Key Words: Nox4 Ⅲ Poldip2 Ⅲ vascular smooth muscle cells Ⅲ reactive oxygen species Ⅲ cytoskeleton R eactive oxygen species (ROS), such as superoxide (O 2 ·Ϫ ) and hydrogen peroxide (H 2 O 2 ), are implicated in the development of multiple cardiovascular disease pathologies, including hypertension, atherosclerosis, and restenosis. 1 Physiologically, ROS mediate many cellular functions such as proliferation, gene expression, migration, differentiation, and cytoskeletal remodeling. 2 One major source of ROS is the NADPH oxidase (Nox) family of enzymes.The catalytic moieties of NADPH oxidases are homologs of the flavin-and NADPH-binding protein gp91phox (Nox2), termed Nox1, Nox3, Nox4, Nox5, Duox1, and Duox2. Most cell types express multiple Nox enzymes that are differentially regulated and have distinct subcellular localizations, suggesting that these oxidases serve unique roles. For example, Nox1 and Nox4 are the predominant homologs in rodent vascular smooth muscle cells (VSMCs) from large vessels. Whereas Nox1 is primarily found in caveolae, Nox4 is found in the nucleus, in focal adhesions and along stress fibers. 3 Nox1 mediates VSMC growth and migration, whereas Nox4 is involved in differentiation. 4 Nox enzymes also differ in their mode of regulation. The Nox2-based oxidase consists of 5 subunits. Together, the membrane proteins Nox2 and p22phox comprise the cytochrome b558 membrane complex, which is localized in submembranous vesicles and the plasma membrane. Catalytic activity is initiated by translocation to the membrane of cytosolic subunits p47phox, p67phox, and the smallmolecular-weight G protein Rac. 4 Nox1 and Nox3 are similarly regulated by the p47phox and p67phox homologs Nox organizer 1 (NoxO1) and Nox activator 1 (NoxA1), respectively, as well as with Rac. 4 However, none of the presently known cytosolic regulatory subunits is required for Nox4 activation. 5 The mechanism by which Nox4 activity is regulated remains unclear. Some studies suggest that the principal mechanism of Nox4 regulation may be induction at the mRNA level, rather than assembly of an enzyme complex or posttranslational protein modifications. 6 However, although it is known that Nox4 requires p22phox, there has been no Original received January 7, 2009; revision received June 19, 2009; accepted June 24, 2009. MethodsAn expanded Materials and Methods section is available in the Online Data Supplement at http://circres.ahajournals.org. Cell CultureRat aortic VSMCs and human aortic smooth muscle cells (passages 6 to 12) were grown in DMEM. HEK293 cells were cultured in DMEM with 10% FBS. Rat VSMCs were used for all VSMC experiments, except where indicated. AntibodiesPoldip2 goat antibody was custom made by GenScript Corporation (Piscataway, NJ) against the peptide sequence NPAGHGSKEVKGKTC. Yeast Two-Hybrid AssayWe used the Matchmaker LexA yeast 2-hybrid system (Clontech) and a VSMC cDNA library constructed in pB42AD. The cytosolic tail of rat p22phox (nucleotides 360 to 579) served as bait. Positive colonies were amplified ...

A critical step toward understanding mitochondrial genetics and its impact on human disease is to identify and characterize the full complement of nucleus-encoded factors required for mitochondrial gene expression and mitochondrial DNA (mtDNA) replication. Two factors required for transcription initiation from a human mitochondrial promoter are h-mtRNA polymerase and the DNA binding transcription factor, h-mtTFA. However, based on studies in model systems, the existence of a second human mitochondrial transcription factor has been postulated. Here we report the isolation of a cDNA encoding h-mtTFB, the human homolog of Saccharomyces cerevisiae mitochondrial transcription factor B (sc-mtTFB) and the first metazoan member of this class of transcription factors to which a gene has been assigned. Recombinant h-mtTFB is capable of binding mtDNA in a non-sequence-specific fashion and activates transcription from the human mitochondrial light-strand promoter in the presence of h-mtTFA in vitro. Remarkably, h-mtTFB and its fungal homologs are related in primary sequence to a superfamily of N6 adenine RNA methyltransferases. This observation, coupled with the ability of recombinant h-mtTFB to bind S-adenosylmethionine in vitro, suggests that a structural, and perhaps functional, relationship exists between this class of transcription factors and this family of RNA modification enzymes and that h-mtTFB may perform dual functions during mitochondrial gene expression.

Human mitochondrial transcription factor B1 (h-mtTFB1) has an unprecedented relationship to RNA methyltransferases. Here, we show that this protein methylates a conserved stem-loop in bacterial 16S rRNA and that the homologous sequence in the human mitochondrial 12S molecule is similarly modified. Thus, h-mtTFB1 appears to be dual-function protein, acting both as a transcription factor and an rRNA-modification enzyme.

The proliferation of vascular smooth muscle cells is important in the pathogenesis of many vascular diseases. Reactive oxygen species (ROS) produced by NADPH oxidases in smooth muscle cells have been shown to participate in signaling cascades regulating proliferation induced by platelet-derived growth factor (PDGF), a powerful smooth muscle mitogen. We sought to determine the role of Nox5 in the regulation of PDGF-stimulated human aortic smooth muscle cell (HASMC) proliferation. Cultured HASMC were found to express four isoforms of Nox5. When HASMC stimulated with PDGF were pretreated with N-acetyl cysteine (NAC), proliferation was significantly reduced. Proliferation induced by PDGF was also heavily dependent on JAK/STAT activation, as the JAK inhibitor, AG490, was able to completely abolish PDGF-stimulated HASMC growth. Specific knockdown of Nox5 with a siRNA strategy reduced PDGF-induced HASMC ROS production and proliferation. Additionally, siRNA to Nox5 inhibited PDGF-stimulated JAK2 and STAT3 phosphorylation. ROS produced by Nox5 play an important role in PDGF-induced JAK/STAT activation and HASMC proliferation. KeywordsNADPH oxidase; reactive oxygen species; Nox5; vascular smooth muscle cells; proliferation In atherosclerosis and restenosis after percutaneous coronary intervention, cytokines elaborated by both vascular cells and cells invading the vessel wall induce vascular smooth muscle proliferation and contribute to lesion formation. In the normal vessel wall, smooth muscle cells (SMCs) maintain and regulate vascular tone. However, VSMCs change their phenotype to a synthetic, proliferative state when stimulated by a number of different growth factors.One such growth factor, platelet-derived growth factor (PDGF), is expressed by all vascular cell types [1,2] and by invading inflammatory cells, such as monocytes and lymphocytes, in atherosclerosis [1]. PDGF has long been recognized as a powerful VSMC mitogen [3] and the induction of PDGF receptors in VSMCs during atherogenesis has been demonstrated in several studies [4,5]. Blockade of PDGF, whether by anti-PDGF antibody [6][7][8], PDGF receptor antisense therapy [9,10], or chimeric knockout in mice [11], reduces lesion formation after vascular injury. Ligand binding to the PDGF receptor causes tyrosine autophosphorylation and Address correspondence to: Kathy K. Griendling, Emory University, Division of Cardiology, 319 WMB, 1639 Pierce Dr., Atlanta, GA 30322,, e-mail: kgriend@emory.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. With regard to PDGF signaling, Nox5 is of particular interest because it...

The steady-state amounts of mitochondrial transcripts and transcription proteins were analyzed during mtDNA depletion and subsequent repletion to gain insight into the regulation of human mitochondrial gene expression. As documented previously, HeLa cells depleted of mtDNA via treatment with ethidium bromide (EB) were found to contain reduced steady-state levels of the mitochondrial transcription factor h-mtTFA. When partially mtDNA-depleted cells were cultured in the absence of EB, h-mtTFA recovered to normal levels at a significantly slower rate than mtDNA. Human mtRNA polymerase exhibited a similar depletion-repletion profile, suggesting that the mitochondrial transcription machinery is coordinately regulated in response to changes in mtDNA copy number. Newly synthesized mitochondrial transcripts were detected early in the recovery phase, despite the fact that mtDNA, h-mtTFA and h-mtRNA polymerase were simultaneously depleted. Although delayed relative to mtDNA, the amounts of h-mtTFA and h-mtRNA polymerase sharply increased during the later stages of the recovery phase, which was accompanied by accelerated rates of transcription and mtDNA replication. Altogether, these data indicate that when mtDNA copy number is low, it is beneficial to prevent accumulation of mitochondrial transcription proteins. In addition, h-mtTFA and h-mtRNA polymerase are either normally present in excess of the amount required for transcription or their activity is up-regulated to ensure continued expression and transcription-dependent replication of the mitochondrial genome during mtDNA-depleted states.

Senescence, a hallmark of mammalian aging, is associated with the onset and progression of cardiovascular disease. Angiotensin II (Ang II) signaling and zinc homeostasis dysfunction are increased with age and are linked to cardiovascular disease, but the relationship among these processes has not been investigated. We used a model of cellular senescence induced by Ang II in vascular smooth muscle cells (VSMCs) to explore the role of zinc in vascular dysfunction. We found that Ang II-induced senescence is a zinc-dependent pathway mediated by the downregulation of the zinc transporters ZnT3 and ZnT10, which work to reduce cytosolic zinc. Zinc mimics Ang II by increasing reactive oxygen species (ROS), activating NADPH oxidase activity and Akt, and by downregulating ZnT3 and ZnT10 and inducing senescence. Zinc increases Ang II-induced senescence, while the zinc chelator TPEN, as well as overexpression of ZnT3 or ZnT10, decreases ROS and prevents senescence. Using HEK293 cells, we found that ZnT10 localizes in recycling endosomes and transports zinc into vesicles to prevent zinc toxicity. Zinc and ZnT3/ZnT10 downregulation induces senescence by decreasing the expression of catalase. Consistently, ZnT3 and ZnT10 downregulation by siRNA increases ROS while downregulation of catalase by siRNA induces senescence. Zinc, siZnT3 and siZnT10 downregulate catalase by a post-transcriptional mechanism mediated by decreased phosphorylation of ERK1/2. These data demonstrate that zinc homeostasis dysfunction by decreased expression of ZnT3 or ZnT10 promotes senescence and that Ang II-induced senescence is a zinc and ROS-dependent process. Our studies suggest that zinc might also affect other ROS-dependent processes induced by Ang II, such as hypertrophy and migration of smooth muscle cells.

In contrast to other cell types, vascular smooth muscle cells modify their phenotype in response to external signals. NADPH oxidase 4 (Nox4) is critical for maintenance of smooth muscle gene expression; however, the underlying mechanisms are incompletely characterized. Using smooth muscle α-actin (SMA) as a prototypical smooth muscle gene and transforming growth factor-β (TGF-β) as a differentiating agent, we examined Nox4-dependent signaling. TGF-β increases Nox4 expression and activity in human aortic smooth muscle cells (HASMC). Transfection of HASMC with siRNA against Nox4 (siNox4) abolishes TGF-β-induced SMA expression and stress fiber formation. siNox4 also significantly inhibits TGF-β-stimulated p38MAPK phosphorylation, as well as that of its substrate, mitogen-activated protein kinase-activated protein kinase-2 (MK-2). Moreover, the p38MAPK inhibitor SB-203580 nearly completely blocks the SMA increase induced by TGF-β. Inhibition of either p38MAPK or NADPH oxidase-derived reactive oxygen species impairs the TGF-β-induced phosphorylation of Ser103 on serum response factor (SRF) and reduces its transcriptional activity. Binding of SRF to myocardin-related transcription factor (MRTF) is also necessary, because downregulation of MRTF by siRNA abolishes TGF-β-induced SMA expression. Taken together, these data suggest that Nox4 regulates SMA expression via activation of a p38MAPK/SRF/MRTF pathway in response to TGF-β. KeywordsVascular smooth muscle; smooth muscle α-actin; transforming growth factor-β; NADPH oxidase 4; serum response factor; reactive oxygen species; p38 mitogen activated protein kinase Vascular smooth muscle cells (VSMC) from adult tissue exhibit a differentiated phenotype, which is defined by the expression of contractile proteins such as smooth muscle alpha actin (SMA), calponin (CNN) and smooth muscle myosin heavy chain (SM-MHC) [1]. In contrast to other cell types, VSMC can modify their phenotype in response to external signals, transforming into a synthetic phenotype that is able to proliferate and migrate and secrete increased amounts of matrix proteins. This modification occurs in different cardiovascular diseases such as atherosclerosis, restenosis or hypertension, contributing to their pathophysiology [1]. Redifferentiation of synthetic VSMC limits lesion formation; however, Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. [5,6]. In addition, Nox4 and its activator Poldip2, appear to regulate stress fiber formation, conceivably by activating RhoA [7]. However, almost nothing is known about the mechanisms by which reactive oxygen species ...

Polymerase-δ-interacting protein 2 (Poldip2) interacts with NADPH oxidase 4 (Nox4) and regulates migration; however, the precise underlying mechanisms are unclear. Here, we investigated the role of Poldip2 in focal adhesion turnover, as well as traction force generation and polarization. Poldip2 overexpression (AdPoldip2) in vascular smooth muscle cells (VSMCs) impairs PDGF-induced migration and induces a characteristic phenotype of long cytoplasmic extensions. AdPoldip2 also prevents the decrease in spreading and increased aspect ratio observed in response to PDGF and slightly impairs cell contraction. Moreover, AdPoldip2 blocks focal adhesion dissolution and sustains H2O2 levels in focal adhesions, whereas Poldip2 knockdown (siPoldip2) significantly decreases the number of focal adhesions. RhoA activity is unchanged when focal adhesion dissolution is stimulated in control cells but increases in AdPoldip2-treated cells. Inhibition of RhoA blocks Poldip2-mediated attenuation of focal adhesion dissolution, and overexpression of RhoA or focal adhesion kinase (FAK) reverses the loss of focal adhesions induced by siPoldip2, indicating that RhoA and FAK mediate the effect of Poldip2 on focal adhesions. Nox4 silencing prevents focal adhesion stabilization by AdPoldip2 and induces a phenotype similar to siPoldip2, suggesting a role for Nox4 in Poldip2-induced focal adhesion stability. As a consequence of impaired focal adhesion turnover, PDGF-treated AdPoldip2 cells are unable to reduce and polarize traction forces, a necessary first step in migration. These results implicate Poldip2 in VSMC migration via regulation of focal adhesion turnover and traction force generation in a Nox4/RhoA/FAK-dependent manner.