Dual Regulation of Myocardin Expression by Tumor Necrosis Factor-α in Vascular Smooth Muscle Cells

De-differentiation of vascular smooth muscle cells (VSMCs) plays a critical role in the development of atherosclerosis, a chronic inflammatory disease involving various cytokines such as tumor necrosis factor-α (TNFα). Myocardin is a co-factor of serum response factor (SRF) and is considered to be the master regulator of VSMC differentiation. It binds to SRF and regulates the expression of contractile proteins in VSMCs. Myocardin is also known to inhibit VSMC proliferation by inhibiting the NF-κB pathway, whereas TNFα is known to activate the NF-κB pathway in VSMCs. NF-κB activation has also been shown to inhibit myocardin expression and smooth muscle contractile marker genes. However, it is not definitively known whether TNFα regulates the expression and activity of myocardin in VSMCs. The current study aimed to investigate the role of TNFα in regulating myocardin and VSMC function. Our studies showed that TNFα down-regulated myocardin expression and activity in cultured VSMCs by activating the NF-κB pathway, resulting in decreased VSMC contractility and increased VSMC proliferation. Surprisingly, we also found that TNFα prevented myocardin mRNA degradation, and resulted in a further significant increase in myocardin expression and activity in differentiated VSMCs. Both the NF-κB and p44/42 MAPK pathways were involved in TNFα regulation of myocardin, which further increased the contractility of VSMCs. These differential effects of TNFα on myocardin seemingly depended on whether VSMCs were in a differentiated or de-differentiated state. Taken together, our results demonstrate that TNFα differentially regulates myocardin expression and activity, which may play a key role in regulating VSMC functions.     

Role of Vascular Smooth Muscle PPARγ in Regulating AT1 Receptor Signaling and Angiotensin II-Dependent Hypertension

Peroxisome proliferator activated receptor γ (PPARγ) has been reported to play a protective role in the vasculature; however, the underlying mechanisms involved are not entirely known. We previously showed that vascular smooth muscle-specific overexpression of a dominant negative human PPARγ mutation in mice (S-P467L) leads to enhanced myogenic tone and increased angiotensin-II-dependent vasoconstriction. S-P467L mice also exhibit increased arterial blood pressure. Here we tested the hypotheses that a) mesenteric smooth muscle cells isolated from S-P467L mice exhibit enhanced angiotensin-II AT₁ receptor signaling, and b) the increased arterial pressure of S-P467L mice is angiotensin-II AT₁ receptor dependent. Phosphorylation of mitogen-activated protein/extracellular signal-regulated kinase (ERK1/2) was robustly increased in mesenteric artery smooth muscle cell cultures from S-P467L in response to angiotensin-II. The increase in ERK1/2 activation by angiotensin-II was blocked by losartan, a blocker of AT₁ receptors. Angiotensin-II-induced ERK1/2 activation was also blocked by Tempol, a scavenger of reactive oxygen species, and correlated with increased Nox4 protein expression. To investigate whether endogenous renin-angiotensin system activity contributes to the elevated arterial pressure in S-P467L, non-transgenic and S-P467L mice were treated with the AT₁ receptor blocker, losartan (30 mg/kg per day), for 14-days and arterial pressure was assessed by radiotelemetry. At baseline S-P467L mice showed a significant increase of systolic arterial pressure (142.0±10.2 vs 129.1±3.0 mmHg, p<0.05). Treatment with losartan lowered systolic arterial pressure in S-P467L (132.2±6.9 mmHg) to a level similar to untreated non-transgenic mice. Losartan also lowered arterial pressure in non-transgenic (113.0±3.9 mmHg) mice, such that there was no difference in the losartan-induced depressor response between groups (−13.53±1.39 in S-P467L vs −16.16±3.14 mmHg in non-transgenic). Our results suggest that interference with PPARγ in smooth muscle: a) causes enhanced angiotensin-II AT₁ receptor-mediated ERK1/2 activation in resistance vessels, b) and may elevate arterial pressure through both angiotensin-II AT₁ receptor-dependent and -independent mechanisms.     

Angiotensin II Type I and Prostaglandin F2α Receptors Cooperatively Modulate Signaling in Vascular Smooth Muscle Cells

The angiotensin II type I (AT1R) and the prostaglandin F2α (PGF2α) F prostanoid (FP) receptors are both potent regulators of blood pressure. Physiological interplay between AT1R and FP has been described. Abdominal aortic ring contraction experiments revealed that PGF2α-dependent activation of FP potentiated angiotensin II-induced contraction, whereas FP antagonists had the opposite effect. Similarly, PGF2α-mediated vasoconstriction was symmetrically regulated by co-treatment with AT1R agonist and antagonist. The underlying canonical Gα_q signaling via production of inositol phosphates mediated by each receptor was also regulated by antagonists for the other receptor. However, binding to their respective agonists, regulation of receptor-mediated MAPK activation and vascular smooth muscle cell growth were differentially or asymmetrically regulated depending on how each of the two receptors were occupied by either agonist or antagonist. Physical interactions between these receptors have never been reported, and here we show that AT1R and FP form heterodimeric complexes in both HEK 293 and vascular smooth muscle cells. These findings imply that formation of the AT1R/FP dimer creates a novel allosteric signaling unit that shows symmetrical and asymmetrical signaling behavior, depending on the outcome measured. AT1R/FP dimers may thus be important in the regulation of blood pressure.     

Control of Adipogenesis by the Autocrine Interplays between Angiotensin 1–7/Mas Receptor and Angiotensin II/AT1 Receptor Signaling Pathways

Angiotensin II (AngII), a peptide hormone released by adipocytes, can be catabolized by adipose angiotensin-converting enzyme 2 (ACE2) to form Ang(1–7). Co-expression of AngII receptors (AT₁ and AT₂) and Ang(1–7) receptors (Mas) in adipocytes implies the autocrine regulation of the local angiotensin system upon adipocyte functions, through yet unknown interactive mechanisms. In the present study, we reveal the adipogenic effects of Ang(1–7) through activation of Mas receptor and its subtle interplays with the antiadipogenic AngII-AT1 signaling pathways. Specifically, in human and 3T3-L1 preadipocytes, Ang(1–7)-Mas signaling promotes adipogenesis via activation of PI3K/Akt and inhibition of MAPK kinase/ERK pathways, and Ang(1–7)-Mas antagonizes the antiadipogenic effect of AngII-AT₁ by inhibiting the AngII-AT₁-triggered MAPK kinase/ERK pathway. The autocrine regulation of the AngII/AT₁-ACE2-Ang(1–7)/Mas axis upon adipogenesis has also been revealed. This study suggests the importance of the local regulation of the delicately balanced angiotensin system upon adipogenesis and its potential as a novel therapeutic target for obesity and related metabolic disorders.     

Tumor Necrosis Factor Receptor-associated Factor 6 (TRAF6) and TGFβ-activated Kinase 1 (TAK1) Play Essential Roles in the C-type Lectin Receptor Signaling in Response to Candida albicans Infection

Tumor necrosis factor receptor-associated factor 6 (TRAF6) and TGFβ-activated kinase 1 (TAK1) are considered as key intermediates in Toll-like receptor (TLR) signaling. However, the role of TRAF6 and TAK1 in C-type lectin receptors (CLRs) in response to fungal infection has not been studied. In this study, we have utilized macrophages derived from TRAF6 knock-out mice and myeloid-specific TAK1-deficient mice and determined the role of TRAF6 and TAK1 in CLR-induced signal transduction events. We demonstrate that TRAF6 and TAK1 are required for NF-κB and JNK activation, and expression of proinflammatory cytokines in response to Candida albicans infection. Our results highlight TRAF6 and TAK1 as key components in the signaling cascade downstream of C-type lectin receptors and as critical mediators of the anti-fungal immune response. Therefore, our studies provide a mechanistic understanding of the host immune response to C. albicans, which has a significant impact for the development of anti-fungal therapeutics and in understanding risk-factors and determining susceptibility to C. albicans infection.     

Independent ��-Arrestin2 and Gq/Protein Kinase C�� Pathways for ERK Stimulated by Angiotensin Type 1A Receptors in Vascular Smooth Muscle Cells Converge on Transactivation of the Epidermal Growth Factor Receptor

Recent studies in receptor-transfected cell lines have demonstrated that extracellular signal-regulated kinase (ERK) activation by angiotensin type 1A receptor and other G protein-coupled receptors can be mediated by both G protein-dependent and β-arrestin-dependent mechanisms. However, few studies have explored these mechanisms in primary cultured cells expressing endogenous levels of receptors. Accordingly, here we utilized the β-arrestin biased agonist for the angiotensin type 1A receptor, SII-angiotensin (SII), and RNA interference techniques to investigate angiotensin II (ANG)-activated β-arrestin-mediated mitogenic signaling pathways in rat vascular smooth muscle cells. Both ANG and SII induced DNA synthesis via the ERK activation cascade. Even though SII cannot induce calcium influx (G protein activation) after receptor stimulation, it does cause ERK activation, although less robustly than ANG. Activation by both ligands is diminished by depletion of β-arrestin2 by small interfering RNA, although the effect is more complete with SII. ERK activation at early time points but not later time points is strongly inhibited by those protein kinase C inhibitors that can block protein kinase Cζ. Moreover, ANG- and SII-mediated ERK activation require transactivation of the epidermal growth factor receptor via metalloprotease 2/9 and Src kinase. β-Arrestin2 facilitates ANG and SII stimulation of Src-mediated phosphorylation of Tyr-845 on the EGFR, a known site for Src phosphorylation. These studies delineate a convergent mechanism by which G protein-dependent and β-arrestin-dependent pathways can independently mediate ERK-dependent transactivation of the EGFR in vascular smooth muscle cells thus controlling cellular proliferative responses.G protein-coupled receptors, also known as seven transmembrane (7TM)² receptors, control virtually all known physiological processes in mammals (1). The various functions of these receptors are mediated and modulated by three families of proteins, which share the property that they interact virtually universally with the receptors in a strictly stimulus-dependent way (1). These three families of proteins are the heterotrimeric G proteins, the G protein-coupled receptor kinases (GRKs), and the β-arrestins. Activation of the receptors stimulates classical G protein-dependent signaling, often involving regulation of levels of second messengers such as cAMP and diacyglycerol. However, as has been known for many years, interaction of activated receptors with GRKs leading to their phosphorylation, and subsequent interaction with β-arrestins leads to desensitization of G protein signaling.In recent years, however, it has become increasingly clear that the β-arrestin-GRK system is in fact bifunctional (2). Thus, even as it desensitizes G protein signaling by the receptors, it also serves as a signal transduction system in its own right, activating a growing list of signaling pathways. These positive signaling functions are often mediated by the ability of β-arrestin to serve as an adaptor or scaffold molecule, bringing elements of diverse signaling pathways into proximity with one another and the receptors and thereby facilitating their activation. This new paradigm for understanding the previously unrecognized signaling properties of the β-arrestin-GRK system has been explored in a wide variety of transfected cultured cell systems.However, to date, relatively little investigation of these novel signaling pathways has been carried out in primary cell culture systems expressing endogenous levels of 7TM receptors. In seeking such a system in which to characterize and compare β-arrestin and G protein-mediated signaling pathways from a typical 7TM receptor, our attention was drawn to cultured rat vascular smooth muscle cells (VSMCs). Several features of rat VSMCs suggest this to be a relevant system for these purposes. Rat VSMCs express a variety of physiologically important 7TM receptors including the angiotensin II type 1A receptor (AT1R) (3). This receptor has been the focus of extensive study in transfected cell systems with respect to its β-arrestin-mediated signaling to a variety of pathways, most particularly extracellular signal-regulated kinase (ERK). Moreover, the AT1R mediates the physiologically important effects of angiotensin II (ANG) on vascular tone as well as on proliferation and chemotaxis (4, 5). Pathophysiologically, ANG stimulation of this receptor has been implicated in VSMC proliferation and chemotaxis, which are thought to play an important role in such important disease processes as atherosclerosis and restenosis after angioplasty (6, 7). Moreover, a ligand has been characterized [Sar¹,Ile⁴,Ile⁸](SII)-angiotensin (SII), a triply mutated angiotensin octapeptide that, in transfected cell systems, acts as a specific agonist for β-arrestin-mediated signaling, although not activating G protein-mediated signaling (8).Accordingly, in the studies described here, we set out to investigate the characteristics of activation of ERK in rat VSMCs that might be mediated through G protein as well as β-arrestin signaling. The results not only demonstrate the importance of β-arrestin-mediated signaling in ERK-mediated proliferative responses of these cells, but also shed new light on the molecular mechanisms and interrelationships between the β-arrestin and classical G protein-mediated activation of these pathways.     

NEU3 Sialidase Is Activated under Hypoxia and Protects Skeletal Muscle Cells from Apoptosis through the Activation of the Epidermal Growth Factor Receptor Signaling Pathway and the Hypoxia-inducible Factor (HIF)-1α

NEU3 sialidase, a key enzyme in ganglioside metabolism, is activated under hypoxic conditions in cultured skeletal muscle cells (C2C12). NEU3 up-regulation stimulates the EGF receptor signaling pathway, which in turn activates the hypoxia-inducible factor (HIF-1α), resulting in a final increase of cell survival and proliferation. In the same cells, stable overexpression of sialidase NEU3 significantly enhances cell resistance to hypoxia, whereas stable silencing of the enzyme renders cells more susceptible to apoptosis. These data support the working hypothesis of a physiological role played by NEU3 sialidase in protecting cells from hypoxic stress and may suggest new directions in the development of therapeutic strategies against ischemic diseases, particularly of the cerebro-cardiovascular system.     

Tetraspanin CD63 Promotes Vascular Endothelial Growth Factor Receptor 2-β1 Integrin Complex Formation,Thereby Regulating Activation and Downstream Signaling in Endothelial Cells in Vitro and in Vivo

CD63 is a member of the transmembrane-4 glycoprotein superfamily (tetraspanins) implicated in the regulation of membrane protein trafficking, leukocyte recruitment, and adhesion processes. We have investigated the involvement of CD63 in endothelial cell (EC) signaling downstream of β1 integrin and VEGF. We report that silencing of CD63 in primary ECs arrested capillary sprouting and tube formation in vitro because of impaired adhesion and migration of ECs. Mechanistically, CD63 associated with both β1 integrin and the main VEGF receptor on ECs, VEGFR2. Our data suggest that CD63 serves to bridge between β1 integrin and VEGFR2 because CD63 silencing disrupted VEGFR2-β1 integrin complex formation identified using proximity ligation assays. Signaling downstream of β1 integrin and VEGFR2 was attenuated in CD63-silenced cells, although their cell surface expression levels remained unaffected. CD63 was furthermore required for efficient internalization of VEGFR2 in response to VEGF. Importantly, systemic delivery of VEGF failed to potently induce VEGFR2 phosphorylation and downstream signaling in CD63-deficient mouse lungs. Taken together, our findings demonstrate a previously unrecognized role for CD63 in coordinated integrin and receptor tyrosine kinase signaling in vitro and in vivo.     

IL-17 Stimulates Migration of Carotid Artery Vascular Smooth Muscle Cells in an MMP-9 Dependent Manner via p38 MAPK and ERK1/2-Dependent NF-κB and AP-1 Activation

Inappropriate vascular remodeling is thought to be the main cause of restenosis following angioplasty. Migration of vascular smooth muscle cells (VSMC) into lumina, which is promoted by degradation of the extracellular matrix by matrix metalloproteinases (MMPs) plays a causal role in pathological vascular remodeling. The aim of the present research is to explore the effects of a novel cytokine, IL-17, on migration of VSMC and MMP-9 secretion. Carotid artery VSMC was isolated from Sprague–Dawley rats. Expression of MMP-9 and cell migration induced by IL-17 and its related signal pathway were detected. The results showed that IL-17-induced migration of VSMC in an MMP-9-dependent manner. IL-17-induced MMP-9 expression was via p38 MAPK and ERK1/2 dependent NF-κB and AP-1 activation. The present results demonstrated that IL-17 may play a role in vascular remodeling and targeting IL-17 or its specific downstream mediators is a potentially novel therapeutic pathway for attenuating the post-angioplastic restenosis.     

Simvastatin Attenuates Oxidative Stress,NF-κB Activation,and Artery Calcification in LDLR-/- Mice Fed with High Fat Diet via Down-regulation of Tumor Necrosis Factor-α and TNF Receptor 1

Simvastatin (SIM) is anti-inflammatory. We used low density lipoprotein receptor knockout (LDLR-/-) mice and human aortic smooth muscle cells (HASMCs) as model systems to study the effect of SIM on arterial calcification and to explore the potential mechanisms contributing to this protective effect. High-fat diet (HFD) caused the LRLR -/- to develop dyslipidemia, diabetics, atherosclerosis and aortic smooth muscle calcification. SIM, N-acetyl cysteine (NAC, a ROS scavenger) and apocynin (APO, a NADPH oxidase inhibitor) did not significantly retard the development of dyslipidemia or diabetic. However, those treatments were still effective in attenuating the HFD-induced atherosclerosis and aortic smooth muscle calcification. These findings suggest that the protective effect of SIM against aortic calcification is not contributed by the cholesterol lowering effect. SIM, NAC and APO were found to attenuate the HFD induced elevation of serum TNF-α, soluble TNFR1 (sTNFR1), 3-nitro-tyrosine. We hypothesized that the pro-inflammatory cytokine, oxidative stress and TNFR1 played a role in inducing aortic calcification. We used HASMC to investigate the role of TNF-α, oxidative stress and TNFR1 in inducing aortic calcification and to elucidate the mechanism contributes the protective effect of SIM against aortic calcification. We demonstrated that treating HASMC with TNF-α induced cell Ca deposit and result in an increase in ALP, NADPH oxidase activity, NF-kB subunit p65, BMP2, MSX2, and RUNX2 expression. SIM suppressed the TNF-α induced activation of NADPH oxidase subunit p47, the above-mentioned bone markers and TNFR1 expression. Furthermore, p65, p47 and TNFR1 siRNAs inhibited the TNF-α-mediated stimulation of BMP-2, MSX2, RUNX2 expression. SIM, APO, and NAC either partially inhibit or completely block the TNF-α induced H₂O₂ or superoxide production. These results suggest that SIM may, independent of its cholesterol-lowering effect, suppresses the progression of vascular diseases through the inhibition of the inflammation mediators TNF-α and TNFR1.