by
Hee Yun Suk;
Chen Zhou;
Teddy T. C. Yang;
Hong Zhu;
Raymond Y.L. Yu;
Opeyemi Olabisi;
XiaoYong Yang;
Deborah Brancho;
Ja-Young Kim;
Philipp E. Scherer;
Philippe G. Frank;
Michael P. Lisanti;
John Calvert;
David J. Lefer;
Jeffery D. Molkentin;
Alessandra Ghigo;
Emilio Hirsch;
Jianping Jin;
Chi-Wing Chow
Insulin resistance, hyperlipidemia, and cardiovascular complications are common dysregulations of metabolic syndrome. Transplant patients treated with immunosuppressant drugs such as cyclosporine A (CsA), an inhibitor of calcineurin phosphatase, frequently develop similar metabolic complications. Although calcineurin is known to mediate insulin sensitivity by regulating β-cell growth and adipokine gene transcription, its role in lipid homeostasis is poorly understood. Here, we examined lipid homeostasis in mice lacking calcineurin Aβ (CnAβ-/-). We show that mice lacking calcineurin Aβ are hyperlipidemic and develop age-dependent insulin resistance. Hyperlipidemia found in CnAβ-/- mice is, in part, due to increased lipolysis in adipose tissues, a process mediated by β-adrenergic G-protein-coupled receptor signaling pathways. CnAβ-/- mice also exhibit additional pathophysiological phenotypes caused by the potentiated GPCR signaling pathways. A cell autonomous mechanism with sustained cAMP/PKA activation is found in CnAβ-/- mice or upon CsA treatment to inhibit calcineurin. Increased PKA activation and cAMP accumulation in CnAβ-/- mice, however, are sensitive to phosphodiesterase inhibitor. Indeed, we show that calcineurin regulates degradation of phosphodiesterase 3B, in addition to phosphodiesterase 4D. These results establish a role for calcineurin in lipid homeostasis. These data also indicate that potentiated cAMP signaling pathway may provide an alternative molecular pathogenesis for the metabolic complications elicited by CsA in transplant patients.
T lymphocyte homeostatic proliferation, driven by the engagement of T cell antigen receptor with self-peptide/major histocompatibility complexes, and signaling through the common γ-chain-containing cytokine receptors, is critical for the maintenance of the T cell compartment and is regulated by the Fas death receptor (Fas, CD95). In the absence of Fas, Fas-deficient lymphoproliferation spontaneous mutation (lpr) mice accumulate homeostatically expanded T cells. The functional consequences of sequential rounds of homeostatic expansion are not well defined. We thus examined the gene expression profiles of murine wild-type and Fas-deficient lpr CD8+T cell subsets that have undergone different amounts of homeostatic proliferation as defined by their level of CD44 expression, and the CD4−CD8−TCRαβ+T cell subset that results from extensive homeostatic expansion of CD8+T cells. Our studies show that recurrent T cell homeostatic proliferation results in global gene expression changes, including the progressive upregulation of both cytolytic proteins such as Fas-Ligand and granzyme B as well as inhibitory proteins such as programmed cell death protein 1 (PD-1) and lymphocyte activating 3 (Lag3). These findings provide an explanation for how augmented T cell homeostatic expansion could lead to the frequently observed clinical paradox of simultaneous autoinflammatory and immunodeficiency syndromes and provide further insight into the regulatory programs that control chronically stimulated T cells.
Intracellular accumulation of wild type tau is a hallmark of sporadic Alzheimer's disease (AD). However, the molecular mechanisms underlying tau toxicity is not fully understood. Here, we detected mitophagy deficits evidenced by the increased levels of mitophagy markers, including COX IV, TOMM20, and the ratio of mtDNA to genomic DNA indexed as mt-Atp6/Rpl13, in the AD brains and in the human wild type full-length tau (htau) transgenic mice. More interestingly, the mitophagy deficit was only shown in the AD patients who had an increased total tau level. Further studies demonstrated that overexpression of htau induced mitophagy deficits in HEK293 cells, the primary hippocampal neurons and in the brains of C57 mice. Upon overexpression of htau, the mitochondrial membrane potential was increased and the levels of PTENinduced kinase 1 (PINK1) and Parkin decreased in the mitochondrial fraction, while upregulation of Parkin attenuated the htau-induced mitophagy deficits. Finally, we detected a dose-dependent allocation of tau proteins into the mitochondrial outer membrane fraction along with its cytoplasmic accumulation. These data suggest that intracellular accumulation of htau induces mitophagy deficits by direct inserting into the mitochondrial membrane and thus increasing the membrane potential, which impairs the mitochondrial residence of PINK1/Parkin. Our findings reveal a novel mechanism underlying the htau-induced neuronal toxicities in AD and other tauopathies.
Recent in vivo studies establish that osteopontin (OPN) expression is hydrogen peroxide (H2O2)-dependent. However, the mechanisms by which H2O2 increases OPN expression remain poorly defined. OPN protein expression increased in an unusual biphasic pattern in response to H2O2. To investigate whether these increases were mediated through transcriptional and/or translational regulation of OPN, smooth muscle cells stimulated with 50 μm H2O2 were used as an in vitro cell system. Early protein increases at 6 h were not preceded by increased mRNA, whereas later increases (18 h) were, suggesting multiple mechanisms of regulation by H2O2. Polyribosomal fractionation assays established that early increases (6 h) in OPN expression were due to increased translation. This increase in translation occurred through phosphorylation of 4E-BP1 at the reactive oxygen species-sensitive Ser-65, which allowed for release and activation of eukaryotic initiation factor eIF4E and subsequent OPN translation. This early increase (6 h) in OPN was blunted in cells expressing a phospho-deficient 4E-BP1 mutant. H2O2 stimulation increased rat OPN promoter activity at 8 and 18 h, and promoter truncation studies established that promoter region −2284 to −795 is crucial for H2O2-dependent OPN transcription. ChIP studies determined that H2O2-dependent transcription is mediated by the reactive oxygen species-sensitive transcription factors NF-κB and AP-1. In conclusion, H2O2 stimulates OPN expression in a unique biphasic pattern, where early increases are translational and late increases are transcriptional.
Two urea transporters, UT-A1 and UT-A3, are expressed in the kidney terminal inner medullary collecting duct (IMCD) and are important for the production of concentrated urine. UT-A1, as the largest isoform of all UT-A urea transporters, has gained much attention and been extensively studied; however, the role and the regulation of UT-A3 are less explored. In this study, we investigated UT-A3 regulation by glycosylation modification. A site-directed mutagenesis verified a single glycosylation site in UT-A3 at Asn279. Loss of the glycosylation reduced forskolin-stimulated UT-A3 cell membrane expression and urea transport activity. UT-A3 has two glycosylation forms, 45 and 65 kDa. Using sugar-specific binding lectins, the UT-A3 glycosylation profile was examined. The 45-kDa form was pulled down by lectin concanavalin A (Con A) and Galant husnivalis lectin (GNL), indicating an immature glycan with a high amount of mannose (Man), whereas the 65-kDa form is a mature glycan composed of acetylglucosamine (GlcNAc) and poly-N-acetyllactosame (poly-LacNAc) that was pulled down by wheat germ agglutinin (WGA) and tomato lectin, respectively. Interestingly, the mature form of UT-A3 glycan contains significant amounts of sialic acid. We explored the enzymes responsible for directing UT-A3 sialylation. Sialyltransferase ST6GalI, but not ST3GalIV, catabolizes UT-A3 α2,6-sialylation. Activation of protein kinase C (PKC) by PDB treatment promoted UT-A3 glycan sialylation and membrane surface expression. The PKC inhibitor chelerythrine blocks ST6GalI-induced UT-A3 sialylation. Increased sialylation by ST6GalI increased UT-A3 protein stability and urea transport activity. Collectively, our study reveals a novel mechanism of UT-A3 regulation by ST6GalI-mediated sialylation modification that may play an important role in kidney urea reabsorption and the urinary concentrating mechanism.
Background: Ciliary neurotrophic factor (CNTF) expression is repressed in astrocytes by neuronal contact in the CNS and is rapidly induced by injury. Here, we defined an inhibitory integrin signaling pathway. Results: The integrin substrates laminin, fibronectin and vitronectin, but not collagen, thrombospondin or fibrinogen, reduced CNTF expression in C6 astroglioma cells. Antibodies against αv and β5, but not α6 or β1, integrin induced CNTF. Together, the ligand and antibody specificity suggests that CNTF is repressed by αvβ5 integrin. Antibodies against Thy1, an abundant neuronal surface protein whose function is unclear, induced CNTF in neuron-astrocyte co-cultures indicating that it is a neuroglial CNTF repressor. Inhibition of the integrin signaling molecule Focal Adhesion Kinase (FAK) or the downstream c-Jun N-terminal kinase (JNK), but not extracellular regulated kinase (ERK) or p38 MAPK, greatly induced CNTF mRNA and protein expression within 4 hours. This selective inhibitory pathway phosphorylated STAT3 on its inhibitory ser-727 residue interfering with activity of the pro-transcription Tyr-705 residue. STAT3 can activate CNTF transcription because it bound to its promoter and FAK antagonist-induced CNTF was reduced by blocking STAT3. Microinjection of FAK inhibitor directly into the brain or spinal cord in adult mice rapidly induced CNTF mRNA and protein expression. Importantly, systemic treatment with FAK inhibitors over 3 days induced CNTF in the subventricular zone and increased neurogenesis. Conclusions: Neuron-astroglia contact mediated by integrins serves as a sensor to enable rapid neurotrophic responses and provides a new pharmacological avenue to exploit the neuroprotective properties of endogenous CNTF.
The yellow fever vaccine YF-17D is one of the most successful vaccines ever developed in humans. Despite its efficacy and widespread use in more than 600 million people, the mechanisms by which it stimulates protective immunity remain poorly understood. Recent studies using systems biology approaches in humans have revealed that YF-17D-induced early expression of general control nonderepressible 2 kinase (GCN2) in the blood strongly correlates with the magnitude of the later CD8+ T cell response. We demonstrate a key role for virus-induced GCN2 activation in programming dendritic cells to initiate autophagy and enhanced antigen presentation to both CD4+ and CD8+ T cells. These results reveal an unappreciated link between virus-induced integrated stress response in dendritic cells and the adaptive immune response.
by
Matthew Jennis;
Che-Pei Kung;
Subhasree Basu;
Anna Budina-Kolomets;
Julia I-Ju Leu;
Sakina Khaku;
Jeremy P. Scott;
Kathy Q. Cai;
Michelle R. Campbell;
Devin K. Porter;
Xuting Wang;
Douglas A. Bell;
Xiaoxian Li;
David S. Garlick;
Qin Liu;
Monica Hollstein;
Donna L. George;
Maureen E. Murphy
A nonsynonymous single-nucleotide polymorphism at codon 47 in TP53 exists in African-descent populations (P47S, rs1800371; referred to here as S47). Here we report that, in human cell lines and a mouse model, the S47 variant exhibits a modest decrease in apoptosis in response to most genotoxic stresses compared with wild-type p53 but exhibits a significant defect in cell death induced by cisplatin. We show that, compared with wild-type p53, S47 has nearly indistinguishable transcriptional function but shows impaired ability to transactivate a subset of p53 target genes, including two involved in metabolism: Gls2 (glutaminase 2) and Sco2. We also show that human and mouse cells expressing the S47 variant are markedly resistant to cell death by agents that induce ferroptosis (iron-mediated nonapoptotic cell death). We show that mice expressing S47 in homozygous or heterozygous form are susceptible to spontaneous cancers of diverse histological types. Our data suggest that the S47 variant may contribute to increased cancer risk in individuals of African descent, and our findings highlight the need to assess the contribution of this variant to cancer risk in these populations. These data also confirm the potential relevance of metabolism and ferroptosis to tumor suppression by p53.
Urea transporters are a family of urea-selective channel proteins expressed in multiple tissues that play an important role in the urine-concentrating mechanism of the mammalian kidney. Previous studies have shown that knockout of urea transporter (UT)-B, UT-A1/A3, or all UTs leads to urea-selective diuresis, indicating that urea transporters have important roles in urine concentration. Here, we sought to determine the role of UT-A1 in the urine-concentrating mechanism in a newly developed UTA1–knockout mouse model. Phenotypically, daily urine output in UT-A1–knockout mice was nearly 3-fold that of WT mice and 82% of all-UT–knockout mice, and the UT-A1–knockout mice had significantly lower urine osmolality than WT mice. After 24-h water restriction, acute urea loading, or high-protein (40%) intake, UT-A1–knockout mice were unable to increase urine-concentrating ability. Compared with all-UT–knockout mice, the UT-A1–knockout mice exhibited similarly elevated daily urine output and decreased urine osmolality, indicating impaired urea-selective urine concentration. Our experimental findings reveal that UT-A1 has a predominant role in urea-dependent urine-concentrating mechanisms, suggesting that UTA1 represents a promising diuretic target.
by
Albert Grinshpun;
Yogev Cohen;
Aviad Zick;
Luna Kadouri;
Tamar Hamburger;
Benjamin Nisman;
Tanir M Allweis;
Gabriela Oprea;
Tamar Peretz;
Beatrice Uziely;
Amir Sonnenblick
The likelihood of recurrence in breast cancer patients with hormone receptor-positive (HR-positive) tumors is influenced by clinical, histopathological, and molecular features. Recent studies suggested that activated STAT3 (pSTAT3) might serve as a biomarker of outcome in breast cancer patients. In the present work, we have analyzed the added value of pSTAT3 to OncotypeDx Recurrence Score (RS) in patient prognostication. We have found that patients with low RS (<26) and low pSTAT3 might represent a population at a higher risk for cancer recurrence. Furthermore, we have observed that a positive pSTAT3 score alone can be a favorable marker for patients with HR-positive breast cancer under the age of 50. In an era of personalized medicine, these findings warrant further appraisal of chemotherapy benefit in this population.