Original article
Activation of proMMP-2 and Src by HHV8 vGPCR in human pulmonary arterial endothelial cells

https://doi.org/10.1016/j.yjmcc.2006.08.004Get rights and content

Abstract

Idiopathic pulmonary arterial hypertension (iPAH) is associated with human herpesvirus 8 (HHV8) infection and demonstrates pathological angiogenesis similar to that observed with another HHV8-linked disease, namely Kaposi Sarcoma (KS). Importantly, the HHV8 encoded viral G-protein-coupled receptor (vGPCR) induces KS lesions in a murine model. Investigating the impact of vGPCR expression on the angiogenic activity of human pulmonary arterial endothelial cells (HPAEC) can yield insight into the pathobiology of HHV8-associated vascular disorders, particularly PAH. Cultured HPAECs were transduced with retroviral vectors carrying either control or vGPCR coding regions. vGPCR expression selectively activated matrix metalloproteinase (MMP)-2, a pivotal matrix modulating enzyme during angiogenesis. A membrane type 1 MMP (MT1-MMP) neutralizing antibody and the tissue inhibitor of metalloproteinases-2 (TIMP-2) independently blocked vGPCR-induced MMP-2 activation. vGPCR expression concordantly promoted MMP-2 activation by increasing MT1-MMP expression while decreasing TIMP-2 expression. vGPCR activated Src kinase as demonstrated by phosphorylation of Src and its substrate focal adhesion kinase (FAK). vGPCR promoted angiogenesis of HPAECs as demonstrated by a substantial increase in tubulogenesis in vitro. The Src inhibitors PP2 and SU6656 significantly diminished vGPCR-induced MMP-2 activation and tubulogenesis. Our findings indicate that vGPCR induces MMP-2 activation in HPAECs through regulation of MT1-MMP and TIMP-2 expression. vGPCR activates Src and inhibition of such activation abrogates proMMP-2 activation and in vitro angiogenesis induced by vGCPR. The current study implicates vGPCR as an etiological agent in iPAH and identifies Src and MMP-2 as potential therapeutic targets in HHV8 associated KS and iPAH.

Introduction

Human herpesvirus 8 (HHV8) is the etiologic agent of Kaposi's sarcoma (KS) with tropism primarily for endothelial cells (EC) and B lymphocytes [1]. One predominant feature of KS is dysregulated angiogenesis that has been associated with viral and cellular angiogenic factors expressed by HHV8-infected cells. Recently, HHV8 has been associated with idiopathic pulmonary arterial hypertension (iPAH), an angioproliferative disorder that exhibits plexogenic lesions that host HHV8 and resemble the cutaneous lesions existing in KS [2]. However attempts to confirm such correlation have not been able to demonstrate a clear association between HHV8 and iPAH [3], [4], [5]. In support of a putative role for HHV8 in PAH, AIDS-related PAH demonstrates KS-like lesions in the pulmonary vasculature, which occur at a rate of one in 200 patients afflicted with HIV, well above the rate of primary iPAH in the general population 1/2,000,000. Similarly, there is an elevated incidence of KS in patients with AIDS.

Several HHV8 latent gene products, such as v-Cyclin, possess EC transforming and angiogenic properties. Ironically, the only viral gene product that suffices to transform EC and initiate angiosarcoma in murine models happens to be a lytic phase protein, viral G-protein-coupled receptor (vGPCR) [6]. vGPCR is structurally and functionally most related to human chemokine receptor CXCR1 and CXCR2 [7]. Unlike endogenous chemokine receptors, vGPCR displays constitutive signaling activity. The concerted action of vGPCR signaling eventuates in the production of VEGF and results in EC transformation.

The angiogenic EC produces matrix metalloproteinases-2 and -9 (MMP) that modulate EC migration and vascular remodeling, two essential steps during angiogenesis [8]. A variety of angiogenic cues activate MMP-2 in EC. MMP-2 is expressed in its inactive form (proMMP-2, 72 kDa) and functional MMP-2 (active MMP-2, 64 and 62 kDa) is generated through cleavage of the prodomain from proMMP-2 by a host of proteolytic proteins. Accumulating evidence indicates a critical role of cell surface-associated membrane type 1 MMP (MT1-MMP) and tissue inhibitor of metalloproteinase 2 (TIMP-2) in the modulation of EC-derived MMP-2 activity and angiogenesis [9]. Early studies have revealed elevated MMP-2 activity in KS and inhibition of KS by endothelin receptor blockade, HIV protease inhibitors, or TIMP-2 overexpression correlates with suppressed MMP-2 activity in KS cells [10], [11], [12]. Interestingly, MMP-2 activity is also elevated in a rat PAH model and correlates with PAH severity [13]. In support of a critical role for MMP-2 in the pathogenesis of PAH, several agents that arrest PAH progression in animal models also concurrently suppress MMP-2 [14], [15].

The proto-oncogene Src kinase integrates signals from a myriad of cell surface receptors and regulate many fundamental cellular processes, including cell growth, survival, differentiation, and migration. Src exists in an autoinhibitory conformation in the absence of activating signals and is activated through autophosphorylation on tyrosine 418 [16]. In addition to its classical role in receptor tyrosine kinase signaling, Src transduces GPCR signals that regulate a host of cellular processes, at least in part through crosstalk with focal adhesion kinase (FAK) [17]. However, the exact role of Src activation by GPCRs in angiogenesis remains unknown.

Angioproliferative pathology and HHV8 infection in PAH together with vGPCR's angiogenic property prompted us to investigate modulation of MMP-2 activity and angiogenesis by vGPCR within the pulmonary endothelium. Using primary cultures of human pulmonary arterial endothelial cells (HPAEC), the current study demonstrates that vGPCR activates MMP-2 in an MT1-MMP-dependent manner and promotes angiogenesis in vitro. Furthermore, vGPCR activates Src and inhibition of Src abrogates proMMP-2 activation and in vitro angiogenesis induced by vGCPR.

Section snippets

Reagents and antibodies

The Protease Inhibitor Cocktail, the Phosphatase Inhibitor Cocktail 2 and Gelatin Type A were purchased from Sigma (St. Louis, MO). Fibrinogen, the Src-selective inhibitors PP2 and SU6656, and an MT1-MMP-specific antibody were purchased from Calbiochem (San Diego, CA). The human recombinant TNF-α protein was purchased from Peprotech (Rocky Hill, NJ). BD Biosciences (Frankline Lakes, NJ) provided growth factor reduced BD Basement Membrane Matrix. Santa Cruz (Santa Cruz, CA) provided Src and

MT1-MMP-dependent selective activation of proMMP-2 by vGPCR in HPAEC

HPAECs were selected as our experimental system to investigate potential modulation of gelatinase activity by vGPCR in the context of HHV8-associated vascular pathobiology in the lung, particularly iPAH. HPAECs were infected with the retroviral vectors either co-expressing vGPCR and GFP or expressing GFP alone as described in the Materials and methods section. Infection efficiency was monitored by visualizing GFP expressing cells, which revealed a comparable efficiency between pBABE and

Discussion

HHV8 causes the development of KS and is associated with iPAH [2]. vGPCR is the sole viral protein known to be capable of promoting pathological angiogenesis in vivo [6]. This study investigated modulation of EC-derived MMP-2 activity by vGPCR as well as the underlying mechanisms. Our findings indicate MT1-MMP-dependent MMP-2 activation by vGPCR in HPAECs. Furthermore, our results reveal Src activation by vGPCR and suggest Src activation mediates MMP-2 activation and angiogenesis in vitro.

Acknowledgments

This work was supported by NIH ES010046, HL063778, and HL083480 awarded to J.A.L. B.S. was supported by an NIH postdoctoral training grant T32HL007973.

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