PYK2 selectively mediates signals for growth versus differentiation in response to stretch of spontaneously active vascular smooth muscle.

Stretch of vascular smooth muscle stimulates growth and proliferation as well as contraction and expression of contractile/cytoskeletal proteins, all of which are also regulated by calcium-dependent signals. We studied the role of the calcium- and integrin-activated proline-rich tyrosine kinase 2 (PYK2) in stretch-induced responses of the rat portal vein loaded by a hanging weight ex vivo. PYK2 phosphorylation at Tyr-402 was increased both by a 10-min stretch and by organ culture with load over several days. Protein and DNA synthesis were reduced by the novel PYK2 inhibitor PF-4594755 (0.5-1 μmol/L), while still sensitive to stretch. In 3-day organ culture, PF-4594755 caused maintained myogenic spontaneous activity but did not affect contraction in response to high-K(+) (60 mmol/L) or to α1-adrenergic stimulation by cirazoline. Basal and stretch-induced PYK2 phosphorylation in culture were inhibited by PF-4594755, closely mimicking inhibition of non-voltage-dependent calcium influx by 2-APB (30 μmol/L). In contrast, the L-type calcium channel blocker, nifedipine (1 μmol/L) eliminated stretch-induced but not basal PYK2 phosphorylation. Stretch-induced Akt and ERK1/2 phosphorylation was eliminated by PF-4594755. PYK2 inhibition had no effect on mRNA expression of several smooth muscle markers, and stretch-sensitive SM22α synthesis was preserved. Culture of portal vein with the Ang II inhibitor losartan (1 μmol/L) eliminated stretch sensitivity of PYK2 and Akt phosphorylation, but did not affect mRNA expression of smooth muscle markers. The results suggest that PYK2 signaling functionally distinguishes effects of voltage- and non-voltage-dependent calcium influx. A small-molecule inhibitor of PYK2 reduces growth and DNA synthesis but does not affect contractile differentiation of vascular smooth muscle

Endothelial protein kinase MAP4K4 promotes vascular inflammation and atherosclerosis

Signalling pathways that control endothelial cell (EC) permeability, leukocyte adhesion and inflammation are pivotal for atherosclerosis initiation and progression. Here we demonstrate that the Sterile-20-like mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4), which has been implicated in inflammation, is abundantly expressed in ECs and in atherosclerotic plaques from mice and humans. On the basis of endothelial-specific MAP4K4 gene silencing and gene ablation experiments in Apoe(-/-) mice, we show that MAP4K4 in ECs markedly promotes Western diet-induced aortic macrophage accumulation and atherosclerotic plaque development. Treatment of Apoe(-/-) and Ldlr(-/-) mice with a selective small-molecule MAP4K4 inhibitor also markedly reduces atherosclerotic lesion area. MAP4K4 silencing in cultured ECs attenuates cell surface adhesion molecule expression while reducing nuclear localization and activity of NFkappaB, which is critical for promoting EC activation and atherosclerosis. Taken together, these results reveal that MAP4K4 is a key signalling node that promotes immune cell recruitment in atherosclerosis

Genome-wide association and Mendelian randomisation analysis provide insights into the pathogenesis of heart failure

Heart failure (HF) is a leading cause of morbidity and mortality worldwide. A small proportion of HF cases are attributable to monogenic cardiomyopathies and existing genome-wide association studies (GWAS) have yielded only limited insights, leaving the observed heritability of HF largely unexplained. We report results from a GWAS meta-analysis of HF comprising 47,309 cases and 930,014 controls. Twelve independent variants at 11 genomic loci are associated with HF, all of which demonstrate one or more associations with coronary artery disease (CAD), atrial fibrillation, or reduced left ventricular function, suggesting shared genetic aetiology. Functional analysis of non-CAD-associated loci implicate genes involved in cardiac development (MYOZ1, SYNPO2L), protein homoeostasis (BAG3), and cellular senescence (CDKN1A). Mendelian randomisation analysis supports causal roles for several HF risk factors, and demonstrates CAD-independent effects for atrial fibrillation, body mass index, and hypertension. These findings extend our knowledge of the pathways underlying HF and may inform new therapeutic strategies

Genome-wide association and Mendelian randomisation analysis provide insights into the pathogenesis of heart failure

Heart failure (HF) is a leading cause of morbidity and mortality worldwide. A small proportion of HF cases are attributable to monogenic cardiomyopathies and existing genome-wide association studies (GWAS) have yielded only limited insights, leaving the observed heritability of HF largely unexplained. We report results from a GWAS meta-analysis of HF comprising 47,309 cases and 930,014 controls. Twelve independent variants at 11 genomic loci are associated with HF, all of which demonstrate one or more associations with coronary artery disease (CAD), atrial fibrillation, or reduced left ventricular function, suggesting shared genetic aetiology. Functional analysis of non-CAD-associated loci implicate genes involved in cardiac development (MYOZ1, SYNPO2L), protein homoeostasis (BAG3), and cellular senescence (CDKN1A). Mendelian randomisation analysis supports causal roles for several HF risk factors, and demonstrates CAD-independent effects for atrial fibrillation, body mass index, and hypertension. These findings extend our knowledge of the pathways underlying HF and may inform new therapeutic strategies

Genome-wide association and Mendelian randomisation analysis provide insights into the pathogenesis of heart failure

Abstract: Heart failure (HF) is a leading cause of morbidity and mortality worldwide. A small proportion of HF cases are attributable to monogenic cardiomyopathies and existing genome-wide association studies (GWAS) have yielded only limited insights, leaving the observed heritability of HF largely unexplained. We report results from a GWAS meta-analysis of HF comprising 47,309 cases and 930,014 controls. Twelve independent variants at 11 genomic loci are associated with HF, all of which demonstrate one or more associations with coronary artery disease (CAD), atrial fibrillation, or reduced left ventricular function, suggesting shared genetic aetiology. Functional analysis of non-CAD-associated loci implicate genes involved in cardiac development (MYOZ1, SYNPO2L), protein homoeostasis (BAG3), and cellular senescence (CDKN1A). Mendelian randomisation analysis supports causal roles for several HF risk factors, and demonstrates CAD-independent effects for atrial fibrillation, body mass index, and hypertension. These findings extend our knowledge of the pathways underlying HF and may inform new therapeutic strategies

Genome-wide association and Mendelian randomisation analysis provide insights into the pathogenesis of heart failure

Abstract: Heart failure (HF) is a leading cause of morbidity and mortality worldwide. A small proportion of HF cases are attributable to monogenic cardiomyopathies and existing genome-wide association studies (GWAS) have yielded only limited insights, leaving the observed heritability of HF largely unexplained. We report results from a GWAS meta-analysis of HF comprising 47,309 cases and 930,014 controls. Twelve independent variants at 11 genomic loci are associated with HF, all of which demonstrate one or more associations with coronary artery disease (CAD), atrial fibrillation, or reduced left ventricular function, suggesting shared genetic aetiology. Functional analysis of non-CAD-associated loci implicate genes involved in cardiac development (MYOZ1, SYNPO2L), protein homoeostasis (BAG3), and cellular senescence (CDKN1A). Mendelian randomisation analysis supports causal roles for several HF risk factors, and demonstrates CAD-independent effects for atrial fibrillation, body mass index, and hypertension. These findings extend our knowledge of the pathways underlying HF and may inform new therapeutic strategies

TPA can overcome the requirement for EIa and together act synergistically in stimulating expression of the adenovirus EIII promoter.

Abstract We have examined the control of gene expression from the adenovirus early region III (Ad-EIII) promoter, which contains two previously defined elements, the AP1 and ATF sites. We found that the AP1 element is capable of mediating activation by the adenovirus immediate early (EIa) gene products. Consistent with studies demonstrating that the AP1 site mediates signal transduction in response to 12-O-tetradecanoylphorbol 13-acetate (TPA) we have shown that TPA can activate Ad-EIII expression and overcome the requirement for EIa. Together TPA and EIa elicited a synergistic response in expression from the Ad-EIII promoter during both transient expression assays and viral infections. This synergistic effect required the AP1 element. An EIII promoter construct, in which sequences upstream of the TATA box had been replaced with four AP1 sites, was responsive to TPA and EIa and in combination promoted the synergistic effect. The analysis of specific factors involved in transcription from the Ad-EIII indicated that proteins recognizing the ATF and AP1 sites were important in expression from this promoter in vitro. Purification of protein factors that specifically stimulated EIII expression resulted in the isolation of a set of factors of the AP1 family. Affinity purified AP1 recognized and activated transcription through both the AP1 and ATF elements. In addition, a protein fraction was identified with DNA binding activity specific for the ATF element. This fraction was dependent on the ATF site for transcriptional activity

The initiator directs the assembly of a transcription factor IID-dependent transcription complex

Abstract Highly purified RNA polymerase II was found to be able to weakly recognize the initiator (Inr) present in the adenovirus IVa2 and major late promoters. The association of RNA polymerase II with the Inr was enhanced by the general transcription factors. The Inr was capable of directing the formation of a DNA-protein complex. Transcription competent complexes on the adenovirus major late and IVa2 promoters appear to be formed by alternative pathways mediated through the Inr and/or "TATA" motif. The presence of both motifs, however, is required for efficient transcription utilizing a discrete start site. Complexes formed at either site required transcription factor TFIID, the TATA binding protein. Consistent with this observation, a TFIID requirement was demonstrated for transcription from a mutant adenovirus major late promoter construct lacking a functional TATA motif

Phosphorylation of cellular proteins regulates their binding to the cAMP response element.

We have studied the protein factors that promote transcription via binding to the cAMP response element (CRE) present in the adenovirus early region III (EIII) and early region IV (EIV) promoters. Three sets of CRE-binding phosphoproteins, ranging in molecular mass from 65-72, 38-43, and 31-37 kDa, were identified in vivo from HeLa cells. Western blot analysis revealed that all three sets of proteins identified were immunologically related to the transcription factor AP1. We found that binding of these proteins to the CRE could be regulated by phosphorylation in vitro. EivF, a 65-72-kDa protein was found to bind specifically to the adenovirus EIV promoter. We have also shown that the smaller molecular mass proteins of 31-37 and 38-43 kDa were able to bind to the CRE present in the adenovirus EIV promoter, as well as to two related DNA elements present in the adenovirus EIII promoter, the ATF and AP1 sites. Phosphorylation of these proteins with the cAMP-dependent protein kinase, affected their transcriptional activity and binding affinity to the three sites. Furthermore, the binding specificity of the 31-37-kDa polypeptides was mediated by cAMP-dependent protein kinase in vitro. Our data suggests that phosphorylation of factors that bind to the CRE may, in part, underlie the cellular response to the adenovirus-encoded Ela protein

Factors involved in specific transcription by mammalian RNA polymerase II. Role of factors IID and MLTF in transcription from the adenovirus major late and IVa2 promoters.

Abstract The role of the adenovirus major late upstream transcription factor (MLTF) in transcription from the adenovirus major late and the IVa2 promoters was studied. The transcription initiation site of the IVa2 promoter is located 210 nucleotides upstream from the CAP site of the major late promoter. Transcription from these two promoters occurs on different DNA strands. Thus, this divergent transcription suggests that the same factor could simultaneously regulate the expression of two different genes. This was investigated utilizing a reconstituted transcription system in vitro. The addition of MLTF to reaction mixtures containing the purified general transcription factors and the major late promoter resulted in a 10-12-fold stimulation of transcription. This stimulation was because of an increase of the stability of the preinitiation complex. MLTF allowed DNA template molecules to undergo multiple rounds of transcription. MLTF also stimulated transcription from the adenovirus-encoded IVa2 promoter. Surprisingly, reconstitution experiments indicated that transcription from the IVa2 promoter which does not have a TATA sequence required all the previously described general transcription factors, including TFIID, the TATA binding protein. The requirement for TFIID was demonstrated by reconstitution experiments as well as by oligonucleotide competition experiments. The implications of this observation are discussed