KS is a highly angiogenic and inflammatory tumor (38
). The growth of early-stage KS heavily depends on various growth factors, cytokines, and chemokines. The fact that the proangiogenic and proinflammatory cytokine Ang-2 is highly expressed in KS tumors suggests that the cytokine plays an important role in KS tumor development (16
). The high expression level of Ang-2 in KS tumors is most likely attributable to KSHV infection. Indeed, KSHV infection of primary endothelial cells upregulates Ang-2 expression (16
). However, significant increase in Ang-2 transcription occurs only 54 h postinfection (16
) and requires the expression of several viral genes (40
). Here, we report that a significant amount of Ang-2 is presynthesized and stored in the WPBs of endothelial cells and that it is rapidly released upon KSHV infection. This finding defines a novel mechanism by which KSHV infection contributes to increased levels of Ang-2 to promote KS tumor growth.
Unlike transcriptional upregulation, KSHV induction of rapid Ang-2 release does not require viral gene expression but viral binding to its cellular integrin receptors. As summarized in , blocking viral binding with soluble heparin sulfate, anti-integrin antibodies, or RGD peptide inhibits KSHV-induced Ang-2 release. KSHV binding to integrins enhances tyrosine phosphorylation of several downstream signaling proteins, including the kinases Src and FAK and the Calα2 subunit of the l
channel. Our results suggest that this KSHV-integrin-transduced protein phosphorylation plays a key role in regulating Ang-2 release, since the protein tyrosine phosphorylation inhibitors genistein and PP2 abolish Ang-2 release. Consistent with previous reports (41
), our data confirm that changes in the intracellular Ca2+
concentration mediate Ang-2 release and that Ca2+
chelators and Ca2+
channel blockers inhibit Ang-2 release. We also demonstrated that KSHV binding to HUVECs induces instantaneous Ca2+
influx, which can be inhibited by protein tyrosine kinase inhibitors. Such Ca2+
influx-transduced “outside-in” signaling appears to play a critical role in mediating Ang-2 release. This may explain why thapsigargin, an agent that raises cytosolic Ca2+
by blocking the ability of the cell to pump Ca2+
into the endoplasmic reticulum, has no effect on KSHV-induced Ang-2 release. Together, these results suggest that the opening of the Ca2+
channel is regulated through its own phosphorylation. Although integrins have been previously found to induce tyrosine phosphorylation-dependent Ca2+
influx in endothelial cells, our finding that KSHV binding to endothelial cells enhances phosphorylation of the Calα2 subunit of the l
channel for induction of Ca2+
influx is new.
Fig 7 Summary of the mechanisms by which KSHV induces rapid Ang-2 release from endothelial cells. KSHV binding to its integrin receptors enhances phosphorylation of tyrosine kinases FAK and Src and the Calα2 subunit of the l-type calcium channel and (more ...)
Our study highlights the importance of the dynamic interaction between KSHV and its integrin receptors in triggering Ang-2 release. In contrast to KSHV, adenovirus, used as a control in our study, does not induce Ang-2 release. Previous studies suggested that integrins facilitate adenovirus entry and internalization (42
). However, mutant adenovirus without an RGD motif can enter cells efficiently (43
), suggesting that the interaction between adenovirus and integrins is RGD independent. This different mechanism may explain why adenovirus does not induce Ang-2 release.
Ang-2 is best known for its role in blood vessel remodeling (44
). It is an antagonist of the endothelial cell-specific tyrosine kinase receptor Tie-2 (44
). In the presence of VEGF, Ang-2 destabilizes existing blood vessels to promote angiogenesis (44
). Ang-2 is highly expressed in most cancers and is a prognosticator of cancer progression. A high level of Ang-2 is associated with aggressive tumor cell migration, invasion, and cancer metastasis (46
). Ang-2 is also a proinflammatory cytokine that plays an essential role in eliciting the host inflammatory response against infection by sensitizing endothelial cells to TNF-α (49
). In addition, Ang-2 promotes infiltration of inflammatory cells, such as monocytes and neutrophils (50
). Previously, we reported that KSHV-induced Ang-2 enhances blood vessel growth in a Matrigel-based in vivo
angiogenesis assay (16
). More recently, we found that the rapidly released Ang-2 induced by KSHV enhances the adhesion of monocytes to endothelial cells (data not shown). The fact that this enhanced monocyte adhesion to endothelial cells can be abolished by soluble Tie-2 suggests that the Ang-2 rapidly released from KSHV-infected HUVECs also plays a role in promoting inflammation.
In summary, we have found that KSHV stimulates rapid release of the proangiogenic and proinflammatory cytokine Ang-2 from endothelial cells through dynamic interaction with its integrin receptors. This finding reveals a novel mechanism of KSHV induction of angiogenesis and inflammation, which might play important roles in the early stages of KS tumor development.