The tight junction protein ZO-2 and Janus kinase 1 mediate intercellular communications in vascular smooth muscle cells.

Recent evidence points to a multifunctional role of ZO-2, the tight junction protein of the MAGUK (membrane-associated guanylate kinase-like) family. Though ZO-2 has been found in cell types lacking tight junction structures, such as vascular smooth muscle cells (VSMC), little is known about ZO-2 function in these cells. We provide evidence that ZO-2 mediates specific homotypic cell-to-cell contacts between VSMC. Using mass spectrometry we found that ZO-2 is associated with the non-receptor tyrosine kinase Jak1. By generating specific ZO-2 constructs we further found that the N-terminal fragment of ZO-2 molecule is responsible for this interaction. Adenovirus-based expression of Jak1 inactive mutant demonstrated that Jak1 mediates ZO-2 tyrosine phosphorylation. By means of RNA silencing, expression of Jak1 mutant form and fluorescently labeled ZO-2 fusion protein we further specified that active Jak1, but not Jak1 inactive mutant, mediates ZO-2 localization to the sites of intercellular contacts. We identified the urokinase receptor uPAR as a pre-requisite for these cellular events. Functional requirement of the revealed signaling complex for VSMC network formation was confirmed in experiments using Matrigel and in contraction assay. Our findings imply involvement of the ZO-2 tight junction independent signaling complex containing Jak1 and uPAR in VSMC intercellular communications. This mechanism may contribute to vascular remodeling in occlusive cardiovascular diseases and in arteriogenesis.

The tight junction protein ZO-2 mediates proliferation of vascular smooth muscle cells via regulation of Stat1.

AIMS: Recent evidence suggests that the zonula occludens protein 2 (ZO-2) might have additional cellular functions, beyond regulation of paracellular permeability of epithelial and endothelial cells. Deregulation of ZO-2 in response to ischaemia, hypertensive stress, and vascular injury implies its involvement in cardiovascular disorders, most likely via regulating the functional behaviour of vascular smooth muscle cells (VSMC). However, a role of ZO-2 in VSMC biology has yet to be established. Our study was designed to understand the specific functions of ZO-2 in human VSMC. METHODS AND RESULTS: The expression of ZO-2 and Stat1 upon vascular injury was studied using ex vivo organ culture of coronary arteries combined with immunohistochemistry. ZO-2 silencing in human primary VSMC was achieved by means of lentiviral gene transfer. Cell proliferation was assessed by analysing DNA synthesis and by cell counting. Stat1 expression was examined using immunoblotting, immunocytochemistry, TaqMan, and fluorescence activated cell sorting (FACS) analysis. Functional relevance of Stat1 up-regulation was studied using a Stat1 promoter-luciferase reporter assay and intracellular microinjections of a Stat1 specific antibody. ZO-2 was highly expressed in the media and neointima of dilated but not of control arteries, whereas expression of the transcription factor Stat1 was inversely regulated upon injury. Analysis of VSMC with down-regulated ZO-2 revealed increased expression of Stat1 in these cells, whereas Stat1 phosphorylation was not affected. Stat1 up-regulation in VSMC with ZO-2 silencing resulted in a coordinate activation of Stat1-specific genes and consequently led to inhibition of cell proliferation. This effect was restored by microinjection of a Stat1 neutralising antibody. CONCLUSION: Our data suggest that the tight junction protein ZO-2 is involved in regulation of VSMC growth control upon vascular injury that is mediated by the transcription factor Stat1. Our findings point to a novel function of ZO-2 in VSMC and implicate ZO-2 as a novel important molecular target in pathological states of vascular remodelling in cardiovascular diseases.

Urokinase induces survival or pro-apoptotic signals in human mesangial cells depending on the apoptotic stimulus.

Deregulated apoptosis of MCs (mesangial cells) is associated with a number of kidney diseases including end-stage diabetic nephropathy. Cell death by apoptosis is a tightly orchestrated event, whose mechanisms are not completely defined. In the present study we show that the uPA (urokinase-type plasminogen activator)/uPAR (uPA receptor) system can initiate both cell survival and pro-apoptotic signals in human MCs in response to different apoptotic stimuli. uPA abrogated MC apoptosis induced by serum withdrawal conditions and enhanced apoptosis initiated in MCs by high glucose. Effects of uPA were independent of its proteolytic activity and required uPAR for both pro- and anti-apoptotic effects. Studies on the uPAR interactome provide evidence that the opposing effects of uPA were directed via different uPAR-interacting transmembrane partners. Exposure of MCs to RGD (Arg-Gly-Asp) peptide led to abrogation of the anti-apoptotic effect of uPA, which implies involvement of integrins in this process. A pro-apoptotic effect of uPA under high-glucose conditions was mediated via association of uPAR and the cation-independent M6P (mannose-6-phosphate)/IGF2R (insulin-like growth factor 2 receptor). Both receptors were co-precipitated and co-localized in MCs. Studies on the underlying signalling indicate that the ERK1/2 (extracellular-signal-regulated kinase 1/2), Akt and BAD (Bcl-2/Bcl-X(L)-antagonist, causing cell death) protein were involved in regulation of apoptosis by uPA in MCs. M6P/IGF2R mediated BAD perinuclear localization during apoptosis initiated by uPA and high glucose. In conclusion, we provide evidence that, in MCs, the uPA/uPAR system regulates survival/apoptosis processes in a stimulus-specific fashion via a mitochondria-dependent mechanism and that BAD protein serves as a downstream molecule.

Urokinase-induced activation of the gp130/Tyk2/Stat3 pathway mediates a pro-inflammatory effect in human mesangial cells via expression of the anaphylatoxin C5a receptor.

Glomerular mesangial cells (MCs) are central to the pathogenesis of progressive glomeruli-associated renal diseases. However, molecular mechanisms underlying changes in MC functions still remain poorly understood. Here, we show that in MCs, the urokinase-type plasminogen activator (uPA) induces, via its specific receptor (uPAR, CD87), upregulated expression of the complement anaphylatoxin C5a receptor (C5aR, CD88), and modulates C5a-dependent functional responses. This effect is mediated via the interaction of the uPA-specific receptor (uPAR, CD87) and gp130, a signal transducing subunit of the receptor complexes for the IL-6 cytokine family. The Janus kinase Tyk2 and the transcription factor Stat3 serve as downstream components in the signaling cascade resulting in upregulation of C5aR expression. In vivo, expression of C5aR and uPAR was increased in the mesangium of wild-type mice in a lipopolysaccharide (LPS)-induced model of inflammation, whereas in uPAR(-/-) animals C5aR expression remained unchanged. This is the first demonstration in vitro and in vivo that uPA acts in MCs as a modulator of immune responses via control of immune-competent receptors. The data suggest a novel role for uPA/uPAR in glomeruli-associated renal failure via a signaling cross-talk between the fibrinolytic and immune systems.

Monocyte-expressed urokinase inhibits vascular smooth muscle cell growth by activating Stat1.

After vascular injury, a remodeling process occurs that features leukocyte migration and infiltration. Loss of endothelial integrity allows the leukocytes to interact with vascular smooth muscle cells (VSMCs) and to elicit "marching orders"; however, the signaling processes are poorly understood. We found that human monocytes inhibit VSMC proliferation and induce a migratory potential. The monocytes signal the VSMCs through the urokinase-type plasminogen activator (uPA). The VSMC uPA receptor (uPAR) receives the signal and activates the transcription factor Stat1 that, in turn, mediates the antiproliferative effects. These results provide the first evidence that monocytes signal VSMCs by mechanisms involving the fibrinolytic system, and they imply an important link between the uPA/uPAR-related signaling machinery and human vascular disease.

Urokinase-induced migration of human vascular smooth muscle cells requires coupling of the small GTPases RhoA and Rac1 to the Tyk2/PI3-K signalling pathway.

Urokinase-type plasminogen activator (uPA) facilitates cell migration by localizing proteolisys on the cell surface and by inducing intracellular signalling pathways. In human vascular smooth muscle cell (VSMC) uPA stimulates migration via the uPA receptor (uPAR) signalling complex containing the Janus kinase Tyk2 and phosphatidylinositol 3-kinase (PI3-K). We report that active GTP-bound forms of small GTPases RhoA and Rac1, but not Cdc42, are directly associated with Tyk2 and PI3-K in an uPA/uPAR-dependent fashion. Endogenous RhoA, but not Rac1 or Cdc42, was significantly activated in response to uPA. RhoA activation was abolished by cell treatment with two unrelated, structurally distinct, specific inhibitors of PI3-K, wortmannin, and LY294002. Downstream of RhoA, phosphorylation of myosin light chain (MLC) was dramatically upregulated by uPA in a Rho kinase- and PI3-K-dependent manner. Thus, selective Rho kinase inhibitor Y27632 and PI3-K inhibitors wortmannin and LY294002 prevented the uPA-induced stimulation of MLC phosphorylation. Rho kinase inhibition also decreased uPA-stimulated VSMC migration as observed in a Boyden chamber. VSMC immunocytochemical staining demonstrated redistribution of RhoA and Rac1 active forms to the newly formed leading edge of migrating cell. VSMC microinjection with antibodies to either Rho or Rac1 decreased uPA-stimulated cell migration, indicating the involvement of both GTPases in the migration process. Our results provide evidence that the small GTPases RhoA and Rac1, together with Rho kinase, are necessary to mediate the uPA/uPAR-directed migration via the Tyk2/PI3-K signalling complex in human VSMC.