NeuroD1 in the endocrine pancreas: localization and dual function as an activator and repressor.

The basic helix-loop-helix transcription factor NeuroD1 regulates cell fate in the nervous system but previously has not been considered to function similarly in the endocrine pancreas due to its reported expression in all islet cell types in the newborn mouse. Because we found that NeuroD1 potently represses somatostatin expression in vitro, its pattern of expression was examined in both strains of mice in which lacZ has been introduced into the NeuroD1 locus by homologous recombination. Analysis of adult transgenic mice revealed that NeuroD1 is predominantly expressed in beta-cells and either absent or expressed below the limit of lacZ detection in mature alpha-, delta-, or PP cells. Consistent with a previous report, NeuroD1 colocalizes with glucagon as well as insulin in immature islets of the newborn mouse. However, no colocalization of NeuroD1with somatostatin was detected in the newborn. In vitro, ectopic expression of NeuroD1 in TRM-6/PDX-1, a human pancreatic delta-cell line, resulted in potent repression of somatostatin concomitant with induction of the beta-cell hormones insulin and islet amyloid polypeptide. Additionally, NeuroD1 induced expression of Nkx2.2, a transcription factor expressed in beta- but not delta-cells. Transfection studies using insulin and somatostatin promoters confirm the ability of NeuroD1 to act as both a transcriptional repressor and activator in the same cell, suggesting a more complex role for NeuroD1 in the establishment and/or maintenance of mature endocrine cells than has been recognized previously.

CCK expression in enteroendocrine cell is regulated by soluble factor(s) from underlying fibroblasts.

Studies on the cross-talk between the intestinal epithelium and the underlying connective tissue have concentrated on enterocytes. In contrast, little is known about the interactions between the mesenchymal compartment and the enteroendocrine cells, scattered among the other cell types of the epithelium. To address this question, a panel of coculture systems between the enteroendocrine STC-1 cell line and three intestinal myofibroblastic cell lines (MIC) was used in order to assess different levels of regulation, namely cell-cell and cell-matrix interactions, and the role of diffusible factors. We demonstrate that the expression of cholecystokinin, a typical intestinal hormone produced by STC-1 cells, is up-regulated in the presence of a fibroblastic environment through a paracrine pathway involving FGF2. Concomitantly, STC-1 cell morphology and proliferation were also modulated, but through distinct mechanisms according to the origin of fibroblasts. The results reveal definite epithelio-mesenchymal interactions that may be critical for the maintenance of phenotype and function of enteroendocrine cells.

Peptones stimulate intestinal cholecystokinin gene transcription via cyclic adenosine monophosphate response element-binding factors.

Cholecystokinin (CCK) is a potent intestinal hormone that regulates several digestive functions. Despite the physiological importance of CCK, the cellular and molecular mechanisms that govern its synthesis and secretion are not completely identified. Peptones, which are fair counterparts of the protein fraction in the intestinal lumen, are good stimulants of CCK secretion. We have previously shown that peptones activate CCK gene transcription in STC-1 enteroendocrine cells. The DNA element(s) necessary to induce the transcriptional stimulation was preliminary, localized in the first 800 bp of the CCK gene promoter. In the present study, we identify a DNA element [peptone-response element (PepRE)] essential to confer peptone-responsiveness to the CCK promoter, and we characterize the transcription factors implicated. Localization of the PepRE between -93 and -70 bp of the promoter was established using serial 5'-3'deletions. Systematic site-directed mutagenesis demonstrated that the core PepRE sequence, spanning from nucleotide -72 to -83, overlapped with the putative AP-1/CRE site. Mutations in the core sequence dramatically decreased peptone-responsiveness of CCK promoter fragments. The PepRE functioned as a low-affinity CRE consensus site, binding only transcription factors of the CREB family. Overexpression, in STC-1 cells, of a dominant-negative protein (A-CREB), that prevented the binding of CREB factors to DNA, completely abolished the peptone-induced transcriptional stimulation. Peptone treatment did not modify the nature and the abundance of proteins bound to the PepRE but led to increased phosphorylation of the CREB factors. In conclusion, the present study first demonstrates that CCK gene expression is under the control of protein-derived nutrients in the STC-1 enteroendocrine cell line.

Role of the amino-terminal domain of simian virus 40 early region in inducing tumors in secretin-expressing cells in transgenic mice.

BACKGROUND & AIMS: The early region of simian virus 40 (SV40) encodes 2 transforming proteins, large T (Tag) and small t antigen, that produce neuroendocrine tumors in the intestine and the pancreas when expressed in secretin cells of transgenic mice. METHODS: Two SV40 early-region transgenes containing a deletion that eliminated expression of the small t antigen were expressed in transgenic mice under control of the secretin gene. The 2 lines of mice, one expressing the native large T antigen and the other T antigen with a mutation in its N-terminal J domain, were examined to determine which biological activities of the SV40 early region were required for tumorigenesis. RESULTS: Most animals expressing wild-type large T antigen developed pancreatic insulinomas and lymphomas and died between 3 and 6 months of age. However, small intestinal neoplasms were extremely rare in the absence of small t antigen expression. Transgenic lines expressing the J domain mutant failed to develop tumors. CONCLUSIONS: Transformation of secretin-producing enteroendocrine cells by SV40 requires functional cooperation between intact large T and small t oncoproteins. In contrast, large T antigen alone is sufficient to induce tumors in the endocrine pancreas and thymus.

Review article: transcriptional events controlling the terminal differentiation of intestinal endocrine cells.

Secretin-producing enteroendocrine cells arise from a multipotential endocrine progenitor in the crypts of the small intestine. As these cells migrate up the crypt-villus axis, they produce secretin and stop dividing as they terminally differentiate and die. Transcription of the secretin gene is controlled by a complex enhancer binding to multiple transcription factors. The basic helix-loop-helix protein, BETA2, binds to an E box sequence and associates with the p300 coactivator to activate transcription of the secretin gene. Basic helix-loop-helix proteins appear to play a pivotal role in the control of cellular differentiation. BETA2 induces cell cycle arrest and apoptosis in addition to activating secretin gene expression. Thus BETA2 may function as a master regulatory gene to coordinate terminal differentiation of secretin cells.

Site-specific epithelial-mesenchymal interactions in digestive neuroendocrine tumors. An experimental in vivo and in vitro study.

Little is known about the functional interactions between digestive neuroendocrine tumor cells and their stromal microenvironment. The focus of our study is whether mesenchymal cells modulate peptide expression, cell proliferation, and invasiveness in digestive neuroendocrine tumor cells. We designed an experimental in vivo and in vitro study using the mouse enteroendocrine cell line STC-1. In vivo, STC-1 cells were injected subcutaneously in 18 immunosuppressed newborn rats. At day 21, all animals presented poorly differentiated neuroendocrine tumors with lung metastases. Subcutaneous tumors were usually limited by a capsule containing basement membrane components and myofibroblasts that presented a low mitotic index. Lung tumors were devoid of capsule and poor in myofibroblasts, and their mitotic index was high. The profile of peptide expression in STC-1 tumors was different from that of cultured STC-1 cells. In vitro, STC-1 cells were cultured with fibroblasts of different origins, including dermis, lung, digestive tract, and liver. Based on their origin, myofibroblasts differentially modulated hormone synthesis, proliferation, spreading, and adhesion of STC-1 cells. In conclusion, our results show that site-specific functional interactions between mesenchymal and neuroendocrine cells may contribute to modulating the behavior of digestive neuroendocrine tumors, depending on their growth site.

Targeted ablation of secretin-producing cells in transgenic mice reveals a common differentiation pathway with multiple enteroendocrine cell lineages in the small intestine.

The four cell types of gut epithelium, enteroendocrine cells, enterocytes, Paneth cells and goblet cells, arise from a common totipotent stem cell located in the mid portion of the intestinal gland. The secretin-producing (S) cell is one of at least ten cell types belonging to the diffuse neuroendocrine system of the gut. We have examined the developmental relationship between secretin cells and other enteroendocrine cell types by conditional ablation of secretin cells in transgenic mice expressing herpes simplex virus 1 thymidine kinase (HSVTK). Ganciclovir-treated mice showed markedly increased numbers of apoptotic cells at the crypt-villus junction. Unexpectedly, ganciclovir treatment induced nearly complete ablation of enteroendocrine cells expressing cholecystokinin and peptide YY/glucagon (L cells) as well as secretin cells, suggesting a close developmental relationship between these three cell types. In addition, ganciclovir reduced the number of enteroendocrine cells producing gastric inhibitory polypeptide, substance-P, somatostatin and serotonin. During recovery from ganciclovir treatment, the enteroendocrine cells repopulated the intestine in normal numbers, suggesting that a common early endocrine progenitor was spared. Expression of BETA2, a basic helix-loop-helix protein essential for differentiation of secretin and cholecystokinin cells was examined in the proximal small intestine. BETA2 expression was seen in all enteroendocrine cells and not seen in nonendocrine cells. These results suggest that most small intestinal endocrine cells are developmentally related and that a close developmental relationship exists between secretin-producing S cells and cholecystokinin-producing and L type enteroendocrine cells. In addition, our work shows the existence of a multipotent endocrine-committed cell type and locates this hybrid multipotent cell type to a region of the intestine populated by relatively immature cells.

[Angiogenesis and endocrine tumors]. / Angiogenèse et tumeurs endocrines.

Endocrine tumors are characteristically hypervascularized. This property recalls that of normal endocrine tissues, which possess a dense and specialized capillary network. The cellular and molecular mechanisms of the angiogenesis process associated with endocrine tumorigenesis are poorly known. Most normal endocrine cells constituvely express high levels of angiogenic factors, such as VEGF, which likely play an important role in the development of the characteristic vascular architecture of normal endocrine tissues. Clinical and experimental data suggest that a surexpression of such angiogenic factors is unlikely to be involved in the induction of the angiogenic process associated with endocrine tumorigenesis. In contrast, according to some experimental observations, the loss of endocrine-specific anti-angiogenic factors may be required for the initiation of the angiogenic process and the transition from endocrine hyperplasia to endocrine neoplasia. Such inhibitory factors remain to be identified and characterized. A better understanding of the mechanisms of angiogenesis in endocrine tumors is important for the delineation of novel therapeutic strategies.

Intestinal-type fibroblasts selectively influence proliferation rate and peptide synthesis in the murine entero-endocrine cell line STC-1.

The intestinal epithelium consists of enterocytes, endocrine cells, goblet cells and Paneth cells, which differentiate from pluripotent stem cells located at the crypt bases. The role of the epithelial-mesenchymal inter-actions has been well documented for the differentiation of enterocytes, but the mechanisms that control endocrine cell differentiation are poorly understood. We have cultured the intestinal endocrine cell line STC-1, which synthesizes most of the intestinal peptide hormones, in media conditioned by several subepithelial fibroblast cell lines from three distinct sites of intestine. The fibroblast Swiss 3T3 cell line was used as a non-intestinal control. Our results show that culture media from intestinal fibroblasts inhibit the proliferation rate of STC-1 cells, while those from Swiss 3T3 fibroblasts do not. As regards peptide hormone gene expression, Swiss 3T3-conditioned media have no effect, whereas media from intestinal fibroblasts variably affect cholecystokinin, glucagon, secretin and somatostatin mRNA levels. In particular, clonal subepithelial myofibroblasts do not exert the same effects as mixed subepithelial fibroblasts from homologous intestinal segment. Taken together, these results suggest that cultured fibroblasts of intestinal origin release soluble factors that inhibit STC-1 cell proliferation and modulate, in a region-specific manner, the expression of hormonal peptide genes in this nonspecialized endocrine cell line.

Regulation of intestinal cholecystokinin gene expression by glucocorticoids.

The effect of glucocorticoids on the expression of intestinal cholecystokinin (CCK) was investigated both in vivo and in cell culture systems. In vivo, 2-day administration of methylprednisolone to adult male rats induced a decrease in CCK-like immunoreactivity (CCK-L1) and CCK mRNA levels in mucosal extracts. In two CCK-producing cell lines, RIN 1056E and STC-1 of pancreatic and intestinal origin respectively, dexamethasone induced dose-dependent decreases in both CCK-L1 and steady-state CCK mRNA levels. The decrease in CCK mRNA was totally prevented by incubation of cells with an excess of RU 38486, a competitive inhibitor for the binding of glucocorticoids to their receptor. Actinomycin D, used to prevent RNA synthesis, did not modify CCK mRNA stability in dexamethasone-pretreated cells as compared with cells not exposed to dexamethasone. When cells were first incubated with actinomycin D, subsequent addition of dexamethasone left the steady-state CCK mRNA levels unaltered in both cell lines. Nuclear run-on assays performed in RIN 1056E cells showed that glucocorticoids decreased the rate of transcription of the CCK gene. In addition, cycloheximide, used to prevent protein synthesis, abolished the inhibitory effects of dexamethasone on steady-state CCK mRNA levels. These results demonstrate that glucocorticoids down-regulate CCK gene expression in the rat intestinal mucosa and in two CCK-producing cell lines. The effect is blocked by a glucocorticoid receptor antagonist. Inhibition of CCK gene expression may result from a decrease in the transcription rate, and probably involves one or several steps that depend on protein synthesis.

Opposite effects of sodium butyrate on CCK mRNA and CCK peptide levels in RIN cells.

The effects of the differentiation-inducing agent sodium butyrate on cholecystokinin (CCK) expression was investigated in the pancreatic islet tumor cell line RIN 1056E, which contains high levels of CCK-like immunoreactivity (CCK-LI). Exposure to butyrate for 24 h dose-dependently inhibited cell proliferation and increased the cell content in CCK-LI over the concentration range 0.1-8 mM. With 2 mM butyrate, cell proliferation was decreased by 50% and CCK-LI content was increased by 300%, whereas the level of steady-state CCK mRNA was reduced by 75%. Cycloheximide (10 µg/mL) abolished the sodium butyrate-induced increase in CCK-LI content. This article reports the novel finding that butyrate exerts opposite effects on CCK mRNA and immunoreactivity. The butyrate-induced increase in cellular CCK-LI content is entirely dependent on continuing protein synthesis.