An NF-kappaB gene expression signature contributes to Kaposi's sarcoma virus vGPCR-induced direct and paracrine neoplasia.

Kaposi's sarcoma (KS) is the most frequent AIDS-associated malignancy, etiologically linked to the infection with the human herpesvirus 8 (HHV-8/KSHV). This member of the gamma-herpesviridae family encodes 81 open reading frames, several bearing oncogenic potential. A constitutively active virally encoded G protein-coupled receptor (vGPCR) readily induces KS-like lesions when expressed in endothelial cells in vivo, and unmasks the oncogenic potential of other HHV-8 genes in a paracrine fashion. How vGPCR causes endothelial cell transformation is still not fully understood. Using full-genome microarray analysis we show here that the expression of nuclear factor-kappaB (NF-kappaB)-regulated genes is a prominent feature triggered by vGPCR in cells expressing this viral oncogene and in cells exposed to vGPCR-induced secretions, thus mimicking its paracrine effect. Indeed, vGPCR activates the NF-kappaB pathway potently, and NF-kappaB activation is a hallmark of both human and experimental KS. Of interest, whereas constitutive NF-kappaB signaling is not sufficient to promote endothelial cells transformation, NF-kappaB function is strictly required for vGPCR-induced direct and paracrine neoplasia. Taken together, these results strongly support the role of NF-kappaB regulated genes in KS pathogenesis, thus providing the rationale for the development of novel mechanism-based therapies for this angioproliferative disease.

MAPK and Akt act cooperatively but independently on hypoxia inducible factor-1alpha in rasV12 upregulation of VEGF.

Oncogenic ras upregulates the expression of VEGF through the activation of the transcriptional enhancer hypoxia inducible factor-1alpha (HIF-1alpha) by a still poorly understood mechanism. Here, we demonstrate that both the Raf/MEK/MAPK and the PI3 kinase/Akt signaling pathways potently and additively stimulate the expression from a hypoxia response element (HRE) within the 5'flanking region of the VEGF promoter. Interestingly, while MAPK appears to specifically upregulate the transactivation activity of HIF-1alpha through direct phosphorylation of its regulatory/inhibitory domain, GSK-3, a downstream target of Akt, directly phosphorylates the HIF-1alpha oxygen-dependent degradation domain. These results suggest a novel mechanism whereby two divergent signaling pathways emerging from Ras may cooperatively but independently regulate the activity of a HIF-1alpha, thereby promoting the expression of a potent angiogenic mediator.

The Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor promotes endothelial cell survival through the activation of Akt/protein kinase B.

The Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor (KSHV-GPCR) is a key molecule in the pathogenesis of Kaposi's sarcoma, playing a central role in the promotion of vascular endothelial growth factor (VEGF)-driven angiogenesis and spindle cell proliferation. We previously have shown that KSHV-GPCR has oncogenic potential when overexpressed in fibroblasts and is responsible for the expression and secretion of VEGF through the regulation of different intracellular signaling pathways (A. Sodhi et al., Cancer Res., 60: 4873-4880, 2000; C. Bais et al., Nature, 391: 86-89, 1998). Here, we describe that this constitutively active G protein-coupled receptor is able to promote cell survival in primary human umbilical vein endothelial cells and that this effect is independent of its ability to secrete VEGF because it is not prevented by the expression of antisense constructs for VEGF or the addition of VEGF-blocking antibodies. Instead we found that ectopic expression of KSHV-GPCR potently induces the kinase activity of Akt/protein kinase B in a dose-dependent manner and triggers its translocation to the plasma membrane. This signaling pathway requires the function of phosphatidylinositol 3'-kinase and is dependent on betagamma subunits released from both pertussis toxin-sensitive and -insensitive G proteins. Furthermore, we found that KSHV-GPCR is able to protect human umbilical vein endothelial cells from the apoptosis induced by serum deprivation and that both wortmannin and the expression of a kinase-deficient Akt K179M mutant are able to block this effect. Finally, we observed that the Akt K179M protein also inhibits the activation of nuclear factor-KB induced by KSHV-GPCR, suggesting that this transcription factor may represent one of the putative downstream targets for Akt in the survival-signaling pathway. These results provide further knowledge in the elucidation of the signal transduction pathways activated by KSHV-GPCR and support its key role in promoting the survival of viral-infected cells. Moreover, the present findings also emphasize the importance of this G protein-coupled receptor in the development of KSHV-related neoplasias.

The Kaposi's sarcoma-associated herpes virus G protein-coupled receptor up-regulates vascular endothelial growth factor expression and secretion through mitogen-activated protein kinase and p38 pathways acting on hypoxia-inducible factor 1alpha.

The elucidation of the molecular mechanisms governing the transition from a nonangiogenic to an angiogenic phenotype is central for understanding and controlling malignancies. Viral oncogenes represent powerful tools for disclosing transforming mechanisms, and they may also afford the possibility of investigating the relationship between transforming pathways and angiogenesis. In this regard, we have recently observed that a constitutively active G protein-coupled receptor (GPCR) encoded by the Kaposi's sarcoma-associated herpes virus (KSHV)/human herpes virus 8 is oncogenic and stimulates angiogenesis by increasing the secretion of vascular endothelial growth factor (VEGF), which is a key angiogenic stimulator and a critical mitogen for the development of Kaposi's sarcoma. Here we show that the KSHV GPCR enhances the expression of VEGF by stimulating the activity of the transcription factor hypoxia-inducible factor (HIF)-1alpha, which activates transcription from a hypoxia response element within the 5'-flanking region of the VEGF promoter. Stimulation of HIF-1alpha by the KSHV GPCR involves the phosphorylation of its regulatory/inhibitory domain by the p38 and mitogen-activated protein kinase (MAPK) signaling pathways, thereby enhancing its transcriptional activity. Moreover, specific inhibitors of the p38 (SKF86002) and MAPK (PD98059) pathways are able to inhibit the activation of the transactivating activity of HIF-1alpha induced by the KSHV GPCR, as well as the VEGF expression and secretion in cells overexpressing this receptor. These findings suggest that the KSHV GPCR oncogene subverts convergent physiological pathways leading to angiogenesis and provide the first insight into a mechanism whereby growth factors and oncogenes acting upstream from MAPK, as well as inflammatory cytokines and cellular stresses that activate p38, can interact with the hypoxia-dependent machinery of angiogenesis. These results may also help to identify novel targets for the development of antiangiogenic therapies aimed at the treatment of Kaposi's sarcoma and other neoplastic diseases.

Activation of serum response factor by RhoA is mediated by the nuclear factor-kappaB and C/EBP transcription factors.

The activity of the transcription factor NF-kappaB can be modulated by members of the Rho family of small GTPases (Perona, R., Montaner, S., Saniger, L., Sánchez-Pérez, I., Bravo, R., and Lacal, J. C. (1997) Genes Dev. 11, 463-475). Ectopic expression of RhoA, Rac1, and Cdc42Hs proteins induces the translocation of NF-kappaB dimers to the nucleus, triggering the transactivation of the NF-kappaB-dependent promoter from the human immunodeficiency virus. Here, we demonstrate that activation of NF-kappaB by RhoA does not exclusively promote its nuclear translocation and binding to the specific kappaB sequences. NF-kappaB is also involved in the regulation of the transcriptional activity of the c-fos serum response factor (SRF), since the activation of a SRE-dependent promoter by RhoA can be efficiently interfered by the double mutant IkappaBalphaS32A/S36A, an inhibitor of the NF-kappaB activity. We also present evidence that RelA and p50 NF-kappaB subunits cooperate with the transcription factor C/EBPbeta in the transactivation of the 4 x SRE-CAT reporter. Furthermore, RhoA increases the levels of C/EBPbeta protein, facilitating the functional cooperation between NF-kappaB, C/EBPbeta, and SRF proteins. These results strengthen the pivotal importance of the Rho family of small GTPases in signal transduction pathways which modulate gene expression and reveal that NF-kappaB and C/EBPbeta transcription factors are accessory proteins for the RhoA-linked regulation of the activity of the SRF.

Multiple signalling pathways lead to the activation of the nuclear factor kappaB by the Rho family of GTPases.

Members of the Rho family of small GTPases activate the nuclear factor kappaB (NF-kappaB) (Perona, R., Montaner, S., Saniger, L., Sánchez-Pérez, I., Bravo, R., and Lacal, J. C. (1997), Genes & Dev. 11, 463-475). We have investigated whether different members of the family of exchange factors specific for Rho proteins (Dbl family) could activate the transcription factor NF-kappaB and have explored both their specificity under in vivo conditions and the mechanisms involved. Activated forms of Dbl, Ost, and Vav proteins induce NF-kappaB activation. While the activation induced by the Vav oncogen was efficiently inhibited by a dominant negative mutant of Rac1, the corresponding mutant of Cdc42Hs was able to block selectively NF-kappaB activation mediated by Dbl. Finally, mutants of RhoA and Cdc42Hs, but not that of Rac1, inhibited the activation of NF-kappaB by Ost. Thus, under in vivo conditions, different members of the Dbl family are related to specific Rho GTPases for the regulation of NF-kappaB. Activation of NF-kappaB by Rho or Ras proteins is mutually independent. However, there is a link between the NF-kappaB and the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) cascades since a dominant negative mutant of MEKK1 is able to inhibit NF-kappaB activation induced by Rac1 and Cdc42Hs proteins, but not by RhoA. These results indicate that, in mammalian cells, multiple pathways coexist for the activation of NF-kappaB, some of which are mediated by specific members of the Ras and Rho families of small GTPases.

The small GTP-binding protein Rho links G protein-coupled receptors and Galpha12 to the serum response element and to cellular transformation.

Receptors coupled to heterotrimeric G proteins can effectively stimulate growth promoting pathways in a large variety of cell types, and if persistently activated, these receptors can also behave as dominant-acting oncoproteins. Consistently, activating mutations for G proteins of the Galphas and Galphai2 families were found in human tumors; and members of the Galphaq and Galpha12 families are fully transforming when expressed in murine fibroblasts. In an effort aimed to elucidate the molecular events involved in proliferative signaling through heterotrimeric G proteins we have focused recently on gene expression regulation. Using NIH 3T3 fibroblasts expressing m1 muscarinic acetylcholine receptors as a model system, we have observed that activation of this transforming G protein-coupled receptors induces the rapid expression of a variety of early responsive genes, including the c-fos protooncogene. One of the c-fos promoter elements, the serum response element (SRE), plays a central regulatory role, and activation of SRE-dependent transcription has been found to be regulated by several proteins, including the serum response factor and the ternary complex factor. With the aid of reporter plasmids for gene expression, we observed here that stimulation of m1 muscarinic acetylcholine receptors potently induced SRE-driven reporter gene activity in NIH 3T3 cells. In these cells, only the Galpha12 family of heterotrimeric G protein alpha subunits strongly induced the SRE, while Gbeta1gamma2 dimers activated SRE to a more limited extent. Furthermore, our study provides strong evidence that m1, Galpha12 and the small GTP-binding protein RhoA are components of a novel signal transduction pathway that leads to the ternary complex factor-independent transcriptional activation of the SRE and to cellular transformation.

Signaling from G protein-coupled receptors to the c-jun promoter involves the MEF2 transcription factor. Evidence for a novel c-jun amino-terminal kinase-independent pathway.

The c-Jun amino-terminal kinases (JNKs) are a subfamily of mitogen-activated protein kinases that phosphorylate c-Jun and ATF2, and it has been postulated that phosphorylated c-Jun enhances its own expression through AP-1 sites on the c-jun promoter. In this study, we asked whether signals activating JNK regulate the c-jun promoter. Using NIH 3T3 cells expressing G protein-coupled m1 acetylcholine receptors as an experimental model, we have recently shown that the cholinergic agonist carbachol, but not platelet-derived growth factor, potently elevates JNK activity. Consistent with these findings, carbachol, but not platelet-derived growth factor, increased the activity of a c-jun promoter-driven reporter gene (for chloramphenicol acetyltransferase). However, coexpression of JNK kinase kinase (MEKK) effectively increased JNK activity, but resulted in surprisingly limited induction of the c-jun promoter. This raised the possibility that pathway(s) distinct from JNK control the c-jun promoter, and prompted us to explore which of its regulatory elements participate in transcriptional control. We observed that deletion of the 3' AP-1 site diminished chloramphenicol acetyltransferase activity in response to carbachol, but only to a limited extent. In contrast, deletion of a MEF2 site dramatically reduced expression, and deletion of both the MEF2 and 3' AP-1 sites abolished induction. Furthermore, cotransfection with MEF2C and MEF2D cDNAs potently enhanced the activity of the c-jun promoter in response to carbachol, and stimulation of m1 receptors, but not direct JNK activation, induced expression of a MEF2-responsive plasmid. Taken together, these data strongly suggest that MEF2 mediates c-jun promoter expression by G protein-coupled receptors through a yet to be identified pathway, distinct from that of JNK.

Activation of the nuclear factor-kappaB by Rho, CDC42, and Rac-1 proteins.

The Rho family of small GTPases are critical elements involved in the regulation of signal transduction cascades from extracellular stimuli to the cell nucleus, including the JNK/SAPK signaling pathway, the c-fos serum response factor, and the p70 S6 kinase. Here we report a novel signaling pathway activated by the Rho proteins that may be responsible for their biological activities, including cytoskeleton organization, transformation, apoptosis, and metastasis. The human RhoA, CDC42, and Rac-1 proteins efficiently induce the transcriptional activity of nuclear factor kappaB (NF-kappaB) by a mechanism that involves phosphorylation of Ikappa Balpha and translocation of p50/p50 and p50/p65 dimers to the nucleus, but independent of the Ras GTPase and the Raf-1 kinase. We also show that activation of NF-kappaB by TNFalpha depends on CDC42 and RhoA, but not Rac-1 proteins, because this activity is drastically inhibited by their respective dominant-negative mutants. In contrast, activation of NF-kappaB by UV light was not affected by Rho, CDC42, or Rac-1 dominant-negative mutants. Thus, members of the Rho family of GTPases are involved specifically in the regulation of NF-kappaB-dependent transcription.

Overexpression of PKC zeta in NIH3T3 cells does not induce cell transformation nor tumorigenicity and does not alter NF kappa B activity.

Signal transduction is the major mechanism by which cells communicate among themselves through extracellular stimuli. Among the different structural components involved in signal transduction, protein kinases are one of the key elements in the process. Protein kinase C is a multimember family of kinases which has been involved in the regulation of diverse cellular functions. Regulation of cell growth in fibroblasts has been reported to be one of such functions. In particular the PKC zeta isoenzyme has been postulated to be transforming to NIH3T3 cells (Berra et al., 1993) and to serve as an effector for Ras proteins through the regulation of the NF kappa B transcription factor (Dominguez et al., 1993) and direct interaction (Díaz-Meco et al., 1994). We have investigated the effects of overexpressing the mouse wild-type PKC zeta in NIH3T3 cells. When compared to the parental NIH3T3 cells, we have found (1) no significant effect on cell morphology; (2) no difference in growth properties in the absence of serum or in the presence of individual growth factors such as insulin, phorbol esters or PDGF; (3) no growth in soft agar nor tumorigenic activity in nude mice. In addition cells stably overexpressing the PKC zeta kinase did not interfere or amplify the induction of NF kappa B activity by tumor necrosis factor alfa (TNF-alpha) nor altered NF kappa B activity in transient expression of cells treated with TNF-alpha. Thus, mammalian PKC zeta is most likely not directly involved in the regulation of cell proliferation in fibroblasts nor affects directly or indirectly the activation of NF kappa B.

Generation of phosphorylcholine as an essential event in the activation of Raf-1 and MAP-kinases in growth factors-induced mitogenic stimulation.

Cell proliferation is regulated by an appropriate combination of intracellular signals involving activation of kinases and the generation of phospholipid metabolites. We report here that growth factors induce a biphasic generation of phosphorylcholine (PCho) in quiescent NIH 3T3 cells, resulting in an early and transient increase at 100 s and a larger and sustained increase after 3 h of stimulation. Generation of PCho at both early and late times of growth factors stimulation results from the consecutive activation of phospholipase D (PLD) and choline kinase (ChoK). Production of PCho by specific growth factors seems an essential requirement for the early signals associated to activation of Raf-1 and MAP kinases, since blockage of choline kinase completely inhibited activation of Raf-1 and MAP kinases by PDGF or FGF. Both the transient early increase and the late sustained increase in PCho are required for the induction of DNA-synthesis, besides completion of the activation of the serine/threonine kinases cascade. Thus, our results strongly suggest that generation of PCho by the PLD/choline kinase pathway is one of the critical steps in regulating cell growth in NIH 3T3 stimulated by growth factors.