Fluid shear stress inhibits TNF-mediated JNK activation via MEK5-BMK1 in endothelial cells.

Steady laminar blood flow protects vessels from atherosclerosis. We showed that flow decreased tumor necrosis factor-alpha (TNF)-mediated VCAM1 expression in endothelial cells (EC) by inhibiting JNK. Here, we determined the relative roles of MEK1, MEK5 and their downstream kinases ERK1/2 and BMK1 (ERK5) in flow-mediated inhibition of JNK activation. Steady laminar flow (shear stress=12dyn/cm(2)) increased BMK1 and ERK1/2 activity in EC. Pre-exposing EC for 10min to flow inhibited TNF activation of JNK by 58%. A key role for BMK1, but not ERK1/2 was shown. (1) Incubation of EC with PD184352, at concentrations that blocked ERK1/2, but not BMK1, had no effect on flow inhibition of TNF-mediated JNK activation. (2) BIX02188, a MEK5-selective inhibitor, completely reversed the inhibitory effects of flow. These findings indicate that flow inhibits TNF-mediated signaling events in EC by a mechanism dependent on activation of MEK5-BMK1, but not MEK1-ERK1/2. These results support a key role for the MEK5-BMK1 signaling pathway in the atheroprotective effects of blood flow.

GIT1 mediates HDAC5 activation by angiotensin II in vascular smooth muscle cells.

OBJECTIVE: The G protein-coupled receptor (GPCR)-kinase2 interacting protein1 (GIT1) is a scaffold protein involved in angiotensin II (Ang II) signaling. Histone deacetylase-5 (HDAC5) has emerged as an important substrate of calcium/calmodulin-dependent protein kinase II (CamK II) in GPCR signaling. Here we investigated the hypothesis that Ang II-mediated vascular smooth muscle cell (VSMC) gene transcription involves GIT1-CamK II-dependent phosphorylation of HDAC5. METHODS AND RESULTS: Ang II rapidly stimulated phosphorylation of HDAC5 at Ser498 in VSMCs. Knockdown of GIT1 significantly decreased HDAC5 phosphorylation induced by Ang II. The involvement of Src, phospholipase gamma (PLCgamma), and CamK II in GIT1-mediated HDAC5 phosphorylation was demonstrated. The association of GIT1 and CamK II was constitutive but increased after stimulation with Ang II. Moreover, the interaction of GIT1 and CamK II through the ARF GTPase-activating protein (ARF-GAP) and coiled-coil domains of GIT1 was essential for the phosphorylation of HDAC5. Finally, knockdown of GIT1 decreased myocyte enhancer factor 2 transcriptional activity induced by Ang II. CONCLUSIONS: This study identifies a novel function for GIT1 as a mediator of Ang II-induced VSMC gene transcription via a Src-PLCgamma-CamK II-HDAC5 signaling pathway.

Crosstalk coregulation mechanisms of G protein-coupled receptors and receptor tyrosine kinases.

G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) are transmembrane receptors that initiate intracellular signaling cascades in response to a diverse array of ligands. Recent studies have shown that signal transduction initiated by GPCRs and RTKs is not organized in distinct signaling cassettes where receptor activation leads to cell division and gene transcription in a linear manner. In fact, signal integration and diversification arises from a complex network involving crosscommunication between separate signaling units. Several different styles of crosstalk between GPCR- and RTK-initiated pathways exist, with GPCRs or components of GPCR-induced pathways being either upstream or downstream of RTKs. Activation of GPCRs sometimes results in a phenomenon known as "transactivation" of RTKs, which leads to the recruitment of scaffold proteins, such as Shc, Grb2, and Sos in addition to mitogen-activated protein kinase activation. In other cases, RTKs use different components of GPCR-mediated signaling, such as beta-arrestin, G protein-receptor kinases, and regulator of G protein signaling to integrate signaling pathways. This chapter outlines some of the more common mechanisms used by both GPCRs and RTKs to initiate intracellular crosstalk, thereby creating a complex signaling network that is important to normal development.

Vitronectin: a possible determinant of adenovirus type 19 tropism for human corneal epithelium.

PURPOSE: Adenoviruses typically demonstrate specific tissue tropisms, as in the association of Ad19 with epidemic keratoconjunctivitis. We sought to determine factors that might influence the apparent tropism of Ad19 for the cornea. DESIGN: Laboratory investigation. METHODS: Adenovirus serotypes Ad2, 5, 9, 10, 11, 13, and 19 were compared for their capacity to replicate in human corneal epithelial cells (HCECs) in culture. Organotypically cultured human corneas were infected with Ad19 or Ad2, and viral titers were compared after 7 days. Replication of both viruses was compared in HCECs cultured on various extracellular matrices. Western blot analysis and immunohistochemistry were applied to human donor corneas and HCECs. RESULTS: One week after infection of HCEC monolayer cultures, Ad2 titers were significantly higher than any of the other viruses tested (P <.05). In organotypic corneal cultures, Ad19 titers were significantly higher than Ad2 (P = .0003). Ad2 replication in HCECs equaled or exceeded that of Ad19 on all extracellular matrices except vitronectin, where Ad2 replication was reduced and Ad19 replication enhanced (P <.0001). Vitronectin was detected by immunohistochemistry within the corneal epithelial basement membranes of human donor corneas. Increased alpha(v) integrin expression and greater tyrosine kinase phosphorylation in HCECs cultured on vitronectin were demonstrated by Western blot analysis. CONCLUSIONS: In vitro, vitronectin enhances growth of Ad19, possibly by up-regulation of receptor alpha(v) integrins and increased activity of tyrosine kinases necessary for adenoviral internalization. We hypothesize that differential tissue tropisms for adenoviruses may derive in part from tissue-specific extracellular matrix expression.

GIT1 mediates thrombin signaling in endothelial cells: role in turnover of RhoA-type focal adhesions.

Thrombin mediates changes in endothelial barrier function and increases endothelial permeability. A feature of thrombin-enhanced endothelial hyperpermeability is contraction of endothelial cells (ECs), accompanied by formation of focal adhesions (FAs). Recently, a G protein-coupled receptor kinase-interacting protein, GIT1, was shown to regulate FA disassembly. We hypothesized that GIT1 modulates thrombin-induced changes in FAs. In human umbilical vein ECs (HUVECs), thrombin recruited GIT1 to FAs, where GIT1 colocalized with FAK and vinculin. Recruitment of GIT1 to FAs was dependent on activation of the small GTPase RhoA, and Rho kinase, as demonstrated by adenoviral transfection of dominant-negative RhoA and treatment with Y-27632. Thrombin stimulated GIT1 tyrosine phosphorylation with a time course similar to FAK phosphorylation in a Rho kinase- and Src-dependent manner. Depletion of GIT1 with antisense GIT1 oligonucleotides had no effect on basal cell morphology, but increased cell rounding and contraction of HUVECs, increased FA formation, and increased FAK tyrosine phosphorylation in response to thrombin, concomitant with increased endothelial hyperpermeability. These data identify GIT1 as a novel mediator in agonist-dependent signaling in ECs, demonstrate that GIT1 is involved in cell shape changes, and suggest a role for GIT1 as a negative feedback regulator that augments recovery of cell contraction.

Corneal IL-8 expression following adenovirus infection is mediated by c-Src activation in human corneal fibroblasts.

Emerging evidence indicates that intracellular signaling cascades mediate entry of pathogenic adenoviruses into target host cells as well as some of the undesirable inflammatory responses to adenoviral gene vectors. We found that Ad19 infection of cultured human corneal fibroblasts induced IL-8 gene transcription independently of IL-1beta, TNF-alpha, and viral gene expression, suggesting that intracellular signaling events might mediate early inflammatory events in adenovirus keratitis. Heat but not UV light inactivation of the virus abrogated the effect of infection on IL-8 mRNA and protein levels, consistent with a viral binding-mediated mechanism. The tyrosine kinase inhibitor herbimycin blocked Ad19-induced IL-8 expression. Western blot analysis revealed tyrosine phosphorylation of the functionally related kinases c-Src and extracellular signal-regulated kinase (ERK) 1/2 in corneal fibroblasts within 15 min after infection. Respective inhibitors of these kinases, PP2 and PD98059, also blocked Ad19-induced IL-8 mRNA and protein expression. Application of inhibitors to Src and ERK kinase assays suggested an upstream relationship of c-Src to ERK. Finally, DNA microarray studies performed 1 h after Ad19 or mock infection of corneal fibroblasts in the presence or absence of the Src-specific inhibitor PP2 confirmed a relationship between c-Src and IL-8 expression in Ad19-infected corneal cells. c-Src may act as a global regulator of early proinflammatory host responses to Ad19 infection of the human cornea.

Activation of focal adhesion kinase in adenovirus-infected human corneal fibroblasts.

PURPOSE: Focal adhesion kinase (FAK), a nonreceptor protein tyrosine kinase with protean downstream influences on cell cycle regulation, cytoskeletal dynamics, and cell attachment, is activated by integrin binding and aggregation. Adenoviruses, including those associated with human keratitis, enter permissive cells by an integrin-mediated mechanism. Hence, a possible relationship between adenovirus infection and tyrosine phosphorylation of FAK in human corneal cells was explored. METHODS: Human corneal fibroblasts (HCFs) derived from donor corneas were infected for various periods with adenovirus type 19 (Ad19) or were mock infected with virus-free medium. Parallel experiments included echistatin, which is a snake venom disintegrin and a partial inhibitor of FAK. For immunoblot analysis, Triton-X-soluble and Triton-X-insoluble fractions were analyzed by SDS-PAGE and immunoblotted with phosphospecific antibodies. Expression of the interleukin (IL)-8 gene was analyzed by RT-PCR and ELISA. RESULTS: Ad19 infection of HCFs induced tyrosine phosphorylation of a protein at 125 kDa, which was evident within 15 minutes after infection and was established by immunoprecipitation to be FAK. Immunoblot with antibody to FAK tyrosine-397 confirmed phosphorylation of this key binding site for downstream signaling proteins. Immunoblot analysis further suggested a shift in the intracellular location of phosphorylated FAK from the cytosol (Triton-X-soluble cell lysate fraction) to the cytoskeleton (Triton-X-insoluble cell lysate pellet) on infection. Finally, treatment of HCFs with echistatin reduced virus-induced expression of the neutrophil chemotaxin IL-8, previously implicated in adenoviral pathogenesis. CONCLUSIONS: Ad19 infection of HCFs induces rapid phosphorylation of FAK, and a dramatic change in its intracellular distribution. Activation of FAK may contribute to the inflammatory response to adenovirus infection of the human cornea.

Innate immunity in th e cornea: a putative role for keratocytes in the chemokine response to viral infection of the human corneal stroma.

Existing evidence suggests that chemokine expression by virus-infected cells is a common response to viral infection. By such a mechanism, non-immunologic cells may participate in the generation of an early innate immune response to infection. In the absence of classic immunologic cells in the corneal stroma, keratocytes may play a similar role in the corneal responses to viral infection.