Although nitric oxide (NO) kills or inhibits the replication of a variety of intracellular pathogens, the antimicrobial mechanisms of NO are unknown. Here, we identify a viral protease as a target of NO. The life cycle of many viruses depends upon viral proteases that cleave viral polyproteins into individual polypeptides. NO inactivates the Coxsackievirus protease 3C, an enzyme necessary for the replication of Coxsackievirus. NO S-nitrosylates the cysteine residue in the active site of protease 3C, inhibiting protease activity and interrupting the viral life cycle. Substituting a serine residue for the active site cysteine renders protease 3C resistant to NO inhibition. Since cysteine proteases are critical for virulence or replication of many viruses, bacteria, and parasites, S-nitrosylation of pathogen cysteine proteases may be a general mechanism of antimicrobial host defenses.

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Abstract-Many forms of vascular disease are characterized by increased transforming growth factor (TGF)-␤ 1 expression and endothelial dysfunction. Smad proteins are a key step in TGF-␤-initiated signal transduction. We hypothesized that NO may regulate endothelial TGF-␤-dependent gene expression. We show that NO inhibits TGF-␤/Smad-regulated gene transactivation in a cGMP-dependent manner. NO effects were mimicked by a soluble analogue of cGMP. Inhibition of cGMP-dependent protein kinase 1 (PKG-1) or overexpression of dominant-negative PKG-1␣ suppressed NO/cGMP inhibition of TGF-␤-induced gene expression. Inversely, overexpression of PKG-1␣ catalytic subunit blocked TGF-␤-induced gene transactivation. Furthermore NO delayed and reduced phosphorylated Smad2/3 nuclear translocation, an effect mediated by PKG-1, whereas N G -nitro-L-arginine methyl ester augmented Smad phosphorylation and gene expression in response to TGF-␤. Aortas from endothelial NO synthase-deficient mice showed enhanced basal TGF-␤ 1 and collagen type I expression; endothelial cells from these animals showed increased Smad phosphorylation and transcriptional activity. Proteasome inhibitors prevented the inhibitory effect of NO on TGF-␤ signaling. NO reduced the metabolic life of ectopically expressed Smad2 and enhanced its ubiquitination. Taken together, these results suggest that the endothelial NO/cGMP/PKG pathway interferes with TGF-␤/Smad2 signaling by directing the proteasomal degradation of activated Smad. Key Words: nitric oxide Ⅲ endothelial cells Ⅲ vascular remodeling Ⅲ transforming growth factor-␤ T ransforming growth factor (TGF)-␤ plays a major role in the vascular response to injury by controlling both cellular proliferation and extracellular matrix turnover through the Smad-signaling pathway. [1][2][3] Ligand binding leads to phosphorylation and nuclear translocation of receptor-activated Smads (R-Smads), Smad2/3, which modulate the transcription of a large number of genes. Smad7 and Smad6, inhibitory Smads (I-Smad), antagonize TGF-␤ signaling. Smad7/6 expression is induced by TGF-␤ in the endothelium, providing an autoregulatory negative feedback loop on TGF-␤ signaling. 4 The response to TGF-␤ in the cardiovascular system is tightly controlled. Mice deficient in TGF-␤ 1 die in utero because of vascular defects. 5 Smad1-deficient mice fail to establish chorion-allantoic circulation, whereas Smad5-deficient embryos have defects in yolk sac vasculature with enlarged blood vessels. 6,7 Mice deficient in the accessory receptor endoglin exhibit embryonic lethality, with cardiovascular and angiogenesis defects associated with abnormal vascular smooth muscle cell development. 8 Smad6-deficient mice develop aortic ossification and elevated blood pressure. 9 TGF-␤ is highly expressed in injured arteries, and TGF-␤-dependent effects play a role in the pathogenesis of atherosclerosis, coronary artery disease, transplant arteriosclerosis, hypertension, diabetes, myocardial remodeling, and restenosis. 10 -14 Blood vessels overexpressing TG...

The endothelial nitric oxide synthase (eNOS) is a critical regulator of cardiovascular homeostasis, whose dysregulation leads to different vascular pathologies. Endoglin is a component of the transforming growth factor beta (TGF-beta) receptor complex present in endothelial cells that is involved in angiogenesis, cardiovascular development, and vascular homeostasis. Haploinsufficient expression of endoglin has been shown to downregulate endothelium-derived nitric oxide in endoglin(+/-) (Eng(+/-)) mice and cultured endothelial cells. Here, we find that TGF-beta1 leads to an increased vasodilatation in Eng(+/+) mice that is severely impaired in Eng(+/-) mice, suggesting the involvement of endoglin in the TGF-beta regulated vascular homeostasis. The endoglin-dependent induction of eNOS occurs at the transcriptional level and is mediated by the type I TGF-beta receptor ALK5 and its downstream substrate Smad2. In addition, Smad2-specific signaling is upregulated in endoglin-induced endothelial cells, whereas it is downregulated upon endoglin gene suppression with small interference RNA (siRNA). The endoglin-dependent upregulation of Smad2 was confirmed using eNOS and pARE promoters, whose activities are known to be Smad2 dependent, as well as with the interference of Smad2 with siRNA, Smurf2, or a dominant negative form of Smad2. Furthermore, increased expression of endoglin in endoglin-inducible endothelial cells or in transfectants resulted in increased levels of Smad2 protein without affecting the levels of Smad2 mRNA. The increased levels of Smad2 appear to be due to a decreased ubiquitination and proteasome-dependent degradation leading to stabilization of Smad2. These results suggest that endoglin enhances Smad2 protein levels potentiating TGF-beta signaling, and leading to an increased eNOS expression in endothelial cells.

Bisphenol A (BPA) is found in human urine and fat tissue. Higher urinary BPA concentrations are associated with arterial hypertension. To shed light on the underlying mechanism, we orally administered BPA (4 nM to 400 μM in drinking water) to 8-wk-old CD11 mice over 30 d. Mice developed dosage-dependent high blood pressure (systolic 130 ± 12 vs. 170 ± 12 mmHg; EC50 0.4 μM), impairment of acetylcholine (AcH)-induced carotid relaxation (0.66 ± 0.08 vs. 0.44 ± 0.1 mm), a 1.7-fold increase in arterial angiotensin II (AngII), an 8.7-fold increase in eNOS mRNA and protein, and significant eNOS-dependent superoxide and peroxynitrite accumulation. AngII inhibition with 0.5 mg/ml losartan reduced oxidative stress and normalized blood pressure and endothelium-dependent relaxation, which suggests that AngII uncouples eNOS and contributes to the BPA-induced endothelial dysfunction by promoting oxidative and nitrosative stress. Microarray analysis of mouse aortic endothelial cells revealed a 2.5-fold increase in expression of calcium/calmodulin-dependent protein kinase II-α (CaMKII-α) in response to 10 nM BPA, with increased expression of phosphorylated-CaMKII-α in carotid rings of BPA-exposed mice, whereas CaMKII-α inhibition with 100 nM autocamptide-2-related inhibitor peptide (AIP) reduced BPA-mediated increase of superoxide. Administration of CaMKII-α inhibitor KN 93 reduced BPA-induced blood pressure and carotid blood velocity in mice, and reverted BPA-mediated carotid constriction in response to treatment with AcH. Given that CaMKII-α inhibition prevents BPA-mediated high blood pressure, our data suggest that BPA regulates blood pressure by inducing AngII/CaMKII-α uncoupling of eNOS.

Bone tissue renovation is a dynamic event in which osteoblasts and osteoclasts are responsible for the turnover between bone formation and bone resorption, respectively. During bone development, extracellular matrix remodeling is required for osteoblast differentiation and the process is largely mediated by the proteolytic activity of extracellular matrix metalloproteinases (MMPs), which play a fundamental role in osteoblast migration, unmineralized matrix degradation, and cell invasion. The recent advances towards investigation in osteogenesis have provided significant information about the transcriptional regulation of several genes, including MMPs, by the expression of crucial transcription factors like NFAT, ATF4, osterix, TAZ, and Cbfa-1–responsive elements. Evidence from gene knock-out studies have shown that bone formation is, at least in part, mediated by nitric oxide (NO), since mice deficient in endothelial nitric oxide synthase (eNOS) and mice deficient in the eNOS downstream effector (cGMP)-dependent protein kinase (PKG) show bone abnormalities, while inducible NOS (iNOS) null mice also show imbalances in bone osteogenesis and abnormalities in bone healing. Recently, in vitro data showed that Cbfa-1 and the MAPK pathways were crucial for osteoblastic cell differentiation, and NO was found to play a significant role. This article sheds light on some of the mechanisms that may influence NO-mediated actions in bone development.

Abstract-Transforming growth factor-␤ (TGF-␤) increases expression of endothelial nitric oxide synthase (eNOS), although the precise mechanism by which it does so is unclear. We report that Smad2, a transcription factor activated by TGF-␤, mediates TGF-␤ induction of eNOS in endothelial cells. TGF-␤ induces Smad2 translocation from cytoplasm to nucleus, where it directly interacts with a specific region of the eNOS promoter. Overexpression of Smad2 increases basal levels of eNOS, and further increases TGF-␤ stimulation of eNOS expression. Key Words: endothelial cell Ⅲ hypoxia Ⅲ atherosclerosis A lthough endothelial nitric oxide synthase (eNOS or NOS3) was originally described as a constitutive NOS, subsequent work showed that eNOS expression is regulated by a variety of physiological and pathophysiological factors. 1-6 Expression of eNOS is increased by exercise, shear stress, hypoxia, hyperthyroidism, cirrhosis, and endothelial cell proliferation. [7][8][9][10] Various mediators can also increase eNOS expression, including estrogens, angiotensin II, glucose, oxidized linoleic acid, lysophosphatidylcholine, hydrogen peroxide, and transforming growth factor-␤. 1-6 Expression of eNOS is decreased by hypoxia, inflammation, and pulmonary hypertension. [11][12][13][14] Mediators that decrease eNOS expression include oxidized LDL, lipopolysaccharide, and tumor necrosis factor-␣. Regulation of eNOS ExpressionThe level of eNOS expression is determined by multiple mechanisms, including posttranscriptional regulation 15-18 and posttranslational regulation. 19 -36 Expression of eNOS is also regulated at the transcriptional level. The 5Ј-flanking region of the eNOS gene lacks a TATA box, but contains elements that mediate constitutive expression of genes in endothelial cells, including Sp1 sites and GATA. [37][38][39][40][41][42] The 5Ј-flanking region also contains many putative sites for further transcriptional regulation of eNOS. However, only a few of these sites have been formally shown to regulate eNOS transcription, including PEA3 and AP-1 binding sites. 43,44 Finally, a region of the eNOS promoter extending from Ϫ1269 and Ϫ935 mediates TGF-␤ induction of eNOS transcription. 45 TGF-␤ and Vascular PathophysiologyTGF-␤ belongs to a superfamily of dimeric growth factors, including bone morphogenetic proteins and activins. 46 -48 TGF-␤ regulates diverse biological processes, including cell proliferation, differentiation, and migration. TGF-␤ also regulates cellular functions within the cardiovascular system, including angiogenesis, fibrosis, and production of extracellular matrix. TGF-␤ Signal Transduction and SmadsAfter its secretion and activation, TGF-␤ forms a complex with type I and type II TGF-␤ receptors on target cells. Once bound to TGF-␤, the type II TGF-␤ receptor phosphorylates the type I receptor, and the type I receptor in turn phosphorylates transcription factors known as Smads. Smad family members mediate TGF-␤ signal transduction, and include receptor Smads (Smad2 and Smad3) that associate with and are ...

Nitric oxide is a radical molecule with antibacterial, -parasitic, and -viral properties. We investigated the mechanism of NO inhibition of Coxsackievirus B3 (