In diabetes, the death of insulin-producing -cells by apoptosis leads to insulin deficiency. The lower prevalence of diabetes in females suggests that female sex steroids protect from -cell injury. Consistent with this hypothesis, 17-estradiol (estradiol) manifests antidiabetic actions in humans and rodents. In addition, estradiol has antiapoptotic actions in cells that are mediated by the estrogen receptor-a (ER␣), raising the prospect that estradiol antidiabetic function may be due, in part, to a protection of -cell apoptosis via ER␣. To address this question, we have used mice that were rendered estradiol-deficient or estradiol-resistant by targeted disruption of aromatase (ArKO) or ER␣ (␣ERKO) respectively. We show here that in both genders, ArKO ؊/؊ mice are vulnerable to -cell apoptosis and prone to insulin-deficient diabetes after exposure to acute oxidative stress with streptozotocin. In these mice, estradiol treatment rescues streptozotocin-induced -cell apoptosis, helps sustain insulin production, and prevents diabetes. In vitro, in mouse pancreatic islets and -cells exposed to oxidative stress, estradiol prevents apoptosis and protects insulin secretion. Estradiol protection is partially lost in -cells and islets treated with an ER␣ antagonist and in ␣ERKO islets. Accordingly, ␣ERKO mice are no longer protected by estradiol and display a gender nonspecific susceptibility to oxidative injury, precipitating -cell apoptosis and insulin-deficient diabetes. Finally, the predisposition to insulin deficiency can be mimicked in WT mice by pharmacological inhibition of ER␣ by using the antagonist tamoxifen. This study demonstrates that estradiol, acting, at least in part, through ER␣, protects -cells from oxidative injury and prevents diabetes in mice of both genders.estradiol ͉ oxidative stress
The BETA2 (neuroD) gene is expressed in endocrine cells during pancreas development and is essential for proper islet morphogenesis. The objective of this study is to identify potential upstream regulators of the BETA2 gene during pancreas development. We demonstrated that the expression of neurogenin 3 (ngn3), an islet-and neuron-specific basic-helix-loop-helix transcription factor, partially overlaps that of BETA2 during early mouse development. More importantly, overexpression of ngn3 can induce the ectopic expression of BETA2 in Xenopus embryos and stimulate the endogenous RNA of BETA2 in endocrine cell lines. Furthermore, overexpression of ngn3 could cause a dose-dependent activation on the 1.0-kb BETA2 promoter in islet-derived cell lines. Deletion and mutation analyses revealed that two proximal E box sequences, E1 and E3, could bind to ngn3-E47 heterodimer and mediate ngn3 activation. Based on these results, we hypothesize that ngn3 is involved in activating the expression of BETA2 at an early stage of islet cell differentiation through the E boxes in the BETA2 promoter.The endocrine pancreas, which is organized as the islets of Langerhans, contains at least four distinct types of endocrine cells (␣, , ␦, and PP). The differentiation and maturation of islet cells during development is a complex process controlled by a unique network of gene regulation. Recently, it has been demonstrated by gene targeting studies that several tissuespecific transcription factors, such as BETA2 (neuroD) (24, 25), PDX-1 (1, 27), Islet-1 (2), Nkx2.2 (42), PAX-6 (41), and PAX-4 (40), are involved in this process. These factors, alone or in concert, can activate the expression of genes encoding hormones, such as glucagon (9, 44), insulin (9, 25, 28), and somatostatin (3, 31). BETA2 (neuroD), a basic helix-loop-helix (bHLH) transcription factor, was isolated both as a transcriptional activator of the insulin gene (25) and as a differentiation factor of neurogenesis (17). BETA2 is selectively expressed in the developing endocrine pancreas, the small intestine, and the nervous system (17). It has been shown that BETA2 transactivates the insulin (25) and glucagon genes (9) by binding to the E box sequences localized in their promoters. Furthermore, the functional importance of BETA2 to pancreatic islet cell development has been demonstrated by loss-of-function studies (24). BETA2-deficient (BETA2 Ϫ/Ϫ ) mice die of severe diabetes caused by a major reduction in the number of  cells and a lack of proper islet formation. These results indicate that BETA2 plays an important role in maintaining the differentiation of endocrine cells and proper islet morphogenesis. Results obtained from BETA2-deficient mice also imply that the upstream factors controlling BETA2 expression are likely to be involved in the early events which determine endocrine cell differentiation. So far, numbers of a novel family of genes, the neurogenin genes (ngn) (19, 39), have been reported to be good candidates for upstream regulators of the BETA2 gene. During n...
NeuroD1(BETA2) and Tpit are cell-specific activators of pituitary proopiomelanocortin (POMC) gene transcription. Expression of both factors slightly precedes that of POMC at embryonic d 12.5 of mouse pituitary development. We now report that NeuroD1(BETA2) is required for early corticotroph differentiation. In agreement with the transcriptional synergism observed between Tpit and basic helix-loop-helix dimers containing NeuroD1(BETA2), POMC expression is delayed in NeuroD1-deficient mice. However, this differentiation defect does not reflect a change of corticotroph commitment as revealed by Tpit expression. The delay of corticotroph terminal differentiation is transient and coincides with the developmental window of NeuroD1 expression in corticotrophs. In contrast to their requirement in other NeuroD1-expressing cells, the neurogenin genes do not appear to be necessary for corticotroph differentiation. Taken together with a similar requirement of Tpit for corticotroph differentiation but not for commitment, the present data indicate that the POMC promoter is a point of convergence for independent corticotroph differentiating signals.
BETA2 (NeuroD1) is a member of the basic helix-loophelix transcription factor family. BETA2 plays an important role in the development of the pancreas and the nervous system. Using microarray technology, we identified neuronatin (Nnat) as differentially expressed between wild-type (WT) and knockout (KO) pancreatic RNA from embryonic day 14 (e14.5). NNAT is a member of the proteolipid family of amphipathic polypeptides and is believed to be involved in ion channel transport or channel modulation. Northern blot and in situ hybridization analysis of WT and KO samples confirmed the downregulation of Nnat in pancreas of mutant BETA2 embryos. Chromatin immunoprecipitation and gel shift assays were performed and demonstrated the presence of BETA2 on the Nnat promoter, thus confirming the direct transcriptional regulation of Nnat by BETA2. To assess NNAT potential function, we performed knockdown studies by siRNA in NIT cells and observed a reduction in the ability of the NIT cells to respond to glucose. These results suggest for the first time an important role for NNAT in insulin secretion and for proper -cell function.
Small heterodimer partner (SHP; NR0B2) is an atypical orphan nuclear receptor that lacks a conventional DNA binding domain (DBD) and represses the transcriptional activity of various nuclear receptors. In this study, we examined the novel cross talk between SHP and BETA2/NeuroD, a basic helix-loop-helix transcription factor. In vitro and in vivo protein interaction studies showed that SHP physically interacts with BETA2/NeuroD, but not its heterodimer partner E47. Moreover, confocal microscopic study and immunostaining results demonstrated that SHP colocalized with BETA2 in islets of mouse pancreas. SHP inhibited BETA2/NeuroD-dependent transactivation of an E-box reporter, whereas SHP was unable to repress the E47-mediated transactivation and the E-box mutant reporter activity. In addition, SHP repressed the BETA2-dependent activity of glucokinase and cyclin-dependent kinase inhibitor p21 gene promoters. Gel shift and in vitro protein competition assays indicated that SHP inhibits neither dimerization nor DNA binding of BETA2 and E47. Rather, SHP directly repressed BETA2 transcriptional activity and p300-enhanced BETA2/NeuroD transcriptional activity by inhibiting interaction between BETA2 and coactivator p300. We also showed that C-terminal repression domain within SHP is also required for BETA2 repression. However, inhibition of BETA2 activity was not observed by naturally occurring human SHP mutants that cannot interact with BETA2/NeuroD. Taken together, these results suggest that SHP acts as a novel corepressor for basic helix-loop-helix transcription factor BETA2/NeuroD by competing with coactivator p300 for binding to BETA2/NeuroD and by its direct transcriptional repression function.
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