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The beta cell protein synthetic machinery is dedicated to the production of mature insulin, which requires the proper folding and trafficking of its precursor, proinsulin. The complete network of proteins that mediate proinsulin folding and advancement through the secretory pathway, however, remains poorly defined. Here we used affinity purification and mass spectrometry to identify for the first time, the proinsulin biosynthetic interaction network in human islets. Stringent analysis established a central node of proinsulin interactions with ER folding factors, including chaperones and oxidoreductases, that is remarkably conserved in both sexes and across three ethnicities. The ER-localized peroxiredoxin PRDX4 was identified as a prominent proinsulin interacting protein. In beta cells, gene silencing of PRDX4 rendered proinsulin susceptible to misfolding, particularly in response to oxidative stress, while exogenous PRDX4 improved proinsulin folding. Moreover, proinsulin misfolding induced by oxidative stress or high glucose was accompanied by sulfonylation of PRDX4, a modification known to inactivate peroxiredoxins. Notably, islets from patients with Type II diabetes (T2D) exhibited significantly higher levels of sulfonylated PRDX4 than islets from healthy individuals. In conclusion, we have generated the first reference map of the human proinsulin interactome to identify critical factors controlling insulin biosynthesis, beta cell function, and T2D.

The β-cell protein synthetic machinery is dedicated to the production of mature insulin, which requires the proper folding and trafficking of its precursor, proinsulin. The complete network of proteins that mediate proinsulin folding and advancement through the secretory pathway, however, remains poorly defined. Here we used affinity purification and mass spectrometry to identify, for the first time, the proinsulin biosynthetic interaction network in human islets. Stringent analysis established a central node of proinsulin interactions with endoplasmic reticulum (ER) folding factors, including chaperones and oxidoreductases, that is remarkably conserved in both sexes and across three ethnicities. The ER-localized peroxiredoxin PRDX4 was identified as a prominent proinsulin-interacting protein. In β-cells, gene silencing of PRDX4 rendered proinsulin susceptible to misfolding, particularly in response to oxidative stress, while exogenous PRDX4 improved proinsulin folding. Moreover, proinsulin misfolding induced by oxidative stress or high glucose was accompanied by sulfonylation of PRDX4, a modification known to inactivate peroxiredoxins. Notably, islets from patients with type 2 diabetes (T2D) exhibited significantly higher levels of sulfonylated PRDX4 than islets from healthy individuals. In conclusion, we have generated the first reference map of the human proinsulin interactome to identify critical factors controlling insulin biosynthesis, β-cell function, and T2D.

Background & Aims: Oncogenic mutations in KRAS, coupled with inactivation of p53, CDKN2A/p16INK4A, and SMAD4, drive progression of pancreatic ductal adenocarcinoma (PDA). Overexpression of MYC and deregulation of retinoblastoma (RB) further promote cell proliferation and make identifying a means to therapeutically alter cell-cycle control pathways in PDA a significant challenge. We previously showed that the basic helix-loop-helix transcription factor E47 induced stable growth arrest in PDA cells in vitro and in vivo. Here, we identified molecular mechanisms that underlie E47-induced growth arrest in low-passage, patient-derived primary and established PDA cell lines. Methods: RNA sequencing was used to profile E47-dependent transcriptomes in 5 PDA cell lines. Gene Ontology analysis identified cell-cycle control as the most altered pathway. Small interfering RNA/short hairpin RNA knockdown, small-molecule inhibitors, and viral expression were used to examine the function of E47-dependent genes in cell-cycle arrest. Cell morphology, expression of molecular markers, and senescence-associated β-galactosidase activity assays identified cellular senescence. Results: E47 uniformly inhibited PDA cell-cycle progression by decreasing expression of MYC, increasing the level of CDKN1B/p27KIP1, and restoring RB tumor-suppressor function. The molecular mechanisms by which E47 elicited these changes included altering both RNA transcript levels and protein stability of MYC and CDKN1B/p27KIP1. At the cellular level, E47 elicited a senescence-like phenotype characterized by increased senescence-associated β-galactosidase activity and altered expression of senescence markers. Conclusions: E47 governs a highly conserved network of cell-cycle control genes, including MYC, CDKN1B/p27KIP1, and RB, which can induce a senescence-like program in PDA cells that lack CDKN2A/p16INK4A and wild-type p53. RNA sequencing data are available at the National Center for Biotechnology Information GEO at https://www.ncbi.nlm.nih.gov/geo/; accession number: GSE100327. Keywords: Pancreatic Ductal Adenocarcinoma, bHLH, Cell Cycle, Senescence

As a key component of the vertebrate neuroendocrine system, the pituitary gland relies on the progressive and coordinated development of distinct hormone-producing cell types and an invading vascular network. The molecular mechanisms that drive formation of the pituitary vasculature, which is necessary for regulated synthesis and secretion of hormones that maintain homeostasis, metabolism, and endocrine function, remain poorly understood. Here, we report that expression of integrin β1 in embryonic pituitary epithelial cells is required for angiogenesis in the developing mouse pituitary gland. Deletion of pituitary epithelial integrin β1 before the onset of angiogenesis resulted in failure of invading endothelial cells to recruit pericytes efficiently, whereas deletion later in embryogenesis led to decreased vascular density and lumen formation. In both cases, lack of epithelial integrin β1 was associated with a complete absence of vasculature in the pituitary gland at birth. Within pituitary epithelial cells, integrin β1 directs a large transcriptional program that includes components of the extracellular matrix and associated signaling factors that are linked to the observed non-cell-autonomous effects on angiogenesis. We conclude that epithelial integrin β1 functions as a critical and canonical regulator of developmental angiogenesis in the pituitary gland, thus providing insight into the long-standing systems biology conundrum of how vascular invasion is coordinated with tissue development.

Homeodomain proteins, described 30 years ago, exert essential roles in development as regulators of target gene expression; however, the molecular mechanisms underlying transcriptional activity of homeodomain factors remain poorly understood. Here investigation of a developmentally required POU-homeodomain transcription factor, Pit1 (also known as Pou1f1), has revealed that, unexpectedly, binding of Pit1-occupied enhancers to a nuclear matrin-3-rich network/architecture is a key event in effective activation of the Pit1-regulated enhancer/coding gene transcriptional program. Pit1 association with Satb1 (ref. 8) and β-catenin is required for this tethering event. A naturally occurring, dominant negative, point mutation in human PIT1(R271W), causing combined pituitary hormone deficiency, results in loss of Pit1 association with β-catenin and Satb1 and therefore the matrin-3-rich network, blocking Pit1-dependent enhancer/coding target gene activation. This defective activation can be rescued by artificial tethering of the mutant R271W Pit1 protein to the matrin-3 network, bypassing the pre-requisite association with β-catenin and Satb1 otherwise required. The matrin-3 network-tethered R271W Pit1 mutant, but not the untethered protein, restores Pit1-dependent activation of the enhancers and recruitment of co-activators, exemplified by p300, causing both enhancer RNA transcription and target gene activation. These studies have thus revealed an unanticipated homeodomain factor/β-catenin/Satb1-dependent localization of target gene regulatory enhancer regions to a subnuclear architectural structure that serves as an underlying mechanism by which an enhancer-bound homeodomain factor effectively activates developmental gene transcriptional programs.

Precise control of transcriptional programmes underlying metazoan development is modulated by enzymatically active co-regulatory complexes, coupled with epigenetic strategies. One thing that remains unclear is how specific members of histone modification enzyme families, such as histone methyltransferases and demethylases, are used in vivo to simultaneously orchestrate distinct developmental gene activation and repression programmes. Here, we report that the histone lysine demethylase, LSD1--a component of the CoREST-CtBP co-repressor complex--is required for late cell-lineage determination and differentiation during pituitary organogenesis. LSD1 seems to act primarily on target gene activation programmes, as well as in gene repression programmes, on the basis of recruitment of distinct LSD1-containing co-activator or co-repressor complexes. LSD1-dependent gene repression programmes can be extended late in development with the induced expression of ZEB1, a Krüppel-like repressor that can act as a molecular beacon for recruitment of the LSD1-containing CoREST-CtBP co-repressor complex, causing repression of an additional cohort of genes, such as Gh, which previously required LSD1 for activation. These findings suggest that temporal patterns of expression of specific components of LSD1 complexes modulate gene regulatory programmes in many mammalian organs.

During mammalian pituitary gland development, distinct cell types emerge from a common primordium. Appearance of specific cell types occurs in response to opposing signaling gradients that emanate from distinct organizing centers. These signals induce expression of interacting transcriptional regulators, including DNA binding–dependent activators and DNA binding–independent transrepressors, in temporally and spatially overlapping patterns. Together they synergistically regulate precursor proliferation and induction of distinct cell types. Terminal cell type differentiation requires selective gene activation strategies and long-term active repression, mediated by cell type–specific and promoter-specific recruitment of coregulatory complexes. These mechanisms imply the potential for flexibility in the ultimate identity of differentiated cell types.

In normal pancreatic acinar cells, acquisition of activating mutations in Kras leads to protective oncogene-induced-senescence (OIS) (Collado et al, Nature 2005). This barrier to malignant transformation is lost upon subsequent mutation and LOH of p53, DNA-methylation based silencing of the p16 locus, and pancreatitis (Guerra et al, Cancer Cell 2011). We have previously shown that pancreatic ductal adenocarcinoma (PDA) tumors express high levels of ID3, an inhibitor of bHLH transcription factor activity. Moreover, overexpression of ID3 alone in human exocrine cells was sufficient to induce cell cycle entry suggesting that the bHLH/ID3 axis is a powerful arbiter of cell cycle status in vitro and in vivo (Lee et al, Molecular Cancer Research 2011). Here we report that restoring bHLH activity in aggressive PDA cells reinstates senescence in the absence of wild-type p53 or p16 in both established PDA lines and patient-derived xenograft lines. Previously we showed that overexpression of the bHLH protein, E47, in established cell lines - Panc-1, Bx-PC3 and MiaPaCa-2 - induced growth arrest through a mechanism that involved upregulation of p21 expression (Kim et al, Pancreas 2015). Here we report that two patient-derived pancreatic cancer cell lines - AA0779E from a primary human PDA, and AA1334E, a liver metastasis from a primary human PDA, also stopped proliferating within 48 hours in response to high levels of E47. We used RNA-sequencing to characterize E47 induced genome-wide changes in the transcriptomes of all five cell lines and found that the most significant and consistent alterations occurred in expression of a large cohort of genes that code for regulators of the cell cycle. In all lines, p21 was among the most highly upregulated. In addition, the RNA-sequencing data revealed that E47 restored normal expression of genes that are specifically linked to senescence, including laminB1, Dec1/bHLHe40 and CENP-A. Moreover, all of the cell lines underwent extensive senescence-associated changes in cell size and morphology concomitant with induction of senescence-associated beta-galactosidase activity (SA-beta-gal). In conclusion, we have shown that overexpressed E47 initiates a senescence program that is independent of wild-type p53 or p16 in all PDA cell lines examined including those derived from a liver metastasis of PDA. Interestingly, Panc-1 and AA0779E cells remained stably arrested for weeks in culture while Bx-PC3, MiaPaCa-2, and AA1334E cells died within 96 hours. This outcome is consistent with a model in which genetic variability among lines determines whether the cells fully execute the cellular senescence program or succumb to cell death after initiation. Importantly, since most patients present with disseminated disease, the data suggest that a therapy aimed at restoring bHLH activity could be effective in halting growth of both primary tumors and metastases. Citation Format: Kathleen M. Scully, Reyhaneh Lahmy, Heejung Kim, Andrew Lowy, Pamela Itkin-Ansari.{Authors}. Overexpression of the basic helix-loop-helix transcription factor, E47, promotes p16-independent senescence in established and patient-derived xenograft lines. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A55.

Targeted insertional disruption of the mouse estrogen receptor-alpha (ER alpha) gene has provided a genetic model in which to test hypotheses that estrogens exert important effects in development and homeostatic functions of the anterior pituitary gland, particularly in the lactotroph and gonadotroph cell types. Analysis of ER alpha gene-disrupted mice reveals a marked reduction in PRL mRNA and a decrease in lactotroph cell number, but normal specification of lactotroph cell phenotype. Gonadotropin mRNA levels in ER alpha gene-disrupted female mice are elevated, consistent with previously described transcriptional suppression of gonadotropin subunit gene expression in response to sustained administration of estrogen in wild type mice. These results provide genetic evidence that ER alpha plays a critical role in PRL and gonadotropin gene transcription and is involved in lactotroph cell growth, but is not required for specification of lactotroph cell phenotype.

Transcriptional activation of the prolactin and growth hormone genes, occurring in a cell-specific fashion, requires short-range synergistic interactions between the pituitary-specific POU domain factor Pit-1 and other transcription factors, particularly nuclear receptors. Unexpectedly, we find that these events involve the gene-specific use of alternative Pit-1 synergy domains. Synergistic activation of the prolactin gene by Pit-1 and the estrogen receptor requires a Pit-1 amino-terminal 25-amino-acid domain that is not required for analogous synergistic activation of the growth hormone promoter. The action of this Pit-1 synergy domain is dependent on the presence of two of three tyrosine residues spaced by 6 amino acids and can be replaced by a comparable tyrosine-dependent trans-activation domain of an unrelated transcription factor (hLEF). The gene-specific utilization of this tyrosine-dependent synergy domain is conferred by specific Pit-1 DNA-binding sites that determine whether Pit-1 binds as a monomer or a dimer. Thus, the critical DNA site in the prolactin enhancer, where this domain is required, binds Pit-1 as a monomer, whereas the Pit-1 sites in the growth hormone gene, which do not utilize this synergy domain, bind Pit-1 as a dimer. The finding that the sequence of specific DNA sites dictates alternative Pit-1 synergy domain utilization based on monomeric or dimeric binding suggests an additional regulatory strategy for differential target gene activation in distinct cell types.

A pituitary LIM homeodomain factor, P-Lim, is expressed as Rathke's pouch forms and as specific pituitary cell phenotypes are established, suggesting functional roles throughout pituitary development. While selectively expressed in both anterior and intermediate pituitary in mature mice, P-Lim is also transiently expressed in the developing ventral neural cord and brainstem. P-Lim binds to and activates the promoter of the alpha-glycoprotein subunit gene, a marker of early pituitary development, and synergizes with Pit-1 in transcriptional activation of genes encoding terminal differentiation markers. The LIM domain of P-Lim specifically interacts with the Pit-1 POU domain and is required for synergistic interactions with Pit-1, but not for basal transcriptional activation events.

We have identified and characterized a new member of the leucine zipper (bZIP) gene family of transcription factors, thyrotroph embryonic factor (TEF). Analysis of the ontogeny of TEF gene expression reveals the presence of TEF transcripts, beginning on embryonic day 14, only in the region of the rat anterior pituitary gland in which thyrotrophs arise. This pattern of gene expression corresponds temporally and spatially to the onset of thyroid-stimulating hormone (TSH beta) gene expression, which defines the thyrotroph phenotype. Coupled with this observation, we find that TEF can bind to and trans-activate the TSH beta promoter. In contrast to this restricted pattern of expression during embryogenesis, TEF transcripts appear in several tissues in the mature organism. We propose that TEF belongs to a new class of bZIP proteins on the basis of the unique homology between TEF and another member of the bZIP gene family, the albumin D box-binding protein (DBP). TEF and DBP transcripts are coexpressed in a pituitary cell line, and these two proteins can readily form heterodimers. The DNA-binding and dimerization domains of TEF correspond to those found in other bZIP proteins. We have however, identified a cluster of basic amino acids, found only in TEF and DBP, that is necessary for the proper DNA-binding site specificity of TEF. A major trans-activation domain of TEF resides outside the region of homology to other bZIP proteins. These data are consistent with a role for a member of a new class of bZIP transcription factors in activating gene expression in the developing thyrotroph.

Reciprocal gene activation and restriction during cell type differentiation from a common lineage is a hallmark of mammalian organogenesis. A key question, then, is whether a critical transcriptional activator of cell type–specific gene targets can also restrict expression of the same genes in other cell types. Here, we show that whereas the pituitary-specific POU domain factor Pit-1 activates growth hormone gene expression in one cell type, the somatotrope, it restricts its expression from a second cell type, the lactotrope. This distinction depends on a two–base pair spacing in accommodation of the bipartite POU domains on a conserved growth hormone promoter site. The allosteric effect on Pit-1, in combination with other DNA binding factors, results in the recruitment of a corepressor complex, including nuclear receptor corepressor N-CoR, which, unexpectedly, is required for active long-term repression of the growth hormone gene in lactotropes.

Pit-1 is a tissue-specific POU domain factor obligatory for the appearance of three cell phenotypes in the anterior pituitary gland. Expression of the pit-1 gene requires the actions of a cell-specific 390-bp enhancer, located 10 kb 5' of the pit-1 transcription initiation site, within sequence that proves essential for effective pituitary targeting of transgene expression during murine development. The enhancer requires the concerted actions of a cell-specific cis-active element, Pit-1 autoregulatory sites, and atypical morphogen response elements. Pituitary ontogeny in the Pit-1-defective Snell dwarf mouse reveals that pit-1 autoregulation is not required for initial activation or continued expression during critical phases of Pit-1 target gene activation but, subsequently, is necessary for maintenance of pit-1 gene expression following birth. A potent 1,25-dihydroxyvitamin D3-responsive enhancer element defines a physiological site in which a single nucleotide alteration in the sequence of core binding motifs modulates the spacing rules for nuclear receptor response elements. Unexpectedly, the major retinoic acid response element is absolutely dependent on Pit-1 for retinoic acid receptor function. On this DNA element, Pit-1 appears to function as a coregulator of the retinoic acid receptor, suggesting an intriguing linkage between a cell-specific transcription factor and the actions of morphogen receptors that is likely to be prototypic of mechanisms by which other cell-specific transcription factors might confer morphogen receptor responsivity during mammalian organogenesis.