Klotho gene delivery suppresses Nox2 expression and attenuates oxidative stress in rat aortic smooth muscle cells via the cAMP-PKA pathway
Yuhong Wang
Department of Physiology, College of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA
Search for more papers by this authorMakoto Kuro-o
Department of Pathology, University of Texas Southwestern Medical School, Dallas, TX 75390, USA
Search for more papers by this authorZhongjie Sun
Department of Physiology, College of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA
Search for more papers by this authorYuhong Wang
Department of Physiology, College of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA
Search for more papers by this authorMakoto Kuro-o
Department of Pathology, University of Texas Southwestern Medical School, Dallas, TX 75390, USA
Search for more papers by this authorZhongjie Sun
Department of Physiology, College of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA
Search for more papers by this authorSummary
Klotho is a recently discovered anti-aging gene. The purpose of this study was to investigate whether klotho gene transfer attenuates superoxide production and oxidative stress in rat aorta smooth muscle (RASM) cells. RASM cells were transfected with AAV plasmids carrying mouse klotho full-length cDNA (mKL) or LacZ as a control. Klotho gene transfer increased klotho expression in RASM cells. Notably, klotho gene expression decreased Nox2 NADPH oxidase protein expression but did not affect Nox2 mRNA expression, suggesting that the inhibition may occur at the posttranscriptional level. Klotho gene transfer decreased intracellular superoxide production and oxidative stress in RASM cells. Klotho gene expression also significantly attenuated the angiotensin II (AngII)-induced superoxide production, oxidative damage, and apoptosis. Interestingly, klotho gene delivery dose dependently increased the intracellular cAMP level and PKA activity in RASM cells. Rp-cAMP, a competitive inhibitor of cAMP, abolished the klotho-induced increase in PKA activity, indicating that klotho activated PKA via cAMP. Notably, inhibition of cAMP-dependent PKA activity by RP-cAMP abolished klotho-induced inhibition of Nox2 protein expression, suggesting an important role of cAMP-dependent PKA in this process. This finding revealed a previously unidentified role of klotho in regulating Nox2 protein expression in RASM cells. Klotho not only downregulated Nox2 protein expression and intracellular superoxide production but also attenuated AngII-induced superoxide production, oxidative damage, and apoptosis. The klotho-induced suppression of Nox2 protein expression may be mediated by the cAMP–PKA pathway.
Supporting Information
Fig. S1 Mouse klotho (mKL) gene delivery led to expression of mKL in rat aortic smooth muscle (RASM) cells.
Fig. S2 Mouse klotho gene delivery suppressed Nox2 expression while Nox4 protein expression was not detectable.
Fig. S3 Suppression of PKA activity by Rp-cAMP did not affect klotho and rat Nox2 mRNA expression.
Data S1 Xxxx xxx.
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