Leptin-mediated differential regulation of microsomal triglyceride transfer protein in the intestine and liver affects plasma lipids.

The hormone leptin regulates fat storage and metabolism by signaling through the brain and peripheral tissues. Lipids delivered to peripheral tissues originate mostly from the intestine and liver via synthesis and secretion of apolipoprotein B (apoB)-containing lipoproteins. An intracellular chaperone, microsomal triglyceride transfer protein (MTP), is required for the biosynthesis of these lipoproteins, and its regulation determines fat mobilization to different tissues. Using cell culture and animal models, here we sought to identify the effects of leptin on MTP expression in the intestine and liver. Leptin decreased MTP expression in differentiated intestinal Caco-2 cells, but increased expression in hepatic Huh7 cells. Similarly, acute and chronic leptin treatment of chow diet-fed WT mice decreased MTP expression in the intestine, increased it in the liver, and lowered plasma triglyceride levels. These leptin effects required the presence of leptin receptors (LEPRs). Further experiments also suggested that leptin interacted with long-form LEPR (ObRb), highly expressed in the intestine, to down-regulate MTP. In contrast, in the liver, leptin interacted with short-form LEPR (ObRa) to increase MTP expression. Mechanistic experiments disclosed that leptin activates signal transducer and activator of transcription 3 (STAT3) and mitogen-activated protein kinase (MAPK) signaling pathways in intestinal and hepatic cells, respectively, and thereby regulates divergent MTP expression. Our results also indicated that leptin-mediated MTP regulation in the intestine affects plasma lipid levels. In summary, our findings suggest that leptin regulates MTP expression differentially by engaging with different LEPR types and activating distinct signaling pathways in intestinal and hepatic cells.

Cardiac lineage protein-1 (CLP-1) regulates cardiac remodeling via transcriptional modulation of diverse hypertrophic and fibrotic responses and angiotensin II-transforming growth factor ß (TGF-ß1) signaling axis.

It is well known that the renin-angiotensin system contributes to left ventricular hypertrophy and fibrosis, a major determinant of myocardial stiffness. TGF-ß1 and renin-angiotensin system signaling alters the fibroblast phenotype by promoting its differentiation into morphologically distinct pathological myofibroblasts, which potentiates collagen synthesis and fibrosis and causes enhanced extracellular matrix deposition. However, the atrial natriuretic peptide, which is induced during left ventricular hypertrophy, plays an anti-fibrogenic and anti-hypertrophic role by blocking, among others, the TGF-ß-induced nuclear localization of Smads. It is not clear how the hypertrophic and fibrotic responses are transcriptionally regulated. CLP-1, the mouse homolog of human hexamethylene bis-acetamide inducible-1 (HEXIM-1), regulates the pTEFb activity via direct association with pTEFb causing inhibition of the Cdk9-mediated serine 2 phosphorylation in the carboxyl-terminal domain of RNA polymerase II. It was recently reported that the serine kinase activity of Cdk9 not only targets RNA polymerase II but also the conserved serine residues of the polylinker region in Smad3, suggesting that CLP-1-mediated changes in pTEFb activity may trigger Cdk9-dependent Smad3 signaling that can modulate collagen expression and fibrosis. In this study, we evaluated the role of CLP-1 in vivo in induction of left ventricular hypertrophy in angiotensinogen-overexpressing transgenic mice harboring CLP-1 heterozygosity. We observed that introduction of CLP-1 haplodeficiency in the transgenic α-myosin heavy chain-angiotensinogen mice causes prominent changes in hypertrophic and fibrotic responses accompanied by augmentation of Smad3/Stat3 signaling. Together, our findings underscore the critical role of CLP-1 in remodeling of the genetic response during hypertrophy and fibrosis.

Characterization of Trypanosoma cruzi infectivity, proliferation, and cytokine patterns in gut and pancreatic epithelial cells maintained in vitro.

Trypanosoma cruzi infects all nucleated cells in both humans and experimental animals. As a prelude to our studies of T. cruzi pathogenesis in the gastrointestinal system, we have initiated in vitro cultures of gut (Caco-2 and HT-29) and pancreatic (Panc-1) epithelial cells. We show that along with primary human fibroblasts, all three cell lines are susceptible to infection and support proliferation of T. cruzi. Infection with T. cruzi modified dramatically the cytokines elaborated by these cells. Substantially greater quantities of IL-5 and TGF-ß1 were produced by fibroblasts and Caco-2 and Panc-1 cells, whereas secretion of IFN-γ and TNF-α was greatly reduced in all three cell types. Since these cells are not known to be the primary sources of IFN-γ, we examined IFN-γ mRNA expression in these cells. Both Caco-2 and Panc-1 cells were found to express IFN-γ mRNA, validating its secretion. These findings may provide insight into signaling pathways that mediate innate immunity to T. cruzi and pathogenesis of gastrointestinal and pancreatic alterations in Chagas disease.

Hexim-1 modulates androgen receptor and the TGF-ß signaling during the progression of prostate cancer.

BACKGROUND: Androgen and TGF-ß signaling are important components during the progression of prostate cancer. However, whether common molecular events participate in the activation of these signaling pathways are less understood. METHOD: Hexim 1 expression was detected by immunohistochemistry of human tissue microarrays and TRAMP mouse models. The in vivo significance of Hexim-1 was established by crossing the TRAMP mouse model of prostate cancer with Hexim-1 heterozygous mice. TRAMP C2 cell line was also modified to delete one copy of Hexim-1 gene to generate TRAMP-C2-Hexim-1+/- cell lines. RESULTS: In this report, we observed that Hexim-1 protein expression is absent in normal prostate but highly expressed in adenocarcinoma of the prostate and a characteristic sub-cellular distribution among normal, benign hyperplasia, and adenocarcinoma of the prostate. Heterozygosity of the Hexim-1 gene in the prostate cancer mice model and the TRAMP-C2 cell line, leads to increased Cdk9-dependent serine phosphorylation on protein targets such as the androgen receptor (AR) and the TGF-ß-dependent downstream transcription factors, such as the SMAD proteins. CONCLUSION: Our results suggest that changes in the Hexim-1 protein expression and cellular distribution significantly influences the AR activation and the TGF-ß signaling. Thus, Hexim-1 is likely to play a significant role in prostate cancer progression.

CLP-1 associates with MyoD and HDAC to restore skeletal muscle cell regeneration.

Emerging evidence suggests that eukaryotic gene transcription is regulated primarily at the elongation stage by association and dissociation of the inhibitory protein cardiac lineage protein 1 (CLP-1/HEXIM1) from the positive transcription elongation factor b (P-TEFb) complex. It was reported recently that P-TEFb interacts with skeletal muscle-specific regulatory factor, MyoD, suggesting a linkage between CLP-1-mediated control of transcription and skeletal myogenesis. To examine this, we produced CLP-1 knockdown skeletal muscle C2C12 cells by homologous recombination, and demonstrated that the C2C12 CLP-1 +/- cells failed to differentiate when challenged by low serum in the medium. We also showed that CLP-1 interacts with both MyoD and histone deacetylases (HDACs) maximally at the early stage of differentiation of C2C12 cells. This led us to hypothesize that the association might be crucial to inhibition of MyoD-target proliferative genes. Chromatin immunoprecipitation analysis revealed that the CLP-1/MyoD/HDAC complex binds to the promoter of the cyclin D1 gene, which is downregulated in differentiated muscle cells. These findings suggest a novel transcriptional paradigm whereby CLP-1, in conjunction with MyoD and HDAC, acts to inhibit growth-related gene expression, a requirement for myoblasts to exit the cell cycle and transit to myotubes.

A Targetable Myeloid Inflammatory State Governs Disease Recurrence in Clear-Cell Renal Cell Carcinoma.

It is poorly understood how the tumor immune microenvironment influences disease recurrence in localized clear-cell renal cell carcinoma (ccRCC). Here we performed whole-transcriptomic profiling of 236 tumors from patients assigned to the placebo-only arm of a randomized, adjuvant clinical trial for high-risk localized ccRCC. Unbiased pathway analysis identified myeloid-derived IL6 as a key mediator. Furthermore, a novel myeloid gene signature strongly correlated with disease recurrence and overall survival on uni- and multivariate analyses and is linked to TP53 inactivation across multiple data sets. Strikingly, effector T-cell gene signatures, infiltration patterns, and exhaustion markers were not associated with disease recurrence. Targeting immunosuppressive myeloid inflammation with an adenosine A2A receptor antagonist in a novel, immunocompetent, Tp53-inactivated mouse model significantly reduced metastatic development. Our findings suggest that myeloid inflammation promotes disease recurrence in ccRCC and is targetable as well as provide a potential biomarker-based framework for the design of future immuno-oncology trials in ccRCC. SIGNIFICANCE: Improved understanding of factors that influence metastatic development in localized ccRCC is greatly needed to aid accurate prediction of disease recurrence, clinical decision-making, and future adjuvant clinical trial design. Our analysis implicates intratumoral myeloid inflammation as a key driver of metastasis in patients and a novel immunocompetent mouse model. This article is highlighted in the In This Issue feature, p. 2221.

Positive transcription elongation factor b activity in compensatory myocardial hypertrophy is regulated by cardiac lineage protein-1.

Emerging evidence illustrates the importance of the positive transcription elongation factor (P-TEF)b in control of global RNA synthesis, which constitutes a major feature of the compensatory response to diverse hypertrophic stimuli in cardiomyocytes. P-TEFb complex, composed of cyclin T and cdk9, is critical for elongation of nascent RNA chains via phosphorylation of the carboxyl-terminal domain of RNA polymerase (Pol) II. We and others have shown that the activity of P-TEFb is inhibited by its association with cardiac lineage protein (CLP)-1, the mouse homolog of human HEXIM1, in various physiological and pathological conditions. To investigate the mechanism of control of P-TEFb activity by CLP-1 in cardiac hypertrophy, we used a transgenic mouse model of hypertrophy caused by overexpression of calcineurin in the heart. We observed that the level of CLP-1 associated with P-TEFb was reduced markedly in hypertrophic hearts. We also generated bigenic mice (MHC-cyclin T1/CLP-1(+/-)) by crossing MHC-cyclin T1 transgenic mice with CLP-1 heterozygote. The bigenic mice exhibit enhanced susceptibility to hypertrophy that is accompanied with an increase in cdk9 activity via an increase in serine 2 phosphorylation of carboxyl-terminal domain and an increase in GLUT1/GLUT4 ratio. These mice have compensated systolic function without evidence of fibrosis and reduced lifespan. These data suggest that the reduced level of CLP-1 introduced in the background of elevated levels of cyclin T1 elevates derepression of P-TEFb activity and emphasizes the importance of the role of CLP-1 in the mechanism governing compensatory hypertrophy in cardiomyocytes.

Physical and Mental Health Impacts of the COVID-19 Pandemic among US Adults with Chronic Respiratory Conditions.

Adults living with chronic respiratory diseases are at higher risk of death due to COVID-19. Our objective was to evaluate the physical and mental health symptoms among US adults living with chronic respiratory conditions. We used data of 10,760 US adults from the nationally representative COVID-19 Impact Survey. Chronic respiratory conditions were self-reported and included asthma (14.7%), chronic obstructive pulmonary disease or COPD (4.7%), and bronchitis/emphysema (11.6%). We used multivariable Poisson regression to evaluate physical health symptoms. We estimated associations of mental health symptoms using multinomial logistic regression. In multivariable models, adults with asthma were more likely to report physical symptoms including runny or stuffy nose, chest congestion, fever, and chills. In addition, adults with COPD were more likely to report several physical symptoms including fever (adjusted prevalence ratio [aPR]: 1.37, 95% confidence interval [CI]: 1.09-1.72), chills (aPR: 2.10, 95% CI: 1.67-2.64), runny or stuffy nose (aPR: 1.78, 95% CI: 1.39-2.27), chest congestion (aPR: 2.14, 95% CI: 1.74-2.61), sneezing (aPR: 1.59, 95% CI: 1.23-2.05), and muscle or body aches (aPR: 1.38, 95% CI: 1.06-1.81). Adults with chronic respiratory conditions are more likely to report physical and mental health symptoms during the COVID-19 pandemic compared to others. Providers should prioritize discussing mental health symptom management as the pandemic continues to be a public health concern in the US.

Cross-talk between calcineurin/NFAT and Jak/STAT signalling induces cardioprotective alphaB-crystallin gene expression in response to hypertrophic stimuli.

Among the stress proteins that are up-regulated in the heart due to imposed biomechanical stress, alphaB-crystallin (CryAB) is the most abundant and pivotal in rendering protection against stress-induced cell damage. Cardiomyocyte-specific expression of the CryAB gene was shown to be dependent upon an intact alphaBE4 cis-element located in the CryAB enhancer. To date, there is no evidence on the identity of regulatory proteins and associated signalling molecules that control CryAB expression in cardiomyocytes. In this study, we define a mechanism by which the calcineurin/NFAT and Jak/STAT pathways regulate CryAB gene expression in response to a hypertrophic agonist endothelin-1 (En-1), in hypertrophic hearts of mice with pressure overload (TAC) and in heart-targeted calcineurin over-expressing mice (MHC-CnA). We observed that in response to various hypertrophic stimuli the transcription factors NFAT, Nished and STAT3 form a dynamic ternary complex and interact with the alphaBE4 promoter element of the CryAB gene. Both dominant negative NFAT and AG490, an inhibitor of the Jak2 phosphorylation, inhibited CryAB gene transcription in transient transfection assays. AG490 was also effective in blocking the nuclear translocation of NFAT and STAT3 in cardiomyocytes treated with En-1. We observed a marked increase in CryAB gene expression in MHC-CnA mouse hearts accompanied with increased phosphorylation of STAT3. We conclude that hypertrophy-dependent CryAB gene expression can be attributed to a functional linkage between the Jak/STAT and calcineurin/NFAT signalling pathways, each of which are otherwise known to be involved independently in the deleterious outcome in cardiac hypertrophy.

Repression of the cardiac myosin light chain-2 gene in skeletal muscle requires site-specific association of antithetic regulator, Nished, and HDACs.

The transcriptional activation mechanisms that regulate tissue-specific expression of cardiac muscle genes have been extensively investigated, but little is known of the regulatory events involved in repression of cardiac-specific genes in non-cardiac cells. We have previously reported that Nished, a ubiquitous transcription factor, interacts with a positive sequence element, the Intron Regulatory Element (IRE) as well as a negatively acting element, the Cardiac-Specific Sequence (CSS), in myosin light chain-2 (MLC2v) gene to promote activation and repression of the gene in cardiac and skeletal muscle cells respectively. Here, we show that the negative regulation of cardiac MLC2v gene in skeletal muscle cells is mediated via the interaction of Nished with histone deacetylase (HDAC) co-repressor. Treatment of cells with the HDAC inhibitor, Trichostatin A (TSA), alleviates the repressor activity of Nished in a dose-dependent manner. Co-transfection studies in primary muscle cells in culture and in Nished expressing stable skeletal muscle cell line demonstrate that Nished down-regulates the cardiac MLC2 gene expression when its association is restricted to CSS alone. Chromatin immunoprecipitation data suggest that the CSS-mediated repression of cardiac MLC2v gene in skeletal muscle cells excludes the participation of the positive element IRE despite the presence of an identical Nished binding site. Taken together, it appears that the negative control of MLC2v transcription is based on a dual mode of regulations, one that affords inaccessibility of IRE to Nished and second that promotes the formation of the transcription repression complex at the inhibitory CSS site to silence the cardiac gene in skeletal muscle cell.

Down-regulation of cardiac lineage protein (CLP-1) expression in CLP-1 +/- mice affords.

In order to understand the transcriptional mechanism that underlies cell protection to stress, we evaluated the role of CLP-1, a known inhibitor of the transcription elongation complex (pTEFb), in CLP-1 +/- mice hearts. Using the isolated heart model, we observed that the CLP-1 +/- hearts, when subjected to ischaemic stress and evaluated by haemodynamic measurements, exhibit significant cardioprotection. CLP-1 remains associated with the pTEFb complex in the heterozygous hearts, where as it is released in the wild-type hearts suggesting the involvement of pTEFb regulation in cell protection. There was a decrease in Cdk7 and Cdk9 kinase activity and consequently in phosphorylation of serine-5 and serine-2 of Pol II CTD in CLP-1 +/- hearts. However, the levels of mitochondrial proteins, PGC-1alpha and HIF-1alpha, which enhance mitochondrial activity and are implicated in cell survival, were increased in CLP-1 +/- hearts subjected to ischaemic stress compared to that in wild-type CLP-1 +/- hearts treated identically. There was also an increase in the expression of pyruvate dehydrogenase kinase (PDK-1), which facilitates cell adaptation to hypoxic stress. Taken together, our data suggest that regulation of the CLP-1 levels is critical to cellular adaptation of the survival program that protects cardiomyocytes against stress due collectively to a decrease in RNA Pol II phosphorylation but an increase in expression of target proteins that regulate mitochondrial function and metabolic adaptation to stress.

Early signaling by vascular endothelial growth factor and placental growth factor in human bone marrow-derived endothelial cells is mediated by superoxide.

AIMS: We investigated the role of superoxide O(2)(-) during the initiation of vascular endothelial growth factor (VEGF)- and placental growth factor (PlGF)-mediated signal transduction in bone marrow-derived endothelial cells. METHODS: BMhTERT cells were treated with VEGF or PlGF in the presence or absence of antioxidants. The signaling pathways downstream were analyzed by immunoprecipitation and Western blotting. Superoxide and reactive oxygen species (ROS) were measured using Superluminol or 2',7'-dichlorofluorescein fluorescence measurements. RESULTS: We show here that VEGF and PlGF generate extracellular and intracellular O(2)(-) that regulates their downstream signaling transduction pathways. Indeed, the extracellular O(2)(-) generated treatment of endothelial cells (using hypoxanthine/xanthine oxidase) was sufficient to initiate receptor phosphorylation of VEGF receptor 2. The PlGF treatment of endothelial cells increased the generation of intracellular ROS in an extracellular O(2)(-) dependent manner. Quenching of intracellular ROS by resveratrol inhibits PlGF- and VEGF-dependent induction of MAP kinase phosphorylation. Additionally, we found that the interaction of VEGF and PlGF with their specific receptors generates O(2)(-) in a cell-free system. Endothelial cells treated with VEGF stop proliferation in the presence of extracellular catalase, superoxide dismutase or peroxiredoxin IV. CONCLUSION: Our studies underscore the role of O(2)(-) as a critical regulator of VEGF and PlGF signal transduction initiation in endothelial cells.

Pivotal role of cardiac lineage protein-1 (CLP-1) in transcriptional elongation factor P-TEFb complex formation in cardiac hypertrophy.

OBJECTIVE: Our aim was to determine if the expression pattern of CLP-1 in developing heart is consistent with its role in controlling RNA transcript elongation by transcriptional elongation factor b (P-TEFb) and if the inhibitory control exerted over P-TEFb by CLP-1 is released under hypertrophic conditions. METHODS: We performed immunoblot and immunofluorescence analysis of CLP-1 and the P-TEFb components cdk9 and cyclin T in fetal mouse heart and 2 day post-natal mouse cardiomyocytes to determine if they are co-localized. We induced hypertrophy in rat cardiomyocytes either by mechanical stretch or treatment with hypertrophic agents such as endothelin-1 and phenylephrine to determine if CLP-1 is released from P-TEFb in response to hypertrophic stimuli. The involvement of the Jak/STAT signal transduction pathway in this process was studied by blocking this pathway with the Jak2 kinase inhibitor, AG490, and assessing the association of CLP-1 with P-TEFb complexes. RESULTS: We found that CLP-1 is expressed along with P-TEFb components in developing heart during the period in which knockout mice lacking the CLP-1 gene develop cardiac hypertrophy and die. Under conditions of hypertrophy induced by mechanical stretch or agonist treatment, CLP-1 dissociates from the P-TEFb complex, a finding consistent with the de-repression of P-TEFb kinase activity seen in hypertrophic cardiomyocytes. Blockage of Jak/STAT signaling by AG490 prevented release of CLP-1 from P-TEFb despite the ongoing presence of hypertrophic stimulation by mechanical stretch. CONCLUSIONS: CLP-1 expression in developing heart and isolated post-natal cardiomyocytes colocalizes with P-TEFb expression and therefore has the potential to regulate RNA transcript elongation by controlling P-TEFb cdk9 kinase activity in heart. We further conclude that the dissociation of CLP-1 from P-TEFb is responsive to hypertrophic stimuli transduced by cellular signal transduction pathways. This process may be part of the genomic stress response resulting in increased RNA transcript synthesis in hypertrophic cardiomyocytes.

Rapidly progressive course of Trypanosoma cruzi infection in mice heterozygous for hexamethylene bis-acetamide inducible 1 (Hexim1) gene.

Infection with Trypanosoma cruzi causes Chagas disease and results in myocardial inflammation and cardiomyopathy. Downregulated Hexim1 expression, as in Hexim1+/- mice, reduces cardiac inflammation and fibrosis following ischemic stress. We asked whether reduced expression of Hexim1 would also afford protection against T. cruzi-induced cardiomyopathy. C57BL/6J (wild type - WT) and Hexim1+/- mice were infected with sub-lethal doses of T. cruzi (Brazil strain), and cardiac function, serologic markers of inflammation and tissue pathology were examined. Infected Hexim1+/- mice had compromised cardiac function, altered expression of both pro- and anti-inflammatory cytokines, and increased inflammation and fibrosis. Cardiac failure was evidenced by severely diminished heart rate, compensatory increase in respiratory rate, and abnormally high left ventricular mass with severe transmural inflammation. Lungs displayed intense peribronchial inflammation and fibrosis extending into the parenchyma. We also observed Smad3-serine208 phosphorylation in hearts and lungs of infected mice, suggesting increased TGF-ß signaling pathway activity. This was more pronounced in Hexim1+/- mice and correlated with increased fibrosis in these tissues. Conspicuous splenomegaly in the Hexim1+/- mice most likely resulted from the observed extensive white pulp expansion. T. cruzi infection induced colonic dilatation and marked villous atrophy in both the WT and Hexim1+/- mice but more so in the latter. The profound exacerbation of pathologic findings suggests a protective role for Hexim1 in T. cruzi infection.

Hexim1 heterozygosity stabilizes atherosclerotic plaque and decreased steatosis in ApoE null mice fed atherogenic diet.

Hexim-1 is an inhibitor of RNA polymerase II transcription elongation. Decreased Hexim-1 expression in animal models of chronic diseases such as left ventricular hypertrophy, obesity and cancer triggered significant changes in adaptation and remodeling. The main aim of this study was to evaluate the role of Hexim1 in lipid metabolism focused in the progression of atherosclerosis and steatosis. We used the C57BL6 apolipoprotein E (ApoE null) crossed bred to C57BL6Hexim1 heterozygous mice to obtain ApoE null - Hexim1 heterozygous mice (ApoE-HT). Both ApoE null backgrounds were fed high fat diet for twelve weeks. Then, we evaluated lipid metabolism, atherosclerotic plaque formation and liver steatosis. In order to understand changes in the transcriptome of both backgrounds during the progression of steatosis, we performed Affymetrix mouse 430 2.0 microarray. After 12 weeks of HFD, ApoE null and ApoE-HT showed similar increase of cholesterol and triglycerides in plasma. Plaque composition was altered in ApoE-HT. Additionally, liver triglycerides and steatosis were decreased in ApoE-HT mice. Affymetrix analysis revealed that decreased steatosis might be due to impaired inducible SOCS3 expression in ApoE-HT mice. In conclusion, decreased Hexim-1 expression does not alter cholesterol metabolism in ApoE null background after HFD. However, it promotes stable atherosclerotic plaque and decreased steatosis by promoting the anti-inflammatory TGFß pathway and blocking the expression of the inducible and pro-inflammatory expression of SOCS3 respectively.

Inhibition of Jak2 phosphorylation attenuates pressure overload cardiac hypertrophy.

RATIONALE: We examined the role of Jak2 kinase phosphorylation in the development of pressure overload hypertrophy in mice subjected to transverse aortic constriction (TAC) and treated with tyrphostin AG490, a pharmacological inhibitor of Jak2. METHODS: Control mice (sham), subjected to TAC for 15 days (TAC) or to TAC and treated with 48 microg/kg/day i.p. of tyrphostin AG490 (TAC+AG490) were evaluated for morphological, physiological, and molecular changes associated with pressure overload hypertrophy. RESULTS: Mice subjected to TAC alone developed concentric hypertrophy that accompanied activation of the components of the Jak/STAT signaling pathway manifested by an increase in phosphorylation of Jak2 and STAT3. We also observed increased phosphorylation of MAPK p44/p42, p38 MAPK and JNK in the TAC group, as well as, an increase in expression of MKP-1 phosphatase which negatively regulates MAPK kinases. Treatment of aortic constricted mice with tyrphostin AG490 failed to develop hypertrophy and showed a marked reduction in phosphorylation of Jak2 and STAT3. There was, however, in TAC and AG490 treated mice, a notable increase in the phosphorylation state of the MAPK p44/42, whereas MKP-1 phosphatase was downregulated. CONCLUSION: These findings suggest that Jak2 kinase plays an important role in left ventricular remodeling during pressure overload hypertrophy. Pharmacological inhibition of Jak2 kinase during pressure overload blocks the development of concentric hypertrophy.

Hexim1, a Novel Regulator of Leptin Function, Modulates Obesity and Glucose Disposal.

Leptin triggers signaling events with significant transcriptional responses that are essential to metabolic processes affecting obesity and glucose disposal. We asked whether hexamethylene bis-acetamide inducible-1 (Hexim1), an inhibitor of RNA II polymerase-dependent transcription elongation, regulates leptin-Janus kinase 2 signaling axis in the hypothalamus. We subjected C57BL6 Hexim1 heterozygous (HT) mice to high-fat diet and when compared with wild type, HT mice were resistant to high-fat diet-induced weight gain and remain insulin sensitive. HT mice exhibited increased leptin-pY(705)Stat3 signaling in the hypothalamus, with normal adipocyte size, increased type I oxidative muscle fiber density, and enhanced glucose transporter 4 expression. We also observed that normal Hexim1 protein level is required to facilitate the expression of CCAAT/enhancer-binding proteins (C/EBPs) required for adipogenesis and inducible suppressor of cytokine signaling 3 (SOCS) expression. Further support on the role of Hexim1 regulating C/EBPs during adipocyte differentiation was shown when HT 3T3L1 fibroblasts failed to undergo adipogenesis. Hexim1 selectively modulates leptin-mediated signal transduction pathways in the hypothalamus, the expression of C/EBPs and peroxisome proliferator-activated receptor-γ (PPAR γ) in skeletal muscle and adipose tissue during the adaptation to metabolic stress. We postulate that Hexim1 might be a novel factor involved in maintaining whole-body energy balance.

Janus kinase-2 signaling mediates apoptosis in rat cardiomyocytes.

We tested the hypothesis that activation Jak2, which is prominently involved in the up-regulation of the renin-angiotensin system (RAS), constitutes a focal point in relaying signals triggered by a Angiotensin II (Ang II) and hypoxia/reoxygenation separately to cause an enhanced susceptibility of cardiac myocyte to apoptotic cell death. Ang II-treated adult cardiomyocytes in culture exhibited an increased level of apoptosis that accompanied activation of pro-apoptotic as well as anti-apoptotic signaling pathways. We observed increased phosphorylation of Jak2 kinase, Stat1, JNK, with increased expression of Bax protein, followed by an increase in caspase-1 and caspase-3 activity. Activation of these pro-apoptotic pathways was blocked by the Jak2 pharmacological inhibitor, Tyrphostin AG490. We also observed an increase in phosphorylation of cardioprotective pathway components, namely S6 ribosomal protein, and heat shock protein 27 (HSP27). Likewise, the oxidative stress, via the hypoxia/reoxygenation treatment of rat adult cardiomyocytes, produced apoptosis that was dependent upon activation of Jak2. The apoptotic response was not only reduced by Losartan, an inverse agonist of the AT1, receptor, but by treatment with AG490 as well. Taken together, these observations provide clear evidence in favor of Jak2 signaling as mediator of the apoptotic response in cardiomyocytes. However, there was a concomitant induction of cytoprotective signaling that presumably provides a negative feed-back to the deleterious effects of the agonist.

Distinct components of Janus kinase/signal transducer and activator of transcription signaling pathway mediate the regulation of systemic and tissue localized renin-angiotensin system.

In an attempt to demonstrate the linkage between the Janus kinase (Jak)/signal transducer and activator of transcription (STAT) signaling and the activity of the systemic or local renin-angiotensin system in vivo, we produced transgenic mice harboring angiotensinogen (ANG) promoter containing the wild-type or mutant STAT target site (St-domain) fused to the luciferase reporter. The ANG-promoter-driven luciferase expression was dependent upon phosphorylation of Jak2, as administration of tyrphostin AG490, a potent inhibitor of Jak2, down-regulated the ANG promoter activity and abolished the stimulated endogenous ANG mRNA level in the liver. Administration of angiotensin II peptide to the mice resulted in prominent expression of luciferase in the liver and heart of animals containing wild type St-domain, but not in transgenes with mutant St-domain. Angiotensin II-induced signaling caused activation of STAT proteins in the liver (systemic), the pattern of which was distinct from that in the heart (local). The inducible expression of ANG promoter appears to be mediated by physical association of p300 with STAT 5B in liver and STAT 3 and STAT 5A in heart. Taken together, these results point to the differences in signaling mechanisms in the circulating and localized renin-angiotensin system and identify at least two molecular steps, the tyrosyl phosphorylation of Jak2 and the STAT/St-domain interaction, as pivotal in the regulation of ANG gene transcription.

The functional role of the JAK-STAT pathway in post-infarction remodeling.

OBJECTIVES: Recently, the Janus kinase/signal transducer and activator of transcription (JAK-STAT) signaling pathway was found to be prominently associated with activation of the autocrine loop of the heart tissue-localized renin angiotensin system (RAS). We investigated if the JAK-STAT pathway is activated in the post-myocardial infarction (MI) non-ischemic myocardium (NIM), destined to undergo remodeling and whether blockade of the pathway in vivo can modify early post-MI remodeling. METHODS: We investigated the time course of tyrosine phosphorylation of JAK-STAT and gp130 proteins in the NIM of post-MI rat heart as well as the binding activity of STAT proteins to the St-domain of the angiotensinogen gene promoter. We further compared the effects of in vivo blockade of RAS by the AT(1) receptor (AT(1)R) blocker losartan with the in vivo blockade of JAK-STAT pathway by the specific JAK2 blocker tyrphostin AG490 on certain aspects of early post-MI remodeling. RESULTS: We showed that JAK2, STATs 1, 3, 5a and 6 and gp130 proteins are tyrosine phosphorylated as early as 5-30 min post-MI and that STATs 1, 3, and 5a remain activated up to 7 days post-MI. Gel mobility shift assay showed a strong binding activity of STAT proteins to the St-domain of angiotensinogen gene promoter in 1-day post-MI NIM. The binding was significantly reduced in rat hearts previously treated with losartan or tyrphostin AG490. Supershift experiments identified STATs 3 and 5a as specifically interacting with the St-domain. Both AT(1)R and JAK2 blockade resulted in significant amelioration of the increase of protein phosphatase 1 activity and decrease in basal level of p16-phospholamban that may underlie early diastolic dysfunction, as well as partial amelioration of early downregulation of Kv4.2 gene expression that may underlie increased arrhythmogenicity of 3-day post-MI heart. On the other hand, while blockade of AT(1)R significantly ameliorated apoptotic changes in 1-day post-MI border zone, blockade of JAK2 increased apoptosis. CONCLUSIONS: The study provides compelling evidence in favor of the linkage of the JAK-STAT pathway with the angiotensin II autocrine loop and uncovers a mechanism by which selective activation of a set of STAT proteins underlies mobilization of the gene activation program intrinsic to post-MI remodeling. It also suggests that drugs that inhibit JAK-STAT phosphorylation may provide a new approach to modify post-MI remodeling. This needs to be confirmed in long term in vivo studies in the post-MI heart.