Hic-5 Mediates TGFß-Induced Adhesion in Vascular Smooth Muscle Cells by a Nox4-Dependent Mechanism.

OBJECTIVE: Focal adhesions (FAs) link the cytoskeleton to the extracellular matrix and as such play important roles in growth, migration, and contractile properties of vascular smooth muscle cells. Recently, it has been shown that downregulation of Nox4, a transforming growth factor (TGF) ß-inducible, hydrogen peroxide (H2O2)-producing enzyme, affects the number of FAs. However, the effectors downstream of Nox4 that mediate FA regulation are unknown. The FA resident protein H2O2-inducible clone (Hic)-5 is H2O2 and TGFß inducible, and a binding partner of the heat shock protein (Hsp) 27. The objective of this study was to elucidate the mechanism, by which Hic-5 and Hsp27 participate in TGFß-induced, Nox4-mediated vascular smooth muscle cell adhesion and migration. APPROACH AND RESULTS: Through a combination of molecular biology and biochemistry techniques, we found that TGFß, by a Nox4-dependent mechanism, induces the expression and interaction of Hic-5 and Hsp27, which is essential for Hic-5 localization to FAs. Importantly, we found that Hic-5 expression is required for the TGFß-mediated increase in FA number, adhesive forces and migration. Mechanistically, Nox4 downregulation impedes Smad (small body size and mothers against decapentaplegic) signaling by TGFß, and Hsp27 and Hic-5 upregulation by TGFß is blocked in small body size and mothers against decapentaplegic 4-deficient cells. CONCLUSIONS: Hic-5 and Hsp27 are effectors of Nox4 required for TGFß-stimulated FA formation, adhesion strength and migration in vascular smooth muscle cell.

Nicotinamide adenine dinucleotide phosphate oxidase-4-dependent upregulation of nuclear factor erythroid-derived 2-like 2 protects the heart during chronic pressure overload.

The transcription factor nuclear factor erythroid-derived 2-like 2 (Nrf2) controls a network of cytoprotective genes. Neither how Nrf2 is activated in the heart under hemodynamic overload nor its role and mechanism of action are known. This study aimed to investigate the activation and role of Nrf2 during chronic cardiac pressure overload. We first compared the responses of Nrf2(-/-) mice and wild-type littermates to chronic pressure overload. Hearts of Nrf2(-/-) mice showed impaired antioxidant gene expression, increased hypertrophy, and worse function compared with those of wild-type littermates after overload. Hearts of Nrf2(-/-) mice had increased mitochondrial DNA damage, a caspase 8/BH3-interacting domain death agonist-related cleavage of mitochondrial apoptosis-inducing factor, nuclear DNA damage, and cell death. Nrf2 activation was under the control of the endogenous reactive oxygen species-generating enzyme nicotinamide adenine dinucleotide phosphate oxidase-4, both in vivo and in vitro. In mice with cardiac-specific overexpression of nicotinamide adenine dinucleotide phosphate oxidase-4, Nrf2 deletion significantly attenuated their protective phenotype during chronic pressure overload. This study identifies nicotinamide adenine dinucleotide phosphate oxidase-4-dependent upregulation of Nrf2 as an important endogenous protective pathway that limits mitochondrial damage and apoptosis-inducing factor-related cell death in the heart under hemodynamic overload.

Activation of NADPH oxidase 4 in the endoplasmic reticulum promotes cardiomyocyte autophagy and survival during energy stress through the protein kinase RNA-activated-like endoplasmic reticulum kinase/eukaryotic initiation factor 2α/activating transcription factor 4 pathway.

RATIONALE: Autophagy is an essential survival mechanism during energy stress in the heart. Oxidative stress is activated by energy stress, but its role in mediating autophagy is poorly understood. NADPH oxidase (Nox) 4 is an enzyme that generates reactive oxygen species (ROS) at intracellular membranes. Whether Nox4 acts as a sensor of energy stress to mediate activation of autophagy is unknown. OBJECTIVE: We investigated whether Nox4 is involved in the regulation of autophagy and cell survival during energy stress in cardiomyocytes. METHODS AND RESULTS: Production of ROS in cardiomyocytes was increased during glucose deprivation (GD) in a Nox4-dependent manner. Protein levels and the ROS-producing activity of Nox4 were increased in the endoplasmic reticulum (ER), but not in mitochondria, in response to GD. Selective knockdown of Nox4, but not Nox2, or selective reduction of ROS in the ER with ER-targeted catalase, but not mitochondria-targeted perioxiredoxin 3, abrogated GD-induced autophagy. Nox4 promoted autophagy during GD through activation of the protein kinase RNA-activated-like ER kinase pathway by suppression of prolyl hydroxylase 4. The decrease in cell survival during GD in the presence of Nox4 knockdown was rescued by reactivation of autophagy by Atg7 overexpression, indicating that the effect of Nox4 on cell survival is critically mediated through regulation of autophagy. Nox4 was activated during fasting and prolonged ischemia in the mouse heart, where Nox4 is also required for autophagy activation and cardioprotection. CONCLUSIONS: Nox4 critically mediates autophagy in response to energy stress in cardiomyocytes by eliciting ROS in the ER and stimulating the protein kinase RNA-activated-like ER kinase signaling pathway.