Andrew B. Leiter

The "normal" endocrine cell of the gut: changing concepts and new evidences.

Abstract: The endocrine cells of the gut are a highly specialized mucosal cell subpopulation. Within the gastrointestinal tract at least 14 different cell types produce a wide range of hormones with a specific regional distribution. The gut endocrine cells belong to the diffuse endocrine system. These cells present two regulated pathways of secretion characterized by large dense core vesicles (LDCV) and synaptic‐like microvesicles (SLMV). Gut endocrine cells are recognized by the expression of several “general” markers, including the LDCV marker chromogranin A and the SLMV marker synaptophysin, in addition to the cytosolic markers neuron‐specific enolase and protein gene product 9.5. The expression of different hormones identifies specific cell types. The gut endocrine cells are reputed to be terminally differentiated and incapable of proliferation. However, some data suggest that the number of gut endocrine cells may adapt in response to tissue‐specific physiological stimuli. Gut endocrine cell differentiation appears to follow a “constitutive” tissue‐specific pathway, which may be disrupted and investigated by genetic manipulation in mice. It is suggested that endocrine cell homeostasis is maintained by the entry of new endocrine‐committed cells along the differentiation pathway and that such intermediate cells may be sensitive to physiological stimuli as well as transforming agents.

Capsaicin causes cell-cycle arrest and apoptosis in ER -positive and -negative breast cancer cells by modulating the EGFR/HER-2 pathway

Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide) is an ingredient of chili peppers with inhibitory effects against cancer cells of different origin. We examined the activity of capsaicin on breast cancer cells in vitro and in vivo. The drug potently inhibited growth of ER-positive (MCF-7, T47D, BT-474) and ER-negative (SKBR-3, MDA-MB231) breast cancer cell lines, which was associated with G0/G1 cell-cycle arrest, increased levels of apoptosis and reduced protein expression of human epidermal growth factor receptor (EGFR), HER-2, activated extracellular-regulated kinase (ERK) and cyclin D1. In contrast, cell-cycle regulator p27KIP1, caspase activity as well as poly-ADP ribose polymerase (PARP) cleavage were increased. Notably, capsaicin blocked breast cancer cell migration in vitro and decreased by 50% the size of MDA-MB231 breast cancer tumors growing orthotopically in immunodeficient mice without noticeable drug side effects. in vivo activation of ERK was clearly decreased, as well as expression of HER-2 and cyclin D1, whereas caspase activity and PARP cleavage products were increased in tumors of drug-treated mice. Besides, capsaicin potently inhibited the development of pre-neoplastic breast lesions by up to 80% without evidence of toxicity. Our data indicate that capsaicin is a novel modulator of the EGFR/HER-2 pathway in both ER-positive and -negative breast cancer cells with a potential role in the treatment and prevention of human breast cancer.

Multiple, temporal-specific roles for HNF6 in pancreatic endocrine and ductal differentiation.

Within the developing pancreas Hepatic Nuclear Factor 6 (HNF6) directly activates the pro-endocrine transcription factor, Ngn3. HNF6 and Ngn3 are each essential for endocrine differentiation and HNF6 is also required for embryonic duct development. Most HNF6(-/-) animals die as neonates, making it difficult to study later aspects of HNF6 function. Here, we describe, using conditional gene inactivation, that HNF6 has specific functions at different developmental stages in different pancreatic lineages. Loss of HNF6 from Ngn3-expressing cells (HNF6(Delta endo)) resulted in fewer multipotent progenitor cells entering the endocrine lineage, but had no effect on beta cell terminal differentiation. Early, pancreas-wide HNF6 inactivation (HNF6(Delta panc)) resulted in endocrine and ductal defects similar to those described for HNF6 global inactivation. However, all HNF6(Delta panc) animals survived to adulthood. HNF6(Delta panc) pancreata displayed increased ductal cell proliferation and metaplasia, as well as characteristics of pancreatitis, including up-regulation of CTGF, MMP7, and p8/Nupr1. Pancreatitis was most likely caused by defects in ductal primary cilia. In addition, expression of Prox1, a known regulator of pancreas development, was decreased in HNF6(Delta panc) pancreata. These data confirm that HNF6 has both early and late functions in the developing pancreas and is essential for maintenance of Ngn3 expression and proper pancreatic duct morphology.

Differential requirements for neurogenin 3 in the development of POMC and NPY neurons in the hypothalamus

The neuroendocrine hypothalamus regulates a spectrum of essential biological processes and underlies a range of diseases from growth failure to obesity. While the exploration of hypothalamic function has progressed well, knowledge of hypothalamic development is poor. In particular, very little is known about the processes underlying the genesis and specification of the neurons in the arcuate and ventromedial nuclei. Recent studies demonstrate that the proneural basic helix-loop-helix transcription factor Mash1 is required for neurogenesis and neuronal subtype specification in the ventral hypothalamus. We demonstrate here that Ngn3, another basic helix-loop-helix transcription factor, is expressed in mitotic progenitors in the arcuate and ventromedial hypothalamic regions of mouse embryos from embryonic days 9.5-17.5. Genetic fate mapping and loss of function studies in mice demonstrate that Ngn3+ progenitors contribute to subsets of POMC, NPY, TH and SF1 neurons and is required for the specification of these neuronal subtypes in the ventral hypothalamus. Interestingly, while Ngn3 promotes the development of arcuate POMC and ventromedial SF1 neurons, it inhibits the development of NPY and TH neurons in the arcuate nuclei. Given the opposing roles of POMC and NPY neurons in regulating food intake, these results indicate that Ngn3 plays a central role in the generation of neuronal populations controlling energy homeostasis in mice.

Characterization of the release of cholecystokinin from a murine neuroendocrine tumor cell line, STC-1

The murine neuroendocrine cell line, STC-1, was found to contain 296.8 +/- 1.8 fmol of cholecystokinin-like immunoreactivity (CCK-LI) per mg cell protein. Immunocytochemical stain of STC-1 cells maintained in monolayer culture indicated that CCK-LI activity was present in 93% of the cells. Analysis by reverse-phase high-performance liquid chromatography indicated that STC-1 cells contained CCK-8 and an unidentified form as the predominant storage form. form. However, only CCK-8 was released into the culture medium upon stimulation by various secretagogues. The release of CCK-LI from STC-1 cells was stimulated by dibutyryl cAMP, forskolin, KCl, A23187, 4 beta-phorbol 12-myristate 13-acetate and luminal stimulants, e.g., sodium oleate, L-tryptophan, camostat and plaunotol. The release of CCK-LI from STC-1 cells was also stimulated by a neuropeptide, bombesin. The stimulatory effects of most of these agents were dose dependent. The stimulatory effects of dibutyryl cAMP, forskolin, and plaunotol were potentiated by 3-isobutyl-1-methyl xanthine, while that of camostat was not. The results obtained in this study indicate that the release of CCK from STC-1 cells shares the same characteristics of CCK release as from the CCK-secreting cells of the intestinal mucosa observed both in the dog and the rat in vitro and in vivo. Thus, the cellular mechanism of CCK release which appears to be cAMP- and Ca(2+)-dependent may be modulated by cellular protein kinase C activity. The STC-1 cell appears to be a suitable model for studying the mechanism of CCK release.

Identification of a transcriptional enhancer important for enteroendocrine and pancreatic islet cell-specific expression of the secretin gene

It is well established that the gene encoding the hormone secretin is expressed in a specific enteroendocrine cell, the S cell. We now show that the secretin gene is transiently expressed in insulin-producing B cells of the developing pancreatic islets in addition to the intestine. Furthermore, secretin is produced by most established islet cell lines. In order to identify and characterize the regulatory elements within the secretin gene that control tissue-specific expression, we have introduced secretin reporter gene constructions into the secretin-producing HIT and STC-1 cell lines as well as the nonexpressing INR1-G9 glucagonoma line. Analysis of deletion mutants revealed that sequences between 174 and 53 bp upstream from the transcriptional start site are required for maximal expression in secretin-producing cells. This positive element functioned independently of position and orientation. Further deletions into the enhancer resulted in a stepwise loss of transcriptional activity, suggesting the presence of several discrete control elements. The sequence CAGCTG within the secretin enhancer closely resembles that of the core of the B-cell-specific enhancer in the insulin gene. Point mutations introduced into this putative element led to greater than 85% reduction in transcriptional activity. Gel mobility shift assays suggested that a factor in B cells closely related or identical to proteins that bind to the insulin enhancer interacts with the CAGCTG motif in the secretin gene.

Basic helix loop helix transcription factors and enteroendocrine cell differentiation

For over 30 years it has been known that enteroendocrine cells derive from common precursor cells in the intestinal crypts. Until recently little was understood about the events that result in commitment to endocrine differentiation or the eventual segregation of over 10 different hormone‐expressing cell types in the gastrointestinal tract. Enteroendocrine cells arise from pluripotent intestinal stem cells. Differentiation of enteroendocrine cells is controlled by the sequential expression of three basic helix‐loop‐helix transcription factors, Math1, Neurogenin 3 (Neurog3) and NeuroD. Math1 expression is required for specification and segregation of the intestinal secretory lineage (Paneth, goblet,and enteroendocrine cells) from the absorptive enterocyte lineage. Neurog3 expression represents the earliest stage of enteroendocrine differentiation and in its absence enteroendocrine cells fail to develop. Subsequent expression of NeuroD appears to represent a later stage of differentiation for maturing enteroendocrine cells. Enteroendocrine cell fate is inhibited by the Notch signalling pathway, which appears to inhibit both Math1 and Neurog3. Understanding enteroendocrine cell differentiation will become increasingly important for identifying potential future targets for common diseases such as diabetes and obesity.

Energy Homeostasis and Gastrointestinal Endocrine Differentiation Do Not Require the Anorectic Hormone Peptide YY

ABSTRACT The gastrointestinal hormone peptide YY is a potent inhibitor of food intake and is expressed early during differentiation of intestinal and pancreatic endocrine cells. In order to better understand the role of peptide YY in energy homeostasis and development, we created mice with a targeted deletion of the peptide YY gene. All intestinal and pancreatic endocrine cells developed normally in the absence of peptide YY with the exception of pancreatic polypeptide (PP) cells, indicating that peptide YY expression was not required for terminal differentiation. We used recombination-based cell lineage trace to determine if peptide YY cells were progenitors for gastrointestinal endocrine cells. Peptide YY+ cells gave rise to all L-type enteroendocrine cells and to islet ∂ and PP cells. In the pancreas, approximately 40% of pancreatic α and rare β cells arose from peptide YY+ cells, suggesting that most β cells and surprisingly the majority of α cells are not descendants of peptide YY+/glucagon-positive/insulin-positive cells that appear during early pancreagenesis. Despite the anorectic effects of exogenous peptide YY3-36 following intraperitoneal administration, mice lacking peptide YY showed normal growth, food intake, energy expenditure, and responsiveness to peptide YY3-36. These observations suggest that targeted disruption of the peptide YY gene does not perturb terminal endocrine cell differentiation or the control of food intake and energy homeostasis.

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 β‐cells and either absent or expressed below the limit of lacZ detection in mature α‐, δ‐, 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 δ‐cell line, resulted in potent repression of somatostatin concomitant with induction of the β‐cell hormones insulin and islet amyloid polypeptide. Additionally, NeuroD1 induced expression of Nkx2.2, a transcription factor expressed in β‐ but not δ‐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. Developmental Dynamics 233:946–953, 2005.

Enteroendocrine precursors differentiate independently of Wnt and form serotonin expressing adenomas in response to active β-catenin

Wnt signaling is required for the maintenance of intestinal stem cells and self-renewal of the intestinal epithelium. Intestinal cancers are frequently associated with mutations that activate the Wnt pathway. The role of Wnt signaling on differentiation of lineage-specific precursors in the intestine is not well characterized. Here, we show that specification of enteroendocrine but not Paneth cells occurs independently of Wnt signals by conditional deletion of β-catenin in immature cells expressing the transcription factor, neurogenin 3. In addition, we determined whether neurogenin 3-expressing cells respond to abnormal Wnt signaling. Activation of the Wnt pathway by conditionally deleting exon 3 of the β-catenin gene at an early stage of enteroendocrine cell differentiation induced small-intestinal adenomas expressing serotonin, a feature not previously described in other tumors induced by Wnt in mice. In contrast, excision of exon 3 of β-catenin at a later stage of enteroendocrine differentiation did not produce tumors. These results provide direct evidence that some intestinal lineages are specified independently of the Wnt pathway and may lead to a better understanding of the spectrum of neuroendocrine differentiation frequently seen in human gastrointestinal cancer.