KSHV activation of MAPKs and associated cellular pathogenesis following de novo
infection has been firmly established. KSHV-encoded IL-6 (vIL-6) and G protein-coupled receptor (vGPCR) upregulate angiopoietin-2 expression through the activation of ERK in lymphatic endothelial cells (14
). vGPCR also induces VEGF expression and secretion through the activation of ERK and p38 (13
). Furthermore, two KSHV genes, K15 and ORF49, initiate ERK and JNK/p38 activation, respectively (7
). Finally, ERK activation appears critically important for the establishment of KSHV gene expression during de novo
). Although these studies underscore the importance of MAPK activation for KS pathogenesis, clinical applications of small molecules targeting intracellular kinases are problematic given the nonselectivity of kinase inhibitors and associated toxicities (17
). Identifying novel mechanisms for KSHV regulation of MAPK activation may, therefore, offer more clinically tractable approaches for the treatment and prevention of KS.
We found that KSHV represses DUSP1 expression during de novo
infection. These data contradict results from one study mentioned previously (24
) but are well supported by our observations of repression of DUSP1 protein and transcript expression under conditions of de novo
infection of HUVEC and HFF by KSHV, repression of DUSP1 expression with ectopic expression of miR-K12-11, miR-K12-1, or xCT, and derepression of DUSP1 with miR-K12-11- or miR-K12-1-specific antagomirs applied to KSHV-infected HUVEC. Additional characterization of DUSP1 expression in a larger array of KSHV-infected primary cell types would help determine the cell type-specific nature of our findings. Although our work has initially focused on DUSP1 for reasons outlined above, “fine-tuning” of MAPK activation may occur in KSHV-infected cells as a result of simultaneous viral regulation of different DUSPs, and additional studies should confirm the functional relevance of these coordinated efforts.
Although our work to date has focused on KSHV miRNA-associated mechanisms for maintaining DUSP1 suppression at later time points during de novo
infection, it is likely that multiple KSHV-associated mechanisms, including viral replication-independent mechanisms, participate in the suppression of DUSP1. This is supported by our observation that miR-K12-11 expression and function are optimal beginning 24 h after KSHV incubation with primary cells in our experiments, along with our kinetic assays that reveal DUSP1 suppression within 10 min of incubation with either UV-KSHV or KSHV. The half-lives of DUSP1 protein and transcripts are relatively short (less than 2 h and as short as 40 min [19
]), so it is plausible that discernible differences in their expression may be observed over a short time frame. One possibility is that DUSP1 suppression in KSHV-infected cells is the result of ERK-dependent ubiquitin-proteasome degradation of DUSP1, as previously observed for other cancer cells (59
). However, DUSP1 suppression with UV-KSHV is not observed at 30 min despite the induction of ERK activation to levels 10-fold greater than those for controls at this time point (the latter result is in accord with previously published data for the kinetics of ERK activation for cells following their incubation with UV-KSHV [12
]). It is also possible that proteasome activity unrelated to ERK influences DUSP1 expression; proteasome activity influences signaling in human KSHV-infected lymphoma cell lines (60
), so we cannot categorically dismiss this possibility.
A second possibility is that virus interactions with the cell surface may trigger DUSP1 downregulation. Our observation that UV-KSHV reduces DUSP1 expression within 10 min, but that heat-inactivated KSHV does not (data not shown), supports this concept. One report indicates that KSHV interactions with toll-like receptor-4 (TLR4) mediate innate immune responses to KSHV (61
), and alterations in DUSP1 expression in response to TLR4 engagement (62
) and dendritic cell interactions with influenza virus (63
) have been reported. It is plausible, therefore, that in the absence of established KSHV gene expression (as with UV-KSHV), TLR-induced, MAPK-dependent activation of cytokines induces subsequent activation of DUSP1 in order to repress MAPK activation in a negative feedback loop.
A recent report indicates that murine herpesvirus-68 miRNAs are packaged within the virion (64
). Therefore, a third possibility is that packaged KSHV miRNAs suppress DUSP1 expression at early time points, and the less-robust DUSP1 suppression observed with UV-KSHV within 10 min of viral incubation may result from an attenuated effect of these miRNAs due to UV-irradiation (and no effect at later time points).
In addition to the likelihood of DUSP1 regulation by replication-independent mechanisms in KSHV-infected cells, it is plausible that DUSP1 expression is regulated by other replication-dependent mechanisms. Previous work has indicated that multiple KSHV miRNAs upregulate xCT, possibly through the targeting of other transcriptional repressors, including c-Maf (32
). In addition, despite our results for luciferase assays indicating a lack of direct interactions between miR-K12-11 or miR-K12-1 and the DUSP1 3′ UTR, we cannot categorically exclude direct interactions between these two miRNAs and DUSP1, since it has been recognized that both cellular and viral miRNAs bind sequences within the 5′ UTR and coding regions of target genes (67
). Moreover, the binding of KSHV miRNA to DUSP1 UTRs may be cell type specific. Nevertheless, our data suggest that multiple KSHV miRNAs coordinately regulate DUSP expression.
Our data are consistent with published data indicating that the upregulation of xCT by miR-K12-11 results in xCT-mediated activation of 14-3-3β expression, which, in turn, leads to 14-3-3β-mediated transcriptional repression of DUSP1 (32
). For additional confirmation, we show that targeting 14-3-3β restores DUSP1 expression and reduces the production of promigratory factors and endothelial cell invasiveness under conditions of either KSHV infection or ectopic miR-K12-11 expression. The expression of FBI1 and its effect on the transcriptional repression of DUSP1 were not directly addressed in our studies. Additionally, our data do not exclude the possibility that KSHV induction of 14-3-3β expression influences other genes which themselves regulate DUSP1 expression, cytokine expression, and/or endothelial cell invasiveness. For example, published data indicate that 14-3-3β facilitates Raf/Ras activation through the coupling of protein kinase C-ζ 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
). Furthermore, the mechanisms through which xCT induces 14-3-3β expression require additional clarification.
DUSP1 expression is induced by growth factors, the initiation of cellular stress, or pharmaceuticals, including retinoids and glucocorticoids (55
). We found that the glucocorticoid dexamethasone induces DUSP1 expression while inhibiting KSHV activation of ERK during de novo
infection. We cannot exclude the possibility that dexamethasone impacts the expression of KSHV genes that regulate ERK activation, or the expression of genes (viral or cellular) that impact cell invasiveness independent of DUSP1. Moreover, although glucocorticoids are used routinely for the treatment of KSHV-associated lymphoma (77
), they also upregulate phosphatidylinositol 3-kinase (PI3K)/Akt activation and nitric oxide synthase activity (78
), both of which are associated with KS progression (32
). Existing clinical data further suggest that glucocorticoids may increase the incidence of KS in organ transplant recipients or may exacerbate existing KS lesions (80
), and it remains controversial whether glucocorticoids induce lytic viral gene expression in latently infected cells (82
). DNA-damaging agents such as doxorubicin may actually induce DUSP1 expression (19
), and a recent study indicated that overexpression of DUSP1 protected breast cancer cells from chemotherapy-induced apoptosis through the repression of caspase activation and DNA fragmentation caused by doxorubicin or paclitaxel (89
). The effects of DUSP1 induction may, therefore, prove undesirable in the context of KS treatment with either of these agents. Additional studies are clearly needed to determine whether pharmacologic upregulation of DUSP1 offers a tractable and safe approach to the treatment of KS.
In summary, our data reveal suppression of DUSP1 expression by miR-K12-11 through indirect mechanisms involving xCT-mediated upregulation of a transcriptional repressor of DUSP1 (14-3-3β) in KSHV-infected cells. We hypothesize that, along with replication-independent mechanisms for KSHV suppression of DUSP1 expression, this contributes to the constitutive MAPK activation and pathogenesis related to KS ().
Proposed schematic representation of DUSP1 regulation by KSHV.