PGE2, mPGES, and EP1-4 receptors are detected in KS lesions
We used immunohistochemistry to first investigate the presence of mPGES, PGE2 and EP1-4 in serial sections of biopsies from 3 healthy subjects and 3 KS+ patients. The presence of KSHV in KS lesions was confirmed by the detection of characteristic nuclear staining of LANA-1 (). Strong cytoplasmic mPGES, PGE2, and EP1-4 reactivity were detected in KS lesions (). However, KS lesion samples exhibited distinct staining for EP2 and EP4 receptors compared to normal samples (). Collectively, these results for the first time demonstrate the presence of inflammation associated EP receptors in KS lesions.
KSHV infection up-regulates EP1, EP3, and EP4 receptors and down-regulates EP2 receptors
We next examined the role of EP receptors in maintaining latent gene expression in TIVE-LTC cells that sustains expression of latency genes (16
). In a separate study, we have observed the up-regulation of COX-2 and mPGES proteins, and PGE2 secretion in TIVE-LTC cells compared to control TIVE cells and down-regulation of LANA-1 expression in TIVE-LTC cells treated with COX-2 inhibitor NS-398 (13
). Western blot analysis demonstrates that compared to TIVE cells, EP1, EP3, and EP4 receptors were significantly up-regulated in TIVE-LTC cells, whereas EP2 receptor was down-regulated in TIVE-LTC cells (). Confocal microscopy also confirmed the presence and cellular localization of all 4 EP receptors in TIVE and TIVE-LTC cells ().
KSHV utilizes EP receptors to maintain LANA-1 and COX-2 gene expression and PGE2 secretion
We measured LANA-1, COX-2, and COX-1 gene expression in TIVE-LTC cells treated with non-cytotoxic concentrations (Supplemental data Fig.1
, Panels A to I) of well-characterized competitive EP receptor blockers SC-51322 (EP1 antagonist; 50μM) or AH6809 (EP2 antagonist; 50μM) or GW 627368X (EP4 antagonist; 5μM) at 2h, 8h and 24h post-treatment and observed no significant change in COX-1 gene expression (). COX-1 gene expression was conducted as a control, since COX-1 promoter is constitutively active. EP1, EP2, and EP4 antagonists down-regulated LANA-1 gene expression significantly by 69%, 82%, and 73%, and by 55%, 64%, and 40% at 8h and 24h post-treatments, respectively (). COX-2 gene expression was down-regulated by EP2 and EP4 antagonists at 2h post-treatment and by EP4 antagonist at 8h post-treatment (). At 2h post-treatment, EP2 and EP4 antagonists down-regulated PGE2 secretion significantly with no significant changes at 8h and 24h post treatments ().
KSHV utilizes EP1 receptor mediated cytoplasmic Ca2+ ([Ca2+]i) signaling to maintain LANA-1 gene expression
From the above results demonstrating the down-regulation of LANA-1 gene expression by EP1 blockade () together with the fact that EP1 receptor is a well-characterized Ca2+
inducing GPCR (10
), we hypothesized that PGE2 secreted in the supernatant of TIVE-LTC cells might be inducing Ca2+
signaling via the EP1 receptor. Therefore, we predicted that if we block the EP1 receptor, the potential of the PGE2 in the supernatant to induce Ca2+
signaling via the EP1 receptor, if any, would also be blocked. The purpose of our experiment was not to test the effect of down regulating PGE2 secretion in the supernatant by EP receptor antagonists and consequently the calcium signal because even at 2h post treatment with EP2 and EP4 antagonists, PGE2 is present with in the range of 80-100pg/ml (). However, our goal was to investigate the effect of blocking EP receptors on the supernatant-induced calcium signal, if any. By doing so, we are answering the role of PGE2 in the supernatant in inducing calcium signal through EP receptors.
To test our hypothesis, TIVE-LTC cells were grown on coverslips and [Ca2+]i was measured as outlined in . To measure [Ca2+]i, cover slip 1 was removed and loaded with the Ca2+ indicator fura-2AM for 30 minutes with and without EP receptor antagonists in Hanks solution. However, while being loaded with fura-2AM, the cells in coverslip 1 are not exposed to the physiological supernatant of TIVE-LTC cells. Therefore, to test whether the supernatant of TIVE-LTC can induce Ca2+ signaling in coverslip 1 cells, we used the supernatant produced by cells grown in parallel on cover slip 2. The supernatant of coverslip 2 TIVE-LTC cells incubated in the presence of DMSO () induced a significant transient elevation in [Ca2+]i. We did not observe any significant change between the observed peak amplitude of transient elevation in [Ca2+]i induced by the supernatant alone or with solvent control (DMSO) (). In contrast, serum free medium used as a negative control had no significant effect on [Ca2+]i () indicating that factors present in the physiological supernatant of TIVE-LTC cells can induce Ca2+ signaling.
Next, we examined whether the transient [Ca2+]i induced by TIVE-LTC cell supernatant is due to activation of EP1 receptor by the supernatant PGE2. Therefore, we treated the cells on coverslip 1 with EP1 or EP2 or EP4 antagonist, while being treated with fura-2AM and the supernatant from coverslip 2 cells, as outlined in . Treatments with EP1 but not EP2 or EP4 antagonists significantly abolished the supernatant mediated Ca2+ signal in TIVE-LTC cells (). These observations clearly demonstrated that PGE2 present in the supernatant of TIVE-LTC cells could induce a Ca2+ signal through the EP1 receptor.
Inhibition of EP receptors down regulates p-Src, p-PI3K, p-PKCζ/λ, p-Akt and p-NFκB and up-regulates p-ERK in KSHV infected TIVE-LTC cells
We next examined the signal cascades regulated by EP receptors. TIVE-LTC cells serum starved for 48h were treated with EP1, EP2, and EP4 antagonists or DMSO. EP2 and EP4 antagonists down-regulated p-Src by 17% and 47% and 29% and 75% at 8h and 24h post-treatments, respectively (). At 24h post-treatment, EP1, EP2, and EP4 antagonists down-regulated PI3K phosphorylation by 23%, 33% and 34%, respectively with no effect at 8h post-treatment (). EP2 and EP4 antagonists down-regulated p-PKCζ/λ by 12% and 20% and 23% and 69% at 8h and 24h post-treatments, respectively (). Compared to DMSO treatment, we did not observe any changes on p-Akt by EP receptor antagonists at 8h and 24h post-treatment ( ()). At 8h post-treatment, p-NFκB was down-regulated by 39%, 46%, and 66% with EP1, EP2, and EP4 antagonists, respectively, and by 41% with EP2 antagonist 24h post-treatment (). With EP4 antagonist, p-ERK 1 and p-ERK 2 was up-regulated by 1.3 and 2.0-folds and 1.2 and 2.0-folds, at 8h and 24h post-treatment, respectively ().
KSHV infection and exogenous PGE2 activates the LANA-1 promoter
Based on our data from TIVE-LTC cells, we hypothesized that PGE2 mediated signaling can up-regulate LANA-1 promoter activity. To test this, 293 cells were transfected with a luciferase reporter gene under the control of 774bp LANA-1 promoter (p-LANA-1-Luc, ) (14
). The efficacy of our system was first demonstrated by the induction of p-LANA-1-Luc activity by primary KSHV infection whereas less induction of p-LANA-1-Luc activity was observed with entry incompetent heparin treated and UV inactivated virus ().
To determine the effect of exogenous PGE2 on p-LANA-1-Luc, we first confirmed the presence of EP1-4 receptors by FACS (). Exogenous PGE2 (10μM) induced p-LANA-1-Luc activity by 8.7-fold at 4h post-treatment with no significant effect on the empty vector (). Treatments with varying concentrations of PGE2 demonstrates that 10μM of PGE2 or more were necessary to activate p-LANA-1-Luc significantly with no significant difference between the effects of 10μM and 100μM of PGE2 (). Well-characterized agonists for EP1-4 receptors were also able to induce p-LANA-1-Luc activity significantly at 4h but not at 24h post-treatment ().
De novo KSHV infection of 293 cells induces the COX-2/PGE2 pathway
To validate whether the 293 cells used above were ideal to study the paradigm, we demonstrated that de novo KSHV infection of 293 cells, induces COX-2 and mPGES proteins (), PGE2 secretion (), and LANA-1 expression (). To determine whether the COX-2/PGE2 pathway is important for maintaining LANA-1 gene expression in 293 cells, we demonstrated that 10μM PGE2 can restore the reduction in LANA-1 expression caused by COX-2 specific inhibitor NS-398 (100μM) ().
Inhibition of Ca2+, p-Src, p-PI3K, p-PKCζ/λ, p-Akt 1/2, p-NFκB, and p-ERK 1/2 blocks PGE2 mediated LANA-1 promoter
We next examined the role of Ca2+, Src, PI3K, PKCζ/λ, Akt 1/2, NFkB, and ERK 1/2 in PGE2 mediated LANA-1 transcriptional regulation by measuring the LANA-1 promoter activity in 293 cells pretreated with specific inhibitors for 2h followed by PGE2 (10μM) treatment for 4h and then incubated with PGE2. We used pharmacological inhibitors of Ca2+ (BAPTA-AM and TMB-8), PI3K (Wortmannin and Ly290042), Src kinase (PP2), PKC (GFX and GO:6976), Akt 1/2 (Akt 1/2 inhibitor), NFκB (Bay 11-7085), and ERK 1/2 (PD98059 and U0126), at the indicated non-cytotoxic concentrations.
Ca2+ chelation (), Src inhibition () and PKC inhibition (, decreased PGE2 mediated p-LANA-1-Luc activity significantly (). Similarly, PI3K inhibitors wortmannin (1.0μM) and Ly290042 (25μM and 50μM) reduced PGE2 induced p-LANA-1-Luc activity significantly (). Akt 1/2 and NFκB inhibition down-regulated PGE2 mediated p-LANA-1-Luc activity significantly (). Though, PGE2 mediated LANA-1 promoter activity was down-regulated by Akt 1/2 inhibitor, we did not observe any effect on Akt phosphorylation by EP receptor antagonists (). Furthermore, exogenous PGE2 was able to induce Akt 1/2 in 293 cells. The dichotomy between TIVE-LTC and 293 cells indicates that in KSHV latent TIVE-LTC cells, Akt phosporylation might also be under the control of EP2/EP4 receptor independent mechanisms. However, in serum starved 293 cells, PGE2 might be acting as a powerful signal inducer through EP receptors to induce Akt that may subsequently activate the LANA-1 promoter. ERK inhibition by PD98059 (10μM and 20μM) and U0126 (5μM and 10μM) reduced PGE2 induced p-LANA-1-Luc activity significantly by 50-55% with no significant inhibition on the basal activity (). Our promoter studies using ERK 1/2 inhibitors are further validated by the observation that de novo KSHV infection and exogenous PGE2 activate ERK 1/2 in 293 cells (). This is in contrast to the EP4 antagonist induced up-regulation of ERK1/2 phosphorylation in TIVE-LTC cells. This could be due to the differences in the cell systems used as the determining factors regulating ERK phosphorylation in TIVE-LTC could be different from that of 293 cells. Under serum-starved conditions, PGE2 might be acting as a power signal inducer in 293 cells through EP receptors (). Therefore, PGE2 induced LANA-1 promote activity is inhibited by ERK inhibitors. However, in TIVE-LTC cells, the presence of viral proteins and a cytokine/chemokine rich supernatant might be altering the signal transduction profile of the cell to such an effect that EP2 and EP4 receptors might be responsible for inhibiting ERK phosphorylation.
To explore further the signal molecules studied here, next we used different combinations of signal inhibitors () at non-cytotoxic concentrations (supplemental data Fig.1
, panel J). Blocking of PI3K and Ca2+
simultaneously demonstrated a significant additive effect of 80% () on the decrease in LANA-1 promoter activity compared with 5μM of BAPTA-AM () and 12.5μM of Ly290042 (), when used alone.
Induction of Ca2+, p-Src, p-PI3K, p-PKCζ/λ, p-Akt 1/2, p-NFκB, and p-ERK 1/2 by PGE2 in 293 cells
To validate the capacity of PGE2 to induce LANA-1 promoter activity through the signal molecules that were blocked in , we examined whether exogenous PGE2 (10μM) and KSHV infection can induce them. To test whether PGE2 can induce Ca2+, we treated serum starved 293 cells loaded with fura-2AM with PGE2 (10μM) that evoked an oscillatory Ca2+ signal for 25 minutes (). As a negative control, we also measured the basal levels of Ca2+ in 293 cells treated with Ca2+ free Hanks BSS and did not observe any intracellular Ca2+ signals (). Compared to untreated cells, LPA treatment (positive control), KSHV infection and PGE2 (10μM) increased the phosphorylation of p-Src, p-PI3K, p-PKCζ/λ, Akt 1/2, NFκB, and ERK 1/2 ().
Identification of candidate PGE2 responsive elements on LANA-1 promoter
To determine the minimal LANA-1 promoter region responsive to exogenous PGE2, a sequential series of LANA-1 promoter deletion constructs were assayed in a luciferase reporter experiment in 293 cells (). We then examined the p-LANA-1-Luc sequence using Alibaba 2.1 TF software to characterize the transcription factor (TF) binding site profile (). Exogenous PGE2 (10μM) activated pGL3.6, pGL3.4, and pGL3.3 promoter constructs at a similar level while, the pGL3.2 and pGL3.1 promoter constructs had significantly lower activities (). Taken together, these results suggested that the promoter region located between −262bp and −159bp with candidate TFs such as YY1, Sp1, Oct-1, Oct-6, C/EBP, and c-Jun is required for PGE2 mediated LANA-1 promoter activity ().