Kyung-Sun Heo, Keigi Fujiwara, and Jun-ichi Abe. Mol. Cells 2014;37:435-40. https://doi.org/10.14348/molcells.2014.0078

Lung injury, whether induced by infection or caustic chemicals, initiates a series of complex wound-healing responses. If uncontrolled, these responses may lead to fibrotic lung diseases and loss of function. Thus, resolution of lung injury must be tightly regulated. The key regulatory proteins required for tightly controlling the resolution of lung injury have yet to be identified. Here we show that loss of deubiquitinase CYLD led to the development of lung fibrosis in mice after infection with Streptococcus pneumoniae. CYLD inhibited transforming growth factor-β-signalling and prevented lung fibrosis by decreasing the stability of Smad3 in an E3 ligase carboxy terminus of Hsc70-interacting protein-dependent manner. Moreover, CYLD decreases Smad3 stability by deubiquitinating K63-polyubiquitinated Akt. Together, our results unveil a role for CYLD in tightly regulating the resolution of lung injury and preventing fibrosis by deubiquitinating Akt. These studies may help develop new therapeutic strategies for preventing lung fibrosis.

Background—: Diabetes mellitus is a major risk factor for cardiovascular mortality by increasing endothelial cell (EC) dysfunction and subsequently accelerating atherosclerosis. Extracellular-signal regulated kinase 5 (ERK5) is activated by steady laminar flow and regulates EC function by increasing endothelial nitric oxide synthase expression and inhibiting EC inflammation. However, the role and regulatory mechanisms of ERK5 in EC dysfunction and atherosclerosis are poorly understood. Here, we report the critical role of the p90 ribosomal S6 kinase (p90RSK)/ERK5 complex in EC dysfunction in diabetes mellitus and atherosclerosis. Methods and Results—: Inducible EC-specific ERK5 knockout (ERK5-EKO) mice showed increased leukocyte rolling and impaired vessel reactivity. To examine the role of endothelial ERK5 in atherosclerosis, we used inducible ERK5-EKO-LDLR ^−/− mice and observed increased plaque formation. When activated, p90RSK associated with ERK5, and this association inhibited ERK5 transcriptional activity and upregulated vascular cell adhesion molecule 1 expression. In addition, p90RSK directly phosphorylated ERK5 S496 and reduced endothelial nitric oxide synthase expression. p90RSK activity was increased in diabetic mouse vessels, and fluoromethyl ketone-methoxyethylamine, a specific p90RSK inhibitor, ameliorated EC-leukocyte recruitment and diminished vascular reactivity in diabetic mice. Interestingly, in ERK5-EKO mice, increased leukocyte rolling and impaired vessel reactivity were resistant to the beneficial effects of fluoromethyl ketone-methoxyethylamine, suggesting a critical role for endothelial ERK5 in mediating the salutary effects of fluoromethyl ketone-methoxyethylamine on endothelial dysfunction. Fluoromethyl ketone-methoxyethylamine also inhibited atherosclerosis formation in ApoE ^−/− mice. Conclusions—: Our study highlights the importance of the p90RSK/ERK5 module as a critical mediator of EC dysfunction in diabetes mellitus and atherosclerosis formation, thus revealing a potential new target for therapeutic intervention.

Rationale:: Disturbed flow induces proinflammatory and apoptotic responses in endothelial cells, causing them to become dysfunctional and subsequently proatherogenic. Objective:: Although a possible link between SUMOylation of p53 and ERK5 detected during endothelial apoptosis and inflammation has been suggested, the mechanistic insights, especially under the proatherogenic flow condition, remain largely unknown. Methods and Results:: SUMOylation of p53 and ERK5 was induced by disturbed flow but not by steady laminar flow. To examine the role of the disturbed flow–induced p53 and ERK5 SUMOylation, we used de-SUMOylation enzyme of sentrin/Small Ubiquitin-like MOdifier (SUMO)-specific protease 2 deficiency ( Senp2 ^+/− ) mice and observed a significant increase in endothelial apoptosis and adhesion molecule expression both in vitro and in vivo. These increases, however, were significantly inhibited in endothelial cells overexpressing p53 and ERK5 SUMOylation site mutants. Senp2 ^+/− mice exhibited increased leukocyte rolling along the endothelium, and accelerated formation of atherosclerotic lesions was observed in Senp2 ^+/− / Ldlr ^−/− , but not in Senp2 ^+/+ /Ldlr ^−/− , mice fed a high-cholesterol diet. Notably, the extent of lesion size in the aortic arch of Senp2 ^+/− / Ldlr ^−/− mice was much larger than that in the descending aorta, also suggesting a crucial role of the disturbed flow–induced SUMOylation of proteins, including p53 and ERK5 in atherosclerosis formation. Conclusions:: These data show the unique role of sentrin/SUMO-specific protease 2 on endothelial function under disturbed flow and suggest that SUMOylation of p53 and ERK5 by disturbed flow contributes to the atherosclerotic plaque formation. Molecules involved in this newly discovered signaling will be useful targets for controlling endothelial cells dysfunction and consequently atherosclerosis formation.

Atherosclerosis is readily observed in regions of blood vessels where disturbed blood flow (d-flow) is known to occur. A positive correlation between protein kinase C ζ (PKCζ) activation and d-flow has been reported, but the exact role of d-flow–mediated PKCζ activation in atherosclerosis remains unclear. We tested the hypothesis that PKCζ activation by d-flow induces endothelial cell (EC) apoptosis by regulating p53. We found that d-flow–mediated peroxynitrite (ONOO⁻) increased PKCζ activation, which subsequently induced p53 SUMOylation, p53–Bcl-2 binding, and EC apoptosis. Both d-flow and ONOO⁻ increased the association of PKCζ with protein inhibitor of activated STATy (PIASy) via the Siz/PIAS-RING domain (amino acids 301–410) of PIASy, and overexpression of this domain of PIASy disrupted the PKCζ–PIASy interaction and PKCζ-mediated p53 SUMOylation. En face confocal microscopy revealed increases in nonnuclear p53 expression, nitrotyrosine staining, and apoptosis in aortic EC located in d-flow areas in wild-type mice, but these effects were significantly decreased in p53^−/− mice. We propose a novel mechanism for p53 SUMOylation mediated by the PKCζ–PIASy interaction during d-flow–mediated EC apoptosis, which has potential relevance to early events of atherosclerosis.