Latent Kaposi's sarcoma-associated herpesvirus infection of endothelial cells activates hypoxia-induced factors

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV or HHV-8) is the etiological agent of Kaposi's sarcoma, a highly vascularized, endothelial-derived tumor. A direct role for KSHV-mediated induction of angiogenesis has been proposed based upon the nature of the neoplasia and various KSHV gene overexpression and infection model systems. We have found that KSHV infection of endothelial cells induces mRNA of hypoxia-induced factor 1alpha (HIF1alpha) and HIF2alpha, two homologous alpha subunits of the heterodimeric transcription factor HIF. HIF is a master regulator of both developmental and pathological angiogenesis, composed of an oxygen-sensitive alpha subunit and a constitutively expressed beta subunit. HIF is classically activated posttranscriptionally with hypoxia, leading to increased protein stability of HIF1alpha and/or HIF2alpha. However, we demonstrate that both alpha subunits are up-regulated at the transcript level by KSHV infection. The transcriptional activation of HIF leads to a functional increase in HIF activity under normoxic conditions, as demonstrated by both luciferase reporter assay and the increased expression of vascular endothelial growth factor receptor 1 (VEGFR1), an HIF-responsive gene. KSHV infection synergizes with hypoxia mimics and induces higher expression levels of HIF1alpha and HIF2alpha protein, and HIF1alpha is increased in a significant proportion of the latently infected endothelial cells. Src family kinases are required for the activation of HIF and the downstream gene VEGFR1 by KSHV. We also show that KS lesions, in vivo, express elevated levels of HIF1alpha and HIF2alpha proteins. Thus, KSHV stimulates the HIF pathway via transcriptional up-regulation of both HIF alphas, and this activation may play a role in KS formation, localization, and progression.

FIG. 1.

KSHV infection increases HIF alpha mRNA in human dermal microvascular endothelial cells. (A) Northern blot analysis of 250 ng of mRNA from mock- and KSHV-infected TIME (lanes 1 and 2) and primary HMVEC-d cells (lanes 3 and 4) probed for HIF1α, HIF2α, and the load control, GAPDH, at 48 h postinfection. −, absence of; +, presence of. (B) Quantitative real-time RT-PCR analysis of the severalfold change in the expression of HIF1α and HIF2α with KSHV infection, normalized to B2M, at 48 h postinfection compared to that with UV-irradiated KSHV treatment of TIME cells (n = 3). Error bars indicate standard deviations.

FIG. 2.

KSHV-infected TIME cells stabilize more HIF alpha subunits when exposed to the hypoxia mimic. Western blot analysis of mock (lanes 1 and 3)- and KSHV-infected (lanes 2 and 4) TIME cell nuclear extracts at 48 h postinfection in the absence (−) (lanes 1 and 2) or presence (+) (lanes 3 and 4) of an HIF-stabilizing hypoxia mimic (150 μM DFO) for the last 8 h. The extracts were probed with antibodies against HIF1α, HIF2α, HIF1β, and the load control, TFIIβ.

FIG. 3.

KSHV-infected TIME cells stabilize more HIF alpha subunits in the presence (+) of a proteosome inhibitor. Western blot analysis of mock (lanes 1 to 3)- and KSHV-infected nuclear extracts (lanes 4 to 6) at 24 h postinfection. The extracts were treated with a proteosome inhibitor (20 μM MG132) for either 1 h (lanes 2 and 5) or 2 h (lane 3 and 6) to stabilize HIF and probed with antibodies against HIF1α, HIF2α, HIF1β, and the load control, TFIIβ. −, absence of KSHV infection; +, presence of KSHV infection.

FIG. 4.

Latent KSHV-infected TIME cells stabilize more HIF1α protein than do uninfected cells. Results show the immunofluorescent detection of nucleic acid (DAPI, blue), KSHV latent nuclear antigen (LANA, red), and HIF1α (green) in mock- and KSHV-infected TIME cells 48 h postinfection in the absence or presence of the proteosome inhibitor, 20 μM MG132, for 1.5 h (magnification, ×200) (A) and 3 h (magnification, ×400) (B). The white arrow indicates an uninfected cell within the KSHV-infected population. (C) Immunofluorescent detection of nucleic acid (DAPI, blue), KSHV lytic switch protein, ORF50 or Rta (red), and HIF1α (green) in KSHV-infected TIME cells 48 h postinfection in the absence or presence of 20 μM MG132 for 2 h. The white arrows indicate the ORF50-positive cells (magnification, ×400). Panels A, B, and C are from separate experiments with similar infection rates (∼80% latent, <0.6% lytic).

FIG. 5.

KSHV infection and hypoxia mimic synergize to increase transcription from an HRE reporter construct and expression of the HRE-driven VEGFR1. (A) HIF-specific DNA-binding luciferase reporter assay in TIME cells (as described in Materials and Methods) shown as relative activity for the control, the mimic (TIME cells treated with 150 μM DFO for 8 h), KSHV at 24 h postinfection, and KSHV with DFO (KSHV+DFO). Error bars indicate standard deviations. (B) Western blot analysis of whole-cell lysates from mock (lanes 1 to 3)- and KSHV-infected TIME cells (lanes 4 to 6) at 48 h postinfection in the absence (lanes 1 and 4) or presence of 150 μM DFO for the last 4 h (lanes 2 and 5) or 8 h (lanes 3 and 6). Lysates were probed for full-length VEGFR1 (upper blot), pan-VEGFR1 antibody, and the load control β-actin. (C) Western blot analysis of HMVEC-d whole-cell lysates from mock-infected (lanes 1), mimic (KSHV-infected TIME cells treated with 150 μM DFO for 8 h) (lane 2), KSHV-infected (lane 3) and KSHV+DFO lysates (lane 4) from 24 h postinfection probed for VEGFR1 and the load control β-actin. −, absence of KSHV infection; +, presence of KSHV infection.

FIG. 6.

KSHV-induced HIF and VEGFR1 can be blocked in a dose-dependent manner by the Src family kinase inhibitor SU6656. (A) Western blot analysis of nuclear extracts from mock (lanes 1 and 3)- or KSHV-infected TIME cells (lanes 2 and 4 to 7) at 48 h postinfection and treated with vehicle (lanes 1 and 2) or 20 μM MG132 (lanes 3 to 7) for the last 2 h and the effect of pretreatment with 5, 10 and 20 μM SU6656 (lanes 5, 6, and 7, respectively) for the last 20 h on MG132 stabilization of HIF by KSHV. The extracts were probed for HIF1α, HIF2α, HIF1β, and the load control TFIIβ. (B) Whole-cell lysates from mock (lane 1)- or KSHV-infected TIME cells (lanes 2 to 5) at 48 h postinfection and the effect of treatment with 5, 10, and 20 μM SU6656 (lanes 3, 4, and 5, respectively) for the last 20 h on KSHV-induced VEGFR1 expression. The lysates were probed for β-actin as a load control. −, absence of indicated virus or drug; +, presence of indicated virus or drug.

FIG. 7.

Lytic induction decreases VEGR1 expression with KSHV infection in a dose-dependent manner. Western blot analysis of whole-cell lysates from mock (lanes 1 to 3)- or KSHV-infected cells (lanes 4 to 6) superinfected with either 500 (lanes 2 and 5) or 1,000 particles per cell (p/c) (lanes 3 and 6) of an adenovirus expressing ORF50 (Ad50) to induce lytic reactivation at 24 h postinfection. The lysates were probed for VEGFR1 and the load control β-actin. The percentage of cells expressing ORF59, a marker of lytic replication, is also listed for each condition. −, absence of KSHV infection; +, presence of KSHV infection.

FIG. 8.

KS lesions express elevated levels of HIF1α and HIF2α. Slides of AIDS-associated dermal KS tissue were obtained from the ACRS, hematoxylin QS counterstained, and stained brown by immunohistochemistry for LANA, HIF1β, and HIF2β as indicated. Shown for each are photos at ×100 (left) and ×400 (right) magnifications from the dashed box of the photo at ×100 magnification.