ERK5 Protein Promotes, whereas MEK1 Protein Differentially Regulates, the Toll-like Receptor 2 Protein-dependent Activation of Human Endothelial Cells and Monocytes

Endothelial cell (EC) Toll-like receptor 2 (TLR2) activation up-regulates the expression of inflammatory mediators and of TLR2 itself and modulates important endothelial functions, including coagulation and permeability. We defined TLR2 signaling pathways in EC and tested the hypothesis that TLR2 signaling differs in EC and monocytes. We found that ERK5, heretofore unrecognized as mediating TLR2 activation in any cell type, is a central mediator of TLR2-dependent inflammatory signaling in human umbilical vein endothelial cells, primary human lung microvascular EC, and human monocytes. Additionally, we observed that, although MEK1 negatively regulates TLR2 signaling in EC, MEK1 promotes TLR2 signaling in monocytes. We also noted that activation of TLR2 led to the up-regulation of intracellularly expressed TLR2 and inflammatory mediators via NF-κB, JNK, and p38-MAPK. Finally, we found that p38-MAPK, JNK, ERK5, and NF-κB promote the attachment of human neutrophils to lung microvascular EC that were pretreated with TLR2 agonists. This study newly identifies ERK5 as a key regulator of TLR2 signaling in EC and monocytes and indicates that there are fundamental differences in TLR signaling pathways between EC and monocytes.     

弱激光对脂质体介导的血管平滑肌细胞基因转染的影响

本研究采用阳离子脂质体介导外源基因转染体外培养的兔血管平滑肌细胞（ＳＭＣ）,在基因转染过程中给予激光照射,用细胞化学染色方法测定基因转染阳性率。结果显示：用５１０．６ｎｍ激光于基因转染前,以功率密度１ｍｗ／ｃｍ２,能量密度２、４、６Ｊ／ｃｍ２和５ｍＷ／ｃｍ２,４、６Ｊ／ｃｍ２;及１０ｍＷ／ｃｍ２,２Ｊ／ｃｍ２进行照射均能显著提高基因转染率（ｐ＜０．０５）;于基因转染后即刻以功率密度１ｍＷ／ｃｍ２、能量密度２Ｊ／ｃｍ２和５ｍＷ／ｃｍ２、６Ｊ／ｃｍ２照射也能提高基因转染率（ｐ＜０．０５）。而用６２７．８ｎｍ激光照射对基因转染率无显著影响     

Steroid-sensitive Gene 1 Is a Novel Cyclic GMP-dependent Protein Kinase I Substrate in Vascular Smooth Muscle Cells

NO, via its second messenger cGMP, activates protein kinase GI (PKGI) to induce vascular smooth muscle cell relaxation. The mechanisms by which PKGI kinase activity regulates cardiovascular function remain incompletely understood. Therefore, to identify novel protein kinase G substrates in vascular cells, a λ phage coronary artery smooth muscle cell library was constructed and screened for phosphorylation by PKGI. The screen identified steroid-sensitive gene 1 (SSG1), which harbors several predicted PKGI phosphorylation sites. We observed direct and cGMP-regulated interaction between PKGI and SSG1. In cultured vascular smooth muscle cells, both the NO donor S-nitrosocysteine and atrial natriuretic peptide induced SSG1 phosphorylation, and mutation of SSG1 at each of the two predicted PKGI phosphorylation sites completely abolished its basal phosphorylation by PKGI. We detected high SSG1 expression in cardiovascular tissues. Finally, we found that activation of PKGI with cGMP regulated SSG1 intracellular distribution.     

Retinoic acid-induced gene-I (RIG-I) associates with nucleotide-binding oligomerization domain-2 (NOD2) to negatively regulate inflammatory signaling

Cytoplasmic caspase recruiting domain (CARD)-containing molecules often function in the induction of potent antimicrobial responses in order to protect mammalian cells from invading pathogens. Retinoic acid-induced gene-I (RIG-I) and nucleotide binding oligomerization domain 2 (NOD2) serve as key factors in the detection of viral and bacterial pathogens, and in the subsequent initiation of innate immune signals to combat infection. RIG-I and NOD2 share striking similarities in their cellular localization, both localize to membrane ruffles in non-polarized epithelial cells and both exhibit a close association with the junctional complex of polarized epithelia. Here we show that RIG-I and NOD2 not only colocalize to cellular ruffles and cell-cell junctions, but that they also form a direct interaction that is mediated by the CARDs of RIG-I and multiple regions of NOD2. Moreover, we show that RIG-I negatively regulates ligand-induced nuclear factor-κB (NF-κB) signaling mediated by NOD2, and that NOD2 negatively regulates type I interferon induction by RIG-I. We also show that the three main Crohn disease-associated mutants of NOD2 (1007fs, R702W, G908R) form an interaction with RIG-I and negatively regulate its signaling to a greater extent than wild-type NOD2. Our results show that in addition to their role in innate immune recognition, RIG-I and NOD2 form a direct interaction at actin-enriched sites within cells and suggest that this interaction may impact RIG-I- and NOD2-dependent innate immune signaling.     

The Prodomain-bound Form of Bone Morphogenetic Protein 10 Is Biologically Active on Endothelial Cells

BMP10 is highly expressed in the developing heart and plays essential roles in cardiogenesis. BMP10 deletion in mice results in embryonic lethality because of impaired cardiac development. In adults, BMP10 expression is restricted to the right atrium, though ventricular hypertrophy is accompanied by increased BMP10 expression in a rat hypertension model. However, reports of BMP10 activity in the circulation are inconclusive. In particular, it is not known whether in vivo secreted BMP10 is active or whether additional factors are required to achieve its bioactivity. It has been shown that high-affinity binding of the BMP10 prodomain to the mature ligand inhibits BMP10 signaling activity in C2C12 cells, and it was proposed that prodomain-bound BMP10 (pBMP10) complex is latent. In this study, we demonstrated that the BMP10 prodomain did not inhibit BMP10 signaling activity in multiple endothelial cells, and that recombinant human pBMP10 complex, expressed in mammalian cells and purified under native conditions, was fully active. In addition, both BMP10 in human plasma and BMP10 secreted from the mouse right atrium were fully active. Finally, we confirmed that active BMP10 secreted from mouse right atrium was in the prodomain-bound form. Our data suggest that circulating BMP10 in adults is fully active and that the reported vascular quiescence function of BMP10 in vivo is due to the direct activity of pBMP10 and does not require an additional activation step. Moreover, being an active ligand, recombinant pBMP10 may have therapeutic potential as an endothelial-selective BMP ligand, in conditions characterized by loss of BMP9/10 signaling.     

Curcumin suppresses T cell activation by blocking Ca2+ mobilization and nuclear factor of activated T cells (NFAT) activation

Curcumin is the active ingredient of the spice turmeric and has been shown to have a number of pharmacologic and therapeutic activities including antioxidant, anti-microbial, anti-inflammatory, and anti-carcinogenic properties. The anti-inflammatory effects of curcumin have primarily been attributed to its inhibitory effect on NF-κB activity due to redox regulation. In this study, we show that curcumin is an immunosuppressive phytochemical that blocks T cell-activation-induced Ca(2+) mobilization with IC(50) = ～12.5 μM and thereby prevents NFAT activation and NFAT-regulated cytokine expression. This finding provides a new mechanism for curcumin-mediated anti-inflammatory and immunosuppressive function. We also show that curcumin can synergize with CsA to enhance immunosuppressive activity because of different inhibitory mechanisms. Furthermore, because Ca(2+) is also the secondary messenger crucial for the TCR-induced NF-κB signaling pathway, our finding also provides another mechanism by which curcumin suppresses NF-κB activation.     

Repression of Smooth Muscle Differentiation by a Novel High Mobility Group Box-containing Protein,HMG2L1

The molecular mechanisms regulating smooth muscle-specific gene expression during smooth muscle development are poorly understood. Myocardin is an extraordinarily powerful cofactor of serum response factor (SRF) that stimulates expression of smooth muscle-specific genes. In an effort to search for proteins that regulate myocardin function, we identified a novel HMG box-containing protein HMG2L1 (high mobility group 2 like 1). We found that HMG2L1 expression is correlated with the smooth muscle cell (SMC) synthetic phenotype. Overexpression of HMG2L1 in SMCs down-regulated smooth muscle marker expression. Conversely, depletion of endogenous HMG2L1 in SMCs increases smooth muscle-specific gene expression. Furthermore, we found HMG2L1 specifically abrogates myocardin-induced activation of smooth muscle-specific genes. By GST pulldown assays, the interaction domains between HMG2L1 and myocardin were mapped to the N termini of each of the proteins. Finally, we demonstrated that HMG2L1 abrogates myocardin function through disrupting its binding to SRF and abolishing SRF-myocardin complex binding to the promoters of smooth muscle-specific genes. This study provides the first evidence of this novel HMG2L1 molecule playing an important role in attenuating smooth muscle differentiation.     

KRIT1 Protein Depletion Modifies Endothelial Cell Behavior via Increased Vascular Endothelial Growth Factor (VEGF) Signaling

Disruption of endothelial cell-cell contact is a key event in many cardiovascular diseases and a characteristic of pathologically activated vascular endothelium. The CCM (cerebral cavernous malformation) family of proteins (KRIT1 (Krev-interaction trapped 1), PDCD10, and CCM2) are critical regulators of endothelial cell-cell contact and vascular homeostasis. Here we show novel regulation of vascular endothelial growth factor (VEGF) signaling in KRIT1-depleted endothelial cells. Loss of KRIT1 and PDCD10, but not CCM2, increases nuclear β-catenin signaling and up-regulates VEGF-A protein expression. In KRIT1-depleted cells, increased VEGF-A levels led to increased VEGF receptor 2 (VEGFR2) activation and subsequent alteration of cytoskeletal organization, migration, and barrier function and to in vivo endothelial permeability in KRIT1-deficient animals. VEGFR2 activation also increases β-catenin phosphorylation but is only partially responsible for KRIT1 depletion-dependent disruption of cell-cell contacts. Thus, VEGF signaling contributes to modifying endothelial function in KRIT1-deficient cells and microvessel permeability in Krit1^+/− mice; however, VEGF signaling is likely not the only contributor to disrupted endothelial cell-cell contacts in the absence of KRIT1.