Hypoxia is a deficiency in oxygen delivery to tissues and is connected to physiological and pathophysiological processes such as embryonic development and cancer. The master regulators of oxygen homeostasis in mammalian cells are the heterodimeric hypoxia-inducible transcription factors 1 and 2 (HIF-1 and HIF-2, respectively). The oxygen-labile HIF-2α subunit has been implicated not only in transcription but also as a regulator of eukaryotic initiation factor 4E2 (eIF4E2)-directed hypoxic translation. Here, we have identified the DEAD box protein family member DDX28 as an interactor and negative regulator of HIF-2α that suppresses HIF-2α's ability to activate eIF4E2-directed translation. Stable silencing of DDX28 via short hairpin RNA (shRNA) in hypoxic human U87MG glioblastoma cells caused an increase of eIF4E2 binding to the m7GTP cap structure and the translation of eIF4E2 target mRNAs (including the HIF-2α mRNA itself). DDX28 depletion elevated nuclear and cytoplasmic HIF-2α protein, but HIF-2α transcriptional activity did not increase, possibly due to its already high nuclear abundance in hypoxic control cells. Depletion of DDX28 conferred a proliferative advantage to hypoxic, but not normoxic, cells. DDX28 protein levels are reduced in several cancers, including gliomas, relative to levels in normal tissue. Therefore, we uncover a regulatory mechanism for this potential tumor suppressor in the repression of HIF-2α- and eIF4E2-mediated translation activation of oncogenic mRNAs.

Cyclase-associated protein 1 (CAP1) is a conserved actin-regulating protein that enhances actin filament dynamics and also regulates adhesion in mammalian cells. We previously found that phosphorylation at the Ser307/Ser309 tandem site controls its association with cofilin and actin and is important for CAP1 to regulate the actin cytoskeleton. Here, we report that transient Ser307/Ser309 phosphorylation is required for CAP1 function in both actin filament disassembly and cell adhesion. Both the phosphomimetic and the nonphosphorylatable CAP1 mutant, which resist transition between phosphorylated and dephosphorylated forms, had defects in rescuing the reduced rate of actin filament disassembly in the CAP1 knockdown HeLa cells. The phosphorylation mutants also had defects in alleviating the elevated focal adhesion kinase (FAK) activity and the enhanced focal adhesions in the knockdown cells. In dissecting further phosphoregulatory cell signals for CAP1, we found that cyclin-dependent kinase 5 (CDK5) phosphorylates both Ser307 and Ser309 residues, whereas cAMP signaling induces dephosphorylation at the tandem site, through its effectors protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac). No evidence supports an involvement of activated protein phosphatase in executing the dephosphorylation downstream from cAMP, whereas preventing CAP1 from accessing its kinase CDK5 appears to underlie CAP1 dephosphorylation induced by cAMP. Therefore, this study provides direct cellular evidence that transient phosphorylation is required for CAP1 functions in both actin filament turnover and adhesion, and the novel mechanistic insights substantially extend our knowledge of the cell signals that function in concert to regulate CAP1 by facilitating its transient phosphorylation.

During spermatogenesis, up to 75% of germ cells in the testes undergo apoptosis and are cleared by Sertoli cells. X-linked XK blood group-related 8 (Xkr8) is a plasma membrane protein that scrambles phospholipids in response to apoptotic signals, exposing phosphatidylserine (PtdSer). Here, we found that Xkr8-/- male mice were infertile due to reduced sperm counts in their epididymides. Apoptotic stimuli could not induce PtdSer exposure in Xkr8-/- germ cells. Consistent with the hypothesis that PtdSer functions as an "eat-me" signal to phagocytes, cells expressing phosphatidylserine receptor TIM4 and MER tyrosine kinase receptor efficiently engulfed apoptotic wild-type male germ cells but not Xkr8-/- germ cells. Fluorescence and electron microscopy revealed Sertoli cells carrying engulfed and degenerated dead cells. However, many unengulfed apoptotic cells and residual bodies and much cell debris were present in Xkr8-/- testes and epididymides. These results indicate that Xkr8-mediated PtdSer exposure is essential for the clearance of apoptotic germ cells by Sertoli cells. There was no apparent inflammation in Xkr8-/- testes, suggesting that the unengulfed apoptotic cells may have undergone secondary necrosis, releasing noxious materials that affected the germ cells. Alternatively, failure to engulf the apoptotic germ cells may have caused the Sertoli cells to starve and lose their ability to support spermatogenesis.

Chemotherapy resistance and tumor relapse are the major contributors to low patient survival, and both have been largely attributed to cancer stem-like cells (CSCs) or tumor-initiating cells (TICs). Moreover, most conventional therapies are not effective against CSCs, which necessitates the discovery of CSC-specific biomarkers and drug targets. Here, we demonstrated that the embryonic transcription factor SOX9 is an important regulator of acquired chemoresistance in non-small-cell lung cancer (NSCLC). Our results show that SOX9 expression is elevated in NSCLC cells after treatment with the chemotherapeutic cisplatin and that overexpression of SOX9 correlates with worse overall survival in lung cancer patients. We further demonstrated that SOX9 knockdown increases cellular sensitivity to cisplatin, whereas its overexpression promotes drug resistance. Moreover, this transcription factor promotes the stem-like properties of NSCLC cells and increases their aldehyde dehydrogenase (ALDH) activity, which was identified to be the key mechanism of SOX9-induced chemoresistance. Finally, we showed that ALDH1A1 is a direct transcriptional target of SOX9, based on chromatin immunoprecipitation and luciferase reporter assays. Taken together, our novel findings on the role of the SOX9-ALDH axis support the use of this CSC regulator as a prognostic marker of cancer chemoresistance and as a potential drug target for CSC therapy.

Brain insulin resistance is a key pathological feature contributing to obesity, diabetes, and neurodegenerative disorders, including Alzheimer's disease (AD). Besides the classic transcriptional mechanism mediated by hormones, posttranscriptional regulation has recently been shown to regulate a number of signaling pathways that could lead to metabolic diseases. Here, we show that microRNA 7 (miR-7), an abundant microRNA in the brain, targets insulin receptor (INSR), insulin receptor substrate 2 (IRS-2), and insulin-degrading enzyme (IDE), key regulators of insulin homeostatic functions in the central nervous system (CNS) and the pathology of AD. In this study, we found that insulin and liver X receptor (LXR) activators promote the expression of the intronic miR-7-1 in vitro and in vivo, along with its host heterogeneous nuclear ribonucleoprotein K (HNRNPK) gene, encoding an RNA binding protein (RBP) that is involved in insulin action at the posttranscriptional level. Our data show that miR-7 expression is altered in the brains of diet-induced obese mice. Moreover, we found that the levels of miR-7 are also elevated in brains of AD patients; this inversely correlates with the expression of its target genes IRS-2 and IDE. Furthermore, overexpression of miR-7 increased the levels of extracellular Aβ in neuronal cells and impaired the clearance of extracellular Aβ by microglial cells. Taken together, these results represent a novel branch of insulin action through the HNRNPK-miR-7 axis and highlight the possible implication of these posttranscriptional regulators in a range of diseases underlying metabolic dysregulation in the brain, from diabetes to Alzheimer's disease.

Aminoacyl-tRNA synthetase cofactors play important roles in coordinating aminoacylation and translation. In this study, we describe an additional function of the fission yeast aminoacyl-tRNA synthetase cofactor 1 (Asc1) in translation. We found that Asc1 directly binds and stabilizes the interaction between small ribosomal protein Rps0A/uS2 and eukaryotic initiation factor 3a (eIF3a). In the absence of Asc1, the interaction between eIF3a and Rps0A/uS2 was compromised. The interaction between Rps0A/uS2 and eIF3a mediated the 40S ribosomal subunit binding of eIF3 in 43S preinitiation complex formation to stimulate translation initiation. Keeping with this idea, in an asc1 mutant, the association of mRNA with the 40S ribosomal subunit was defective and protein synthesis was compromised. To show that Asc1 is directly involved in translation, we demonstrate that the addition of recombinant Asc1 is able to rescue the translation defect of the asc1 mutant in a cell-free system. Furthermore, this function of Asc1 is likely to be evolutionarily conserved, as a similar interaction with eIF3a and Rps0A/uS2 could be identified in the budding yeast Saccharomyces cerevisiae and human aminoacyl-tRNA synthetase cofactors. Together, these results identify a function of aminoacyl-tRNA synthetase cofactors in translation preinitiation complex formation, which adds significantly to the expanded functions associated with aminoacyl-tRNA synthetases and their cofactors.

This article refers to:MafB Is Important for Pancreatic β-Cell Maintenance under a MafA-Deficient ConditionCREB Promotes Beta Cell Gene Expression by Targeting Its Coactivators to Tissue-Specific Enhancers

This article refers to:The Role of Metabolic Flexibility in the Regulation of the DNA Damage Response by Nitric OxideCCN1–Yes-Associated Protein Feedback Loop Regulates Physiological and Pathological Angiogenesis