Constitutive Activation of NF- (cid:1) B and Secretion of Interleukin-8 Induced by the G Protein-coupled Receptor of Kaposi’s Sarcoma-associated Herpesvirus Involve G (cid:2) 13 and RhoA*

The Kaposi’s sarcoma herpesvirus (KSHV) open reading frame 74 encodes a G protein-coupled receptor (GPCR) for chemokines. Exogenous expression of this constitutively active GPCR leads to cell transformation and vascular overgrowth characteristic of Kaposi’s sarcoma. We show here that expression of KSHV-GPCR in transfected cells results in constitutive transactivation of nuclear factor (cid:1) B (NF- (cid:1) B) and secretion of interleu-kin-8, and this response involves activation of G (cid:2) 13 and RhoA. The induced expression of a NF- (cid:1) B luciferase reporter was partially reduced by pertussis toxin and the G (cid:3)(cid:4) scavenger transducin, and enhanced by co-expres-sion of G (cid:2) 13 and to a lesser extent, G (cid:2) q . These results indicate coupling of KSHV-GPCR to multiple G proteins for NF- (cid:1) B activation. Expression of KSHV-GPCR led to stress fiber formation in NIH 3T3 cells. To examine the involvement of the G (cid:2) 13 -RhoA pathway in KSHV-GPCR- mediated NF- (cid:1) B activation, HeLa

membrane domain structure. Recent studies have demonstrated that chemokine receptors play important roles in lymphocyte homing, migration of phagocytes to inflammatory sites, and entry of HIV-1 into host cells (reviewed in Refs. 1 and 2). These receptors bind a large number of chemokines, peptides of typically 8 -10 kDa and contain cysteine residues at defined positions. Activation of chemokine receptors results in the dissociation of G␣ from G␤␥ proteins, triggering a series of signaling events leading to chemotaxis and other cellular functions (1,2).
Human herpesvirus 8 is a recently identified ␥-herpesvirus associated with Kaposi's sarcoma and hence is also named Kaposi's sarcoma-associated herpesvirus (KSHV) (3,4). Kaposi's sarcoma is characterized by angiogenic proliferation of mesenchymal cells, resulting in abundant vascular spaces filled with red blood cells and surrounded by spindle cells. KSHV DNA is found in spindle cells as well as endothelial cells in the lesions of Kaposi's sarcoma, suggesting that the viral DNA encodes proteins that contribute to overgrowth of vascular endothelial cells (4). Analysis of the KSHV sequence has led to the identification of an open reading frame (ORF-74) that encodes a putative GPCR, which bears structural similarity to CXCR1 and CXCR2, the two human receptors for IL-8 (5,6). When expressed in transfected cell lines, KSHV-GPCR binds a large number of chemokines (7). Unlike most other chemokine receptors that depend on agonist binding for activation, the KSHVderived receptor is a constitutively active GPCR. Exogenous expression of KSHV-GPCR results in cell proliferation and foci formation (7). Furthermore, KSHV-GPCR induces oncogenic transformation of NIH 3T3 cells leading to tumor growth in nude mice (8). The transformed cells promote a switch of surrounding endothelial cells to an angiogenic phenotype due to increased secretion of vascular endothelial growth factor (8), which stimulates endothelial growth in Kaposi's sarcoma lesions through a paracrine mechanism (9). Expression of KSHV-GPCR in transgenic mice produces the same pathological changes as seen in Kaposi's sarcoma, indicating a causal relationship between KSHV-GPCR and certain lesions of Kaposi's sarcoma (10).
There has been a great deal of interest in the signaling pathways activated by KSHV-GPCR. Whereas some chemokines bind to and further activate this receptor (7,11), others inhibit its function (12)(13)(14). As with many GPCRs whose activation is negatively regulated by G protein-coupled receptor kinases, signaling by the KSHV-GPCR is blocked by G proteincoupled receptor kinases 5 but not G protein-coupled receptor kinases 2 (15). Like other chemokine receptors, KSHV-GPCR stimulates activation of several protein kinases, including related adhesion focal tyrosine kinase, extracellular signal-related protein kinases, and p38 (8,16,17). However, KSHV-GPCR differs from most chemokine receptors in that it is a viral oncogene that transforms host cells and stimulates tumor growth. Therefore it is important to determine the proximal signaling events including the G proteins that couple to this constitutively active GPCR.
Recent studies conducted in this and other laboratories have demonstrated activation of nuclear factor B (NF-B) by GPCRs (18 -23). NF-B is a ubiquitously expressed and highly regulated dimeric transcription factor (24). Numerous environmental signals can induce NF-B activation, which in turn regulates the expression of a large number of genes coding for cytokines, chemokines, and growth factors (25). Activation of NF-B has been suggested and recently demonstrated to play an important function in autocrine production of several CXC chemokines through the IL-8 receptors (26 -28). In melanoma cells, autocrine production of growth-regulated oncogene-␣ (GRO-␣; also termed melanoma growth stimulatory activity or MGSA) is associated with cell proliferation similar to that observed in KSHV-GPCR-transfected cells (27,29). This finding prompted us to determine whether KSHV-GPCR, like its mammalian homologs, can also activate NF-B. Here we report that KSHV-GPCR stimulates constitutive NF-B activation and induces IL-8 secretion in transfected HeLa cells. We further demonstrate that the heterotrimeric G protein, G␣ 13 , together with G␣ q , G␣ i/o , and G␤␥ proteins, plays an important role in KSHV-GPCR-mediated activation of NF-B and secretion of IL-8. NF-B activation by KSHV-GPCR was reported by others while this work was being completed (30) and after initial submission of the manuscript (31,32).

EXPERIMENTAL PROCEDURES
Reagents-The anti-G␣ q and anti-G␣ 13 Ab were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-G␣ i2 antibody was prepared against a synthetic peptide with the sequence CAKNNLKDCGLF. The G␣ i2 and G␣ q expression vectors were kindly provided by Drs. Cindy Knall and Gary Johnson (University of Colorado, Denver, CO) and were described elsewhere (33). The G␣ 13 and p115RhoGEF constructs were described in previous publications (34 -36). A HA-tagged G␣ 13 construct was obtained from Dr. Silvio Gutkind (National Institutes of Health, Bethesda, MD). The IL-8 luciferase reporter was a generous gift from Dr. Naofumi Mukaida (Kanazawa University, Japan). Other reagents were described in a recent publication from our laboratory (37).
Cell Culture, Transfection, and Luciferase Reporter Assay-HeLa cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 IU/ml penicillin, and 50 g/ml streptomycin. Cells (ϳ40% confluence) in 6-well plates were transfected with plasmid expression vectors coding for a 3 ϫ B-directed luciferase reporter (37) or an IL-8 luciferase reporter (Ϫ272-Luc) (38), KSHV-GPCR and other expression constructs as indicated. The total DNA was brought to 1 g/well, and pCMV␤ (␤-galactosidase expression vector) was included for standardization of expression and expression efficiency. Transient transfection was performed as described (37) using the LipofectAMINE Plus reagent (Life Technologies, Rockville, MD) according to manufacturer's instructions. Twenty-eight hours after transfection, cells were serum-starved for 16 -18 h, washed twice with phosphate-buffered saline, and assayed with or without agonist stimulation. Reporter lysis buffer (Promega, Madison, WI) was added to the cells, and the expressed luciferase activity was measured in a Femtomaster FB12 luminometer (Zylux, Maryville, TN). Relative expression level of the transfected constructs was standardized against the expressed ␤-galactosidase. Luciferase assays were done with duplicate or triplicate samples, and two to four independent experiments were usually conducted. Normalized data were plotted using the Prism software (Version 3.0; GraphPad, San Diego, CA).
Immunodetection-Cell surface expression of an AU5-tagged KSHV-GPCR was measured on a Coulter Elite flow cytometer, using a monoclonal Ab against AU5 (1:500, Covance, Denver, PA) and a secondary, fluorescein isothiocyanate-conjugated goat anti-mouse Ab (1:200). The AU5 tag (TDFYLK) was added to the NH 2 terminus of KSHV-GPCR by polymerase chain reaction (30 cycles with Pfu Turbo polymerase). The resulting polymerase chain reaction fragment was subcloned into the pRK5 vector (BD Pharmingen) and its sequence confirmed by DNA sequencing. The final construct contains a methonine preceding the tag, which was fused to KSHV-GPCR minus the initiation codon. In transfection experiments, the AU5-tagged KSHV-GPCR gave the same constitutive NF-B activation data as with untagged KSHV-GPCR.
For Western blotting, proteins from whole cell extracts were separated on 6 to 10% acrylamide SDS-polyacrylamide electrophoresis gels by electrophoresis at 30 mA. Proteins were electrotransferred to nitrocellulose membrane at 100 V for 1 h at 4°C. The membrane was pretreated with 5% nonfat milk in TTBS (20 mM Tris-HCl, pH 7.5, 120 mM NaCl, 0.05% Tween 20) for 1-2 h at room temperature. Incubation with primary Ab was done at 4°C in TTBS with 5% bovine serum albumin, for 16 h. The membrane was then washed 3 times with TTBS, for 10 min each, and incubated with horseradish peroxidase-conjugated secondary Ab for 1 h at room temperature (23°C). After 3 washes with TTBS, the bound Ab was detected by enhanced chemiluminescence (Pierce, Rockford, IL).
Fluorescent Microscopy-A stable NIH 3T3 cell line was established by transfection with KSHV-GPCR cDNA in the pSFFV.neo expression vector (39). The G418-resistant clones were pooled and the expression of KSHV-GPCR was confirmed by flow cytometry using a rabbit polyclonal Ab against the amino-terminal 41 residues of the receptor (Torrey Pines Biolabs, Pearland, TX). The cells were grown on gelatin-coated glass coverslips. Two days later, the cells were fixed with glutaraldehyde (4%), permeabilized with Triton X-100 (0.1%), and stained for 20 min with rhodamine-phalloidin (0.5 M; Sigma). The stained samples were viewed using a Nikon Eclipse TE300 inverted microscope equipped with Hoffman optics and appropriate filter sets for epifluorescence microscopy. The images were captured with a Hamamatsu cooled CCD camera and SimplePCI software (C-Imaging Systems, Cranberry Township, PA).
Detection of IL-8 Secretion-IL-8 secreted from cultured HeLa cells was detected using an ELISA kit and following the protocol supplied by the manufacturer (BIOSOURCE, Camarello, CA). For each experiment, duplicate samples were taken and standard curves were generated using manufacturer-supplied reagents.
Data Analysis-Luciferase reporter activities were normalized against ␤-galactosidase activities from the coexpressed pCMV␤ (luc/␤gal), and expressed as relative luciferase activities over basal (set as 1). Data were plotted using the Prism software. Where indicated, statistical analysis was conducted with one-way ANOVA using the same software.

Expression of KSHV-GPCR Induces Constitutive Activation of NF-B-Recent
studies have shown that certain GPCRs can activate NF-B, leading to transcription of genes coding for cytokines and growth factors. To investigate whether the constitutively active KSHV-GPCR plays a role in NF-B activation and cytokine secretion, HeLa cells were transiently transfected with expression vectors coding for an NH 2 -terminal tagged KSHV-GPCR and a B-driven luciferase reporter (37). HeLa was chosen because the cell line has been extensively characterized for NF-B activation by cytokine receptors, and also because the cell does not contain SV40 large T antigen that can cause overexpression of transfected genes when certain vectors are used. To correct variations in transfection efficiency, an expression vector coding for ␤-galactosidase under a CMV promoter was co-transfected with the above constructs, and the expressed ␤-galactosidase activity was used for normalization of B luciferase data. Forty-eight hours after transfection, cells were collected and the induced B luciferase reporter activities were measured.
In a typical experiment ( Fig. 1), expression of KSHV-GPCR in HeLa cells induced an increase in the B luciferase activity indicating transactivation of NF-B. KSHV-GPCR-induced B luciferase activities ranged from 2.5-27-fold over baseline, and correlated with the amount of input receptor DNA (from 20 to 800 ng, Fig. 1A). KSHV-GPCR had no effect on the expression of a luciferase reporter lacking functional NF-B-binding sites (kB-). To determine whether the induced NF-B luciferase activity is a function of cell surface receptor expression, the transfected HeLa cells were subjected to flow cytometry anal-ysis using an anti-AU5 monoclonal Ab, that recognizes the NH 2 -terminal AU5-tagged-KSHV-GPCR (see "Experimental Procedures"). As shown in Fig. 1B, the AU5 tag could be detected when the DNA construct was used at Ն400 ng. Transfection of the cells with 800 ng of DNA resulted in a cell surface expression that is nearly twice as much as transfecting with 400 ng of DNA (mean fluorescent channel number of 0.21 versus 0.11). Thus, expression of KSHV-GPCR leads to dosedependent activation of NF-B in the transfected cells.
The ability of TNF␣ to stimulate NF-B activation in HeLa cells has been well recognized. We therefore compared KSHV-GPCR-induced NF-B luciferase activity with that of TNF␣.
Transfection of the cells with 200 ng of the KSHV-GPCR expression vector resulted in slightly higher B luciferase activity than the activity induced by 10 ng/ml TNF␣ (Fig. 1C). Furthermore, the NF-B activity induced by KSHV-GPCR was additive to the TNF␣-induced NF-B activation (Fig. 1C), suggesting that potentially different signaling pathways are utilized by TNF␣ and KSHV-GPCR.
KSHV-GPCR Couples to Multiple G Proteins for NF-B Activation-KSHV-GPCR is a structural homolog of the human chemokine receptors CXCR1 and CXCR2. These receptors have been shown to mediate chemotaxis and other leukocyte functions through functional coupling to pertussis toxin-sensitive G proteins (40). Although nearly all chemokine receptors are known for coupling to G␣ i proteins, the identity of the G proteins that mediate the cellular functions of KSHV-GPCR remained unclear when this study was initiated. To determine whether KSHV-GPCR also couples to G i/o , we treated the transfected cells with pertussis toxin (PTX, 500 ng/ml, 4 h). This treatment resulted in a 20 -25% reduction in the ability of KSHV-GPCR to induce the B luciferase reporter activity ( Fig.  2A), suggesting that G i/o is one of the G proteins that are activated by KSHV-GPCR. To identify other G proteins that contribute to the remainder of KSHV-GPCR-induced NF-B activation, we used a gain of function approach that has been proven effective by recent studies (37,41). HeLa cells were transfected to express selected wild type G␣ proteins including G␣ i2 , G␣ q , and G␣ 13 , in addition to the B reporter and the KSHV-GPCR expression construct. These G proteins are endogenously expressed in HeLa cells (Fig. 2B), and it was predicted that coexpression of a given G␣ protein that couples to the receptor would further enhance the induced B reporter activity. Our results demonstrate that coexpression of G␣ i2 did not further increase the B luciferase activity, but coexpression of G␣ q and G␣ 13 potentiated the KSHV-GPCR-induced response by 25% (p Յ 0.05) and 60% (p Յ 0.01), respectively (Fig. 2C). These effects were not due to variation of transfection efficiency since such differences were overcome by normalization of data with the co-transfected ␤-galactosidase construct. In comparison, expression of these G␣ proteins without KSHV-GPCR did not significantly alter the B luciferase activity. As an additional control, experiments were conducted under the same conditions to examine the effect of these G proteins on B 2 bradykinin receptor-mediated NF-B activation. Our results indicate that G␣ q produced a more potent enhancement than G␣ 13 of the BK-induced response (Fig. 2D). These results combined suggest that KSHV-GPCR preferentially couples to G␣ 13 , in addition to G␣ i/o and G␣ q , for NF-B activation.
Previous studies have demonstrated the ability of G␣ 13 to activate serum response factor (42), but its function in NF-B activation was not known at the time these experiments were conducted. We therefore examined the ability of G␣ 13 to mediate NF-B activation by co-transfection of a construct encoding a GTPase-deficient (constitutively active) form of G␣ 13 (Q226L) together with the B luciferase reporter. As shown in Fig. 3A, the constitutively active G␣ 13 induced a potent and dose-dependent increase of the B luciferase activity, in the absence of KSHV-GPCR. Like KSHV-GPCR, the activated G␣ 13 (Q226L) also enhanced TNF␣-induced NF-B transactivation (Fig. 3B).
The above findings suggest that G␣ 13 is involved in KSHV-GPCR-induced NF-B activation. To test this possibility further, a number of loss-of-function experiments were conducted. We compared the abilities of relevant inhibitors to block NF-B activation by KSHV-GPCR and G␣ 13 (Q226L). Regulators of G protein signaling (RGS) are a group of recently identified proteins that contain GTPase-activating protein domains. RGS proteins are effective inhibitors for G protein activation (43). The p115RhoGEF has been identified as a guanine nucleotide exchange factor that mediates activation of RhoA by G␣ 13 (35,44). p115RhoGEF contains an RGS domain that exhibits specificity for G␣ 13 , and has been used as an inhibitor of this G␣ protein (45). We therefore speculated that the RGS domain of p115RhoGEF (p115RGS) would inhibit KSHV-GPCR-induced NF-B activation if G␣ 13 were involved in coupling this receptor. Our experimental results demonstrated that p115RGS indeed partially (ϳ50%) blocked the induced NF-B activation (Fig. 4A). The inhibition was targeted primarily to G␣ 13 as p115RGS also inhibited NF-B activation by G␣ 13 (Q226L).
We have shown above that KSHV-GPCR couples to more than one G␣ protein. Activation of G␣ results in the release of G␤␥, which can directly stimulate several downstream effectors and lead to NF-B activation (37). To determine the involvement of G␤␥, we co-transfected HeLa cells with a G␤␥ scavenger, bovine transducin. Expression of transducin led to a partial inhibition of KSHV-GPCR-mediated NF-B activation while having no effect on NF-B activation by the constitutively active G␣ 13 (Q226L) (Fig. 4B). These results combined indicate that KSHV-GPCR-induced NF-B activation involves multiple G proteins including G␣ i/o , G␣ q , G␣ 13 , as well as G␤␥ proteins that are released when G␣ is activated by the receptor. 13 activates the small GTPase RhoA through p115RhoGEF, a guanine nucleotide exchange factor (35,44). To determine whether RhoA is downstream of KSHV-GPCR-induced signaling pathway, we took advantage of the ability of RhoA to induce formation of stress fibers in serum-starved fibroblast (46). A stable transfectant of NIH 3T3 cells was generated that expressed moderate levels of KSHV-GPCR (Fig. 5A). This cell line exhibited NF-B-activating property similar to the transiently transfected HeLa cells (Fig. 5A, inset). The cells were serum-starved, fixed, and stained with rhodamine-phalloidin for the detection of actin cytoskeleton. Most of the receptor-transfected cells displayed actin stress fibers indicative of RhoA activation (Fig. 5B, left panels), as compared with the control NIH 3T3 cells which lacked stress fiber formation (Fig. 5B, right panels). This result suggests RhoA activation by KSHV-GPCR.

RhoA Is a Downstream Effector of KSHV-GPCR and Contributes to the Induced NF-B Activation-G␣
Given that RhoA is a downstream effector of G␣ 13 , we speculated that RhoA might function downstream of KSHV-GPCR to activate NF-B. To test this hypothesis, HeLa cells were co-transfected with KSHV-GPCR and a dominant negative form of RhoA, RhoA(T19N). Expression of RhoA(T19N) par-

FIG. 2. KSHV-GPCR couples to multiple G proteins for NF-B activation.
A, effect of PTX on KSHV-GPCR-induced NF-B transactivation. PTX was added to 500 ng/ml for 4 h. B, Western blotting showing the expression of G␣ proteins in HeLa cells without (Ϫ) and with (ϩ) transfection of the individual wild type G proteins. Antibodies against G␣ 13 , G␣ q , and G␣ i2 were used for immunoblotting (I.B.). Arrowhead marks G␣ 13 . Because only about 30% of the cells were transfected and all the cells were collected for Western blotting, actually G protein expression level in the transfected cells could be higher. C and D, expression of G␣ proteins differentially alters the induced B luciferase activity in cells transfected with the KSHV-GPCR expression plasmid (C) or a B 2 bradykinin receptor expression plasmid (D). The G␣ proteins were used at 200 ng/sample. The B 2 bradykinin receptortransfected cells were either left unstimulated (Ϫ) or stimulated (ϩ) with bradykinin (BK) at 10 nM for 4 h. All experiments were conducted 3 times, and a representative set of data was shown. Data were collected in triplicate, normalized against the coexpressed ␤-galactosidase to overcome variations in transfection efficiency, and expressed as mean Ϯ S.E.

FIG. 3. A constitutively active G␣ 13 induces NF-B activation.
A, expression of G␣ 13 (Q226L) induces dose-dependent increases of the B luciferase activity. B, G␣ 13 (Q226L) potentiated the induction of NF-B activation by TNF␣. The expression plasmid of G␣ 13 (Q226) was used at 200 ng/sample (B), or at the indicated amount (A). TNF␣ was added to 10 ng/ml for 4 h. Data were collected in duplicate, normalized against the coexpressed ␤-galactosidase activity, and expressed as mean Ϯ S.D. A total of three experiments were conducted, and a representative set of results is shown. tially inhibited KSHV-GPCR-induced NF-B activation (Fig.  6A). This mutant RhoA also inhibited NF-B activation by G␣ 13 (Q226L) to a greater extent. Moreover, the C3 exoenzyme from Clostritium botulinum, a Rho-specific inhibitor (47), inhibited NF-B activation induced by KSHV-GPCR and G␣ 13 (Q226L) (Fig. 6B). In contrast, the C3 exoenzyme had no effect on TNF␣-induced NF-B activation (not shown). Taken together, these data suggest that KSHV-GPCR activate the G␣ 13 -RhoA pathway.
The G␣ 13

-RhoA Pathway Is Involved in KSHV-GPCR-induced IL-8 Secretion-
The expression of a number of chemokines, including IL-8 and GRO-␣/MGSA, are controlled in part by NF-B (25, 48 -50). IL-8 and GRO-␣/MGSA also bind and stimulate KSHV-GPCR (11). To determine whether KSHV-GPCR-induced NF-B activation affects IL-8 expression, HeLa cells were co-transfected with a luciferase reporter driven by the human IL-8 promoter (Ϫ272 base pairs upstream of transcription initiation site) that contains functional sites for NF-B, NF-IL6, and AP-1 (38). KSHV-GPCR induced potent expression of the IL-8 luciferase reporter, which was dependent on the input DNA concentration (Fig. 7A). The KSHV-GPCRinduced IL-8 gene expression was accompanied by increased production of IL-8 as detected in the culture medium by ELISA (Fig. 7B).
In Fig. 2 we showed a ϳ20% reduction of the B luciferase activity when the KSHV-GPCR-transfected cells were treated with PTX. Similar reductions (ϳ16%) were observed in IL-8 luciferase reporter assay (Fig. 8A) and in IL-8 secretion as determined by ELISA (Fig. 8B), indicating that a PTX-sensitive G protein is partially responsible for KSHV-GPCR-induced IL-8 gene expression and protein synthesis. A longer treatment with PTX (100 ng, 16 h) reduced the IL-8 luciferase activity by 24% (data not shown).
KSHV-GPCR has been reported to activate a PKC-responsive promoter that contains an AP-1 binding motif (7). In addition, the receptor induces inositol phosphate accumulation in transfected COS-1 cells (7). These findings suggest that KSHV-GPCR couple to G␣ q . Since the IL-8 promoter also contains an AP-1 binding motif (49), and G␣ q is known to activate NF-B (37), we sought to determine the relative contribution of G␣ q and G␣ 13 to KSHV-GPCR-induced IL-8 gene expression and IL-8 secretion. In the transfected cells, a constitutively active G␣ q (Q209L) induced nearly twice as much expression of the IL-8 luciferase reporter as did either KSHV-GPCR or G␣ 13 (Q226L) (Fig. 8A). This activity, however, did not translate into a potent IL-8 production as cells transfected with G␣ q (Q209L) secreted little IL-8 (Fig. 8B). In comparison, IL-8 secretion was detected from cells transfected to express KSHV-GPCR and the activated G␣ 13 (Fig. 8B). There is a good correlation between the abilities of these two constructs to induce the IL-8 luciferase reporter and their abilities to stimulate IL-8 secretion.
The above observation suggests the involvement of G␣ 13 in KSHV-GPCR-induced IL-8 secretion. To test whether RhoA is part of the G␣ 13 signaling pathway for this function and HeLa cells were transfected with the Ϫ272 IL-8 luciferase reporter, together with an expression vector for KSHV-GPCR, G␣ 13 (Q226L), or G␣ q (Q209L). Some samples were treated with PTX (500 M, 4 h) prior to assays. After 48 h, the induced luciferase activities were measured, normalized against the coexpressed ␤-galactosidase activities, and expressed as fold induction over basal (A). In B, IL-8 secreted into the culture media was determined by ELISA. The data were normalized against the coexpressed ␤-galactosidase activities and are shown as mean Ϯ S.D. All expression constructs were used at 200 ng/sample. C, inhibition of IL-8 secretion by a dominant negative RhoA and by a IB␣ super repressor. The cells were transfected similarly as above, with 200 ng each of the RhoA(T19N) and IB␣M. Expression of these molecules were determined by Western blotting. Expression of the G␣ 13 (Q226L) protein was determined using a hemagglutinin-tagged construct. Duplicate data were collected from the above experiments and are shown as values of mean Ϯ S.D. from one of the three experiments, all giving similar results.
whether NF-B is required for KSHV-GPCR-induced IL-8 expression, HeLa cells were co-transfected with the dominant negative RhoA(T19N) or an IB␣ "super repressor" which is devoid of inducible serine phosphorylation (51). As shown in Fig. 8C, expression of RhoA(T19N) partially inhibited IL-8 secretion induced by KSHV-GPCR and by the activated G␣ 13 , whereas expression of IB␣M nearly completely blocked the induced IL-8 secretion. This result is consistent with the notion that NF-B is essential for IL-8 expression (49). It also suggests the presence of a G␣ 13 activated pathway that may not involve RhoA.
KSHV-GPCR binds a large number of chemokines (7), including both agonists (11) and antagonists for this receptor (12,13). We investigated whether these chemokines affect KSHV-GPCR-induced secretion of IL-8 by treatment of the transfected HeLa cells with GRO-␣/MGSA, an activator of KSHV-GPCR, or with interferon-␥-inducible protein 10 (IP-10), a negative antagonist for the receptor. As shown in Fig. 9, KSHV-GPCRinduced IL-8 secretion was further enhanced by stimulating the cells with GRO-␣/MGSA, and diminished by treating the cells with IP-10. The two chemokines did not affect G␣ 13 (Q226L)-induced IL-8 secretion, indicating that these chemokines regulate IL-8 secretion at the receptor level. Because expression of GRO-␣/MGSA, like IL-8, is also controlled by NF-B, these findings suggest a possible autocrine or paracrine regulatory mechanism for KSHV-GPCR-induced NF-B activation and chemokine production. DISCUSSION In this study we have demonstrated that expression of KSHV-GPCR results in constitutive NF-B transactivation. This function is mediated by several G proteins including the PTX-sensitive G␣ i/o , the PTX-insensitive G␣ q and G␣ 13 , and G␤␥ proteins. Because the roles of G␣ i/o , G␣ q , and G␤␥ in NF-B activation have been characterized previously with another GPCR (37) and recently with KSHV-GPCR (30,32), our study is focused on the activation of G␣ 13 and RhoA by KSHV-GPCR. The involvement of G␣ 13 and RhoA in KSHV-GPCR signaling is evidenced by the following observations. 1) Expression of wild type G␣ 13 enhances KSHV-GPCR-induced NF-B activation. 2) Inhibition of G␣ 13 by p115RGS, a RGS specific for G␣ 13 , reduces NF-B activation by KSHV-GPCR. 3) Stress fiber formation has been observed in stably transfected NIH 3T3 cells that express KSHV-GPCR, indicating RhoA activation. 4) A dominant negative mutant of RhoA partially inhibits KSHV-GPCR-induced NF-B activation. 5) The C3 exoenzyme, a specific inhibitor of RhoA, reduces NF-B activation by KSHV-GPCR. 6) The above inhibitors and dominant negative constructs also effectively inhibit NF-B activation stimulated by a constitutively active G␣ 13 mutant. We did not observe complete inhibition by any of the above agents except IB␣M, indicating the involvement of multiple G proteins in KSHV-GPCR-induced NF-B activation.
The observation that activated G␣ 13 , but not activated G␣ q , stimulates IL-8 secretion provides additional evidence for a role of G␣ 13 in functional coupling with KSHV-GPCR. The underlying mechanism is not clear at this time, but may include the possibilities of translational inhibition of the IL-8 message by G␣ q and lack of a necessary component for its efficient translation. This finding is similar to an earlier report indicating that C5a induces transcription of the IL-1 message but does not provide a translational signal (52). The C5a receptor is known for coupling to G␣ i and G␣ 16 , a member of the G␣ q family, but not to G␣ 13 . Further experiments will be necessary to thoroughly investigate the differences between G␣ q and G␣ 13 in stimulating IL-8 biosynthesis. G␣ q has been shown to mediate KSHV-GPCR-induced accumulation of inositol phosphates as this function is resistant to PTX treatment (7). Using the Ϫ272 IL-8 luciferase reporter, we demonstrated that G␣ q is a potent inducer of IL-8 transcription. Therefore, G␣ q may indirectly participate in IL-8 biosynthesis by up-regulating the IL-8 message.
Results obtained from this study suggest activation of RhoA by KSHV-GPCR. RhoA is a member of the Rho small GTPases that regulate cytoskeleton rearrangement and other important cellular functions such as cell cycle progression through G 1 (53). Thus, the stimulated cell proliferation by KSHV-GPCR may be attributed in part to Rho-mediated alteration of cell cycle. Consistent with a previous observation that the Rho GTPases activate c-Jun NH 2 -terminal kinase (JNK) and p38 MAP kinases but not extracellular signal-regulated kinase (ERK) (53), KSHV-GPCR has been shown to activate JNK and p38 but not ERK (8). These findings combined suggest that KSHV-GPCR stimulates cell proliferation through activation of Rho GTPases, but probably not Ras, which has been known to stimulate ERK activation through Raf and ERK kinase. We are currently investigating the role of other Rho GTPases, Cdc42 and Rac1, in KSHV-GPCR-mediated functions.
RhoA is an effector of G␣ 13 , and GPCRs that couple to G␣ 13 activate cellular functions in part through this small GTPase. A recent study demonstrates that G2A, a stress-inducible GPCR expressed in immature T and B lymphocyte progenitors (54), couples to G␣ 13 and activates RhoA (55). Expression of G2A in NIH 3T3 cells results in oncogenic transformation characterized by loss of contact inhibition, anchorage-independent growth, and tumorigenicity in mice (45). These studies indicate that G2A is a GPCR that utilizes G␣ 13 and RhoA for oncogenic transformation. Like G2A, KSHV-GPCR also induces cellular proliferation, cell transformation, and tumorigenicity in nude mice (7)(8)(9). In addition, both receptors are absent from cells under resting conditions, but are induced to express by either environmental stress or viral infection. These similarities suggest that the two receptors share certain components of their signaling pathways.
NF-B is a ubiquitous transcription factor that regulates the expression of a large number of cytokine and growth factor genes (24). NF-B also regulates the expression of several genes coding for anti-apoptotic factors such as Bcl-2 (24). Thus, activation of NF-B may be partially responsible for the accelerated proliferation of cells that express KSHV-GPCR (7). Of potential interest is that NF-B activation by KSHV-GPCR leads to expression of IL-8, a CXC chemokine that binds KSHV-GPCR as well as the chemokine receptors CXCR1 and CXCR2 (11). Expression of another CXC chemokine, GRO-␣/MGSA, is also induced by NF-B (50,56), although the cells used in the current study did not secrete GRO-␣/MGSA when stimulated by a variety of NF-B activators including TNF␣ (data not shown). In several cell models, binding of the above two CXC chemokines to CXCR2 induces activation of the receptor and is responsible for the proliferation of these cells through an autocrine mechanism (29,(57)(58)(59). Since both IL-8 and GRO-␣/ MGSA have been shown to bind and activate KSHV-GPCR, a potential function of the secreted IL-8 could be autocrine stimulation through KSHV-GPCR similar to that seen with CXCR2 (59). Thus, it is likely that KSHV-GPCR-induced activation of NF-B and secretion of IL-8 contribute to the proliferation of cells that express this constitutively active receptor.
A characteristic feature of Kaposi's sarcoma is overgrowth of vascular endothelial cells. It has been shown in transgenic mice that KSHV-GPCR is primarily responsible for the vascular overgrowth found in Kaposi's sarcoma (10). KSHV-GPCR activates hypoxia-inducing factor 1␣, resulting in increased production of vascular endothelial growth factor (17). Vascular endothelial growth factor has been shown to play an important role in KSHV-GPCR-stimulated angiogenesis (8,10). In this regard, it is notable that IL-8 and a number of CXC chemokines bearing the NH 2 -terminal Glu-Leu-Arg (ELR) sequence can stimulate proliferation of vascular endothelial cells and therefore are angiogenic factors (60,61). These CXC chemokines bind and activate CXCR2, expressed in vascular endothelial cells, and stimulate the growth of these cells (62). Therefore, KSHV-GPCR-stimulated activation of NF-B and the resultant secretion of IL-8 may contribute to angiogenesis and vascular overgrowth as seen in Kaposi's sarcoma. Interestingly, the ELR ϩ GRO-␣/MGSA could further stimulate IL-8 secretion, whereas the angiostatic and ELR Ϫ CXC chemokine IP-10 (61) inhibited the induced IL-8 secretion. These results suggest that chemokines can positively and negatively regulate angiogenic factor production in cells that express KSHV-GPCR.
While this paper was in review, Schwarz and Murphy (31) reported secretion of several proinflammatory cytokines, chemokines, and growth factors in cells transfected to express KSHV-GPCR. Although the study does not lead to identification of the G proteins that couples KSHV-GPCR, the findings suggest that more than one G protein is involved in KSHV-GPCR signaling. This work also demonstrates that the ability of KSHV-GPCR to activate NF-B and AP-1 is preserved in several cell lines. In another recent publication, Couty et al. (32) demonstrated with strong evidence that KSHV-GPCR can couple to G i/o and G q . They also displayed constitutive NF-B activation in the transfected mouse lung endothelial cells, that could be further increased by the KSHV-GPCR agonist GRO-␣ (32). Since G␣ q and the phospholipase C␤ pathway is not con-stitutively activated by this receptor in mouse lung endothelial cells, the inability of PTX and transducin to completely block constitutive activation of NF-B in mouse lung endothelial cells suggests the presence of another signaling molecule, possibly a G protein, in coupling the receptor for this function. Furthermore, Couty et al. (32) reported that KSHV-GPCR stimulates phosphatidylinositol 3-kinase via a PTX-insensitive mechanism. Thus, our finding that G␣ 13 functionally couples KSHV-GPCR for NF-B activation and IL-8 secretion complements these recent reports and demonstrates for the first time that a chemokine receptor can utilize G␣ 13 for transcriptional regulation. With the recent identification of US28 as another constitutively active chemokine receptor of viral origin (41), it will be interesting to determine whether expression of a constitutively active GPCR is a general mechanism by which viruses and environmental stress regulate homeostasis of host cells.