B-Raf–dependent expression of vascular endothelial growth factor–A in Kaposi sarcoma–associated herpesvirus-infected human B cells

Abstract

IntroductionKaposi sarcoma-associated herpesvirus/human herpesvirus 8 (KSHV/HHV-8) is the latest addition to the list of human herpesviruses. KSHV was first isolated from Kaposi sarcoma (KS) lesions in persons suffering from acquired immunodeficiency syndrome (AIDS) in 1994. 1 The cancerous conditions that are etiologically associated with KSHV are KS, primary effusion lymphoma (PEL), and multicentric Castleman disease (MCD). 2 Apart from the inflammatory cytokines (ICs)/growth factors (GFs), lytic KSHV infection plays an instrumental role in the progression of KS lesions. 3 The lytic cycle of infection is also critical for the spread of KSHV to different organs. Successful KSHV infection is characterized by both virus entry and the ability of the virus to establish latency.In our earlier study, we provided evidence to show that the overexpression of Raf specifically enhanced KSHV infection of target cells at the level of entry. 4 Regulation of Raf is crucial for the proper maintenance of cell growth, proliferation, apoptosis, and differentiation. 5 Of the 3 isoforms, B-Raf has gained a lot of focus over the last couple of years due to the detection of B-Raf somatic missense mutations in malignant melanomas (66%), colon cancers (15%), and at lower frequencies in a wide variety of human cancers. [6][7][8] The ability of Raf to regulate vascular endothelial growth factor-A (VEGF-A) has also been demonstrated. 4 In separate studies, we found VEGF-A to enhance KSHV infection of fibroblasts and epithelial cells. 9,10 Interestingly, constitutive activation of the components (Ras/Raf) of the mitogen-activated protein kinase (MAPK) pathway of signaling as well as VEGF expression has been a common feature with KSHV pathogenesis. 11,12 Hence, in this study we attempted to analyze the relationship between the expression of B-Raf and VEGF-A in KSHV-infected hematopoietic cells. We provide evidences for the existence of a Raf-dependent VEGF-A expression in KSHV-infected hematopoietic cells. Materials and methods Cell cultureHuman foreskin fibroblasts (HFFs; Clonetics, Walkersville, MD), BCBL-1, BC-1 (American Type Culture Collection [ATCC], Manassas, VA; CRL-2230), BC-2 (ATCC CRL-223), BCP-1 (ATCC CRL-2294), and BJAB cells were used in this study. Target cells were grown in either phenol red-free Dulbecco modified Eagle medium (DMEM) or RPMI medium (Invitrogen, Carlsbad, CA) containing 10% charcoal-stripped fetal bovine serum (FBS; Atlanta Biologicals, Lawrenceville, GA), L-glutamine, and antibiotics. 13,14 Dermal microvascular endothelial cells (HMVEC-Ds; CC-2543; Clonetics) were propagated in EGM MV-microvascular endothelial cell medium (Clonetics) as per standard protocols. 13 The passage numbers for HFFs and HMVEC-Ds used in this study ranged between 6 and 10, and 5 and 9, respectively. HFF/pBabePuro3, HFF/⌬B-Raf [DD] :ER, and HFF/⌬B-Raf [FF] : ER cells were cultured as per standard protocols. 4,10 ␤-Estradiol (1 M) stimulation of these cells results in the activation of ⌬B-Raf:ER fusion proteins. 4Inhibitors PD98059 w...

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“…Similarly, Akula et al (31) showed that 1 h of treatment of HHV8-infected B cells with 50 Amol/L PD98059, another MEK1/2 inhibitor (32), causes a substantial decrease in levels of VEGF mRNA and protein. These observations indicate that, although MKK signaling is an important regulator of VEGF release, some aspects of its control may be cell type specific.…”

Castleman's Disease: From Basic Mechanisms to Molecular Therapeutics

“…Akula et al [36] demonstrated that VEGF expression by HHV-8 -infected human B cells is mediated by the RAF pathway and that VEGF production can be aborted by using a mitogen-activated protein kinase/extracellular signalrelated kinase kinase inhibitor [36]. Understanding whether the RAF pathway plays an essential role in the mechanism of CD requires further research.…”

Kaposi's Sarcoma-Associated Herpesvirus Suppression of DUSP1 Facilitates Cellular Pathogenesis following De Novo Infection

“…Additionally, our data do not exclude the possibility that KSHV induction of 14-3-3␤ expression influences other genes which themselves regulate DUSP1 expression, cytokine expression, and/or endothelial cell invasiveness. For example, published data indicate that 14-3-3␤ facilitates Raf/Ras activation through the coupling of protein kinase C-to Raf-1 (71), and we and others have demonstrated that KSHV induction of ERK-dependent VEGF expression and endothelial cell invasiveness are due, in part, to KSHV regulation of Ras/Raf activation (34,72). Furthermore, the mechanisms through which xCT induces 14-3-3␤ expression require additional clarification.…”