Cellular stimuli generate reactive oxygen species (ROS) via the local action of NADPH oxidases (Nox) to modulate cytoskeletal organization and cell migration through unknown mechanisms. Cofilin is a major regulator of cellular actin dynamics whose activity is controlled by phosphorylation/dephosphorylation at Ser3. Here we show that Slingshot-1L (SSH-1L), a selective cofilin regulatory phosphatase, is involved in H₂O₂-induced cofilin dephosphorylation and activation. SSH-1L is activated by its release from a regulatory complex with 14-3-3ζ protein through the redox-mediated oxidation of 14-3-3ζ by H₂O₂. The ROS-dependent activation of the SSH-1L-cofilin pathway stimulates the SSH-1L–dependent formation of cofilin-actin rods in cofilin-GFP–expressing HeLa cells. Similarly, the formation of endogenous ROS stimulated by angiotensin II (AngII) also activates the SSH-1L-cofilin pathway via oxidation of 14-3-3ζ to increase AngII-induced membrane ruffling and cell motility. These results suggest that the formation of ROS by NADPH oxidases engages a SSH-1L-cofilin pathway to regulate cytoskeletal organization and cell migration.

Brief treatment with transforming growth factor (TGF)–β1 stimulated the migration of macrophages, whereas long-term exposure decreased their migration. Cell migration stimulated by TGF-β1 was markedly inhibited by 10 μg/mL Tat-C3 exoenzyme. TGF-β1 increased mRNA and protein levels of macrophage inflammatory protein (MIP)–1α in the initial period, and these effects also were inhibited by 10 μg/mL Tat-C3 and a dominant-negative (DN)–RhoA (N19RhoA). Cycloheximide, actinomycin D, and antibodies against MIP-1α and monocyte chemoattractant protein-1 (MCP-1) abolished the stimulation of cell migration by TGF-β1. These findings suggest that migration of these cells is regulated directly and indirectly via the expression of chemokines such as MIP-1α and MCP-1 mediated by RhoA in response to TGF-β1. TGF-β1 activated RhoA in the initial period, and thereafter inactivated them, suggesting that the inactivation of RhoA may be the cause of the reduced cell migration in response to TGF-β1 at later times. We therefore attempted to elucidate the molecular mechanism of the inactivation of RhoA by TGF-β1. First, TGF-β1 phosphorylated RhoA via protein kinase A, leading to inactivation of RhoA. Second, wild-type p190 Rho GTPase activating protein (p190RhoGAP) reduced and DN-p190RhoGAP reversed the reduction of cell migration induced by TGF-β, suggesting that it inactivated RhoA via p190 Rho GAP.

Nox activator 1 (NoxA1) is a homologue of p67^phox that acts in conjunction with Nox organizer 1 (NoxO1) to regulate reactive oxygen species (ROS) production by the NADPH oxidase Nox1. The phosphorylation of cytosolic regulatory components by multiple kinases plays important roles in assembly and activity of the phagocyte NADPH oxidase (Nox2) system, but little is known about regulation by phosphorylation in the Nox1 system. Here we identify Ser¹⁷² and Ser⁴⁶¹ of NoxA1 as phosphorylation sites for protein kinase A (PKA). A consequence of this phosphorylation was the enhancement of NoxA1 complex formation with 14-3-3 proteins. Using both a transfected human embryonic kidney 293 cell Nox1 model system and endogenous Nox1 in colon cell lines, we showed that the elevation of cAMP inhibits, whereas the inhibition of PKA enhances, Nox1-dependent ROS production through effects on NoxA1. Inhibition of Nox1 activity was intensified by the availability of 14-3-3ζ protein, and this regulatory interaction was dependent on PKA-phosphorylatable sites at Ser¹⁷² and Ser⁴⁶¹ in NoxA1. We showed that phosphorylation and 14-3-3 binding induce the dissociation of NoxA1 from the Nox1 complex at the plasma membrane, suggesting a mechanism for the inhibitory effect on Nox1 activity. Our data establish that PKA-phosphorylated NoxA1 is a new binding partner of 14-3-3 protein(s) and that this forms the basis of a novel mechanism regulating the formation of ROS by Nox1 and, potentially, other NoxA1-regulated Nox family members.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China, in December 2019 and has been rapidly spreading worldwide. Although the causal relationship among mutations and the features of SARS-CoV-2 such as rapid transmission, pathogenicity, and tropism, remains unclear, our results of genomic mutations in SARS-CoV-2 may help to interpret the interaction between genomic characterization in SARS-CoV-2 and infectivity with the host. A total of 4,254 genomic sequences of SARS-CoV-2 were collected from the Global Initiative on Sharing all Influenza Data (GISAID). Multiple sequence alignment for phylogenetic analysis and comparative genomic approach for mutation analysis were conducted using Molecular Evolutionary Genetics Analysis (MEGA), and an in-house program based on Perl language, respectively. Phylogenetic analysis of SARS-CoV-2 strains indicated that there were 3 major clades including S, V, and G, and 2 subclades (G.1 and G.2). There were 767 types of synonymous and 1,352 types of non-synonymous mutation. ORF1a, ORF1b, S, and N genes were detected at high frequency, whereas ORF7b and E genes exhibited low frequency. In the receptor-binding domain (RBD) of the S gene, 11 non-synonymous mutations were observed in the region adjacent to the angiotensin converting enzyme 2 (ACE2) binding site. It has been reported that the rapid infectivity and transmission of SARS-CoV-2 associated with host receptor affinity are derived from several mutations in its genes. Without these genetic mutations to enhance evolutionary adaptation, species recognition, host receptor affinity, and pathogenicity, it would not survive. It is expected that our results could provide an important clue in understanding the genomic characteristics of SARS-CoV-2.

Rationale:: Neointimal hyperplasia is characterized by excessive accumulation of vascular smooth muscle cells (SMCs) leading to occlusive disorders, such as atherosclerosis and stenosis. Blood vessel injury increases growth factor secretion and matrix synthesis, which promotes SMC proliferation and neointimal hyperplasia via FAK (focal adhesion kinase). Objective:: To understand the mechanism of FAK action in SMC proliferation and neointimal hyperplasia. Methods and Results:: Using combined pharmacological FAK catalytic inhibition (VS-4718) and SMC-specific FAK kinase-dead (Myh11-Cre-ER ^T2 ) mouse models, we report that FAK regulates SMC proliferation and neointimal hyperplasia in part by governing GATA4- (GATA-binding protein 4) cyclin D1 signaling. Inhibition of FAK catalytic activity facilitates FAK nuclear localization, which is required for proteasome-mediated GATA4 degradation in the cytoplasm. Chromatin immunoprecipitation identified GATA4 binding to the mouse cyclin D1 promoter, and loss of GATA4-mediated cyclin D1 transcription diminished SMC proliferation. Stimulation with platelet-derived growth factor or serum activated FAK and redistributed FAK from the nucleus to cytoplasm, leading to concomitant increase in GATA4 protein and cyclin D1 expression. In a femoral artery wire injury model, increased neointimal hyperplasia was observed in parallel with elevated FAK activity, GATA4 and cyclin D1 expression following injury in control mice, but not in VS-4718-treated and SMC-specific FAK kinase-dead mice. Finally, lentiviral shGATA4 knockdown in the wire injury significantly reduced cyclin D1 expression, SMC proliferation, and neointimal hyperplasia compared with control mice. Conclusions:: Nuclear enrichment of FAK by inhibition of FAK catalytic activity during vessel injury blocks SMC proliferation and neointimal hyperplasia through regulation of GATA4-mediated cyclin D1 transcription.

The NADPH oxidase (Nox) enzyme family generates reactive oxygen species (ROS) that contribute to cell signaling, innate immune responses, proliferation, and transcription. The signaling mechanisms that regulate this important enzyme family are only beginning to be understood. Evidence is emerging which suggests that phosphorylation of Nox and/or their regulatory components may be important means of modulating their activity. We describe here the evidence for Nox regulation through the action of kinases, and speculate on how such regulatory mechanisms might contribute to the development of pathological disease states. Antioxid. Redox Signal. 11, 2429–2441.