Extracellular Signal-Regulated Kinase 5 SUMOylation Antagonizes Shear Stress–Induced Antiinflammatory Response and Endothelial Nitric Oxide Synthase Expression in Endothelial Cells

Abstract

Abstract-Shear stress-induced extracellular signal-regulated kinase (ERK)5 activation and the consequent regulation of Kruppel-like factor 2 and endothelial nitric oxide synthase expression represents one of the antiinflammatory and vascular tone regulatory mechanisms maintaining normal endothelial function. Endothelial dysfunction is a major initiator of atherosclerosis, a vascular pathology often associated with diabetes. Small ubiquitin-like modifier (SUMO) covalently attaches to certain residues of specific target transcription factors and could inhibit its activity. We investigated whether H 2 O 2 and AGE (advanced glycation end products), 2 well-known mediators of diabetes, negatively regulated ERK5 transcriptional activity and laminar flow-induced endothelial nitric oxide synthase expression through ERK5 SUMOylation. H 2 O 2 and AGE induced endogenous ERK5 SUMOylation. In addition, ERK5 SUMOylation was increased in the aortas from diabetic mice. ERK5 transcriptional activity, but not kinase activity, was inhibited by expression of Ubc9 (SUMO E2 conjugase) or PIAS1 (E3 ligase), suggesting the involvement of ERK5 SUMOylation on its transcriptional activity. Point-mutation analyses showed that ERK5 is covalently modified by SUMO at 2 conserved sites, Lys6 and Lys22, and that the SUMOylation defective mutant of ERK5, dominant negative form of Ubc9 (DN-Ubc9), and small interfering RNA PIAS1 reversed H 2 O 2 and AGE-mediated reduction of shear stress-mediated ERK5/myocyte enhancer factor 2 transcriptional activity, as well as promoter activity of Kruppel-like factor 2. Finally, PIAS1 knockdown reversed the inhibitory effect of H 2 O 2 in shear stress-induced Kruppel-like factor 2 and endothelial nitric oxide synthase expression. These data clearly defined SUMOylation-dependent ERK5 transcriptional repression independent of kinase activity and suggested this process as among the molecular mechanisms of diabetes-mediated endothelial dysfunction. (Circ Res. 2008;102:538-545.)Key Words: ERK5 Ⅲ SUMOylation Ⅲ KLF2 Ⅲ diabetes Ⅲ shear stress L aminar flow-induced extracellular signal-regulated kinase (ERK)5 activation has a critical role in regulating peroxisome proliferator-activated receptor (PPAR)␥ and Kruppel-like factor (KLF)2 (a recently identified transcriptional inhibitor of endothelial cell [EC] inflammation) and in inhibiting tumor necrosis factor-␣-mediated adhesion molecule expression, 1 demonstrating an antiinflammatory role of ERK5 activation in ECs. The upstream kinase that phosphorylates ERK5 has been identified as MEK5. 2,3 Activation of ERK5 is documented to have an antiapoptotic effect in neuronal and ECs. 4,5 Recently we, along with Kasler et al, reported that ERK5 is not only a kinase enzyme but also possesses transcriptional activity. 1,6 The NH 2 -terminal ERK5 kinase domain acts as a negative regulator of these transactivation domains and, when activated, releases the NH 2 -terminal ERK5 inhibitory effect, resulting in increased COOH-terminal ERK5 transcriptional activity. Both the association o...

Akt SUMOylation Regulates Cell Proliferation and Tumorigenesis

“…Accumulating evidence indicates that SUMOylation can target a wide variety of proteins including nuclear transcription factors, membrane, and cytosolic proteins (4,7). Compared with a large amount of nuclear transcription factor, only a few protein kinases, such as Aurora-B kinase, MEK, FAK, and ERK5, have been identified as SUMO targets (8)(9)(10)(11). Moreover, regulation of various protein kinases by SUMOylation and the related biologic functions remains unclear.…”

Disturbed flow-activated p90RSK kinase accelerates atherosclerosis by inhibiting SENP2 function

“…However, this inflammatory cell infiltration into the lesion was significantly impaired in In addition, we have already reported that s-flow increased and d-flow decreased ERK5 transactivation via regulating ERK5-SUMOylation (8,11). These data suggest crucial roles played by p53 and ERK5 SUMOylation in differentiating EC responses to s-flow and d-flow.…”

“…Indeed, d-flow, not steady laminar flow (s-flow), activates proinflammatory and proapoptotic genes in ECs, causing them to become dysfunctional and proatherogenic (3)(4)(5). SUMOylation plays an important role in regulating actin filament remodeling (6), migration (6,7), inflammation (8), and apoptosis (3) that occur in ECs in response to flow stimulation (3,4,8). SUMOylation is one of the most dynamic posttranslational modifications, with its diverse repertoire of effects, such as the localization, transcriptional activity, and DNA binding of modified proteins (9)(10)(11).…”

Laminar Flow Activation of ERK5 Protein in Vascular Endothelium Leads to Atheroprotective Effect via NF-E2-related Factor 2 (Nrf2) Activation

“…Immunoprecipitation Analysis-Cell lysates were incubated with anti-ERK5 antibody (Cell Signaling Technology) or anti-rabbit IgG (Cell Signaling Technology) overnight at 4°C as described previously (14). Protein A/G-agarose beads were added and incubated for 1 h at 4°C, and unbound proteins were removed by centrifugation.…”

“…Parmar et al (13) reported that KLF2 transcription is induced in endothelium under atheroprotective laminar flow via MAPK/ERK kinase 5 (MEK5)-extracellular signal-regulated protein kinase 5 (ERK5)-MEF2 signaling pathway. Transcriptional activation of KLF2 triggers induction of anti-inflammatory genes including eNOS and reduction of proinflammatory genes including cytokines (13,14). In addition to KLF2, ERK5 activation induces the expression of KLF4-dependent genes, which are also important in flow-mediated EC-protective responses (15,16).…”