Ovarian clear-cell carcinoma (OCCC) is an aggressive form of ovarian cancer with high ARID1A mutation rates. Here we present a mutant mouse model of OCCC. We find that ARID1A inactivation is not sufficient for tumor formation, but requires concurrent activation of the phosphoinositide 3-kinase catalytic subunit, PIK3CA. Remarkably, the mice develop highly penetrant tumors with OCCC-like histopathology, culminating in hemorrhagic ascites and a median survival period of 7.5 weeks. Therapeutic treatment with the pan-PI3K inhibitor, BKM120, prolongs mouse survival by inhibiting tumor cell growth. Cross-species gene expression comparisons support a role for IL-6 inflammatory cytokine signaling in OCCC pathogenesis. We further show that ARID1A and PIK3CA mutations cooperate to promote tumor growth through sustained IL-6 overproduction. Our findings establish an epistatic relationship between SWI/SNF chromatin remodeling and PI3K pathway mutations in OCCC and demonstrate that these pathways converge on pro-tumorigenic cytokine signaling. We propose that ARID1A protects against inflammation-driven tumorigenesis.

Gordon Holmes syndrome (GHS) is a rare Mendelian neurodegenerative disorder characterized by ataxia and hypogonadism. Recently, it was suggested that disordered ubiquitination underlies GHS though the discovery of exome mutations in the E3 ligase RNF216 and deubiquitinase OTUD4. We performed exome sequencing in a family with two of three siblings afflicted with ataxia and hypogonadism and identified a homozygous mutation in STUB1 (NM_005861) c.737C→T, p.Thr246Met, a gene that encodes the protein CHIP (C-terminus of HSC70-interacting protein). CHIP plays a central role in regulating protein quality control, in part through its ability to function as an E3 ligase. Loss of CHIP function has long been associated with protein misfolding and aggregation in several genetic mouse models of neurodegenerative disorders; however, a role for CHIP in human neurological disease has yet to be identified. Introduction of the Thr246Met mutation into CHIP results in a loss of ubiquitin ligase activity measured directly using recombinant proteins as well as in cell culture models. Loss of CHIP function in mice resulted in behavioral and reproductive impairments that mimic human ataxia and hypogonadism. We conclude that GHS can be caused by a loss-of-function mutation in CHIP. Our findings further highlight the role of disordered ubiquitination and protein quality control in the pathogenesis of neurodegenerative disease and demonstrate the utility of combining whole-exome sequencing with molecular analyses and animal models to define causal disease polymorphisms.

The large Maf family of basic leucine-zipper-containing transcription factors are known regulators of key developmental and functional processes in various cell types, including pancreatic islets. Here, we demonstrate that within the adult pancreas, MafB is only expressed in islet ␣-cells and contributes to cell type-specific expression of the glucagon gene through activation of a conserved control element found between nucleotides ؊77 to ؊51. MafB was also shown to be expressed in developing ␣-and ␤-cells as well as in proliferating hormone-negative cells during pancreatogenesis. In addition, MafB expression is maintained in the insulin ؉ and glucagon ؉ cells remaining in mice lacking either the Pax4 or Pax6 developmental regulators, implicating a potentially early role for MafB in gene regulation during islet cell development. These results indicate that MafB is not only important to islet ␣-cell function but may also be involved in regulating genes required in both endocrine ␣-and ␤-cell differentiation. Diabetes 55:297-304, 2006 T he pancreatic islets of Langerhans are composed of ␣-, ␤-, ␦-, and pancreatic polypeptide cells, which independently produce the hormones glucagon, insulin, somatostatin, and pancreatic polypeptide, respectively. Collectively, these hormones regulate both fuel and energy metabolism, with insulin and glucagon key to controlling glucose homeostasis (1). Thus, glucagon secreted from ␣-cells stimulates the mobilization of glucose through gluconeogenesis and glycogenolysis to prevent hypoglycemia, whereas ␤-cell-secreted insulin promotes glucose storage. Physiological glucose levels are maintained through the counter-regulatory actions of glucagon and insulin in peripheral tissues, with defects in ␣-and ␤-cell function playing a significant part in the ability of diabetic patients to maintain glycemic control.The identification and characterization of the transcription factors regulating insulin and glucagon expression have not only revealed their significance in islet function but also during pancreatogenesis. The pancreas develops from dorsal and ventral epithelial bud evaginations from the foregut, with glucagon-producing cells first appearing at mouse embryonic day (E) 9.5 in the dorsal bud (2-4). Insulin-producing cells emerge at E10.5 (3), whereas somatostatin and pancreatic polypeptide ϩ cells are not detected until E15.5 and E18.5, respectively (5). Insulin-and glucagon-producing cells appear in waves during development, with the functional ␣-and ␤-cells that will populate the islet produced starting at ϳE13.5 (5). This latter phase is termed the "secondary transition," and these cells continue to proliferate but are only organized into islet structures and become glucose-responsive shortly after birth (6).The transcription factors associated with controlling cell-specific expression of the insulin and glucagon genes are principal regulators of islet cell formation, including Pdx1 (7-10), Pax6 (11,12), Pax4 (13,14), and NeuroD1 (15,16). Pdx1 is necessary for the growth of the en...

Highlights d Multi-omics analysis and techniques with NASA's GeneLab platform d The largest cohort of astronaut data to date utilized for analysis d Mitochondrial dysregulation driving spaceflight health risks d NASA Twin Study data validates mitochondrial dysfunction during space missions

We have previously described rat insulinoma INS-1-derived cell lines with robust or poor glucose-stimulated insulin secretion (GSIS). In the current study, we have further resolved these lines into three classes: class 1, glucose-unresponsive͞glucagon-expressing; class 2, glucose-unresponsive͞glucagon-negative; and class 3, glucose-responsive͞glucagon-negative. The transcription factor Nkx2.2 was expressed with relative abundance of 3.3, 1.0, and 1.0 in class 1, class 2, and class 3 cells, respectively, whereas Nkx6.1 expression had the opposite trend: 1.0, 2.6, and 6.4 in class 1, class 2, and class 3 cells, respectively. In class 1 cells, overexpressed Nkx6.1 suppressed glucagon expression but did not affect the levels of several other prominent beta cell transcription factors. RNA interference (RNAi)-mediated suppression of Nkx6.1 in class 3 cells resulted in a doubling of glucagon mRNA, with no effect on Pdx1 levels, whereas suppression of Pdx1 in class 3 cells caused a 12-fold increase in glucagon transcript levels, demonstrating independent effects of Nkx6.1 and Pdx1 on glucagon expression in beta cell lines. RNAi-mediated suppression of Nkx6.1 expression in class 3 cells also caused a decrease in GSIS from 13.9-to 3.7-fold, whereas suppression of Pdx1 reduced absolute amounts of insulin secretion without affecting fold response. Finally, RNAi-mediated suppression of Nkx6.1 mRNA in primary rat islets was accompanied by a significant decrease in GSIS relative to control cells. In sum, our studies have revealed roles for Nkx6.1 in suppression of glucagon expression and control of GSIS in islet beta cells.

The homeodomain transcription factor Nkx6.1 plays an important role in pancreatic islet ␤-cell development, but its effects on adult ␤-cell function, survival, and proliferation are not well understood. In the present study, we demonstrated that treatment of primary rat pancreatic islets with a cytomegalovirus promoter-driven recombinant adenovirus containing the Nkx6.1 cDNA (AdCMV-Nkx6.1) causes dramatic increases in [methyl-3 H] thymidine and 5-bromo-2-deoxyuridine (BrdU) incorporation and in the number of cells per islet relative to islets treated with a control adenovirus (AdCMV-␤GAL), whereas suppression of Nkx6.1 expression reduces thymidine incorporation. Immunocytochemical studies reveal that >80% of BrdU-positive cells in AdCMVNkx6.1-treated islets are ␤ cells. Microarray, real-time PCR, and immunoblot analyses reveal that overexpression of Nkx6.1 in rat islets causes concerted upregulation of a cadre of cell cycle control genes, including those encoding cyclins A, B, and E, and several regulatory kinases. Cyclin E is upregulated earlier than the other cyclins, and adenovirus-mediated overexpression of cyclin E is shown to be sufficient to activate islet cell proliferation. Moreover, chromatin immunoprecipitation assays demonstrate direct interaction of Nkx6.1 with the cyclin A2 and B1 genes. Overexpression of Nkx6.1 in rat islets caused a clear enhancement of glucosestimulated insulin secretion (GSIS), whereas overexpression of Nkx6.1 in human islets caused an increase in the level of [ 3 H]thymidine incorporation that was twice the control level, along with complete retention of GSIS. We conclude that Nkx6.1 is among the very rare factors capable of stimulating ␤-cell replication with retention or enhancement of function, properties that may be exploitable for expansion of ␤-cell mass in treatment of both major forms of diabetes.Type 1 diabetes results from autoimmune destruction of insulin-producing ␤ cells in the islets of Langerhans, whereas type 2 diabetes involves loss of glucose-stimulated insulin secretion (GSIS) and a gradual diminution of ␤-cell mass (45). Insulin injection therapy has been the standard treatment for type 1 diabetes since the discovery of the hormone more than 80 years ago. Islet transplantation has been investigated as an alternative to insulin injection, but a major obstacle to broad application of this approach has been an inadequate supply of human islets (21). Pharmacotherapy of type 2 diabetes includes administration of agents that enhance insulin secretion, but these drugs often lose efficacy over time and cause complications such as hypoglycemia (30). Moreover, no controlled strategy for restoration of ␤-cell mass has been identified for the type 2 disease. Thus, a more complete understanding of the mechanisms that control islet ␤-cell growth and function is required in order to develop more effective therapies for both major forms of diabetes.Several members of the homeodomain family of transcription factors, including Pdx1, Hb9/Hlxb9, Nkx2.2, Nkx6.1, Isl-1, Pax6, an...

Summary Deterioration of functional islet β-cell mass is the final step in progression to Type 2 diabetes. We previously reported that overexpression of Nkx6.1 in rat islets has the dual effects of enhancing glucose-stimulated insulin secretion (GSIS) and increasing β-cell replication. Here we show that Nkx6.1 strongly upregulates the prohormone VGF in rat islets and that VGF is both necessary and sufficient for Nkx6.1-mediated enhancement of GSIS. Moreover, the VGF-derived peptide TLQP-21 potentiates GSIS in rat and human islets and improves glucose tolerance in vivo. Chronic injection of TLQP-21 in pre-diabetic ZDF rats preserves islet mass and slows diabetes onset. TLQP-21 prevents islet cell apoptosis by a pathway similar to that used by GLP-1, but independent of the GLP-1, GIP, or VIP receptors. Unlike GLP-1, TLQP-21 does not inhibit gastric emptying or increase heart rate. We conclude that TLQP-21 is a targeted agent for enhancing islet β-cell survival and function.

Abstract-Muscle ring finger (MuRF)1 is a muscle-specific protein implicated in the regulation of cardiac myocyte size and contractility. MuRF2, a closely related family member, redundantly interacts with protein substrates and heterodimerizes with MuRF1. Mice lacking either MuRF1 or MuRF2 are phenotypically normal, whereas mice lacking both proteins develop a spontaneous cardiac and skeletal muscle hypertrophy, indicating cooperative control of muscle mass by MuRF1 and MuRF2. To identify the unique role that MuRF1 plays in regulating cardiac hypertrophy in vivo, we created transgenic mice expressing increased amounts of cardiac MuRF1. Adult MuRF1 transgenic (Tg ϩ ) hearts exhibited a nonprogressive thinning of the left ventricular wall and a concomitant decrease in cardiac function. Experimental induction of cardiac hypertrophy by transaortic constriction (TAC) induced rapid failure of MuRF1 Tg ϩ hearts. Microarray analysis identified that the levels of genes associated with metabolism (and in particular mitochondrial processes) were significantly altered in MuRF1 Tg ϩ hearts, both at baseline and during the development of cardiac hypertrophy. Surprisingly, ATP levels in MuRF1 Tg ϩ mice did not differ from wild-type mice despite the depressed contractility following TAC. In comparing the level and activity of creatine kinase (CK) between wild-type and MuRF1 Tg ϩ hearts, we found that mCK and CK-M/B protein levels were unaffected in MuRF1 Tg ϩ hearts; however, total CK activity was significantly inhibited. We conclude that increased expression of cardiac MuRF1 results in a broad disruption of primary metabolic functions, including alterations in CK activity that leads to increased susceptibility to heart failure following TAC. This study demonstrates for the first time a role for MuRF1 in the regulation of cardiac energetics in vivo. Key Words: muscle ring finger-1 Ⅲ MuRF1 Ⅲ ubiquitin ligase Ⅲ cardiac hypertrophy Ⅲ heart failure Ⅲ creatine kinase T he muscle ring finger (MuRF) proteins are striated muscle-specific proteins that have been implicated in various aspects of contractile regulation and myogenic responses. 1 MuRF1 is a well-characterized RING finger-dependent ubiquitin ligase that targets sarcomere proteins, such as cardiac troponin (cTn)I, during the process of skeletal muscle atrophy. 2,3 MuRF1 has also been implicated in the regulation of cardiac myocyte size and contractility [3][4][5] and inhibits the development of cardiac hypertrophy, 6,7 a dynamic process commonly thought of as a precursor to heart failure. 8 To date, the study of the regulation of cardiac muscle mass by MuRF1 has centered around its involvement in the regulation of sarcomere protein degradation. Although this is certainly an important function, in this report, we propose that MuRF1 operates in a broader capacity that encompasses both protein turnover as well as control of cardiac metabolism.Soon after the discovery of MuRF1, the related proteins MuRF2 and MuRF3 were identified as interacting proteins capable of forming hetero...