RhoA/Rho kinase mediates TGF-β₁-induced kidney myofibroblast activation through Poldip2/Nox4-derived reactive oxygen species

Abstract

The small G proteins Rac1 and RhoA regulate actin cytoskeleton, cell shape, adhesion, migration, and proliferation. Recent studies in our laboratory have shown that NADPH oxidase Nox4-derived ROS are involved in transforming growth factor (TGF)-β1-induced rat kidney myofibroblast differentiation assessed by the acquisition of an α-smooth muscle actin (α-SMA) phenotype and expression of an alternatively spliced fibronectin variant (Fn-EIIIA). Rac1 and RhoA are essential in signaling by some Nox homologs, but their role as effectors of Nox4 in kidney myofibroblast differentiation is not known. In the present study, we explored a link among Rac1 and RhoA and Nox4-dependent ROS generation in TGF-β1-induced kidney myofibroblast activation. TGF-β1 stimulated an increase in Nox4 protein expression, NADPH oxidase activity, and abundant α-SMA and Fn-EIIIA expression. RhoA but not Rac1 was involved in TGF-β1 induction of Nox4 signaling of kidney myofibroblast activation. TGF-β1 stimulated active RhoA-GTP and increased Rho kinase (ROCK). Inhibition of RhoA with small interfering RNA and ROCK using Y-27632 significantly reduced TGF-β1-induced stimulation of Nox4 protein, NADPH oxidase activity, and α-SMA and Fn-EIIIA expression. Treatment with diphenyleneiodonium, an inhibitor of NADPH oxidase, did not decrease RhoA activation but inhibited TGF-β1-induced α-SMA and Fn-EIIIA expression, indicating that RhoA is upstream of ROS generation. RhoA/ROCK also regulated polymerase (DNA-directed) δ-interacting protein 2 (Poldip2), a newly discovered Nox4 enhancer protein. Collectively, these data indicate that RhoA/ROCK is upstream of Poldip2-dependent Nox4 regulation and ROS production and induces redox signaling of kidney myofibroblast activation and may broader implications in the pathophysiology of renal fibrosis.

Myocardin-related Transcription Factor Regulates Nox4 Protein Expression

“…Such a hypothesis is supported by the recent finding that RhoA acts upstream of Nox4-mediated ROS generation (31). Moreover, we noted that the Nox4 promoter contains a CArG box.…”

“…A recent elegant paper reported that TGF␤-induced Rho/Rho kinase activation is an essential upstream mechanism for Nox4 expression and activity in renal fibroblasts and their transition to myofibroblasts (31). Mechanistically, the authors have implicated polymerase (DNA-directed) ␦-interacting protein 2 (Poldip2), a recently described Nox4 enhancer (68), which was also induced by Rho.…”

Salvianolic acid B attenuates lipopolysaccharide-induced acute lung injury in rats through inhibition of apoptosis, oxidative stress and inflammation

“…In cytokines participating in radiation-induced lung injury, TGF-β has a predominant role and is a major cytokine, which has been most thoroughly researched. It may induce inflammatory cells and fibroblasts to synthesize IL-1 and IL-6 (27). Furthermore, it may self-induce the production of more TGF-β and inhibit the degradation of extracellular matrix (ECM).…”

“…It stimulates fibroblasts to proliferate and induces the expression of collagen RNAI and III. The fastigium of TGF-β expression corresponds to the vigorous stage of fibroblast proliferation and collagen synthesis, suggesting that TGF-β and other cytokines mediate the early inflammatory response (27). α-SMA, which is the primary molecule of ALI, mainly exists in the cytoplasm.…”

Transforming Growth Factor β1 Function in Airway Remodeling and Hyperresponsiveness. The Missing Link?

“…Evidence suggests that TGF-b1 affects HASM shortening by Smadmediated contractile protein expression and ROS production. In addition, numerous studies demonstrate a role for TGF-b1 in ROCK activation (98)(99)(100)(101). Although the role of TGF-b1 in mediating classic E-C coupling remains unclear, TGF-b1 modulates Ca 21 homeostasis in a variety of cell types.…”