Blockade of myeloid differentiation protein 2 prevents obesity-induced inflammation and nephropathy.

Obesity is a major and independent risk factor of kidney diseases. The pathogenic mechanisms of obesity-associated renal injury are recognized to at least involve a lipid-rich and pro-inflammatory state of the renal tissues, but specific mechanisms establishing causal relation remain unknown. Saturated fatty acids are elevated in obesity, and known to induce chronic inflammation in kidneys. Myeloid differentiation protein 2 (MD2) is an important protein in lipopolysaccharide-induced innate immunity response and inflammation. We suggested that obesity-associated renal injury is regulated by MD2 thereby driving an inflammatory renal injury. The used three mouse models for in vivo study: MD2 knockout mice (KO) maintained on high fat diet (HFD), wild-type mice on HFD plus L6H21, a specific MD2 inhibitor and KO mice given palmitic acid (PA) by IV injection. The in vitro studies were carried out in cultured renal tubular epithelial cells, mouse mesangial cells and primary macrophages, respectively. The HFD mice presented with increased hyperlipidemia, serum creatinine and proteinuria. Renal tissue from HFD mice had increased fibrosis, inflammatory cytokines, macrophage infiltration, and activation of NF-κB and MAPKs. This HFD-induced renal injury profile was not observed in KO mice or L6H21-treated mice. Mice given PA mimmicked the HFD-induced renal injury profiles, which were prevented by MD2 knockout. The in vitro data further confirmed MD2 mediates PA-induced inflammation. MD2 is causally related with obesity-associated renal inflammatory injury. We believe that MD2 is an attractive target for future therapeutic strategies in obesity-associated kidney diseases.

Loss of p120 catenin upregulates transcription of pro-inflammatory adhesion molecules in human endothelial cells.

P120 catenin (p120ctn) is an adherens junction protein recognized to regulate barrier function, but emerging evidence indicates that p120ctn may also exert control on other cellular functions such as transcriptional suppression of genes. We investigated the hypothesis that loss of p120ctn in human endothelial cells activates transcription of pro-inflammatory adhesion molecules. For study, siRNA targeted to p120ctn was transfected into brain microvascular (HBMECs) or pulmonary artery endothelial cells (HPAECs) for 24-120h, which depleted 50-80% of endogenous p120ctn. This loss of p120ctn resulted in increased promoter reporter activity of transcription factors, NFκB, AP-1, and Kaiso, as well as of target genes, MMP-1 and ICAM-1. Real-time RT-PCR analysis indicated that the mRNA for ICAM-1, VCAM-1, and E- and P-selectins were all upregulated during the period of 24-120h of p120ctn depletion, although the time-course and extent of the expression profiles differed. The upregulated mRNA of adhesion molecules corresponded with increased PMN adhesion to the EC surface and elevated ICAM-1 protein expression. We further explored the role of ERK1/2 as a potential signaling mechanism responsible for regulation of transcriptional activities by p120ctn. Results indicated that loss of p120ctn increased phosphorylated ERK1/2, and a MEK1 inhibitor (PD98059) prevented NFκB nuclear translocation. This implicates ERK1/2 in signaling the NFκB activation induced by p120ctn loss. The findings provide strong evidence that deficiency in p120ctn expression in endothelial cells is a potent stimulus for transcriptional upregulation of multiple adhesion molecules. We conclude that p120ctn functions to suppress transcription, which is an important and novel regulation in vascular endothelium.

P120 catenin represses transcriptional activity through Kaiso in endothelial cells.

P120 catenin (p120ctn) belongs to the family of Armadillo repeat-containing proteins, which are believed to have dual functions of cell-cell adhesion and transcriptional regulation. In vascular endothelium, p120ctn is mostly recognized for its cell-cell adhesion function through its ability to regulate VE-cadherin. The current study investigated whether p120ctn in endothelial cells also has the capability to signal transcription events. Examination of several endothelial cell types indicated that Kaiso, a p120ctn-binding transcription factor, was abundantly expressed, with a predominant localization to the perinuclear region. Immunoprecipitation of endothelial cell lysates with a p120ctn antibody resulted in p120ctn-Kaiso complex formation, confirming the interactions of the two proteins. Transfection of the KBS (Kaiso-binding sequence) luciferase reporter plasmid into endothelial cells resulted in a 40% lower reporter activity compared to the mutant Kaiso-insensitive construct or empty vector pGL3, indicating that the suppressed reporter activity was attributed to endogenous Kaiso. The knock-down of p120ctn increased the KBS reporter activity 2-fold over control, but had no effects on the mutant KBS reporter activity. Furthermore, p120ctn knock-down also reduced Kaiso expression, suggesting that p120ctn functioned to stabilize Kaiso. Overall, the findings provide evidence that in endothelial cells, p120ctn has a transcription repression function through regulation of Kaiso, possibly as a cofactor with the transcription factor.

MD2 activation by direct AGE interaction drives inflammatory diabetic cardiomyopathy.

Hyperglycemia activates toll-like receptor 4 (TLR4) to induce inflammation in diabetic cardiomyopathy (DCM). However, the mechanisms of TLR4 activation remain unclear. Here we examine the role of myeloid differentiation 2 (MD2), a co-receptor of TLR4, in high glucose (HG)- and diabetes-induced inflammatory cardiomyopathy. We show increased MD2 in heart tissues of diabetic mice and serum of human diabetic subjects. MD2 deficiency in mice inhibits TLR4 pathway activation, which correlates with reduced myocardial remodeling and improved cardiac function. Mechanistically, we show that HG induces extracellular advanced glycation end products (AGEs), which bind directly to MD2, leading to formation of AGEs-MD2-TLR4 complex and initiation of pro-inflammatory pathways. We further detect elevated AGE-MD2 complexes in heart tissues and serum of diabetic mice and human subjects with DCM. In summary, we uncover a new mechanism of HG-induced inflammatory responses and myocardial injury, in which AGE products directly bind MD2 to drive inflammatory DCM.

MD2 blockade prevents oxLDL-induced renal epithelial cell injury and protects against high-fat-diet-induced kidney dysfunction.

There is a strong epidemiological link between obesity, a growing worldwide concern, and kidney disease. Emerging evidence indicates that the pathogenic basis of obesity-related kidney disease may be attributed to Toll-like receptor 4 (TLR4) of the innate immune system. We hypothesized that renal epithelial cell injury in response to oxidized low-density lipoprotein (oxLDL) requires myeloid differentiation factor 2 (MD2), a co-receptor of TLR4. Moreover, we also hypothesized that renal dysfunction is MD2-dependent in the high-fat diet (HFD) mouse model. Results indicated that the MD2 selective inhibitor (L6H21) abrogated the oxLDL-induced formation of MD2-TLR4 dimerization in the renal proximal tubular epithelial cell line NRK-52E. Further, MD2 blockade in NRK-52E cells using siRNA target sequences or L6H21 prevented oxLDL-induced cell injury as indicated by expression of profibrotic molecules, autophagic activity and apoptosis. Similarly, TLR4 knockdown in NRK-52E cells using siRNA target sequences prevented oxLDL-induced cell injury. In the HFD mouse model, MD2 knockout protected against development of kidney dysfunction and renal tissue injury, corroborating the observations observed in NRK-52E cells. Thus, the oxLDL-induced renal tubular epithelial cell profibrotic responses, autophagy and apoptosis were dependent on MD2, as were the renal dysfunction and tissue impairment in HFD mice. These are new findings indicating that the MD2-TLR4 immune signaling complex is a critical pathogenic factor in the development of kidney disease related to obesity or metabolic syndrome.

Inhibition of myeloid differentiation factor 2 by baicalein protects against acute lung injury.

BACKGROUND: ALI/ARDS is characterized by severe hypoxemic respiratory failure attributed to inflammatory tissue injury. There are no treatment modalities able to prevent/reverse the dire pathological sequelae in these patients. Evidence links the inflammatory lung injury to uncontrolled activation of the immune signaling complex, TLR4-MD2 (Toll-like receptor-myeloid differentiation factor 2). Baicalein, a natural flavonoid, is reported to have robust anti-inflammatory properties, but its inhibition mechanism remains unclear. HYPOTHESIS/PURPOSE: This study investigated the protective mechanisms of baicalein on ALI/ARDS. METHODS: We used two experimental mouse models of LPS-induced ALI, pulmonary infection model (intratracheal LPS), and systemic infection model (intravenous LPS). Blood, BALF, lung and liver tissues were analyzed using routine methods. In vitro studies using peritoneal mouse macrophages or recombinant proteins were designed to elucidate inhibition mechanisms of baicalein. RESULTS: Our critical new findings revealed that Baicalein was an MD2 inhibitor, directly bound to MD2, effectively suppressing TLR4-MD2 activation and the subsequent MAPK and NF-κB signaling. The inhibited MD2 prevented development of inflammatory tissue injury and improved survival. The importance of MD2 in the inflammatory injury in ALI was corroborated by data obtained from MD2-/- mice, which did not develop the characteristic LPS-induced lung tissue damage. Thus, the findings indicated that MD2 was critical for development of ALI, functioning as an early upstream signal driving the progression of inflammatory injury. CONCLUSION: Baicalein, as a direct and selective MD2 inhibitor, inhibited the early upstream TLR4-MD2 signaling and is a promising therapeutic agent for the treatment of ALI/ARDS.

New MD2 inhibitors derived from curcumin with improved anti-inflammatory activity.

An overactive Toll-like receptor (TLR) signaling complex is a significant pathogenic factor of acute and chronic inflammatory diseases. The natural product curcumin is reported to inhibit the TLR4 co-receptor, MD2 (myeloid differentiation protein 2), but its low in vivo bioavailability limits its therapeutic potential. We developed new curcumin analogs (MACs) with removal of the ß-diketone moiety and substituted residues in benzene rings, and identify these as potential MD2 inhibitors with improved inhibition potency and stability over that of curcumin. Specifically, MAC 17 and 28 showed the highest anti-inflammatory activity, with >90% inhibition of LPS-stimulated cytokine secretion from macrophages, and protected against LPS-induced acute lung injury and sepsis. The MACs inhibited the TLR4-MD2 signaling complex through competition with LPS for binding on MD2, likely at Arg90. Our findings indicated that MAC 17 and 28 are promising candidates for future development as therapeutic drugs for inflammatory diseases with an endotoxin etiology.

A novel lysophospholipid- and pH-sensitive receptor, GPR4, in brain endothelial cells regulates monocyte transmigration.

Abundant evidence documents the highly proinflammatory actions of lysophosphatidylcholine (LPC). Further, LPC, found in high amounts in oxidized low-density lipoprotein (LDL), is implicated as an atherogenic factor. In endothelial cells, LPC impairs endothelial barrier function through GPR4, a novel receptor hypothesized to be sensitive to LPC and protons. The authors investigated the stimulation by LPC or low pH of GPR4 in human brain microvascular endothelial cells (HBMECs) and whether the activated GPR4 regulates in vitro monocyte transmigration. The results indicated that HBMECs stimulated by LPC (5 microM), but not low pH, showed a twofold increase in monocyte transmigration. Using retroviruses containing siRNA to GPR4, a > 60% reduction of GPR4 expression resulted in blockade of the LPC-stimulated transmigration. The inhibited response was restored by co-expression with an small interference RNA (siRNA)-resistant, but functional, GPR4 mutant construct. To investigate potential signaling mechanisms, the siRNA-mediated knockdown of GPR4 also prevented LPC-induced RhoA activation. C3 transferase, a Rho inhibitor, prevented approximately approximately 65% of the LPC-stimulated transmigration. LPC also increased MLC phosphorylation by 5 min, which was inhibited by the Rho kinase inhibitor, Y-27632 (10 microM) or ML-7 (myosin light chain kinase (MLCK) inhibitor). The findings indicate that the proinflammatory and atherogenic LPC stimulated endothelial GPR4, which promoted monocyte transmigration through a RhoA-dependent pathway.

Angiotensin II induces kidney inflammatory injury and fibrosis through binding to myeloid differentiation protein-2 (MD2).

Growing evidence indicates that angiotensin II (Ang II), a potent biologically active product of RAS, is a key regulator of renal inflammation and fibrosis. In this study, we tested the hypothesis that Ang II induces renal inflammatory injury and fibrosis through interaction with myeloid differentiation protein-2 (MD2), the accessory protein of toll-like receptor 4 (TLR4) of the immune system. Results indicated that in MD2-/- mice, the Ang II-induced renal fibrosis, inflammation and kidney dysfunction were significantly reduced compared to control Ang II-infused wild-type mice. Similarly, in the presence of small molecule MD2 specific inhibitor L6H21 or siRNA-MD2, the Ang II-induced increases of pro-fibrotic and pro-inflammatory molecules were prevented in tubular NRK-52E cells. MD2 blockade also inhibited activation of NF-κB and ERK. Moreover, MD2 blockade prevented the Ang II-stimulated formation of the MD2/TLR4/MyD88 signaling complex, as well as the increased surface binding of Ang II in NRK-52E cells. In addition, Ang II directly bound recombinant MD2 protein, rather than TLR4 protein. We conclude that MD2 is a significant contributor in the Ang II-induced kidney inflammatory injury in chronic renal diseases. Furthermore, MD2 inhibition could be a new and important therapeutic strategy for preventing progression of chronic renal diseases.

GPR4 plays a critical role in endothelial cell function and mediates the effects of sphingosylphosphorylcholine.

Angiogenesis is critical for many physiological and pathological processes. We show here that the lipid sphingosylphosphorylcholine (SPC) induces angiogenesis in vivo and GPR4 is required for the biological effects of SPC on endothelial cells (EC). In human umbilical vein EC, down-regulation of GPR4 specifically inhibits SPC-, but not sphingosine-1-phosphate-, or vascular endothelial growth factor (VEGF)-induced tube formation. Re-introduction of GPR4 fully restores the activity of SPC. In microvascular EC, GPR4 plays a pivotal role in cell survival, growth, migration, and tube formation through both SPC-dependent and -independent pathways. The biological effects resulting from SPC/GPR4 interactions involve the activation of both phosphatidylinositol-3 kinase and Akt. Moreover, the effects of SPC on EC require SPC induced trans-phosphorylation and activation of the VEGF receptor 2. These results identify SPC and its receptor, GPR4, as critical regulators of the angiogenic potential of EC.

Potential genetic therapies for acute lung injury.

Acute lung injury (ALI) is a common, highly lethal acquired disorder that affects over one hundred thousand people each year and for which there are no specific therapies. Extensive investigations in experimental models and humans with ALI have identified several maladaptive host responses and dysregulated protein systems that offer therapeutic opportunities for genetic intervention. Several lines of evidence suggest that gene transfer can be used to deliver protective proteins that improve alveolar epithelial and/or endothelial cell function or immunomodulators that augment lung defense mechanisms and speed clearance of infection. In many instances, gene transfer is the only avenue for producing localized expression of these pharmaceuticals. This article reviews recent translational, animal-based studies that tested the use of gene and cell based therapies to ameliorate or prevent ALI. The lack of effective therapies for ALI and the approachability of the lung for local gene transfer suggest that ALI is a unique example of an acute disease process that is suitable for gene therapy.

Stimulated bronchial epithelial cells release bioactive lysophosphatidylcholine 16:0, 18:0, and 18:1.

PURPOSE: In human subjects and animal models with acute and chronic lung injury, the bioactive lysophosphatidylcholine (LPC) is elevated in lung lining fluids. The increased LPC can promote an inflammatory microenvironment resulting in lung injury. Furthermore, pathological lung conditions are associated with upregulated phospholipase A2 (PLA2), the predominant enzyme producing LPC in tissues by hydrolysis of phosphatidylcholine. However, the lung cell populations responsible for increases of LPC have yet to be systematically characterized. The goal was to investigate the LPC generation by bronchial epithelial cells in response to pathological mediators and determine the major LPC species produced. METHODS: Primary human bronchial epithelial cells (NHBE) were challenged by vascular endothelial growth factor (VEGF) for 1 or 6 h, and condition medium and cells collected for quantification of predominant LPC species by high performance liquid chromatography-tandem mass spectrometry (LC-MS-MS). The cells were analyzed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) for PLA2. The direct effects of LPC in inducing inflammatory activities on NHBE were assessed by transepithelial resistance as well as expression of interleukin-8 (IL-8) and matrix metalloproteinase-1 (MMP-1). RESULTS: VEGF stimulation of NHBE for 1 or 6 h, significantly increased concentrations of LPC16:0, LPC18:0, and LPC18:1 in condition medium compared to control. The sPLA2-selective inhibitor (oleyloxyethyl phosphorylcholine) inhibited the VEGF-induced release of LPC16:0 and LPC18:1 and PLA2 activity. In contrast, NHBE stimulated with TNF did not induce LPC release. VEGF did not increase mRNA of PLA2 subtypes sPLA2-X, sPLA2-XIIa, cPLA2-IVa, and iPLA2-VI. Exogenous LPC treatment increased expression of IL-8 and MMP-1, and reduced the transepithelial resistance in NHBE. CONCLUSIONS: Our findings indicate that VEGF-stimulated bronchial epithelial cells are a key source of extracellular LPCs, which can function as an autocrine mediator with potential to induce airway epithelial inflammatory injury.

MicroRNA-146 inhibits thrombin-induced NF-κB activation and subsequent inflammatory responses in human retinal endothelial cells.

PURPOSE: Nuclear factor-κB (NF-κB), a key regulator of immune and inflammatory responses, plays important roles in diabetes-induced microvascular complications including diabetic retinopathy (DR). Thrombin activates NF-κB through protease-activated receptor (PAR)-1, a member of the G-protein-coupled receptor (GPCR) superfamily, and contributes to DR. The current study is to uncover the roles of microRNA (miRNA) in thrombin-induced NF-κB activation and retinal endothelial functions. METHODS: Target prediction was performed using the TargetScan algorithm. Predicted target was experimentally validated by luciferase reporter assays. Human retinal endothelial cells (HRECs) were transfected with miRNA mimics or antimiRs and treated with thrombin. Expression levels of miR-146 and related protein-coding genes were analyzed by quantitative (q)RT-PCR. Functional changes of HRECs were analyzed by leukocyte adhesion assays. RESULTS: We identified that caspase-recruitment domain (CARD)-containing protein 10 (CARD10), an essential scaffold/adaptor protein of GPCR-mediated NF-κB activation pathway, is a direct target of miR-146. Thrombin treatment resulted in NF-κB-dependent upregulation of miR-146 in HRECs; while transfection of miR-146 mimics resulted in significant downregulation of CARD10 and prevented thrombin-induced NF-κB activation, suggest that a negative feedback regulation of miR-146 on thrombin-induced NF-κB through targeting CARD10. Furthermore, overexpression of miR-146 prevented thrombin-induced increased leukocyte adhesion to HRECs. CONCLUSIONS: We uncovered a novel negative feedback regulatory mechanism on thrombin-induced GPCR-mediated NF-κB activation by miR-146. In combination with the negative feedback regulation of miR-146 on the IL-1R/toll-like receptor (TLR)-mediated NF-κB activation in RECs that we reported previously, our results underscore a pivotal, negative regulatory role of miR-146 on multiple NF-κB activation pathways and related inflammatory processes in DR.

Bioactive lysophosphatidylcholine 16:0 and 18:0 are elevated in lungs of asthmatic subjects.

PURPOSE: Asthma is a chronic inflammatory disease of the airways, and is associated with upregulation of phospholipase A2 (PLA2), the enzyme that hydrolyzes phosphatidylcholine, producing lysophosphatidylcholine (LPC) and free fatty acids. LPC is a lipid mediator with known pro-inflammatory and pro-atherogenic properties, and is believed to be a critical factor in cardiovascular diseases. We postulate that asthmatic subjects have an elevated content of LPC in the lung lining fluids. METHODS: Eight non-asthmatic controls and seven asthmatic subjects were recruited for broncho-alveolar lavage fluids (BALF) collection for analysis of LPC by high performance liquid chromatography-tandem mass spectrometry. RESULTS: LPC16:0 and LPC18:0 were significantly elevated in the BALF of asthmatics with impaired lung function characteristic of moderate asthma, but not mild asthma. The increased LPC content in BALF was accompanied by increased PLA2 activity. Furthermore, qRT-PCR analysis of the BALF cell fraction indicated increased secretory PLA2-X (sPLA2-X). CONCLUSIONS: The increased LPC content in the lung lining fluids is a potential critical lipid mediator in the initiation and/or progression of airway epithelial injury in asthma.

Phytoestrogen genistein up-regulates endothelial nitric oxide synthase expression via activation of cAMP response element-binding protein in human aortic endothelial cells.

We previously reported that genistein, a phytoestrogen, up-regulates endothelial nitric oxide synthase (eNOS) and prevents hypertension in rats that are independent of estrogen signaling machinery. However, how genistein regulates eNOS expression is unknown. In the present study, we show that genistein enhanced eNOS expression and NO synthesis in primary human aortic endothelial cells. Inhibition of extracellular signal regulated kinase, phosphoinositol-3 kinase, or protein kinase C did not affect genistein-enhanced eNOS expression and NO synthesis. However, chemical inhibition of protein kinase A (PKA) or adenoviral transfer of the specific endogenous PKA inhibitor gene completely abolished PKA activity and genistein-stimulated eNOS expression and NO production. Accordingly, genistein induced PKA activity and subsequent phosphorylation of cAMP response element (CRE)-binding protein (CREB) at Ser133. Suppression of CREB by small interfering RNA transfection abolished genistein-enhanced eNOS expression and NO production. Consistently, deletion of the CRE site within human eNOS promoter eliminated genistein-stimulated eNOS promoter activity. These findings provide the first evidence to our knowledge that genistein may play a beneficial role in vascular function through targeting the PKA/CREB/eNOS/NO signaling pathway.

Genistein induces pancreatic beta-cell proliferation through activation of multiple signaling pathways and prevents insulin-deficient diabetes in mice.

Genistein, a flavonoid in legumes and some herbal medicines, has various biological actions. However, studies on whether genistein has an effect on pancreatic beta-cell function are very limited. In the present study, we investigated the effect of genistein on beta-cell proliferation and cellular signaling related to this effect and further determined its antidiabetic potential in insulin-deficient diabetic mice. Genistein induced both INS1 and human islet beta-cell proliferation after 24 h of incubation, with 5 mum genistein inducing a maximal 27% increase. The effect of genistein on beta-cell proliferation was neither dependent on estrogen receptors nor shared by 17beta-estradiol or a host of structurally related flavonoid compounds. Pharmacological or molecular intervention of protein kinase A (PKA) or ERK1/2 completely abolished genistein-stimulated beta-cell proliferation, suggesting that both molecules are essential for genistein action. Consistent with its effect on cell proliferation, genistein induced cAMP/PKA signaling and subsequent phosphorylation of ERK1/2 in both INS1 cells and human islets. Furthermore, genistein induced protein expression of cyclin D1, a major cell-cycle regulator essential for beta-cell growth. Dietary intake of genistein significantly improved hyperglycemia, glucose tolerance, and blood insulin levels in streptozotocin-induced diabetic mice, concomitant with improved islet beta-cell proliferation, survival, and mass. These results demonstrate that genistein may be a natural antidiabetic agent by directly modulating pancreatic beta-cell function via activation of the cAMP/PKA-dependent ERK1/2 signaling pathway.

Phosphorylation of GTP dissociation inhibitor by PKA negatively regulates RhoA.

The cAMP-PKA cascade is a recognized signaling pathway important in inhibition of inflammatory injury events such as endothelial permeability and leucocyte trafficking, and a critical target of regulation is believed to be inhibition of Rho proteins. Here, we hypothesize that PKA directly phosphorylates GTP dissociation inhibitor (GDI) to negatively regulate Rho activity. Amino acid analysis of GDIalpha showed two potential protein kinase A (PKA) phosphorylation motifs, Ser(174) and Thr(182). Using in vitro kinase assay and mass spectrometry, we found that the purified PKA catalytic subunit phosphorylated GDIalpha-GST fusion protein and PKA motif-containing GDIalpha peptide at Ser(174), but not Thr(182). Transfection of COS-7 cells with mutated full-length GDIalpha at Ser(174) to Ala(174) (GDIalpha-Ser(174A)) abrogated the ability of cAMP to phosphorylate GDIalpha. However, mutation of Thr(182) to Ala(182) (GDIalpha-Thr(182A)) did not abrogate, and cAMP increased phosphorylation of GDIalpha to a similar extent as wild-type GDIalpha transfectants. The mutant GDIalpha-Ser(174A), but not GDIalpha-Thr(182A), was unable to prevent cAMP-mediated inhibition of Rho-dependent serum-response element reporter activity. Furthermore, the mutant GDIalpha-Ser(174A) was unable to prevent the thrombin-induced RhoA activation. Coprecipitation studies indicated that neither mutation of the PKA consensus sites nor phosphorylation alter GDIalpha binding with RhoA, suggesting that phosphorylation of Ser(174) regulated preformed GDIalpha-RhoA complexes. The findings provide strong support that the selective phosphorylation at Ser(174) by PKA is a signaling pathway in the negative regulation of RhoA activity and therefore could be a potential protective mechanism for inflammatory injury.

Inhibition of endothelial barrier dysfunction by P21-activated kinase-1.

We investigated the activity of P21-activated kinase-1 (Pak1) on myosin light chain phosphorylation and on thrombin-induced barrier dysfunction in human endothelial cells (HMEC). HMEC were infected with recombinant adenoviruses that express constitutively active Pak1, LacZ, wild-type, and a mutant myosin regulatory light chain, mMLC20 (Thr18Ala, Ser19Ala). Expression of the recombinant Pak1 mediated by adenovirus in HMEC was regulated. Active Pak1 induced dephosphorylation of MLC20 in HMEC, but not in smooth muscle cells. Active Pak1 significantly inhibited thrombin-induced endothelial barrier dysfunction. Expression of the unphosphorylatable MLC20 also inhibited thrombin-induced endothelial barrier dysfunction. Constitutively active Pak1 associated with phosphatase 2A and induced a post-translational modification of the phosphatase. Our data provide novel evidence indicating that Pak1 regulates endothelial barrier function through activation of phosphatase 2A.

Lysophosphatidylcholine impairs endothelial barrier function through the G protein-coupled receptor GPR4.

Abundant evidence indicates that lysophosphatidylcholine (LPC) is proinflammatory and atherogenic. In the vascular endothelium, LPC increases permeability and expression of proinflammatory molecules such as adhesion molecules and cytokines. Yet, mechanisms by which LPC mediates these activities remain unclear and controversial. Recent evidence implicates involvement of a novel subfamily of G protein-coupled receptors (GPR4, G2A, OGR1, and TDAG8) that are sensitive to lysolipids and protons. We previously reported that one of these receptors, GPR4, is selectively expressed by a variety of endothelial cells and therefore hypothesize that the LPC-stimulated endothelial barrier dysfunction is mediated through GPR4. We developed a peptide Ab against GPR4 that detected GPR4 expression in transfected COS 7 cells and endogenous GPR4 expression in endothelial cells by Western blot. Endothelial cells infected with a retrovirus containing small interference RNA (siRNA) to GPR4 resulted in 40-50% decreased GPR4 expression, which corresponded with partial prevention of the LPC-induced 1) decrease in transendothelial resistance, 2) stress fiber formation, and 3) activation of RhoA. Furthermore, coexpression of the siRNA-GPR4 with a siRNA-resistant mutant GPR4 fully restored the LPC-induced resistance decrease. However, extracellular pH of <7.4 did not alter baseline or LPC-stimulated resistances. The results provide strong evidence that the LPC-mediated endothelial barrier dysfunction is regulated by endogenous GPR4 in endothelial cells and suggest that GPR4 may play a critical role in the inflammatory responses activated by LPC.