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Background and objective: Type 2 Diabetes Mellitus (T2DM) with insulin resistance (IR) is prone to damage the vascular endothelial, leading to the formation of vulnerable carotid plaques and increasing ischemic stroke (IS) risk. The purpose of this study is to develop a nomogram model based on carotid ultrasound radiomics for predicting IS risk in T2DM patients. Methods: 198 T2DM patients were enrolled and separated into study and control groups based on IS history. After manually delineating carotid plaque region of interest (ROI) from images, radiomics features were identified and selected using the least absolute shrinkage and selection operator (LASSO) regression to calculate the radiomics score (RS). A combinatorial logistic machine learning model and nomograms were created using RS and clinical features like the triglyceride-glucose index. The three models were assessed using area under curve (AUC) and decision curve analysis (DCA). Results: Patients were divided into the training set and the testing set by the ratio of 0.7. 4 radiomics features were selected. RS and clinical variables were all statically significant in the training set and were used to create a combination model and a prediction nomogram. The combination model (radiomics + clinical nomogram) had the largest AUC in both the training set and the testing set (0.898 and 0.857), and DCA analysis showed that it had a higher overall net benefit compared to the other models. Conclusions: This study created a carotid ultrasound radiomics machine-learning-based IS risk nomogram for T2DM patients with carotid plaques. Its diagnostic performance and clinical prediction capabilities enable accurate, convenient, and customized medical care. [ABSTRACT FROM AUTHOR]

N6-metyladenosine (m6A) RNA methylation has been proven to be involved in diverse biological processes, but its potential roles in the development of lipopolysaccharide (LPS) induced retinal pigment epithelium (RPE) inflammation have not been revealed. In this study, we explored the effects and underlying mechanisms of methyltransferase-like 3 (METTL3) in LPS stimulated RPE cells. Proliferation of METTL3-silenced RPE cells was examined by Cell counting kit-8 (CCK8) and 5-Ethynyl-2'-Deoxyuridine (Edu). Expression of tight junction proteins ZO-1 and Occludin, and secretion of inflammatory factors interleukins (IL)-1, 6 and 8 were detected by Western blotting or Enzyme-linked immunosorbent assay (ELISA). RNA sequencing and methylated RNA immunoprecipitation (MeRIP) sequencing were used to analyze the target gene nuclear receptor subfamily 2 group F member 1 (NR2F1) of METTL3. Our results showed that both human RPE (hRPE) cells and ARPE19 cells exhibited inhibited proliferation, tight junction protein expression, and increased inflammatory factor secretion after METTL3 silencing. Mechanistically, we found that NR2F1, as a METTL3-methylated target gene, inhibits Occludin level and promotes IL-6 secretion of RPE cells in an m6A-dependent manner. Interestingly, NR2F1 deficiency reversed the decreased Occludin expression and increased IL-6 secretion in METTL3-defective RPE cells. In conclusion, our study revealed that METTL3 attenuates RPE cell inflammation by methylating NR2F1, suggesting the critical role of METTL3 in RPE cells. [ABSTRACT FROM AUTHOR]

Many lung diseases are caused by an excessive inflammatory response, and inflammatory lung diseases are often modeled using lipopolysaccharide (LPS) in mice. Cyclooxygenase-2 (COX-2) encoded by the Ptgs2 gene is induced in response to inflammatory stimuli including LPS. The objective of this study was to test the hypothesis that mice deficient in COX-2 (Ptgs2-/-) will be protected from LPS-induced lung injury. Wild-type (WT; CD1 mice) and Ptgs2-/- mice (on a CD1 background) were treated with LPS or vehicle for 24 h. LPS treatment resulted in histological evidence of lung injury, which was attenuated in the Ptgs2-/- mice. LPS treatment increased the mRNA levels for tumor necrosis factor-α, interleukin-10, and monocyte chemoattractant protein-1 in the lungs of WT mice, and the LPS-induced increases in these levels were attenuated in the Ptgs2-/- mice. The protein levels of active caspase-3 and caspase-9 were lower in the LPS-treated lungs of Ptgs2-/- mice than in LPS-treated WT mice, as were the number of terminal deoxynucleotide transferase dUTP nick end labeling-positive cells in lung sections. LPS exposure resulted in a greater lung wet-to-dry weight ratio (W/D) in WT mice, suggestive of pulmonary edema, while in LPS-treated Ptgs2-/- mice, the W/D was not different from controls and less than in LPS-treated WT mice. These results demonstrate that COX-2 is involved in the inflammatory response to LPS and suggest that COX-2 not only acts as a downstream participant in the inflammatory response, but also acts as a regulator of the inflammatory response likely through a feed-forward mechanism following LPS stimulation. [ABSTRACT FROM AUTHOR]

Tropical cyclone (TC) over the North Atlantic is an important extreme weather event, causing great social and economic damages to the American coastal regions. In this study, we find that the North Atlantic TC activity (genesis frequency and accumulated cyclone energy) has significant multidecadal variability, which exhibits an out-of-phase relationship with that over the eastern North Pacific, referring to as the inter-basin seesaw in TC activity. By using five indicators of the Atlantic Meridional Overturning Circulation (AMOC) strength, we link this TC activity seesaw pattern to the AMOC and the associated North Atlantic sea surface temperature anomalies. Both observation and Atlantic pacemaker experiment indicate that the AMOC induces the North Atlantic sea surface temperature warming and eastern North Pacific cooling, which lead to contrasting backgrounds (vertical wind shear, maximum potential intensity, and humidity) for the TC formation and development, resulting in the seesaw pattern in TC activity. Our results provide additional evidence for the reversed relationship in TC activity between the two ocean basins and attribute this seesaw to the variations in the AMOC. More importantly, the atmospheric-NAO-based AMOC indicator shows a leading role in depicting the multidecadal seesaw, which has great potential in improving the decadal prediction of North Atlantic/eastern North Pacific TCs. [ABSTRACT FROM AUTHOR]

Endothelial cell apoptosis is an early event in the development of acute lung injury (ALI). We have previously found that the Src family tyrosine kinase (STK) Yes activates caspase-3, whereas the STK Fyn inhibits caspase-3 activation in cultured pulmonary endothelial cells. We hypothesized that deficiency in Yes or Fyn in mice would have differential effects on lipopolysaccharide (LPS)-induced ALI. Mice were treated with LPS (10 mg/kg ip) for 24 h. Histological evidence of lung injury was greater in LPS-treated wild-type mice than in vehicle-treated wild-type mice, and the LPS-induced histological evidence of lung injury was attenuated in yes-/- mice and enhanced in fyn-/- mice. In wild-type or fyn-/- mice, LPS resulted in greater lung wet-to-dry weight ratios than in controls, whereas in yes-/- mice lung, wet-to-dry weight was similar between LPS and controls. LPSexposed fyn-/- mice had greater respiratory system resistance and lower respiratory system compliance than did LPSexposed wild-type mice. TUNEL positive cells in the lung following LPS treatment were greater in the fyn-/- mice and lower in the yes-/- mice compared with that in the wild-type mice. Following LPS treatment lung protein levels of PECAM-1 were lower in fyn-/- mice than in controls or yes-/- mice. LPS treatment increased cleaved caspase-3 protein levels in wild-type mice, whereas LPS-induced caspase-3 activation was attenuated in yes-/- mice and enhanced in fyn-/- mice. These results indicate that LPS-induced ALI is positively mediated via Yes-related mechanisms and negatively mediated by Fyn-related mechanisms. [ABSTRACT FROM AUTHOR]

The pathology of acute lung injury (ALI) involves inducible nitric oxide (NO) synthase (iNOS)-derived NO-induced apoptosis of pulmonary endothelial cells. In vitro, iNOS-derived NO production has been shown to depend on the uptake of L-arginine by the cationic amino acid transporters (CAT). To test the hypothesis that mice deficient in CAT-2 (slc7a2-/- a C57BL/6 background) would be protected from hyperoxia-induced ALI, mice (slc7a2-/- or wild-type) were placed in >95% oxygen (hyperoxia) or 21% oxygen (control) for 60 h. In wild-type mice exposed to hyperoxia, the exhaled nitric oxide (exNO) was twofold greater than in wild-type mice exposed to normoxia (P < 0.005), whereas in slc7a2-/- mice there was no significant difference between exNO in animals exposed to hyperoxia or normoxia (P = 0.95). Hyperoxia-exposed wild-type mice had greater (P < 0.05) lung resistance and a lower (P < 0.05) lung compliance than did hyperoxia-exposed slc7a2-/- mice. The lung wet-to-dry weight ratio was greater (P < 0.005) in the hyperoxiaexposed wild-type mice than in hyperoxia-exposed slc7a2-/- mice. Neutrophil infiltration was lower (P < 0.05) in the hyperoxia-exposed slc7a2-/- mice than in the hyperoxia-exposed wild-type mice as measured by myeloperoxidase activity. The protein concentration in bronchoalveolar lavage fluid was lower (P < 0.001) in the hyperoxiaexposed slc7a2-/- mice than in similarly exposed wild-type mice. The percent of TUNEL-positive cells in the lung following hyperoxia exposure was significantly lower (P < 0.001) in the slc7a2-/- mice than in the wild-type mice. These results are consistent with our hypothesis that lack of CAT-2 protects mice from acute lung injury. [ABSTRACT FROM AUTHOR]

We have previously shown that hypoxia-induced proliferation of human pulmonary microvascular endothelial cells (hPMVEC) depends on arginase II, and that epidermal growth factor receptor (EGFR) is necessary for hypoxicinduction of arginase II. However, it remains unclear how hypoxia activates EGFR-mediated signaling in hPMVEC. We hypothesized that hypoxia results in epidermal growth factor (EGF) production and that EGF binds to EGFR to activate the signaling cascade leading to arginase II induction and proliferation in hPMVEC. We found that hypoxia significantly increased the mRNA levels of EGF, EGFR, and arginase in hPMVEC. Hypoxia significantly increased pEGFR(Tyr845) protein levels and an EGF neutralizing antibody prevented the hypoxic induction of pEGFR. Inhibiting EGFR activation prevented hypoxia-induced arginase II mRNA and protein induction. Treatment of hPMVEC with exogenous EGF resulted in greater levels of arginase II protein both in normoxia and hypoxia. An EGF neutralizing antibody diminished hypoxic induction of arginase II and resulted in fewer viable cells in hPMVEC. Similarly, siRNA against EGF prevented hypoxic induction of arginase II and resulted in fewer viable cells. Finally, conditioned media from hypoxic hPMVEC induced proliferation in human pulmonary artery smooth muscle cells (hPASMC), however, conditioned media from a group of hypoxic hPMVEC in which EGF were knocked down did not promote hPASMC proliferation. These findings demonstrate that hypoxia-induced arginase II expression and cellular proliferation depend on autocrine EGF production leading to EGFR activation in hPMVEC. We speculate that EGF-EGFR signaling may be a novel therapeutic target for pulmonary hypertensive disorders associated with hypoxia. [ABSTRACT FROM AUTHOR]

We have previously shown that hypoxic proliferation of human pulmonary microvascular endothelial cells (hPMVECs) depends on epidermal growth factor receptor (EGFR) activation. To determine downstream signaling leading to proliferation, we tested the hypothesis that hypoxia-induced proliferation in hPMVECs would require EGFR-mediated activation of extracellular signal-regulated kinase (ERK) leading to arginase II induction. To test this hypothesis, hPMVECs were incubated in either normoxia (21% O2, 5% CO2) or hypoxia (1% O2, 5% CO2) and Western blotting was performed for EGFR, arginase II, phosphorylated- ERK (pERK), and total ERK (ERK). Hypoxia led to greater EGFR, pERK, and arginase II protein levels than did normoxia in hPMVECs. To examine the role of EGFR in these hypoxia-induced changes, hPMVECs were transfected with siRNA against EGFR or a scrambled siRNA and placed in hypoxia. Inhibition of EGFR using siRNA attenuated hypoxia-induced pERK and arginase II expression as well as the hypoxia-induced increase in viable cell numbers. hPMVECs were then treated with vehicle, an EGFR inhibitor (AG1478), or an ERK pathway inhibitor (U0126) and placed in hypoxia. Pharmacologic inhibition of EGFR significantly attenuated the hypoxia-induced increase in pERK level. Both AG1478 and U0126 also significantly attenuated the hypoxia-induced increase in viable hPMVECs numbers. hPMVECs were transfected with an adenoviral vector containing arginase II (AdArg2) and overexpression of arginase II rescued the U0126-mediated decrease in viable cell numbers in hypoxic hPMVECs. Our findings suggest that hypoxic activation of EGFR results in phosphorylation of ERK, which is required for hypoxic induction of arginase II and cellular proliferation. [ABSTRACT FROM AUTHOR]

Solid tumors can often be hypoxic in regions, and cancer cells can respond to hypoxia with an increase in proliferation and a decrease in apoptosis, leading to a net increase in viable cell numbers. We have recently found that in an osteosarcoma cell line, hypoxia-induced proliferation depends on arginase II induction. Epidermal growth factor receptor (EGFR) has been shown to mediate the hypoxia-induced cellular proliferation in some cancer cell lines. We hypothesized that hypoxia-induced proliferation of HeLa cells would depend on arginase II induction and that this induction of arginase II would occur through EGFR activation. Exposure of HeLa cells to hypoxia resulted in an upregulation of arginase II mRNA and protein levels, with no effect on arginase I expression. Hypoxia also resulted in significantly greater viable cell numbers than did normoxia. The hypoxia-induced increase in viable cell numbers was prevented by either a small molecule inhibitor of arginase or siRNA targeting arginase II. Overexpression of arginase II resulted in an increase in viable cell numbers both in normoxia and hypoxia. Hypoxia caused a substantial induction of both epidermal growth factor (EGF) and EGFR. Preventing hypoxia-induced EGFR expression using siRNA abolished hypoxia-induced arginase II expression and the increase in viable cell numbers. Treatment with EGF in normoxia not only induced arginase II expression but also resulted in an increase in viable cell numbers. Blocking EGF interactions with EGFR using either an EGF neutralizing antibody or an EGFR antibody prevented the hypoxia-induced increase in viable cell numbers. These results demonstrate an EGF/EGFR/arginase II pathway that is necessary for hypoxic proliferation in HeLa cells. [ABSTRACT FROM AUTHOR]

Arginase and nitric oxide synthase (NOS) share a common substrate, l‐arginine, and have opposing effects on vascular remodeling. Arginase is the first step in polyamine and proline synthesis necessary for cellular proliferation, while NO produced from NOS promotes apoptosis. Previously, we identified a single nucleotide polymorphism (SNP) in the arginase‐1 (ARG1) gene, rs2781666 (T‐allele) that was associated with a decreased risk for developing pulmonary hypertension (PH) in a cohort of infants with bronchopulmonary dysplasia (BPD). In this study, we utilized lymphocytes from neonates (the only readily available cells from these patients expressing the two genotypes of interest) with either the rs2781666 SNP (TT) or wild type (GG) to test the hypothesis that the protection of the ARG1 SNP against the development of PH in BPD would involve augmented NO production leading to more apoptosis. Lymphocytes were stimulated with IL‐4, IL‐13, and phorbol myristate acetate (PMA). We found that TT lymphocytes had similar levels of arginase I and arginase II expression, but there was a tendency for lower urea production (a surrogate marker of arginase activity), than in the GG lymphocytes. The TT lymphocytes also had significantly greater NO production than did GG lymphocytes despite no differences in iNOS expression between genotypes. Furthermore, the TT lymphocytes had lower numbers of viable cells, and higher levels of cleaved caspase‐3 than did GG lymphocytes. Inhibiting NOS activity using Nω‐Nitro‐l‐arginine methyl ester hydrochloride (l‐NAME) significantly decreased cleaved caspase‐3 levels in the TT lymphocytes. These data demonstrate that the TT genotype results in greater levels of NO production leading to more apoptosis, which is consistent with the concept that BPD patients with the TT genotype are protected against the development of PH by producing greater basal levels of endogenous NO. [ABSTRACT FROM AUTHOR]

Endothelial cells are essential for normal lung function: they sense and respond to circulating factors and hemodynamic alterations. In inflammatory lung diseases such as acute respiratory distress syndrome, endothelial cell apoptosis is an inciting event in pathogenesis and a prominent pathological feature. Endothelial cell apoptosis is mediated by circulating inflammatory factors, which bind to receptors on the cell surface, activating signal transduction pathways, leading to caspase-3-mediated apoptosis. We hypothesized that yes and src have differential effects on caspase-3 activation in human pulmonary microvascular endothelial cells (hPMVEC) due to differential downstream signaling effects. To test this hypothesis, hPMVEC were treated with siRNA against src (siRNAsrc), siRNA against yes (siRNAyes), or their respective scramble controls. After recovery, the hPMVEC were treated with cytomix (LPS, IL-1β, TNF-α, and IFN-γ). Treatment with cytomix induced activation of the extracellular signal-regulated kinase (ERK) pathway and caspase-3-mediated apoptosis. Treatment with siRNAsrc blunted cytomix-induced ERK activation and enhanced cleaved caspase-3 levels, while treatment with siRNAyes enhanced cytomix-induced ERK activation and attenuated levels of cleaved caspase-3. Inhibition of the ERK pathway using U0126 enhanced cytomix-induced caspase-3 activity. Treatment of hPMVEC with cytomix induced Akt activation, which was inhibited by siRNAsrc. Inhibition of the phosphatidylinositol 3-kinase/Akt pathway using LY294002 prevented cytomix-induced ERK activation and augmented cytomix-induced caspase-3 cleavage. Together, our data demonstrate that, in hPMVEC, yes activation blunts the ERK cascade in response to cytomix, resulting in greater apoptosis, while cytomixinduced src activation induces the phosphatidylinositol 3-kinase pathway, which leads to activation of Akt and ERK and attenuation of apoptosis. [ABSTRACT FROM AUTHOR]

Profound cardiovascular dysfunction is an important cause of mortality from septic shock. The molecular underpinnings of cardiac dysfunction during the inflammatory surge of early sepsis are not fully understood. MAPKs are important signal transducers mediating inflammation whereas G-protein signaling pathways modulate the cardiac response to stress. Using H9c2 cardiomyocytes, we investigated the interaction of MAPK and G-protein signaling in a sepsis model to test the hypothesis that the cardiomyocyte inflammatory response is controlled by MAPKs via G-protein-mediated events. We found that LPS stimulated proinflammatory cytokine production was markedly exacerbated by siRNA knockdown of the MAPK negative regulator Mkp-1. Cytokine production was blunted when cells were treated with p38 inhibitor. Two important cellular signaling molecules typically regulated by G-protein-coupled receptors, cAMP and PKC activity, were also stimulated by LPS and inflammatory cytokines TNF-α and IL-6, through a process regulated by Mkp-1 and p38. Interestingly, neutralizing antibodies against Gαs and Gαq blocked the increase in cellular cAMP and PKC activation, respectively, in response to inflammatory stimuli, indicating a critical role of G-protein coupled receptors in this process. LPS stimulation increased COX-2 in H9c2 cells, which also express prostaglandin receptors. Blockade of G-protein-coupled EP4 prostaglandin receptor by AH 23848 prevented LPS-induced cAMP increase. These data implicate MAPKs and G-proteins in the cardiomyocyte inflammatory response to LPS as well as crosstalk via COX-2-generated PGE2. These data add to our understanding of the pathogenesis of septic shock and have the potential to guide the selection of future therapeutics. [ABSTRACT FROM AUTHOR]

Il-10-deficient mice develop colitis associated with exaggerated Th1/Th17 responses and are a valuable model of inflammatory bowel disease. Mkp-1 is a major negative regulator of MAPKs, and its expression is enhanced by IL-10. To understand the role of Mkp-1 in the regulation of intestinal mucosal immune responses, we studied the effect of Mkp-1 deletion on the pathogenesis of colitis in Il-10-/- mice. We found that knockout of Mkp-1 on an Il-10-/- background accelerated the development of colitis. Compared with Il-10-/- mice, colitis not only appeared earlier but also was more severe in Il-10-/-/Mkp-1-/- mice. Il-10-/- mice exhibited a mild intestinal inflammation in the specific pathogen-free environment, and rectal prolapse rarely appeared before 6 mo of age. In contrast, the majority of Il-10-/-/Mkp- 1-/- mice developed severe colitis rapidly and presented with rectal prolapse after only 2-3 mo. The colon of Il-10-/-/Mkp-1-/- mice showed diffuse transmural chronic inflammation and mucosal hyperplasia, with significantly more proliferating crypt epithelial cells than those of Il-10-/- mice. In addition to the severe colitis, Il-10-/-/ Mkp-1-/-/ mice also developed conjunctivitis and blepharitis. The colon of Il-10-/-/Mkp-1-/- mice contained significantly higher levels of proinflammatory cytokines and exhibited greater MAPK activities than did the colon of Il-10-/- mice. Splenocytes and lymphocytes from Il-10-/- /Mkp-1-/- mice produced higher levels of Th1 cytokines ex vivo upon activation than did cells from Il-10-/- mice. Our studies support a pivotal role of Mkp-1 as a negative regulator of mucosal immune responses and highlight its protective function against inflammatory bowel disease. [ABSTRACT FROM AUTHOR]

Inducible nitric oxide (NO) synthase (iNOS) is a stress response protein upregulated in inflammatory conditions, and NO may suppress cellular proliferation. We hypothesized that preventing l-arginine (l-arg) uptake in endothelial cells would prevent lipopolysaccharide/tumor necrosis factor-a (LPS/TNF)-induced, NO-mediated suppression of cellular proliferation. Bovine pulmonary arterial endothelial cells (bPAEC) were treated with LPS/TNF or vehicle (control), and either 10 mM l-leucine [l-leu; a competitive inhibitor of l-arg uptake by the cationic amino acid transporter (CAT)] or its vehicle. In parallel experiments, iNOS or arginase II were overexpressed in bPAEC using an adenoviral vector (AdiNOS or AdArgII, respectively). LPS/TNF treatment increased the expression of iNOS, arginase II, CAT-1, and CAT-2 mRNA in bPAEC, resulting in greater NO and urea production than in control bPAEC, which was prevented by l-leu. LPS/TNF treatment resulted in fewer viable cells than in controls, and LPS/TNF-stimulated bPAEC treated with l-leu had more viable cells than LPS/TNF treatment alone. LPS/TNF treatment resulted in cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase expression, which was attenuated by l-leu. AdiNOS reduced viable cell number, and treatment of AdiNOS transfected bPAEC with l-leu preserved cell number. AdArgII increased viable cell number, and treatment of AdArgII transfected bPAEC with l-leu prevented the increase in cell number. These data demonstrate that iNOS expression in pulmonary endothelial cells leads to decreased cellular proliferation, which can be attenuated by preventing cellular l-arg uptake. We speculate that CAT activity may represent a novel therapeutic target in inflammatory lung diseases characterized by NO overproduction. [ABSTRACT FROM AUTHOR]

Background: Germline mutations in a tumor suppressor gene FLCN lead to development of fibrofolliculomas, lung cysts and renal cell carcinoma (RCC) in Birt-Hogg-Dubé syndrome. TFE3 is a member of the MiTF/TFE transcription factor family and Xp11.2 translocations found in sporadic RCC involving TFE3 result in gene fusions and overexpression of chimeric fusion proteins that retain the C-terminal DNA binding domain of TFE3. We found that GPNMB expression, which is regulated by MiTF, was greatly elevated in renal cancer cells harboring either TFE3 translocations or FLCN inactivation. Since TFE3 is implicated in RCC, we hypothesized that elevated GPNMB expression was due to increased TFE3 activity resulting from the inactivation of FLCN. Methodology/Principal Findings: TFE3 knockdown reduced GPNMB expression in renal cancer cells harboring either TFE3 translocations or FLCN inactivation. Moreover, FLCN knockdown induced GPNMB expression in FLCN-restored renal cancer cells. Conversely, wildtype FLCN suppressed GPNMB expression in FLCN-null cells. FLCN inactivation was correlated with increased TFE3 transcriptional activity accompanied by its nuclear localization as revealed by elevated GPNMB mRNA and protein expression, and predominantly nuclear immunostaining of TFE3 in renal cancer cells, mouse embryo fibroblast cells, mouse kidneys and mouse and human renal tumors. Nuclear localization of TFE3 was associated with TFE3 posttranslational modifications including decreased phosphorylation. Conclusions/Significance: Increased TFE3 activity is a downstream event induced by FLCN inactivation and is likely to be important for renal tumor development. This study provides an important novel mechanism for induction of TFE3 activity in addition to TFE3 overexpression resulting from Xp11.2 translocations, suggesting that TFE3 may be more broadly involved in tumorigenesis. [ABSTRACT FROM AUTHOR]

IL-12 and IL-23 are produced by activated antigen-presenting cells but the two induce distinct immune responses by promoting Th1 and Th17 cell differentiation, respectively. IL-23 is a heterodimeric cytokine consisting of two subunits: p40 that is shared with IL-12 and p19 unique to 11-23. In this study, we showed that the production of IL-23 but not IL-12 was negatively regulated by protein phosphatase 2A (PP2A) in dendritic cells (DC). PP2A inhibits IL-23 production by suppressing the expression of the IL-23p19 gene. Treating DC with okadaic acid that inhibits the PPM activity or knocking down the catalytic subunit of PPM with siRNA enhanced 11-23 but not lL-12 production. Unlike PPM, MAP kinase phosphatase-1 or CYLD did not show an effect on IL-23 production supporting the specificity of PP2A. PPM-mediated inhibition requires a newly made protein that is likely responsible for bringing PP2A and IKKβ together upon LPS stimulation, which then results in the termination of IKK phosphorylation. Thus, our results uncovered an important role of the protein phosphatase in the regulation of IL-23 production and identified PP2A as a previously uncharacterized inhibitor of IL-23p19 expression in DC. [ABSTRACT FROM AUTHOR]

The mitogen-activated protein (MAP) kinases are involved in cellular responses to many stimuli, including hypoxia. MAP kinase signaling is regulated by a family of phosphatases that include MAP kinase phosphatase-1 (MKP-1). We hypothesized that mice lacking the Mkp-1 gene would have exaggerated chronic hypoxia-induced pulmonary hypertension. Wild-type (WT) and Mkp-1-/- mice were exposed to either 4 wk of normoxia or hypobaric hypoxia. Following chronic hypoxia, both genotypes demonstrated elevated right ventricular pressures, right ventricular hypertrophy as demonstrated by the ratio of the right ventricle to the left ventricle plus septum weights [RV(LV + S)], and greater vascular remodeling. However, the right ventricular systolic pressures, the RV/(LV + S), and the medial wall thickness of 100- to 300-μm vessels was significantly greater in the Mkp-1-/- mice than in the WT mice following 4 wk of hypobaric hypoxia. Chronic hypoxic exposure caused no detectable change in eNOS protein levels in the lungs in either genotype; however, Mkp-1-/- mice had lower levels of eNOS protein and lower lung NO production than did WT mice. No iNOS protein was detected in the lungs by Western blotting in any condition in either genotype. Both arginase I and arginase 11 protein levels were greater in the lungs of hypoxic Mkp-1-/- mice than those in hypoxic WT mice. Lung levels of proliferating cell nuclear antigen were greater in hypoxic Mkp-1-/- than in hypoxic WT mice. These data are consistent with the concept that MKP-1 acts to restrain hypoxia-induced arginase expression and thereby reduces vascular remodeling and the severity of pulmonary hypertension. [ABSTRACT FROM AUTHOR]

We hypothesized that the Src family tyrosine kinases (STKs) are involved in the upregulation of arginase and inducible nitric oxide synthase (iNOS) expression in response to inflammatory stimuli in pulmonary endothelial cells. Treatment of bovine pulmonary arterial endothelial cells (bPAEC) with lipopolysaccharide and tumor necrosis factor-α (LIT) resulted in increased urea and nitric oxide (NO) production, and this increase in urea and NO production was inhibited by the STK inhibitor PP1 (10 µM). The STK inhibitors PP2 (10 µM) and herbimycin A (10 µM) also prevented the L/T-induced expression of both arginase II and iNOS mRNA in bPAEC. Together, the data demonstrate a central role of STK in the upregulation of both arginase II and iNOS in bPAEC in response to L/T treatment. To identify the specific kinase(s) required for the induction of urea and NO production, we studied human pulmonary microvascular endothelial cells (hPMVEC) so that short interfering RNA (siRNA) techniques could be employed. We found that hPMVEC express Fyn, Yes, c-Src, Lyn, and BIk and that the protein expression of Fyn, Yes, c-Src, and Lyn could be inhibited with specific siRNA. The siRNA targeting Fyn prevented the cytokine-induced increase in urea and NO production, whereas siRNAs specifically targeting Yes, c-Src, and Lyn had no appreciable effect on cytokine-induced urea and NO production. These findings support our hypothesis that inflammatory stimuli lead to increased urea and NO production through a STK-mediated pathway. Furthermore, these results indicate that the STK Fyn plays a critical role in this process. [ABSTRACT FROM AUTHOR]

Nef-induced podocyte proliferation and dedifferentiation via mitogen-activated protein kinase 1,2 (MAPK1,2) activation plays a role in human immunodeficiency virus (HIV) nephropathy pathogenesis. All-trans retinoic acid (atRA) reverses the HIV-induced podocyte phenotype by activating cyclic AMP (cAMP)/protein kinase A (PKA) and inhibiting MAPK1,2. Here we show that atRA, through cAMP and PKA, triggers a feed-forward loop involving CREB and USF1 to induce biphasic stimulation of MKP1. atRA stimulated CREB and USF1 binding to the MKP1 gene promoter, as shown by gel shifting and chromatin immunoprecipitation assays. CREB directly mediated the early phase of atRA-induced MKP1 stimulation; whereas the later phase was mediated by CREB indirectly through induction of USF1. These findings were confirmed by a reporter gene assay using the MKP1 promoter with mutation of CRE or Ebox binding sites. Consistent with these findings, the biological effects of atRA on podocytes were inhibited by silencing either MKP1, CREB, or USF1 with small interfering RNA. atRA also induced CREB phosphorylation and MKP1 expression and reduced MAPK1,2 phosphorylation in kidneys of HIV type 1-infected transgenic mice. We conclude that atRA induces sustained activation of MKP1 to suppress Nef-induced activation of the Src-MAPK1,2 pathway, thus returning the podocyte to a more differentiated state. The mechanism involves a feed-forward loop where activation of one transcription factor (TF) (CREB) leads to induction of a second TF (USF1). [ABSTRACT FROM AUTHOR]

High NaCl rapidly activates p38 MAPK by phosphorylating it, the phosphorylation presumably being regulated by a balance of kinases and phosphatases. Kinases are known, but the phosphatases are uncertain. Our initial purpose was to identify the phosphatases. We find that in HEK293 cells transient overexpression of MAPK phosphatase-1 (MKP-1), a dual-specificity phosphatase, inhibits high NaCl-induced phosphorylation of p38, and that over-expression of a dominant negative mutant of MKP-1 does the opposite. High NaCl lowers MKP-1 activity by increasing reactive oxygen species, which directly inhibit MKP-1, and by reducing binding of MKP-1 to p38. Because inhibition of p38 is reported to reduce hypertonicity-induced activation of the osmoprotective transcription factor, TonEBP/OREBP, we anticipated that MKP-1 expression might also. However, overexpression of MKP-1 has no significant effect on Ton EBP/OREBP activity. This paradox is explained by opposing effects of p38α and p38δ, both of which are activated by high NaCl and inhibited by MKP-1. Thus, we find that overexpression of p38α increases high NaCI-induced TonEBP/OREBP activity, but overexpression of p38δ reduces it. Also, siRNA-mediated knockdown of p38δ enhances the activation of TonEBP/OREBP. We conclude that high NaCl inhibits MKP-1, which contributes to the activation of p38. However, opposing actions of p38α and p38δ negate any effect on TonEBP/OREBP activity. Thus, activation of p38 isoforms by hypertonicity does not contribute to activation of TonEBP/OREBP because of opposing effects of p38α and p38δ, and effects of inhibitors of p38 depend on which isoform is affected, which can be misleading. [ABSTRACT FROM AUTHOR]

L-Arginine (L-arg) is metabolized to nitric oxide (NO) by inducible NO synthase (iNOS) or to urea and L-ornithine (L-orn) by arginase. NO is involved in the inflammatory response, whereas arginase is the first step in polyamine and proline synthesis necessary for tissue repair and wound healing. Mitogen-activated protein kinases (MAPK) mediate LPS-induced iNOS expression, and MAPK phosphatase-1 (MKP-1) plays a crucial role in limiting MAPK signaling in macrophages. We hypothesized that MKP-1, by attenuating iNOS expression, acts as a switch changing L-arg metabolism from NO production to L-orn production after endotoxin administration. To test this hypothesis, we performed studies in RAW264.7 macrophages stably transfected with an MKP-1 expression vector in thioglyollate-elicited peritoneal macrophages harvested from wild-type and Mkp-1-/- mice, as well as in vivo in wild-type and Mkp-1-/- mice. We found that overexpression of MKP-1 resulted in lower iNOS expression and NO production but greater urea production in response to LPS. Although deficiency of MKP-1 resulted in greater iNOS expression and NO production and lower urea production in response to LPS, neither the overexpression nor the deficiency of MKP-1 had any substantial effect on the expression of the arginases. [ABSTRACT FROM AUTHOR]

Mitogen-activated protein kinase (MAPK) phosphatases (MKPs) are protein phosphatases that dephosphorylate both the phosphothreonine and phosphotyrosine residues on activated MAPKs. Removal of the phosphates renders MAPKs inactive, effectively halting their cellular function. In recent years, evidence has emerged that, similar to MAPKs, MKPs are pivotal in the regulation of immune responses. By deactivating MAPKs, MKPs can modulate both innate and adaptive immunity. A number of immunomodulatory agents have been found to influence the expression of MKP1 in particular, highlighting the central role of this phosphatase in immune regulation. This Review discusses the properties, function and regulation of MKPs during immune responses. [ABSTRACT FROM AUTHOR]

Triptolide is a compound extracted from the Chinese herb Tripterygium wilfordii Hook. f. Triptolide has potent anticancer activity. However, the mechanisms by which triptolide exerts its anticancer activities remain unclear. To explore the molecular mechanisms involved in the anticancer activity of triptolide, we have examined the effect of triptolide on the growth of pancreatic carcinoma PANC-1 and cervical adenocarcinoma HeLa cells. We found that treatment of both HeLa and PANC-1 cells with triptolide potently suppressed cell growth and induced apoptosis, indicated by nuclear fragmentation and blebbing. In both HeLa and PANC-1 cells, apoptosis induced by triptolide was associated with activation of both caspase-3 and caspase-8, and cleavage of poly(ADP-ribose) polymerase and Bid. Moreover, in HeLa cells, caspase-9 is also significantly activated in response to triptolide. Overexpression of Bcl-2 in HeLa cells substantially attenuated triptolide-induced apoptosis. Interestingly, substitution of the 14-OH of triptolide with an acetyl group abrogated both its anticancer and its antiinflammatory activities. Our studies suggest that triptolide may exert its anticancer effects by initiating apoptosis through both death-receptor- and mitochondria-mediated pathways. Our results indicate that both the apoptosis-promoting and the antiinflammatory activities of triptolide depend on the 14-OH group. [ABSTRACT FROM AUTHOR]

Nitric oxide (NO) is a vasodilator produced from L-arginine (L-Arg) by NO synthase (NOS). Gene therapy for hypertensive disorders has been proposed using the inducible isoform of NOS (iNOS). L-Arg also can be metabolized to urea and L-ornithine (L-Orn) by arginase, and L-Orn can be metabolized to proline and/or polyamines, which are vital for cellular proliferation. To determine the effect of iNOS gene transfer on arginase, we transfected bovine pulmonary arterial endothelial cells (bPAEC) with an adenoviral vector containing the gene for iNOS (AdiNOS). As expected, NO production in AdiNOS bPAEC was substantially greater than in control bPAEC. Although urea production was significantly less in the AdiNOS bPAEC than in the control bPAEC, despite similar levels of arginase I protein, AdiNOS transfection of bPAEC had no effect on the uptake of L-Arg. Inhibiting NO production with Nω-nitro-L-arginine methyl ester increased urea production, and inhibiting urea production with L-valine increased nitrite production, in AdiNOS bPAEC. The addition of L-Arg to the medium increased urea production by AdiNOS bPAEC in a concentration-dependent manner. Thus, in these iNOS-transfected bPAEC, the transfected iNOS and native arginase compete for a common intracellular pool of L-Arg. This competition for substrate resulted in impaired proliferation in the AdiNOS-transfected bPAEC. These findings suggest that the use of iNOS gene therapy for pulmonary hypertensive disorders may not only be beneficial through NO-mediated pulmonary vasodilation but also may decrease vascular remodeling by limiting L-Orn production by native arginase. [ABSTRACT FROM AUTHOR]

Presents information on a study which investigated the role of phosphatidylinositol 3-kinase/Akt signaling pathway in oncogenic-TC21-mediated transformation and cell survival. Results; Discussion; Methodology.

Cardiac dysfunction is a major contributor to sepsis related organ failure and mortality and is related to systemic and/or cardiac innate immune responses. Mitogen activated protein kinase phosphatase (MKP)-1 is a key negative regulator of the innate immune responses. Its role in cardiac myocyte dysfunction during sepsis is not defined. We tested the hypothesis that MKP-1 deficiency promotes cardiac dysfunction during sepsis. Five-week old MKP-1-/- and wild-type control mice were treated with a single dose of LPS (1.5 mg/kg, ip). Cardiovascular phenotyping was measured at 0, 4 and 24 h after LPS challenge, via echocardiography and 4- lead ECG analyses. Blood pressure was measured with a tail cuff monitor. In wild-type mice LPS caused transient reductions in LV fractional shortening and BP, which returned to baseline values at 24 h (FS%: 55.1, 51.8, and 60.0% for 0, 4, 24 h respectively) (BP: 82.1, 65.1, 70.0; 0, 4, 24 h respectively). MKP-1-/- mice had more pronounced hemodynamic impairment (FS%: 57.1, 43.2*, and 40.2* for 0, 4, 24 h respectively; *, p<0.01) (BP: 82.1, 41.5*, 40.2*, for 0, 4,24 h respectively; *, p<0.01). These data demonstrate that cardiac myocyte MAPK signaling plays an important role in sepsis-related cardiac dysfunction and it might be related to enhanced cardiac myocyte inflammatory and oxidant mediated injury. [ABSTRACT FROM AUTHOR]