Prostaglandin E2 stimulates COX-2 expression via mitogen-activated protein kinase p38 but not ERK in human follicular dendritic cell-like cells

Mitogen-activated protein kinases (MAPKs) are important intracellular signaling molecules responsible for the various cellular functions under normal or pathologic conditions. These serine/threonine-specific protein kinases transduce signals coming from a plethora of extracellular stimuli including cytokines and prostaglandins (PGs). There are three major families of MAPKs, ERK, p38, and JNK, and subfamilies in each type [1]. ERK and p38, in particular, appear to be critical molecules based on the results obtained from knockout (KO) animals. Mice with single KO of certain subfamily of either ERK or p38 undergo embryonic death. For example, Erk2 KO mice and p38α KO mice are embryonic lethal [2, 3] while the single KO of any Jnk gene does not lead to the embryonic lethality [4]. These earlier studies underscore the physiological importance of ERK and p38 MAPKs.

Follicular dendritic cells (FDCs) are peculiar stromal cells observed in the B cell follicles of peripheral lymphoid organs [5]. Their cellular origin of mesenchymal stromal cells is a distinction of FDCs from other cellular components in the secondary lymphoid tissues most of which derive from hematopoietic stem cells [6]. The biological roles of FDC include B cell recruitment to the follicles, presentation of native antigens on the surface, and provision of survival, proliferation, and differentiation signals to germinal center (GC) B cells [7‐10]. In the course of efforts to understand the GC reactions at the molecular level, we have recently suggested another interacting pathway between FDC and B cells. We demonstrated the expression of cyclooxygenase-2 (COX-2) molecule in FDC-like cells in vitro and further verified FDCs as the major cell type expressing COX-2 in situ [11]. COX-2 is a well-known enzyme induced by various factors including inflammatory stimuli and serves the rate-determining role in the production of PGE₂ [12]. Using the experimental system containing FDC-like cells, we showed that PGs promote the survival of GC B cells by preventing apoptosis [13], augment the antigen-presenting ability of B cells by increasing CD86 expression [14, 15], and exert a positive feedback effect on COX-2 expression [16]. These in vitro results and our previous results with COX-2 KO mouse imply the important role of COX-2 molecule in the inflammation taking place in the immune tissues [17].

We have previously observed that ERK and p38 MAPKs are involved in COX-2 expression in FDC-like cells. For example, LPS-induced COX-2 expression was inhibited by ERK and p38 inhibitors, which was verified by the actual induction of phosphorylation of these MAPKs by LPS [18]. TGF-β-stimulated COX-2 induction also required ERK and p38 [19]. In the present study, we extended our previous reports and explored the intracellular pathway of PGE₂-induced COX-2 expression in FDC-like cells. PGE₂ treatment resulted in a rapid increase of p38 but not ERK phosphorylation. In contrast, IL-1β, whose effect was compared in parallel with PGE₂, induced phosphorylation of both MAPKs. Knockdown of these MAPKs revealed that p38 is essential for PGE₂ to induce COX-2 expression in FDC-like cells, in line with the phosphorylation results. Our data provide a potential rationale for the pharmacologic or genetic approaches to regulate p38 MAPK in the treatment of various inflammatory disorders.

We have recently demonstrated that PGE₂ stimulates COX-2 expression in human FDC-like cells via EP2 and EP4 surface receptors on the cell surface [16, 20]. In this study, we further investigated the underlying intracellular mechanism by examining the potential role of ERK and p38 MAPKs in this process. Our earlier results suggest that both ERK and p38 molecules are involved in the signaling pathway to COX-2 expression [19]. First, the effects of PGE₂ on the phosphorylation degrees of ERK and p38 proteins were analyzed by immunoblotting. The signaling molecule would display increased levels of phosphorylation since ERK and p38 are phosphorylated by MAP kinase/ERK kinase (MEK) to act on the target molecules [21]. PGE₂ did not increase phosphorylated forms of ERK but rather reduced ERK phosphorylation at 60 and 120 min post-stimulation by approximately 50% compared to the control maintained without PGE₂ (Fig. 1a). In contrast, p38 phosphorylation increase was evident from 15 min and continued until 60 min. For instance, more than 2-fold increase of p38 phosphorylation was observed at 30 min compared to the vehicle control. The elevated levels of p38 phosphorylation returned to background levels at 120 min. The enhancing effect on p38 phosphorylation was triggered by PGE₂ because such an activation was not observed in control cultures carried out collaterally in the absence of PGE₂ (Fig. 1b). To explore whether the differential phosphorylation induction is specific to PGE₂, we performed the phosphorylation kinetics of ERK and p38 after stimulation with IL-1β. IL-1β is a strong inducer of COX-2 in FDC-like cells [20]. Different from PGE₂, IL-1β treatment resulted in increased phosphorylation of both ERK and p38. For example, phosphorylation levels of both proteins were approximately 6-fold higher at 15 min after IL-1β stimulation (Fig. 2). Both PGE₂ and IL-1β did not significantly modulate the total protein levels of ERK and p38 molecules. Taken together, these results imply that ERK and p38 molecules are differentially involved in the COX-2 induction pathways by PGE₂ and IL-1β.

To determine the role of ERK and p38 in PGE₂- and IL-1β-triggered COX-2 expression, the impact of ERK and p38 protein knockdown was analyzed. As shown in Fig. 3a, PGE₂-induced COX-2 expression was significantly inhibited by p38 knockdown. About 65% reduced COX-2 protein was obtained in the cells transfected with p38 siRNA. Administration of FDC-like cells with siRNA against ERK did not significantly modulate the COX-2 induction. Similar results were obtained in experiments performed with pharmacologic inhibitors of ERK and p38 (Fig. 3b). PGE₂-induced COX-2 expression was significantly inhibited by SB203580 but not by PD098059. SB203580 is an inhibitor of p38 and PD098059 is a specific inhibitor of MEK-1, an upstream kinase of ERK. These inhibitors did not affect the expression of COX-1. COX-2 induction by other PGs, beraprost and PGF_2α, was affected similarly by p38 knockdown (data not shown). Next, we conducted the siRNA transfection experiment to investigate the effects of ERK and p38 protein knockdown in IL-1β-induced COX-2 expression. As presented in Fig. 4, IL-1β-induced COX-2 expression was suppressed approximately 50% by p38 knockdown and almost completely by SB203580. ERK knockdown or PD098059 treatment did not affect the COX-2 induction stimulated by IL-1β. The modulation of COX-2 protein expression either by IL-1β stimulation or p38 siRNA inhibition was reflected in the actual production of prostaglandins (Fig. 4b).

Using primary human FDC-like cells, we demonstrate here the essential requirement for p38 MAPK in COX-2 protein expression instigated by PGE₂. PGE₂ selectively induced phosphorylation of p38, and p38 knockdown gave rise to a marked suppression of PGE₂-induced COX-2 expression. The current data suggest another intracellular mechanism on the positive effect of PGE₂ on COX-2 expression. We have previously reported that PGE₂ promotes COX-2 expression in FDC-like cells by inhibiting Akt phosphorylation [16]. Therefore, PGE₂ that might be produced by autocrine or paracrine fashion in the GC appears to bind to EP2 or EP4 receptors on the surface of FDC [20] and enhances COX-2 expression by activating p38 MAPK and by suppressing Akt activity simultaneously. The upregulation of p38 phosphorylation and downregulation of Akt phosphorylation occurred with comparable kinetics in FDC-like cells. The elevated COX-2 expression and resultant PG production may contribute to the GC reactions in part by promoting the survival and antigen-presenting capability of B cells [13‐15]. Figure 5 shows our working model for COX-2 induction mechanisms in FDC that is based upon our experimental results presented here and previously.

p38 MAPK was required while ERK was dispensable for COX-2 induction in FDC-like cells. Compared with siRNA knockdown that did not deplete all the p38 proteins in target cells, almost complete inhibition of COX-2 was obtained with p38 inhibitor treatment, implying the essential requirement of p38 MAPK. Similar to this result, many investigators report that p38 MAPK plays the essential role in the IL-1β-stimulated COX-2 expression in human lung fibroblasts and MDA-MB-231 breast cancer cells [22, 23]. However, unlike FDC-like cells, not only p38 but also ERK mediates COX-2 induction by IL-1β in human colorectal cancer cells [24]. In a sharp contrast, IL-1β-treated COX-2 induction in human endometrial stromal cells was inhibited by ERK but not p38 inhibitors [25]. These data imply that COX-2 expression is regulated by distinct MAPKs in different cell types. In our hands, MDA-MB-231 breast epithelial cells did not respond to IL-1β, and COX-2 expression was not modulated as reported elsewhere [26].

Both PGE₂ and IL-1β displayed the common feature of p38 activation and p38 requirement for COX-2 induction. This result may be regarded as a molecular mechanism for our recent observation of a synergistic effect of PGE₂ and IL-1β in COX-2 induction [20], implying that the synergy of PGE₂ and IL-1β may be achieved partly by synergistic augmentation of p38 activity. Inasmuch as FDC is localized in the microenvironment of GC, the synergy suggests that PGE₂ takes part in the early stage of inflammatory response by providing more PGs to the ongoing responses initially triggered by pro-inflammatory cytokines such as IL-1β. IL-1β is produced at the initial stage of inflammation [27]. This speculation for the inflammation-amplifying role for PGE₂ is supported by several reports from other research groups. In response to endogenous PGE₂, pro-inflammmatory cytokines are released from monocytes and synovial fibroblasts [28, 29].

Although the physiological relevance of the current data is unclear, the results are in line with the general concept that MAPK p38 is involved in inflammation while ERK plays pivotal roles in cellular proliferation [30]. PGE₂ did not modulate the proliferation of FDC-like cells in the range of 0.01~10 μM concentrations (Supplementary Fig. 1). In this context, the increased ERK phosphorylation after IL-1β treatment is enigmatic since this cytokine does not significantly affect the growth of FDC-like cells. The target molecules and biological function of IL-1β-induced phosphorylated ERK remain to be investigated.

In conclusion, we demonstrate in this study the requirement of p38 MAPK in PGE₂-stimulated COX-2 protein in FDC-like cells. Considering the pivotal role of PGE₂ in the inflammatory microenvironment, p38 MAPK regulation via pharmacologic or genetic approaches may be taken in the treatment of various inflammatory disorders.