K13 is one of the few KSHV-encoded latent proteins and is consistently expressed in the KS spindle cells, the hallmark of KS lesions . Ectopic expression of K13 in human vascular endothelial cells is sufficient to make them acquire a spindle-shaped morphology that is associated with NF-κB activation and increased production of a number of genes known to be upregulated in KSHV-infected cells [21, 22]. In this study, we provide a comprehensive picture of global transcriptional changes induced by K13 in HUVECs. Our results provide the starting point for future detailed analysis of the K13-induced genes in the pathogenesis of KS.
As K13-induced spindle cell transformation of HUVECs is accompanied by their loss of proliferating potential, it is not possible to generate stable cultures of long-term proliferating HUVECs expressing K13 for the purpose of global gene expression analysis. To circumvent this problem, we took advantage of our previously characterized HUVEC-K13-ERTAM model system in which the K13 activity is dormant in the absence of 4OHT treatment . The HUVEC-K13-ERTAM maintains their normal cobblestone appearance and are indistinguishable from the control vector-expressing cells in their appearance and growth characteristics in the absence of 4OHT treatment. The lack of leakiness in this system was further confirmed by our microarray and qRT-PCR analysis, which revealed no significant difference in the gene expression profile between the vector and K13-ERTAM-expressing HUVECs in the absence of 4OHT. While 4OHT treatment significantly changed the expression of a number of genes in K13-ERTAM cells, it had no major effect on gene expression in the control vector cells, thereby confirming that that 4OHT treatment had no major effect on gene expression on its own. Another advantage of the use of this inducible model system is that allowed us to compare the effect of K13 activity on gene expression in the same cell population, thereby avoiding any artifacts due to cell-to-cell (clonal) variation.
It has been proposed that KSHV infection plays a major role in the recruitment of inflammatory cells to the KS lesions by upregulating the expression of chemotactic chemokines and, consistent with this notion, latent infection of vascular endothelial cells with KSHV has been known to upregulate the expression of several cellular chemokines, such as IL-8, GRO-1, MCP-1, NAP-2, CCL5/RANTES and CXCL16 [32, 15, 21, 18, 33, 22]. The chemokines induced by KSHV infection, such as IL-8, have been also postulated to contribute to neoangiogenesis characteristic of KS lesions. Our analysis revealed that chemokines were the most upregulated genes upon induction of K13 activity in HUVECs, which is consistent with the previous studies [21, 22] and two recent studies that were published while this manuscript was under review [34, 35]. Notable chemokines genes whose expression was increased significantly by K13 included CCL2 (71 fold), CCL20 (53 fold), CCL5/RANTES (48 fold), CXCL10 (47 fold), CCL3 (33 fold), IL8 (31 fold), CX3CL1 (22 fold), CXCL1 (7) and CXCL5 (13 fold). In addition, expression of genes encoding a number of cytokines (e.g. IL1a, IL6, IL15, IL32, CSF1, CSF2, CSF3 and EBI3) and TNF family ligands (TNFSF13B and Lymphotoxin beta) was significantly increased upon induction of K13 activity. Since chemokines and cytokines collectively represented the most upregulated genes in K13 expressing HUVECs, we confirmed their increased secretion in the supernatant of 4OHT treated K13-ERTAM cells. More importantly, a comparison with KSHV-infected cells revealed that ectopic expression of K13 is sufficient to induce most chemokines/cytokines that are induced by KSHV infection. Since K13 is one of the few KSHV genes that are expressed in latently-infected KS spindle cells, the above results support the hypothesis that K13, either alone or in combination with other viral genes, plays a key role in the up-regulation of chemokines and cytokines and subsequent recruitment of inflammatory cells to the KS lesions.
We observed significant upregulation of adhesion molecules (e.g. ICAM-1, VCAM-1 and E-Selectin), MHC-I, TAP1, TAP2 and tapasin upon induction of K13 activity, which is consistent with recent reports [22, 36]. Increased expression of adhesion molecules on vascular endothelial cells expression by K13 might synergize with chemokines to promote the recruitment of inflammatory and blood cells into KS lesions. On the other hand, increased MHC-I expression has been postulated to ensure controlled viral dissemination during latency by promoting cytotoxic T lymphocyte (CTL) proliferation . K13-induced upregulation of adhesion molecules and MHC-1 molecules during natural infection with KSHV may be modulated by concomitant expression of viral lytic proteins, such as the K5 gene product and vIRF1, which have been shown to down-regulate the expression ICAM-1 and MHC-I molecules [36, 37].
Infection with KSHV has been reported to contribute to neoangiogenesis, another characteristic feature of KS lesions, by upregulating the expression of several genes involved in the control of vascular modeling and angiogenesis, such as VEGF-A, VEGF-C, angiopoietin-related protein 4, thrombomodulin, and matrix metalloproteinase (MMP-1) . The expression of VEGF-A, VEGF-C and angiopoietin-related protein 4 was not significantly increased, and expression of thrombomodulin was decreased 2-fold upon induction of K13 activity. Additionally, the expression of tissue factor pathway inhibitor 2 (TFPI2), a gene which is a negative inhibitor of aberrant angiogenesis associated with tumor development was increased 5-fold by K13 . However, the expression of semaphorin 3C (SEMA3C) and TNF-alpha-induced protein 2 (TNFAIP2), two invasion and angiogenic factors [39–41], was induced 2 and 21 fold upon activation of K13 activity, respectively. Similarly, the expression of matrix metallopeptidase 10 (MMP10 or Stromolysin 2), an enzyme implicated in the breakdown of extracellular matrix during tumor invasion, metastases and angiogenesis , was highly induced (11 fold) upon induction of K13 activity. Finally, K13 activity strongly (21 fold) induced the expression of TNF alpha-induced protein 6 (TNFAIP6, also known as TSG-6), a member of the Link module superfamily that regulates extracellular matrix remodeling and inflammatory response . Thus, K13 activity may contribute to neoangiogenesis in KS lesions via increased production of angiogenic factors, such as IL-8, SEMA3C and TNFAIP2, and to invasion and metastases by stimulating extracellular matrix remodeling through increased production of MMP10 and TNFAIP6.
K13 strongly induced two stress response genes, cyclooxygenase-2 (COX-2) and manganese superoxide dismutase (SOD2), which have been reported previously to be strongly induced by KSHV infection . COX-2 is an angiogenic stress response gene that was recently shown to facilitate latent KSHV gene expression and the establishment and maintenance of latency . SOD2 plays an important role against mitochondrial oxidative stress by diminishing reactive oxygen species , and may promote survival of KSHV-infected cells. Furthermore, while this manuscript was under review, an independent study reported upregulation of SOD2 expression by K13 in vascular endothelial cells, which correlated with decreased intracellular superoxide accumulation and increased resistance to superoxide-induced death . Two other anti-apoptotic genes, baculoviral IAP repeat containing 3 (BIRC3/cIAP2) and BCl2-related protein A1 (BCL2A1), were also strongly upregulated upon induction of K13 activity in our study, and may contribute to the survival of KSHV-infected cells.
The genes belonging to the interferon response pathway represented another class of genes whose expression was upregulated upon induction of K13 activity. Notable genes in this class included interferon γ-inducible protein 30 (IFI30), 28 kDa interferon responsive protein (IFRG28), interferon stimulated exonuclease gene 20 kDa (ISG20), guanylate binding protein 1 (GBP1), interferon induced transmembrane protein 1 (IFITM1), interferon regulatory factor-1, -2 and -7, interferon-induced protein 35 (IFI35), interferon-induced protein with tetratricopeptide repeats 3 (IFIT3), interferon omega 1, interferon induced with helicase C domain 1 (IFIHI), and interferon, beta 1.
K13 is a powerful activator of the NF-κB pathway and others and we have previously reported that K13-induced upregulation of proinflammatory cytokines in vascular endothelium cells is associated with NF-κB activation and can be blocked by genetic and pharmacological inhibitors of this pathway [21, 22]. Consistent with the ability of K13 to activate NF-κB, most of the genes induced by K13 in the present study are known targets of the NF-κB pathway [46, 47], including NFKBIA (IκBα), which is not only a direct target gene of the NF-κB pathway but also a key negative regulator of this pathway. The involvement of the NF-κB pathway in K13-induced transcriptional activation of chemokine genes was further supported by our studies using the CXCL10 promoters. It needs to be noted, however, that the transcriptional activation of genes is usually complex and it is conceivable that NF-κB pathway cooperates with other signaling pathways in the transcriptional activation of some genes.
Finally, we observed that K13 also down-regulated the expression of several genes in HUVECs. In contrast to the upregulated genes, the down-regulated genes were diverse and did not belong to particular functional class. Nevertheless, these genes are known to act as tumor suppressor (e.g. ADAMTS18 and Periostin) [48, 49], apoptosis-inducer (e.g. insulin like growth factor binding protein 5) , and regulator of vascular integrity (regulator of G-protein signaling 5) , suggesting that their down-regulation by K13 may have a causal role in the pathogenesis of KS lesions.