Yumei Zhan

Ets-1 is a critical regulator of Ang II-mediated vascular inflammation and remodeling

Ang II is a central mediator of vascular inflammation and remodeling. The transcription factor Ets-1 is rapidly induced in vascular smooth muscle and endothelial cells of the mouse thoracic aorta in response to systemic Ang II infusion. Arterial wall thickening, perivascular fibrosis, and cardiac hypertrophy are significantly diminished in Ets1-/- mice compared with control mice in response to Ang II. The induction of 2 known targets of Ets-1, cyclin-dependent kinase inhibitor p21CIP and plasminogen activator inhibitor-1 (PAI-1), by Ang II is markedly blunted in the aorta of Ets1-/- mice compared with wild-type controls. Expression of p21CIP in VSMCs leads to cellular hypertrophy, whereas expression of p21CIP in endothelial cells is associated with cell cycle arrest, apoptosis, and endothelial dysfunction. PAI-1 promotes the development of perivascular fibrosis. We have identified monocyte chemoattractant protein-1 (MCP-1) as a novel target for Ets-1. Expression of MCP-1 is similarly reduced in Ets1-/- mice compared with control mice in response to Ang II, which results in significantly diminished recruitment of T cells and macrophages to the vessel wall. In summary, our results support a critical role for Ets-1 as a transcriptional mediator of vascular inflammation and remodeling in response to Ang II.

Ets-1 Is a Critical Transcriptional Regulator of Reactive Oxygen Species and p47phox Gene Expression in Response to Angiotensin II

Angiotensin (Ang) II is a potent mediator of vascular inflammation. A central mechanism by which Ang II promotes inflammation is through the generation of reactive oxygen species (ROS). In the current study, we investigated the role of the transcription factor Ets-1 in regulating Ang II–induced ROS generation. ROS generation was measured in the thoracic aorta of Ets-1−/− mice compared with littermate controls after continuous infusion of Ang II. H2O2 and superoxide anion (O2−) production were significantly blunted in the Ets-1−/− mice. Inhibition of Ets-1 expression by small interfering RNA in primary human aortic smooth muscle cells also potently inhibited ROS production and the induction of the NAD(P)H oxidase subunit p47phox in response to Ang II. To evaluate the therapeutic potential of inhibiting Ets-1 in wild-type mice, dominant negative Ets-1 membrane-permeable peptides were administered systemically. Ang II–induced ROS production and medial hypertrophy in the thoracic aorta were markedly diminished as a result of blocking Ets-1. In summary, Ets-1 functions as a critical downstream transcriptional mediator of Ang II ROS generation by regulating the expression of NAD(P)H oxidase subunits such as p47phox.

TGF-β Induces Acetylation of Chromatin and of Ets-1 to Alleviate Repression of miR-192 in Diabetic Nephropathy

Acetylation of chromatin and Ets-1 contributes to induction of miR-192, a microRNA involved in kidney pathogenesis. Kidney Fibrosis Through Acetylation Epigenetic changes alter gene expression and can induce pathogenesis. MicroRNA-192 (miR-192) mediates the transcription of genes involved in kidney fibrosis in response to high glucose–induced signaling by TGF-β (transforming growth factor–β). Kato et al. found that in basal conditions, Ets-1 was bound to chromatin upstream of miR-192 and suppressed its expression in murine kidney mesangial cells. After treatment with high glucose or TGF-β, Ets-1 was acetylated in an Akt- and p300-dependent manner and dissociated from miR-192. Although Ets-1 was dispensable for transiently increased expression of miR-192, Ets-1 deficiency in mice or murine mesangial cells prevented the sustained expression of miR-192 in response to TGF-β. Activation of p300 and acetylation of Ets-1 and of histone H3 at lysines 9 and 14 were increased in diabetic db/db mice compared with wild-type mice, suggesting that alleviation of Ets-1 repression may contribute to diabetic nephropathy. MicroRNAs (miRNAs), such as miR-192, mediate the actions of transforming growth factor–β1 (TGF-β) related to the pathogenesis of diabetic kidney diseases. We found that the biphasic induction of miR-192 expression by TGF-β in mouse renal glomerular mesangial cells initially involved the Smad transcription factors, followed by sustained expression that was promoted by acetylation of the transcription factor Ets-1 and of histone H3 by the acetyltransferase p300, which was activated by the serine and threonine kinase Akt. In mesangial cells from Ets-1–deficient mice or in cells in which Ets-1 was knocked down, basal amounts of miR-192 were higher than those in control cells, but sustained induction of miR-192 by TGF-β was attenuated. Furthermore, inhibition of Akt or ectopic expression of dominant-negative histone acetyltransferases decreased p300-mediated acetylation and Ets-1 dissociation from the miR-192 promoter and prevented miR-192 expression in response to TGF-β. Activation of Akt and p300 and acetylation of Ets-1 and histone H3 were increased in glomeruli from diabetic db/db mice compared to nondiabetic db/+ mice, suggesting that this pathway may contribute to diabetic nephropathy. These findings provide insight into the regulation of miRNAs through signaling-mediated changes in transcription factor activity and in epigenetic histone acetylation under normal and disease states.

E74-like Factor 3 (ELF3) Impacts on Matrix Metalloproteinase 13 (MMP13) Transcriptional Control in Articular Chondrocytes under Proinflammatory Stress

Matrix metalloproteinase (MMP)-13 has a pivotal, rate-limiting function in cartilage remodeling and degradation due to its specificity for cleaving type II collagen. The proximal MMP13 promoter contains evolutionarily conserved E26 transformation-specific sequence binding sites that are closely flanked by AP-1 and Runx2 binding motifs, and interplay among these and other factors has been implicated in regulation by stress and inflammatory signals. Here we report that ELF3 directly controls MMP13 promoter activity by targeting an E26 transformation-specific sequence binding site at position −78 bp and by cooperating with AP-1. In addition, ELF3 binding to the proximal MMP13 promoter is enhanced by IL-1β stimulation in chondrocytes, and the IL-1β-induced MMP13 expression is inhibited in primary human chondrocytes by siRNA-ELF3 knockdown and in chondrocytes from Elf3−/− mice. Further, we found that MEK/ERK signaling enhances ELF3-driven MMP13 transactivation and is required for IL-1β-induced ELF3 binding to the MMP13 promoter, as assessed by chromatin immunoprecipitation. Finally, we show that enhanced levels of ELF3 co-localize with MMP13 protein and activity in human osteoarthritic cartilage. These studies define a novel role for ELF3 as a procatabolic factor that may contribute to cartilage remodeling and degradation by regulating MMP13 gene transcription.

ETS-related Gene (ERG) Controls Endothelial Cell Permeability via Transcriptional Regulation of the Claudin 5 (CLDN5) Gene

Background: Disruption of endothelial cell (EC) junction-associated proteins is a major contributing factor to inflammation-induced barrier dysfunction. Results: Knockdown of ERG in EC led to markedly increased EC permeability and reduced expression of the tight junction protein CLDN5. Conclusion: ERG regulates EC barrier function via transcriptional regulation of CLDN5. Significance: ERG is a transcriptional regulator of EC barrier function. ETS-related gene (ERG) is a member of the ETS transcription factor family. Our previous studies have shown that ERG expression is highly enriched in endothelial cells (EC) both in vitro and in vivo. ERG expression is markedly repressed in response to inflammatory stimuli. It has been shown that ERG is a positive regulator of several EC-restricted genes including VE-cadherin, endoglin, and von Willebrand factor, and a negative regulator of other genes such as interleukin (IL)-8 and intercellular adhesion molecule (ICAM)-1. In this study we have identified a novel role for ERG in the regulation of EC barrier function. ERG knockdown results in marked increases in EC permeability. This is associated with a significant increase of stress fiber and gap formation in EC. Furthermore, we identify CLDN5 as a downstream target of ERG in EC. Thus, our results suggest that ERG plays a pivotal role in regulating EC barrier function and that this effect is mediated in part through its regulation of CLDN5 gene expression.

ESE-1 is a potent repressor of type II collagen gene (COL2A1) transcription in human chondrocytes.

The epithelium‐specific ETS (ESE)‐1 transcription factor is induced in chondrocytes by interleukin‐1β (IL‐1β). We reported previously that early activation of EGR‐1 by IL‐1β results in suppression of the proximal COL2A1 promoter activity by displacement of Sp1 from GC boxes. Here we report that ESE‐1 is a potent transcriptional suppressor of COL2A1 promoter activity in chondrocytes and accounts for the sustained, NF‐κB‐dependent inhibition by IL‐1β. Of the ETS factors tested, this response was specific to ESE‐1, since ESE‐3, which was also induced by IL‐1β, suppressed COL2A1 promoter activity only weakly. In contrast, overexpression of ETS‐1 increased COL2A1 promoter activity and blocked the inhibition by IL‐1β. These responses to ESE‐1 and ETS‐1 were confirmed using siRNA‐ESE1 and siRNA‐ETS1. In transient cotransfections, the inhibitory responses to ESE‐1 and IL‐1β colocalized in the −577/−132 bp promoter region, ESE‐1 bound specifically to tandem ETS sites at −403/−381 bp, and IL‐1‐induced binding of ESE‐1 to the COL2A1 promoter was confirmed in vivo by ChIP. Our results indicate that ESE‐1 serves a potent repressor function by interacting with at least two sites in the COL2A1 promoter. However, the endogenous response may depend upon the balance of other ETS factors such as ETS‐1, and other IL‐1‐induced factors, including EGR‐1 at any given time. Intracellular ESE‐1 staining in chondrocytes in cartilage from patients with osteoarthritis (OA), but not in normal cartilage, further suggests a fundamental role for ESE‐1 in cartilage degeneration and suppression of repair. J. Cell. Physiol. 215: 562–573, 2008.

Antiinflammatory Effects of the ETS Factor ERG in Endothelial Cells Are Mediated Through Transcriptional Repression of the Interleukin-8 Gene

ERG (Ets-related gene) is an ETS transcription factor that has recently been shown to regulate a number of endothelial cell (EC)-restricted genes including VE-cadherin, von Willebrand factor, endoglin, and intercellular adhesion molecule-2. Our preliminary data demonstrate that unlike other ETS factors, ERG exhibits a highly EC-restricted pattern of expression in cultured primary cells and several adult mouse tissues including the heart, lung, and brain. In response to inflammatory stimuli, such as tumor necrosis factor-&agr;, we observed a marked reduction of ERG expression in ECs. To further define the role of ERG in the regulation of normal EC function, we used RNA interference to knock down ERG. Microarray analysis of RNA derived from ERG small interfering RNA– or tumor necrosis factor-&agr;–treated human umbilical vein (HUV)ECs revealed significant overlap (P<0.01) in the genes that are up- or downregulated. Of particular interest to us was a significant change in expression of interleukin (IL)-8 at both protein and RNA levels. Exposure of ECs to tumor necrosis factor-&agr; is known to be associated with increased neutrophil attachment. We observed that knockdown of ERG in HUVECs is similarly associated with increased neutrophil attachment compared to control small interfering RNA–treated cells. This enhanced adhesion could be blocked with IL-8 neutralizing or IL-8 receptor blocking antibodies. ERG can inhibit the activity of the IL-8 promoter in a dose dependent manner. Direct binding of ERG to the IL-8 promoter in ECs was confirmed by chromatin immunoprecipitation. In summary, our findings support a role for ERG in promoting antiinflammatory effects in ECs through repression of inflammatory genes such as IL-8.

The Counter-Regulatory Effects of ESE-1 During Angiotensin II-Mediated Vascular Inflammation and Remodeling

BACKGROUND Angiotensin II (Ang II) is a critical mediator vascular inflammation and remodeling in a number of diseases including hypertension and atherosclerosis. The purpose of this study was to evaluate the role of the epithelium-specific ETS transcription factor-1 (ESE-1), a member of E26 transformation-specific sequence (ETS) transcription factors, as a mediator of Ang II-mediated vascular responses. METHODS ESE-1 knockout mice were used to evaluate the role of ESE-1 in regulating Ang II-mediated vascular inflammation and remodeling. RESULTS ESE-1 levels are low to undetectable under basal conditions but rapidly increase in response to Ang II. Intimal medial thickness and perivascular fibrosis of the aorta were significantly greater in ESE-1 knockout mice compared with the wild-type littermate controls. Proliferating cell nuclear antigen (PCNA) staining was also greater in the aorta of the Ang II-infused ESE-1 knockout mice compared with the controls. The infiltration of T cells and macrophage into the vessel wall of the aorta was dramatically enhanced in the ESE-1 knockout mice compared with the controls. Finally, Ang II-induced expression of a known downstream target of ESE-1, nitric oxide synthase 2 (NOS2), was significantly blunted in ESE-1 knockout mice compared to littermate controls. The alterations in vascular inflammation and remodeling were associated with an exaggerated systolic blood pressure response to Ang II in ESE-1 knockout mice. CONCLUSIONS ESE-1 is an Ang II-inducible transcription factor that plays an important counter-regulatory role in the setting of vascular inflammation and remodeling.

Alterations in transcriptional responses associated with vascular aging.

Vascular aging is an independent risk factor for cardiovascular disease that can occur in the absence of other traditional risk factors. Inflammation is a hallmark of vascular aging that ultimately leads to structural changes in the vessel wall including an increase in medial thickness and perivascular fibrosis. Several classes of transcription factors have been identified that participate in the regulation of cellular responses associated with vascular aging. Nuclear factor (NF)-κB is the prototypic example of a transcriptional activator in the setting of inflammation, being activated in response to multiple inflammatory mediators including pro-inflammatory cytokines and bacterial endotoxin. In contrast, the activation of the nuclear hormone receptor and transcription factor peroxisome proliferator-activated receptor-alpha (PPAR-α) results in its translocation from the cell surface to the nucleus where it exerts anti-inflammatory effects. Vascular aging is also associated with endothelial dysfunction. One important repair mechanism for improving endothelial function is the recruitment of endothelial progenitor cells (EPCs). In the setting of aging the number of EPCs diminishes which has been linked to a decrease in the activity and/or expression of the transcription factor hypoxia inducible factor (HIF)-1 alpha. A change in the balance of the activity of pro-inflammatory transcription factors versus those that inhibit inflammation likely contributes to the process of vascular aging. The purpose of this review is to summarize our current knowledge of these age-related changes in transcriptional responses, and to discuss the therapeutic potential of targeting some of these factors.