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-/os 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 KB (NF-KB) by a mechanism that involves phosphorylation of iKBa 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-KB by TNFa 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-KB 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-KB-dependent transcription.

The Kaposi's sarcoma herpesvirus (KSHV) has been identified as the etiologic agent of Kaposi's sarcoma (KS), but initial events leading to KS development remain unclear. Characterization of the KSHV genome reveals the presence of numerous potential oncogenes. To address their contribution to the initiation of the endothelial cell-derived KS tumor, we developed a novel transgenic mouse that enabled endothelial cell-specific infection in vivo using virus expressing candidate KSHV oncogenes. Here we show that transduction of one gene, vGPCR, was sufficient to induce angioproliferative tumors that strikingly resembled human KS. Endothelial cells expressing vGPCR were further able to promote tumor formation by cells expressing KSHV latent genes, suggestive of a cooperative role among viral genes in the promotion of Kaposi's sarcomagenesis.

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 G␣ s and G␣ i2 families were found in human tumors; and members of the G␣ q and G␣ 12 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 G␣ 12 family of heterotrimeric G protein ␣ subunits strongly induced the SRE, while G␤ 1 ␥ 2 dimers activated SRE to a more limited extent. Furthermore, our study provides strong evidence that m1, G␣ 12 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.

The Kaposi's sarcoma-associated herpesvirus (KSHV), the infectious causative agent of Kaposi's sarcoma (KS), encodes a G protein-coupled receptor (vGPCR) implicated in the initiation of KS. Here we demonstrate that Kaposi's sarcomagenesis involves stimulation of tuberin (TSC2) phosphorylation by vGPCR, promoting the activation of mTOR through both direct and paracrine mechanisms. Pharmacologic inhibition of mTOR with rapamycin prevented vGPCR sarcomagenesis, while overactivation of this pathway was sufficient to render endothelial cells oncogenic. Moreover, mice haploinsufficient for TSC2 are predisposed to vascular sarcomas remarkably similar to KS. Collectively, these results implicate mTOR in KS initiation and suggest that the sarcomagenic potential of KSHV may be a direct consequence of the profound sensitivity of endothelial cells to vGPCR dysregulation of the TSC2/mTOR pathway.

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We have recently engineered an in vivo endothelial cell-specific retroviral gene transfer system and found that a single Kaposi's sarcoma (KS)-associated herpesvirus͞human herpesvirus 8 gene encoding a G protein-coupled receptor (vGPCR), is sufficient to induce KS-like tumors in mice. By using this system, we show here that the Akt signaling pathway plays a central role in vGPCR oncogenesis. Indeed, a constitutively active Akt was sufficient to induce benign hemangiomas in mice, whereas heterozyogosity for PTEN (the phosphatase and tension homologue deleted on chromosome 10), modestly enhancing basal Akt activity, dramatically enhanced vGPCR sarcomagenesis. Examination of KS biopsies from AIDS patients revealed active Akt as a prominent feature, supportive of a role for Akt in human Kaposi's sarcomagenesis. By using a vGPCR agonist-dependent mutant, we further establish constitutive activity as a requirement for vGPCR sarcomagenesis, validating targeted inhibition of key vGPCR signaling pathways as an approach for preventing its oncogenic potential. These observations prompted us to explore the efficacy of inhibiting Akt activation as a molecular approach to KS treatment. Pharmacological inhibition of the Akt pathway with the chemotherapeutic agent 7-hydroxystaurosporine prevented proliferation of vGPCR-expressing endothelial cells in vitro and inhibited their tumorigenic potential in vivo. Both were associated with a decrease in Akt activity. These results identify Akt as an essential player in vGPCR sarcomagenesis and demonstrate the therapeutic potential of drugs targeting this pathway in the treatment of KS.human herpesvirus 8 ͉ Kaposi's sarcoma-associated herpesvirus ͉ 7-hydroxystaurosporine

Viruses use a surprising diversity of approaches to hijack G-protein-coupled receptors and harness their activated intracellular signalling pathways. All of these approaches ultimately function to ensure viral replicative success and often contribute to their pathogenesis. Indeed, a single virus might deploy a repertoire of these strategies to regulate key intracellular survival, proliferative and chemotactic pathways. Understanding the contribution of these biochemical routes to viral pathogenesis might facilitate the development of effective target-specific therapeutic strategies against viral diseases.

Vision loss from ischemic retinopathies commonly results from the accumulation of fluid in the inner retina [macular edema (ME)]. Although the precise events that lead to the development of ME remain under debate, growing evidence supports a role for an ischemia-induced hyperpermeability state regulated, in part, by VEGF. Monthly treatment with anti-VEGF therapies is effective for the treatment of ME but results in a major improvement in vision in a minority of patients, underscoring the need to identify additional therapeutic targets. Using the oxygen-induced retinopathy mouse model for ischemic retinopathy, we provide evidence showing that hypoxic Müller cells promote vascular permeability by stabilizing hypoxia-inducible factor-1α (HIF-1α) and secreting angiogenic cytokines. Blocking HIF-1α translation with digoxin inhibits the promotion of endothelial cell permeability in vitro and retinal edema in vivo. Interestingly, Müller cells require HIF-but not VEGF-to promote vascular permeability, suggesting that other HIF-dependent factors may contribute to the development of ME. Using gene expression analysis, we identify angiopoietinlike 4 (ANGPTL4) as a cytokine up-regulated by HIF-1 in hypoxic Müller cells in vitro and the ischemic inner retina in vivo. ANGPTL4 is critical and sufficient to promote vessel permeability by hypoxic Müller cells. Immunohistochemical analysis of retinal tissue from patients with diabetic eye disease shows that HIF-1α and ANGPTL4 localize to ischemic Müller cells. Our results suggest that ANGPTL4 may play an important role in promoting vessel permeability in ischemic retinopathies and could be an important target for the treatment of ME.diabetes | retinal vein occlusion | angiogenesis | transcription factor I schemic retinopathies include a diverse group of retinal diseases, in which immature retinal vasculature (e.g., retinopathy of prematurity or incontinentia pigmenti) or damage to mature retinal vessels (e.g., diabetic retinopathy, retinal vein occlusion, or sickle cell retinopathy) leads to retinal ischemia (1). Although diverse (and poorly understood) etiologies may lead to insufficient perfusion of the retina, all lead to a common sequelae: the formation of abnormal leaky blood vessels that can manifest clinically with the accumulation of fluid in the inner retina [i.e., macular edema (ME)] and often, a profound loss of vision (2). Indeed, ME in patients with ischemia-induced retinopathies remains the leading cause of vision loss in the working age population in the developed world (3).The concept that ischemic retinopathies are driven by ischemia-induced angiogenic factors was proposed over half a century ago (4). A single transcriptional activator, hypoxia-inducible factor-1 (HIF-1), has recently emerged as the master regulator of these angiogenic mediators. HIF-1 is a heterodimeric protein composed of an exquisitely oxygen-sensitive α-subunit and a ubiquitous β-subunit. Under hypoxic conditions, degradation of the oxygensensitive HIF-1α subunit is reduced, whereas its trans...