold in GHR(-/-) mice, significantly more than their body size reduction. This reduction in pancreatic islet mass appears to be related to decreases in proliferation and cell growth. GHR(-/-) mice were different from the human Laron syndrome in serum insulin level, insulin responsiveness, and obesity. We conclude that growth hormone signaling is essential for maintaining pancreatic islet size, stimulating islet hormone production, and maintaining normal insulin sensitivity and glucose homeostasis.     

Enhanced expression of VEGF-A in β cells increases endothelial cell number but impairs islet morphogenesis and β cell proliferation

There is a reciprocal interaction between pancreatic islet cells and vascular endothelial cells (EC) in which EC-derived signals promote islet cell differentiation and islet development while islet cell-derived angiogenic factors promote EC recruitment and extensive islet vascularization. To examine the role of angiogenic factors in the coordinated development of islets and their associated vessels, we used a "tet-on" inducible system (mice expressing rat insulin promoter-reverse tetracycline activator transgene and a tet-operon-angiogenic factor transgene) to increase the β cell production of vascular endothelial growth factor-A (VEGF-A), angiopoietin-1 (Ang1), or angiopoietin-2 (Ang2) during islet cell differentiation and islet development. In VEGF-A overexpressing embryos, ECs began to accumulate around epithelial tubes residing in the central region of the developing pancreas (associated with endocrine cells) as early as embryonic day 12.5 (E12.5) and increased dramatically by E16.5. While α and β cells formed islet cell clusters in control embryos at E16.5, the increased EC population perturbed endocrine cell differentiation and islet cell clustering in VEGF-A overexpressing embryos. With continued overexpression of VEGF-A, α and β cells became scattered, remained adjacent to ductal structures, and never coalesced into islets, resulting in a reduction in β cell proliferation and β cell mass at postnatal day 1. A similar impact on islet morphology was observed when VEGF-A was overexpressed in β cells during the postnatal period. In contrast, increased expression of Ang1 or Ang2 in β cells in developing or adult islets did not alter islet differentiation, development, or morphology, but altered islet EC ultrastructure. These data indicate that (1) increased EC number does not promote, but actually impairs β cell proliferation and islet formation; (2) the level of VEGF-A production by islet endocrine cells is critical for islet vascularization during development and postnatally; (3) angiopoietin-Tie2 signaling in endothelial cells does not have a crucial role in the development or maintenance of islet vascularization.     

Conditional ablation of Notch signaling in pancreatic development

The role of the Notch signaling members Notch1, Notch2 and Rbpj in exocrine pancreatic development is not well defined. We therefore analyzed conditional pancreas-specific Rbpj and combined Notch1/Notch2 knockout mice using Ptf1a(+/Cre(ex1)) mice crossed with floxed Rbpj or Notch1/Notch2 mice. Mice were analyzed at different embryonic stages for pancreatic exocrine and endocrine development. The absence of Rbpj in pancreatic progenitor cells impaired exocrine pancreas development up to embryonic day 18.5 and led to premature differentiation of pancreatic progenitors into endocrine cells. In Rbpj-deficient pancreata, amylase-expressing acini and islets formed during late embryonic and postnatal development, suggesting an essential role of Rbpj in early but not late development. Contrary to this severe phenotype, the concomitant inactivation of Notch1 and Notch2 only moderately disturbed the proliferation of pancreatic epithelial cells during early embryonic development, and did not inhibit pancreatic development. Our results show that, in contrast to Rbpj, Notch1 and Notch2 are not essential for pancreatogenesis. These data favor a Notch-independent role of Rbpj in the development of the exocrine pancreas. Furthermore, our findings suggest that in late stages of pancreatic development exocrine cell differentiation and maintenance are independent of Rbpj.     

Tertiary lymphoid structures in the pancreas promote selection of B lymphocytes in autoimmune diabetes

Autoimmune diabetes occurs when invading lymphocytes destroy insulin-producing beta cells in pancreatic islets. The role of lymphocytic aggregates at this inflammatory site is not understood. We find that B and T lymphocytes attacking islets in NOD mice organize into lymphoid structures with germinal centers. Analysis of BCR L chain genes was used to investigate selection of B lymphocytes in these tertiary lymphoid structures and in draining pancreatic lymph nodes. The pancreatic repertoire as a whole was found to be highly diverse, with the profile of L chain genes isolated from whole pancreas differing from that observed in regional lymph nodes. A Vkappa14 L chain predominated within the complex pancreatic repertoire of NOD mice. Skewing toward Vkappa4 genes was observed in the pancreas when the repertoire of NOD mice was restricted using a fixed Ig H chain transgene. Nucleotide sequencing of expressed Vkappas identified shared mutations in some sequences consistent with Ag-driven selection and clonal expansion at the site of inflammation. Isolated islets contained oligoclonal B lymphocytes enriched for the germinal center marker GL7 and for sequences containing multiple mutations within CDRs, suggesting local T-B interactions. Together, these findings identify a process that selects B lymphocyte specificities within the pancreas, with further evolution of the selected repertoire at the inflamed site. This interpretation is reinforced by Ag-binding studies showing a large population of insulin-binding B lymphocytes in the pancreas compared with draining lymph nodes.     

Impaired differentiation of endocrine and exocrine cells of the pancreas in transgenic mouse expressing the truncated type II activin receptor

Activin A is expressed in endocrine precursor cells of the fetal pancreatic anlage. To determine the physiological significance of activins in the pancreas, a transgenic mouse line expressing the truncated type II activin receptor under the control of beta-actin promoter was developed. Histological analyses of the pancreas revealed that the pancreatic islets of the transgenic mouse were small in size and were located mainly along the pancreatic ducts. Immunoreactive insulin was detected in islets, some acinar cells, and in some epithelial cells in the duct. In addition, there were abnormal endocrine cells outside the islets. The shape and the size of the endocrine cells varied and some of them were larger than islets. These cells expressed immunoreactive insulin and glucagon. In the exocrine portion, there were morphologically abnormal exocrine cells, which did not form a typical acinar structure. The cells lacked spatial polarity characteristics of acinar cells but expressed immunoreactive amylase, which was distributed diffusely in the cytoplasm. Plasma glucose concentration was normal in the transgenic mouse before and after the administration of glucose. The insulin content of the pancreas in transgenic and normal mice was nearly identical. These results suggest that activins or related ligands regulate the differentiation of the pancreatic endocrine and exocrine cells.     

GDF11 modulates NGN3+ islet progenitor cell number and promotes beta-cell differentiation in pancreas development

Identification of endogenous signals that regulate expansion and maturation of organ-specific progenitor cells is a major goal in studies of organ development. Here we provide evidence that growth differentiation factor 11 (GDF11), a member of the TGF-beta ligand family, governs the number and maturation of islet progenitor cells in mouse pancreas development. Gdf11 is expressed in embryonic pancreatic epithelium during formation of islet progenitor cells that express neurogenin 3. Mice deficient for Gdf11 harbor increased numbers of NGN3+ cells, revealing that GDF11 negatively regulates production of islet progenitor cells. Despite a marked expansion of these NGN3+ islet progenitors, mice lacking Gdf11 have reduced beta-cell numbers and evidence of arrested beta-cell development, indicating that GDF11 is also required for beta-cell maturation. Similar precursor and islet cell phenotypes are observed in mice deficient for SMAD2, an intracellular signaling factor activated by TGF-beta signals. Our data suggest that Gdf11 and Smad2 regulate islet cell differentiation in parallel to the Notch pathway, which previously has been shown to control development of NGN3+ cells. Thus, our studies reveal mechanisms by which GDF11 regulates the production and maturation of islet progenitor cells in pancreas development.     

Function and regulation of gastrin in transgenic mice: a review.

The gastrin gene is expressed in fetal pancreatic islet cells, but in the adult is expressed mainly in the gastric antrum. To study the regulation of the gastrin promoter, we created several transgenes containing the human and rat gastrin 5' flanking regions joined to the coding sequences of the human gastrin gene. The human gastrin transgene contained 1,300 bp of 5' flanking DNA, while the rat gastrin transgene contained 450 bp of 5' flanking DNA. The human gastrin transgene was expressed in fetal islets, but was not expressed in adult gastric antrum. In contrast, the rat gastrin transgene was expressed in adult antral G cells, but no expression was observed in fetal islets. To study the possible role of gastrin as an islet growth factor, a chimeric insulin-gastrin (INS-GAS) transgene was created, in which the expression of the human gastrin gene is driven from the rat insulin I promoter. These INS-GAS mice were mated with mice overexpressing TGF alpha, transcribed from a mouse metallothionein-transforming growth factor alpha (MT-TGF alpha) transgene. While overexpression of gastrin or TGF alpha alone had no effect on islet mass, overexpression of both transgenes resulted in a twofold increase in islet mass. In conclusion, these data indicate that (1) gastrin can interact synergistically with TGF alpha to stimulate islet growth; (2) the human gastrin transgene contains the islet specific enhancer; (3) the rat gastrin transgene contains the antral specific enhancer.     

Dickkopf-3 is expressed in a subset of adult human pancreatic beta cells

The Dickkopf (Dkk) gene family of secretory modulators of canonical Wnt/beta catenin signals is involved in the control of stem cell proliferation, homeostasis and differentiation. Bioinformatic data on dkk-1/3 gene expression, indicating high expression levels in the human pancreas, led us to analyze these two proteins in adult human pancreatic tissue. Dkk-1/3 mRNA levels and protein distribution were analyzed in isolated human islets vs. the exocrine/ductal pancreatic cells and in paraffin sections of adult human pancreata. Using real time PCR only lowest amounts of dkk-1 mRNA were detectable in the endocrine fractions. Immunohistochemistry did not reveal any Dkk-1 protein in adult human pancreatic tissue. Interestingly, Dkk-3 mRNA and protein were clearly present in adult human pancreatic islets. Messenger RNA levels for Dkk-3 were significantly higher in isolated islets as compared to the exocrine/ductal fraction. Co-staining with an antibody against insulin identified the beta cells of the pancreas as the Dkk-3-positive cells. Notably, only a subset of beta cells contained Dkk-3. As shown by western blot analysis Dkk-3 seems to be proteolytically processed in beta cells. To our knowledge, this is the first study describing a molecule with which the pool of pancreatic beta cells can be further subdivided. Future studies will show whether this sub-classification of beta cells translates into functional differences.