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The Journal of Immunology, 2007, 179, 7147 -7156
Copyright © 2007 by The American Association of Immunologists, Inc.
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Jak2 Dampens the Induction by IL-1β of Prostaglandin Endoperoxide H Synthase 2 Expression in Human Orbital Fibroblasts: Evidence for Divergent Influence on the Prostaglandin E2 Biosynthetic Pathway1

Rui Han2, Beiling Chen2 and Terry J. Smith3

Department of Medicine, Division of Molecular Medicine, Harbor-University of California Los Angeles Medical Center, Torrance, CA 90502 and the David Geffen School of Medicine at University of California Los Angeles, Los Angeles, CA 90095


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Prostaglandin endoperoxide H synthase 2 (PGHS-2) catalyzes the rate-limiting steps in the synthesis of PGE2. It is substantially but transiently induced in human orbital fibroblasts treated with IL-1β. In this study, we report that the induction of PGHS-2 by IL-1β is dramatically enhanced and prolonged when Jak2 signaling is abrogated, either with the specific inhibitor AG490 or by transiently transfecting fibroblasts with a dominant negative mutant Jak2. Attenuating Jak2 increases PGHS-2 steady-state mRNA levels, a consequence of increased gene transcription and mRNA survival in IL-1β-treated cultures. Surprisingly, interrupting Jak2 function also blocked the expected increase in PGE2 synthesis usually provoked by IL-1β. This resulted from the rapid loss of IL-1β-dependent arachidonate release and by attenuation of group IIA secreted PLA2 (sPLA2) gene induction. Supplying Jak2-compromised cultures with exogenous arachidonate failed to increase PGE2 production in response to IL-1β until cells were mechanically disrupted. However, transiently transfecting them with wild-type sPLA2 fully restored prostanoid production to anticipated levels. sPLA2 expression following transfection resulted in increased IL-1β-dependent PGHS-2 and microsomal PGE2 synthase levels. Thus, sPLA2 plays important roles in PGE2 synthesis in addition to its release of arachidonate. Our findings suggest that Jak2 ordinarily dampens and limits the duration of the PGHS-2 induction by IL-1β. Moreover, it is required for IL-1β-dependent signaling to sPLA2, the expression and activity of which are necessary for up-regulating PGE2 synthesis in orbital fibroblasts.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Prostaglandin E2 synthesis results from the coordinate activities of multiple enzymes. The process begins with the release of arachidonate from membrane lipids through the actions of phospholipases A2 (secreted phospholipase A2 (sPLA21)4 (1) and cytoplasmic PLA2 (cPLA2)) that hydrolyze acyl groups from the sn-2 position of glycerophospholipids (1). Group IIA sPLA2 is highly expressed in synovial fluid and plasma from patients with inflammatory diseases and neoplasms (2, 3). Moreover, this enzyme may exert some of its biological actions independent of its role in hydrolyzing phospholipids. This may relate to binding a 180-kDa M-type receptor (4). sPLA2 liberates arachidonate and therefore supports the catalytic activities of downstream enzymes such as the inflammatory cyclooxgenase, PG endoperoxide H synthase (PGHS-2; EC 1.14.99.1) (5). PGHS-2 is highly inducible in many cells (6, 7). Its expression can be transiently up-regulated by proinflammatory cytokines and blocked with glucocorticoids (8, 9). Microsomal PGE2 synthase (mPGES; EC 5.3.99.3) is also induced by cytokines (10) and is functionally coupled to PGHS-2 for efficient PGE2 generation.

Fibroblasts participate in tissue reactivity and coordinate immune responses (11). Those derived from the orbit and synovial membrane produce extraordinarily high levels of PGE2 when activated. In synovial fibroblasts derived from patients with rheumatoid arthritis, coordinate induction of PLA2, PGHS-2, and mPGES by cytokines such as IL-1β and TNF-{alpha} results in the generation of this prostanoid (5, 12, 13). Evidence exists for the involvement of both sPLA2 and cPLA2 in cytokine-mediated PGE2 production in these cells (5, 12). Orbital fibroblasts from patients with Graves’ disease manifesting thyroid-associated ophthalmopathy (TAO) also produce high PGE2 levels when treated with proinflammatory cytokines such as CD154, IL-1β, or leukoregulin (14, 15, 16). It is currently believed that the induction of PGHS-2 is critical to the inflammatory response in TAO.

Multiple signaling pathways have been implicated in the regulation by cytokines of PGE2 synthesis. For instance, MAPK plays a critical role in the activation of cPLA2 (17). Both JNK and p38 MAPK pathways are involved in the activation of PGHS-2 gene expression (18, 19, 20). Blockade of the p38 and ERK MAPK pathways can attenuate the induction by IL-1β of PGHS-2 and mPGES (16). In general, it would appear that the signaling upstream from PGHS-2 in most cell types exhibits substantial redundancy (21). The Janus protein tyrosine kinase family (Jak), comprises a group of important ~130-kDa receptor-associating cytoplasmic signaling proteins. Jak1, Jak2, Jak3, and Tyk2 contain well-conserved tyrosine kinase domains located in the carboxyl-terminal adjacent to pseudo-kinase domains (22). These latter regions may help regulate activities of catalytic function (23). Jak2 participates in a number of cellular processes by virtue of its central role in mediating cytokine-dependent signaling. Through the autophosphorylation of multiple tyrosine residues and by provoking tyrosine phosphorylation of residues on STAT proteins, a number of transcriptional events are influenced through Jak2 activity (24). Jak2 knockout mice exhibit a peculiar phenotype dominated by dysfunctional erythropoiesis and embryonic lethality (25, 26). Jak2 activity can be inhibited by AG490, a specific chemical inhibitor (27). Its potential for influencing the expression of IL-1β-driven gene expression such as that of PGHS-2 has not been previously explored. Although several cytokines activate Jak2, evidence for its primacy in mediating IL-1β-dependent signaling has been supported by relatively few reports.

We have found recently that IFN-{gamma} and IL-4, examples of Th1 and Th2 cytokines, respectively, can block the induction of hyaluronan and PGE2 production by IL-1β (28). Cell signaling relevant to these cytokine actions intersects at Jak2. Thus, this kinase might play an important role in regulating the action of IL-1β on PGHS-2 expression (28). In this study, we characterize the role of Jak2 in modulating the induction by IL-1β of PGHS-2 by showing how its interruption prolongs and substantially up-regulates the expression of the cyclooxygenase. This results from enhanced PGHS-2 gene promoter activity and PGHS-2 mRNA stability. Surprisingly, the increased PGE2 production expected following treatment with IL-1β is blocked with AG490 or by transiently transfecting cells with a dominant negative (DN) mutant Jak2. This results from attenuation of relatively early, IL-1β-dependent release of arachidonate and later from a blockade of sPLA2 induction. When wild-type sPLA2 is transiently transfected into fibroblasts where Jak2 is blocked, PGE2 production reaches anticipated levels. Thus, Jak2 may represent an important endogenous regulator of PGHS-2 expression and PGE2 synthesis in inflammatory orbital diseases such as TAO.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Materials

5,6-dichlorobenzimidazole (DRB), arachidonate, and cycloheximide were purchased from Sigma-Aldrich. IL-1β, IL-4, and IFN-{gamma} were purchased from BioSource International. AG490 was supplied by Calbiochem. The human mPGES cDNA and Abs directed against that protein were provided by Dr. P.-J. Jakobsson (Karolinska Institute, Stockholm, Sweden). Full-length human PGHS-2 cDNA was a gift from Dr. K. O’Banion (University of Rochester, Rochester, NY), and the plasmid designated –1800pGL2, containing a 1.8-kb fragment of the human PGHS-2 promoter, was generously supplied by Dr. S. M. Prescott (University of Utah, Salt Lake City, UT). Dr. D. E. Levy (New York University, New York, NY) provided the DN mutant Jak2 expression vector. mAbs directed against human PGHS-2 and sPLA2 and a specific ELISA for sPLA2 were purchased from Cayman. [3H]Arachidonate (214 Ci/mmol) and PGE2 enzyme immunoassay (EIA) kits were obtained from Amersham Biosciences. Abs against Jak2 and phospho-Jak2 were purchased from Santa Cruz Biotechnology.

Cell culture

Orbital fibroblast cultures were initiated from tissue explants obtained as surgical waste during decompression surgery performed for severe TAO or from normal-appearing orbital tissues in patients with noninflammatory conditions. Tissue collection occurred following informed consent. The Institutional Review Board of Harbor-University of California, Los Angeles Medical Center and the Center for Health Sciences at the University of California, Los Angeles approved these activities. Tissue fragments were generated by mechanical disruption of explants and fibroblasts were then allowed to adhere to plastic culture plates. They were covered with Eagle’s medium to which 10% FBS, glutamine (435 µg/ml), and penicillin/streptomycin were added as described previously (29). Medium was changed every 3–4 days, and monolayers were maintained in a 5% CO2, humidified incubator at 37°C. Culture strains were used between the 2nd and 12th passages from initiation. We have determined that the phenotype of these fibroblasts remains stable over that interval. All experimental manipulations, except those involving transient transfection of DNA, were conducted after a state of confluence had been reached. We have previously established the purity of these cultures and found them to be essentially devoid of contamination by endothelial, epithelial, and smooth muscle cells (30).

Northern blot analysis and RT-PCR

Total cellular RNA was extracted using the method of Chomczynski and Sacchi (31) from confluent 100-mm diameter plastic plates. Monolayers were covered with ULTRASPEC solution (Biotecx Laboratories) and harvested with a rubber policeman. Lysates were transferred to 1.5-ml RNase-free centrifuge tubes. A total of 0.2 ml of chloroform was added to each tube that was then vortexed. Tubes were cooled on ice for 15 min and centrifuged at 4°C. RNA was precipitated from the aqueous phase by addition of isopropanol, washed with 75% ethanol, and solubilized in diethyl pyrocarbonate-treated water. Equal amounts of RNA (10–20 µg) were subjected to electrophoresis in 1% agarose-formaldehyde gels and transferred to Zeta-Probe (Bio-Rad) membranes. The integrity of the electrophoresed RNA was verified by UV inspection following ethidium bromide staining. The [32P]dCTP random-primed (Bio-Rad) PGHS-2, mPGES, and sPLA2 probes were allowed to hybridize to immobilized RNA in ExpressHyb hybridization solution (BD Clontech) at 68°C for 1 h. Membranes were washed under high stringency conditions and exposed to X-Omat AR film (Eastman Kodak) at –70°C with intensifier screens. To normalize the amounts of RNA transferred, membranes were stripped according to the manufacturer’s instructions and rehybridized with a radiolabeled human GAPDH probe. Radioactive DNA/RNA hybrids were quantified by subjecting autoradiographs to densitometric analysis.

For PGHS-2 mRNA stability studies, cultures were treated with IL-1β for 3 h as a pretreatment. Cells were washed and incubated in growth medium for 4 h. At time 0, DRB (20 µg/ml), an inhibitor of gene transcription, was added to the medium of all plates without or with IL-1β (10 ng/ml) for the intervals indicated (see Fig. 3B). Abundance of mRNAs for the enzyme was quantified by Northern blot hybridization. PGHS-2 mRNA signals were normalized to their respective GAPDH levels. For sPLA2 mRNA stability analysis, semiquantitative RT-PCR was performed using an Eppendorf MasterCycler. Primer sequences for the reaction were: forward, 5'- ATTTGTCACCCAAGAACTCTTACC-3' and reverse, 5'-AATTCAGCACTGGGTGGAAG-3'. The linear phase for PCR was determined using 100 ng of cDNA and 1 µl of each primer (20 µM) in 50 µl of Master Mix (Qiagen). The optimized condition used was 95°C for 5 min, 95°C for 45 s, 60°C for 45 s, and 72°C for 45 s for 25 cycles. A final incubation for 10 min at 72°C allowed primer extension. PCR products were analyzed in 1% agarose gels and signal intensity was determined by densitometry using an Alpha DigiDoc instrument (Alpha InnoTech). The results were normalized to β-actin using forward primer 5'-CCAAGGCCAACCGCGAGAAGATGAC-3' and reverse primer 5'-AGGGTACATGGTGGTGCCGCCAGAC-3'.


Figure 3
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  		FIGURE 3. Effects of AG490 on (A) activation by IL-1β of the PGHS-2 gene promoter, (B) IL-1β-provoked PGHS-2 mRNA stability, and (C) IL-1β-dependent PGHS-2 3'-UTR/luciferase reporter gene activity in orbital fibroblasts. A and C, Semiconfluent fibroblast cultures were transiently transfected with either an 1800-bp fragment of the human PGHS-2 gene promoter fused to pGL2 or a fragment of the PGHS-2 3'-UTR fused to the pGL3 luciferase reporter gene. Cultures were then left untreated or were treated with IL-1β (10 ng/ml) for 3 or 7 h for the studies involving the gene promoter and 3'-UTR, respectively. None of the treatments affected the activity of the control reporters. Data are expressed as the mean ± SD of three replicates. A, In three experiments, treatment with IL-1β + AG490 resulted in an increase of 3.22 ± 0.83-fold (mean ± SD, n = 3) above those receiving IL-1β alone. B, Confluent cultures were pretreated with IL-1β for 3 h. At time 0, they were treated with nothing (control), IL-1β (10 ng/ml), AG490 (75 µM), or the combination indicated for the duration along the abscissa. Cell layers were harvested and RNA was extracted and subjected to Northern blot hybridization. Normalized data were plotted from a single representative experiment. In three identical experiments, each using a fibroblast strain from a different donor, PGHS-2 mRNA levels were 2.57 ± 0.11-fold (mean ± SD, n = 3) higher in cultures treated with IL-1β + AG490 compared with those receiving IL-1β alone.

 
Western blot analysis of fibroblast proteins

Cellular proteins were solubilized from rinsed fibroblast monolayers following the treatments indicated in the figure legends. The ice-cold harvest buffer contained 0.5% Nonidet P-40, 50 mM Tris-HCl (pH 8.0), and 10 µM PMSF. Lysates were taken up in Laemmli buffer and subjected to SDS-PAGE, and the separated proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad). Primary mAbs directed against PGHS-2 (10 µg/ml; Cayman) were incubated with the membranes for 2 h at room temperature. Following washes, membranes were reincubated with secondary, peroxidase-labeled Abs. In other experiments, primary Abs directed against human mPGES were used. The ECL (Amersham Biosciences) chemiluminescence detection system was used to generate signals, and the resulting bands were analyzed with a densitometer.

PGE2 assay

Fibroblasts were grown to confluence in 24-well plastic cluster plates in medium containing 10% FBS. Monolayers were shifted to serum-free medium for the final 24 h of incubation. IL-1β and the other test compounds were added for the duration and at the concentrations indicated in the figure legends. Medium was removed from cultures, and monolayers were covered with PBS in the presence of respective agents for the final 30 min of the treatment period. PBS was collected quantitatively, clarified by centrifugation, and subjected to PGE2 EIA using a commercially available kit (Amersham Biosciences). For studies involving disrupted cells, confluent cultures were incubated in serum-free DMEM and treated with nothing (control), IL-1β (10 ng/ml), AG490 (75 µM), or the combinations indicated for 16 h. Monolayers were harvested and intact cells were washed twice and incubated in 0.1 M Tris-HCl (pH 8.0) containing 2 mM glutathione. Cells were either kept intact or disrupted by sonication and the supernatant was collected. An aliquot was incubated without or with 10 µM arachidonate at 37°C with agitation for 10 min and then centrifuged for 5 min at 4°C and subjected to PGE2 EIA.

sPLA2 ELISA

Fibroblasts were allowed to proliferate to confluence. They were then shifted to medium containing 1% FBS and treated for the time intervals indicated with IL-1β (10 ng/ml) without or with AG490 (75 µM). Culture medium was collected and processed according to the instructions provided by the kit manufacturer (Cayman).

[3H]Arachidonate release assay

Cultures were allowed to proliferate to 90% confluence and then were incubated for 16 h in medium supplemented with [3H]arachidonate (1 µCi/ml). Cells were washed extensively and then treated with the test compounds indicated. Medium was harvested, centrifuged at 1000 x g for 5 min at 4°C, and counted in a liquid scintillation counter. Data are expressed as mean ± range of dpm. IL-1β-dependent release was determined by subtracting at each treatment time point the radioactivity released in the absence of the cytokine.

Transient transfection of orbital fibroblasts with plasmids containing sPLA2 and PGHS-2 gene promoters, PGHS-2 3'-untranslated region (UTR), sPLA2 expression vector, and DN mutant Jak2

Studies involving assessing activity of the human PGHS-2 promoter used the plasmid designated –1800pGL2, which contains the sequence –1840 to +123 and is thus located 5 bp upstream from the ATG. With regard to the human sPLA2 gene promoter, the following primers were synthesized and used to generate a 327-bp DNA fragment: forward; 5'-ACCGTTGATCACACCCAGAG-3' and reverse; 5'-CTCTCAGAGGACTCCAGAGTTGTATCC-3'. The amplified fragment was TA cloned and subcloned into the pGL2 reporter vector. This fragment extends from –307 bp to +20 bp relative to the putative transcription start site reported by Seilhamer et al. (32). The full-length PGHS-2 3'- UTR was amplified by PCR using the following primers: forward, 5'-TCTAGAAGTCTAATGATCATATTTAT-3' and reverse, 5'-TCTAGATAAGAAAATATAGGCAGAGT-3'. The fragment generated with the primers included an Xba cutting site at the 5' end. PCR were conducted under the following conditions: 95°C for 5 min, 95°C for 90 s, 60°C for 90 s, and 72°C for 90 s for 30 cycles. PCR products were gel-purified and cloned into the TOPO-CLONE vector (Invitrogen Life Technologies). The insert was then cut with Xba and subcloned into the Xba site located downstream from the luciferase coding sequence of the pGL3 vector. With regard to generation of an expression vector containing the coding region of the sPLA2 cDNA, the following primers were generated and used for PCR: forward 5'-ATTTGTCACCCAAGAACTCTTACC-3' and reverse; 5'-AATTCAGCACTGGGTGGAAG-3'. The resulting fragment was TA cloned and subcloned into pcDNA 3.1 expression vector (Promega). This construct was transiently transfected into cells under the conditions described below. The expression of sPLA2 was documented using a specific ELISA for sPLA2 (Cayman).

For studies involving the transient transfection of human fibroblasts, cultures were allowed to proliferate to 80–90% confluence in medium containing 10% FBS. DNA constructs were transfected using the LipofectAMINE PLUS system (Invitrogen Life Technologies). In brief, 0.75 µg of pGL2 promoter DNA and 0.1 µg of pRL-TK vector DNA (Promega), serving as a transfection efficiency control, were mixed with PLUS reagent for 15 min before being combined with LipofectAMINE for another 15 min. The DNA-lipid mixture was added to culture medium for 3 h at 37°C. DMEM containing 10% FBS replaced the transfection mixture overnight. Transfected cultures were then serum-starved and some received either IL-1β (10 ng/ml) for 2 h or nothing (control) as indicated in the figure legends. Cellular material was harvested in buffer provided by the manufacturer (Promega) and stored at –80°C until assayed. Luciferase activity was monitored with the Dual-Luciferase Reporter Assay System (Promega) in an FB12 tube luminometer (Zylux). Values were normalized to internal controls and each experiment was performed at least three times.

Statistics

Data were analyzed using Student’s t test and are presented either as the mean ± range of duplicates or the mean ± SD of triplicates performed in each assay described.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Blocking Jak2 activity results in a dramatically enhanced and prolonged induction by IL-1β of PGHS-2 expression

We assessed the pattern of IL-1β-dependent PGHS-2 expression in confluent orbital fibroblasts. PGHS-2 protein is detectable after 6 h of IL-1β treatment and is near-maximal at 12 and 24 h (Fig. 1A, upper panel). The signal decays to levels close to that in the control by 48 h. Similarly, PGHS-2 mRNA is first detectable at 6 h and remains substantially above control levels at 12 h (Fig. 1A, lower panel). By 24 h, the transcript is barely detectable. This pattern of induction is identical to that seen previously in orbital fibroblasts (16). The impact of inhibiting Jak2 activity on IL-1β-dependent PGHS-2 expression was ascertained. In the presence of AG490 (75 µM), PGHS-2 protein levels are barely detectable after 3 h and continue to climb at 6, 12, and 24 h. They are maximal at 48 h, the duration of the study (Fig. 1B, upper panel). PGHS-2 mRNA levels are also highly induced at 12 h and are maximal at 24 and 48 h when they are at least 40-fold higher than at baseline (Fig. 1B, lower panel). The inhibition by AG490 of Jak2 activity was documented by demonstrating that the compound markedly attenuated the actions of IL-4 on STAT6 phosphorylation (Fig. 1B, inset). Thus, Jak2 inhibition results in a dramatically enhanced and sustained induction of PGHS-2, the duration of which extends far beyond the pattern ordinarily observed. We then examined four orbital fibroblast strains, each from a different donor. As the Western blot analysis in Fig. 1C indicates, by 48 h, PGHS-2 protein expression had disappeared in cultures treated with IL-1β alone. Addition of AG490 to the cytokine dramatically enhanced and prolonged the induction of PGHS-2 by IL-1β. Thus, the impact of AG490 can be generalized to multiple strains of orbital fibroblasts.


Figure 1
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  		FIGURE 1. Effects of IL-1β (10 ng/ml) on PGHS-2 protein and mRNA expression without or with AG490 (75 µM). Confluent culture monolayers of orbital fibroblasts from a patient with severe TAO were treated with IL-1β without (A) or with AG490 (B) for the intervals indicated. Monolayers were harvested and subjected to Western blot analysis for PGHS-2 protein content and normalized by reprobing with anti-β-actin Abs (upper panels) or RNA was extracted and analyzed by Northern blot analysis with a 32P-labeled probe generated from the human PGHS-2 cDNA (lower panels). Relative band densities were quantified with a densitometer. Data in the columns represent the mean ± SD of three experiments. The inset in B contains a Western blot analysis of the effects of IL-4 (10 ng/ml) without or with AG490 for 30 min on phosphorylated STAT6 in orbital fibroblasts. C, Confluent cultures of orbital fibroblasts from four different donors were treated with the compounds indicated for 48 h and then analyzed for PGHS-2 protein levels by Western blot analysis.

 
As an alternative strategy for interfering with Jak2 function in orbital fibroblasts, a DN mutant kinase was transiently transfected into cultures and its impact on IL-1β-induced PGHS-2 expression was determined. In studies using AG490 to inhibit Jak2 activity, levels of PGHS-2 are increased by at least 20-fold over cultures receiving IL-1β alone for 16 h (Fig. 2A). Cells transfected with the Jak2 DN construct exhibit a 3-fold greater induction by IL-1β of the cyclooxygenase compared with those transfected with the empty vector (Fig. 2B). The transfection efficiency of these cultured cells is ~30%. Thus, results obtained with the chemical inhibitor and in cultures following transfection with the mutant Jak2 are congruent.


Figure 2
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  		FIGURE 2. A, Impact of Jak2 inhibition with AG490 on the induction by IL-1β of PGHS-2 protein. Confluent fibroblast cultures, generated from a patient with severe TAO, were treated with nothing, IL-1β (10 ng/ml), AG490 (75 µM), or a combination of these agents for 16 h and cell layers were harvested and subjected to Western blot analysis of PGHS-2 protein levels. B, Transient transfection of orbital fibroblasts with DN Jak2 enhances PGHS-2 induction by IL-1β. Semiconfluent cultures were transiently transfected with empty vector DNA or that containing a DN mutant Jak2. Cultures were treated with nothing or IL-1β for 16 h and monolayers were harvested and subjected to Western blot analysis as in A. Data in the columns are expressed as the mean ± SD of three experiments performed and used culture strains from three different donors.

 
Interfering with Jak2 function enhances IL-1β-dependent PGHS-2 gene promoter activity and mRNA stability

PGHS-2 can be regulated at both transcriptional and posttranscriptional levels, depending upon the cell type (6). In orbital fibroblasts, both levels of regulation are exerted by IL-1β and related cytokines (14, 15, 33). We next set out to identify the mechanism involved in the exaggerated induction of PGHS-2 in cells where Jak2 activity had been attenuated. IL-1β can up-regulate PGHS-2 gene promoter activity modestly at 3 h, a treatment period well after the peak effect seen at 2 h (16) (control, 7.8 ± 0.1 arbitrary units (AU); IL-1β 8.74 ± 0.6 AU; Fig. 3A). Addition of AG490 (75 µM) to medium containing IL-1β enhanced the cytokine effect on promoter activity by 4-fold (IL-1β plus AG490, 33.7 ± 0.5 AU, p < 0.05 vs IL-1β alone). Fig. 3B contains data showing the stability of PGHS-2 mRNA under basal culture conditions and following addition of IL-1β, AG490, or the combination of the two to the culture medium. This transcript is known to decay rapidly in many different cell types, the consequence of instability sequences found in its 3'-UTR (34). As Fig. 3B indicates, there is nearly complete disappearance of the mRNA by 24 h in control medium or medium supplemented with AG490. Addition of IL-1β resulted in a small residual mRNA signal at 24 h which was sustained for the duration of the study (48 h). When IL-1β and AG490 were combined, mRNA levels had dropped by only 38 and 63% at 24 and 48 h, respectively. Thus, the impact of IL-1β on prolonging PGHS-2 mRNA stability is greatly enhanced by inhibiting Jak2 activity.

We subsequently cloned a 1374-bp nearly full-length fragment of the human PGHS-2 3'-UTR containing 22 AUUUA instability elements and spanning the sequence immediately 3' from the stop codon. It was fused to pGL3 and then transfected into orbital fibroblasts. As the data shown in Fig. 3C demonstrate, addition of the 3'-UTR fragment lowered the basal activity of pGL3 by 50%. Addition of IL-1β restored ~20% of the reduced activity. When AG490 was added to the cytokine, the reporter activity was enhanced further so that >50% of this activity had been restored.

Although it enhances the induction by IL-1β of PGHS-2, AG490 treatment blocks the anticipated cytokine-dependent increase in PGE2 production

PGHS-2 catalyzes the rate-limiting steps in PGE2 biosynthesis (6). IL-1β dramatically up-regulates the production of PGE2 in orbital fibroblasts after 16 h (control, 42 ± 1.6 pg/ml; IL-1β, 1162 ± 144 pg/ml, n = 3, p < 0.01). AG490 was then examined for its impact on time-dependent IL-1β-provoked PGE2 synthesis. It completely blocked the expected increase in PGE2 synthesis in IL-1β-treated cultures (Fig. 4A). Levels of the prostanoid remained very low during the early phase of the IL-1β treatment (0–24 h) in the presence of AG490. PGE2 accumulation in the culture medium becomes barely detectable at 48 h when it is 431 ± 180 pg/ml vs 49 ± 2 pg/ml at time 0 (p < 0.01). This sharply contrasts with cultures receiving the cytokine alone, where PGE2 levels were elevated above controls at 3 h, reached a maximum at 12 h when they were at least 500-fold higher than controls (p < 0.001), and were sustained for the duration of the study. Transiently transfecting the fibroblasts with a DN mutant Jak2 also results in a substantially decreased PGE2 level in IL-1β-treated cultures compared with those transfected with empty vector (Fig. 4B). From these studies, it became apparent that despite the dramatic induction of PGHS-2, orbital fibroblasts in which Jak2 signaling is interrupted fail to produce the anticipated high levels of PGE2. Thus, it appears that Jak2 might ordinarily play some critical role in supporting cytokine-provoked PGE2 synthesis while at the same time dampening the magnitude of the induction by IL-1β of PGHS-2 expression.


Figure 4
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  		FIGURE 4. Effects of disrupting Jak2 signaling on PGE2 synthesis. A, Cultures were incubated with IL-1β (10 nM) alone or with AG490 (75 µM) for the times indicated along the abscissa. At 48 h, PGE2 production was 9.4 ± 5.4-fold higher in cultures treated with IL-1β than in those treated with IL-1β + AG490 (p < 0.001, n = 9). B, Semiconfluent cultures were transiently transfected with empty vector (left) or a plasmid containing the DN mutant Jak2 construct (right) as described in Materials and Methods. Transfected cultures were then treated with IL-1β for 16 h. PBS with the respective additives replaced medium for the final 30 min of incubation and was assayed for PGE2 content. Data are expressed as the mean ± SD of triplicate independent determinations. These studies involved culture strains from three different donors.

 
Finding that interrupting the Jak2 pathway caused divergent effects on PGHS-2 expression and PGE2 production suggested that another component of this biosynthetic cascade might be dependent on Jak2 signaling. Thus, we assessed the impact of AG490 on sPLA2 and mPGES expression and their induction by IL-1β. We have reported previously that mPGES forms an efficient enzyme pair with PGHS-2 in orbital fibroblasts (16) as others have reported in different experimental models (10). Moreover, sPLA2 is coupled to PGE2 synthesis in human fibroblasts (5). As the data in Fig. 5A demonstrate, mPGES levels are increased by IL-1β in a time-dependent manner, consistent with our previously reported findings (16). Addition of AG490 in combination with the cytokine inhibited these effects. IL-1β also provokes a time-dependent increase in sPLA2 protein expression (Fig. 5B) that became detectable at 24 h (2-fold) and then at 48 h became elevated by 19-fold (p < 0.001 vs control). AG490 blunts this induction completely. But IL-1β induction of sPLA2 occurs well after rapid increases in PGE2 production and the rapid blockade imposed by AG490. These were evident by 3 h. As the data in Fig. 5C indicate, IL-1β enhanced [3H]arachidonate release within 3 h. This rapid effect was completely blocked by AG490 after 3 h, as the data in Fig. 5D demonstrate. Thus, the impact of Jak2 inhibition on cytokine-dependent PGE2 production can be explained potentially by two discrete actions: 1) blockade of the very rapid effects of IL-1β on arachidonate release occurring within 3 h and 2) attenuation of a slowly evolving induction of sPLA2 by the cytokine after 24–48 h.


Figure 5
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  		FIGURE 5. Impact of AG490 (75 µM) on the induction by IL-1β (10 ng/ml) of mPGES (A) and sPLA2 (B) protein expression. C, Time-dependent effects of IL-1β on [3H]arachidonate release from orbital fibroblasts. D, The impact of AG490 on IL-1β-dependent [3H]arachidonate release. E, IL-4 and IFN-{gamma} can enhance the effects of IL-1β on sPLA2 expression which can be blocked with AG490. Confluent cultures of orbital fibroblasts, in this case from a patient with severe TAO, were subjected to the treatment indicated. A, Cell layers were solubilized and subjected to Western blot analysis for mPGES protein. Data shown are from one experiment. In a total of three experiments, mPGES levels in cultures treated with IL-1β + AG490 were 75 ± 5% (mean ± SD) lower than those treated with IL-1β alone at 48 h (B and E) Media were collected and subjected to an ELISA specific for sPLA2. In three experiments, IL-1β increased sPLA2 levels by 30.6 ± 2-fold (mean ± SD) compared with those treated for 48 h with IL-1β + AG490. (C,D) Cultures were labeled with [3H]arachidonate (1 µCi/ml) for 16 h, as described in Materials and Methods. Cell layers were washed extensively and treated with the test compounds indicated for the intervals indicated along the abscissa (C) or for 3 h (D), medium was harvested, and an aliquot was subjected to liquid scintillation counting. Data in C represent the difference between radioactivity released without and with IL-1β and are expressed as the mean dpm ± SD of triplicate independent determinations. Experiments used cultures from two different donors with severe TAO.

 
Because blocking Jak2 activity abolished the induction by IL-1β of sPLA2, we next tested whether increasing its activity would actually enhance that induction by IL-1β. IL-4 and IFN-{gamma} both augment the induction by IL-1β of sPLA2 (Fig. 5E). In those cultures receiving IL-1β, sPLA2 was 35 ± 2 pg/ml. Addition of IL-4 or IFN-{gamma} resulted in levels of 152 ± 45 pg/ml (p < 0.05 vs IL-1β) and 81 ± 0.9 pg/ml (p < 0.001 vs IL-1β), respectively. In contrast, neither cytokine induced sPLA2 protein expression as single agents (data not shown).

We next determined the mechanisms though which the induction by IL-1β of sPLA2 expression was mediated. Steady-state sPLA2 mRNA was modestly up-regulated by IL-1β at 48 h, the duration of the study, when the transcript was 2.4-fold above control (Fig. 6A). Inhibition of Jak2 activity resulted in a complete suppression of the increased transcript level. IL-1β enhanced the activity of a 327-bp fragment of the human sPLA2 gene promoter fused to a luciferase reporter gene and transfected into orbital fibroblasts (Fig. 6B) when assessed 3 h after cytokine treatment. The magnitude of the increase was ~2.3-fold above activity in untreated cultures. AG490 attenuated this increase (IL-1β, 2.74 ± 0.1 AU; IL-1β plus AG490; 1.82 ± 0.03 AU, p < 0.05). sPLA2 mRNA appears relatively stable under basal culture conditions (Fig. 6C). After 20 h, the transcript levels are depleted by 47%. IL-1β treatment enhances stability modestly. In contrast, AG490 accelerates mRNA loss so that by 20 h of its addition to the medium, sPLA2 mRNA had essentially become undetectable (control, 113 ± 4 AU; IL-1β,152 ± 2 AU; AG490, 8.5 ± 0.5 AU; IL-1β plus AG490, 12 ± 0.01, p < 0.01 vs IL-1β). Addition of IL-1β to the inhibitor resulted in a more modest reduction in transcript loss at 7 h and the mRNA was undetectable at 20 h.


Figure 6
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  		FIGURE 6. A, Effects of AG490 on the induction by IL-1β (10 ng/ml) of steady-state sPLA2 mRNA levels. Confluent orbital fibroblast cultures were treated as indicated, cell layers were harvested, and RNA was extracted and subjected to RT-PCR. sPLA2 signal densities (AU), corrected for respective β-actin signals were: IL-1β: 0 h, 1243; 6 h, 1629; 16 h, 1335; 24 h, 1405; 48 h, 2994; IL-1β + AG490: 0 h, 1012; 6 h, 1006; 16 h, 975; 24 h, 1129; 48 h, 678. Data from three separate experiments revealed sPLA2 mRNA levels 8 ± 1.5-fold (mean ± SD) higher with IL-1β treatment compared with IL-1β + AG490 at 48 h. B, Effect of IL-1β without or with AG490 on sPLA2 promoter/luciferase reporter gene activity. Semiconfluent cultures were transiently transfected with empty vector DNA or that fused to a 327-bp fragment of the sPLA2 gene promoter. Transfected cultures were then treated as indicated for 3 h, harvested, and luciferase activity was assessed. Data are expressed as the mean ± range of duplicates from a single experiment. Results from three experiments demonstrated a 41 ± 3% decrease with IL-1β combined with AG490 compared with IL-1β alone (mean ± SD, p < 0.01). C, Impact of IL-1β, AG490, or the combinations indicated on sPLA2 mRNA stability. Confluent cultures were pretreated with IL-1β for 3 h and medium was removed and replaced with fresh medium containing DRB (20 µg/ml) with the additives indicated. Cultures were harvested and RNA was extracted and subjected to RT-PCR for sPLA2 mRNA levels. These were corrected for their respective β-actin mRNA levels. Data are expressed as the mean ± SD. In other studies, the results were: control, 84 ± 1%; IL-1β, 86 ± 2%; AG490, 51 ± 2%; IL-1β + AG490, and 64 ± 3% at 7 h compared with time 0 (mean ± SD, n = 3). Studies used fibroblast strains from three different donors.

 
To address the mechanism involved in AG490-mediated knock-down of IL-1β-dependent PGE2 production, we determined whether providing an exogenous pool of arachidonate would allow the exaggerated PGHS-2 levels to culminate in enhanced generation of the prostanoid. In a study where cultures receiving IL-1β alone reached a PGE2 level of 77 ± 23 pg/106 cells, addition of exogenous arachidonate (10 µM) to the medium in addition to the cytokine resulted in a PGE2 level of 981 ± 92 pg/106 cells (Fig. 7A). When AG490 was also added, levels of PGE2 became undetectable. Thus, addition of exogenous arachidonate was inadequate to restore PGE2 synthesis in cultures of intact cells where Jak2 was inhibited. Control cultures and those treated with IL-1β with or without AG490 were then incubated with exogenous arachidonate (10 µM) as intact cells or following fibroblast disruption. As expected (35), breaking open the cells substantially restored PGE2 production despite the addition of AG490 (Fig. 7A).


Figure 7
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  		FIGURE 7. A, Exogenous arachidonate failed to compensate for the blockade imposed by AG490 of IL-1β-dependent PGE2 production in intact orbital fibroblasts but disruption restored enhancement. Confluent cultures were treated for 16 h with nothing, IL-1β (10 ng/ml), AG490 (75 µM), arachidonate (10 µM), or the combinations indicated. Media samples were removed 30 min before the end of the incubation and replaced with PBS with the respective additives. Some cells were disrupted by sonication and the supernatant was collected. An aliquot was incubated without or with 10 µM arachidonate and subjected to the PGE2 EIA. Data are expressed as the mean ± range of two independent replicates. B, Transfecting AG490-treated cultures with wild-type sPLA2 rescues PGE2 production in response to IL-1β. Semiconfluent cultures were transiently transfected with empty (control) vector or an expression vector containing a wild-type sPLA2 open reading frame. Cultures were then treated for 16 h with nothing, IL-1β, AG490, or the combination indicated. Media aliquots were assayed for PGE2 content. Sister cultures transfected with sPLA2 were subjected to an assay for sPLA2 protein expression (inset). Data are from a single experiment and are expressed as mean ± SD of three independent replicates. Results from three experiments, each using cells from a different donor, demonstrated a 8 ± 2-fold increase in PGE2 following transfection with wild-type sPLA2 compared with the control vector at 48 h (mean ± SD, p < 0.01).

 
Subsequently, we transiently transfected fibroblasts with wild-type sPLA2 to determine whether increasing the expression of this protein could rescue PGE2 production in AG490-treated cells. Cultures received an expression plasmid containing the sPLA2 reading frame or the empty vector (control), and then they were treated with nothing or IL-1β (10 ng/ml) for 16 h in the absence or presence of AG490. As the data in Fig. 7B demonstrate, cultures transfected with the empty vector or with sPLA2 and challenged with IL-1β in the absence of AG490 responded with a robust increase in PGE2 levels. In contrast, those transfected with the empty vector and treated with AG490 plus the cytokine mounted a dramatically reduced response. In contrast, those transfected with sPLA2 exhibited PGE2 levels similar to cultures without AG490 (empty vector vs sPLA2, p < 0.001). The effects of sPLA2 overexpression on other enzymes in the PGE2 synthetic cascade were then determined in cells treated with AG490. Levels of IL-1β-dependent PGHS-2 (Fig. 8A) and mPGES (Fig. 8B) were both substantially enhanced following transfection of sPLA2 into the orbital fibroblasts. Levels of the former were increased within 3 h (p < 0001 vs vector control) while those of mPGES were affected only after 48 h (p < 0.002 vs vector control). These data indicate that introduction of sPLA2 expression in Jak2-inhibited fibroblasts completely restores PGE2 synthetic function while exogenous arachidonate does not. This finding implies strongly that sPLA2 contributes to PGE2 biosynthesis in orbital fibroblasts through actions apart from arachidonate generation.


Figure 8
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  		FIGURE 8. Transient transfection of AG490-treated orbital fibroblasts with sPLA2 enhanced the induction by IL-1β of PGHS-2 (A) and mPGES (B). Fibroblast cultures were transiently transfected with either empty vector or vector containing sPLA2. They were then treated with nothing or IL-1β (10 ng/ml) and all received AG490 (75 µM) for 3 h (A) or 48 h (B), and monolayers were subjected to Western blot analysis. Data are normalized to their respective β-actin signals. The columns represent the mean ± SD of data from three separate experiments performed using two strains of fibroblasts, each from a different donor. (PGHS-2, IL-1β + vector vs IL-1β + sPLA2, p < 0.0001; mPGES, IL-1β + vector vs IL-1β + sPLA2, p < 0.002).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Jak2 functions in a divergent manner with regard to PGHS-2 expression and PGE2 production in orbital fibroblasts. It may ordinarily serve to dampen this pathway by limiting the amplitude and duration of PGHS-2 induction. In contrast, the current findings also support the participation of Jak2 in mediating the up-regulation by IL-1β of sPLA2 activity and expression. IL-4 and IFN-{gamma} enhance synergistically the up-regulation by IL-1β of sPLA2, effects which intersect at Jak2, consistent with their other actions (36, 37). These cytokines up-regulate sPLA2 expression. Moreover, analysis of the sPLA2 gene promoter reveals CAAT and TATA boxes and numerous transcription factor response elements including those for STAT, NF-{kappa}B, CREB, C/EBP, and AP-1 (38, 39, 40). The 327-bp fragment used in our studies contains STAT, SP-1, AP-1, and CREB sites. Thus, it is not surprising that a complex array of cytokines and the multiple signaling pathways they use contribute to the expression of sPLA2.

Insinuating Jak2 into the induction by IL-1β of PGHS-2 represents a new association between this cytokine and Jak/STAT signaling. STAT3-NF-{kappa}B interactions are promoted by IL-1 through a TRAF6- and p65-dependent mechanism (41). Tsukada et al. (42) reported some time ago that IL-6 and IL-1β might share signaling pathways through a common activation of STATs. IL-1β can modulate the induction of acute-phase response genes by IL-6 (43). The former promotes the dephosphorylation of IL-6-activated STAT1. Jak2 may also play a role in the activation of ERK2, an important component of signaling to PGHS-2 by IL-1β (16), as well as STAT3, in cultured human multiple myeloma cells (44). Our findings suggest that Jak2 plays divergent roles, mediating the induction by IL-1β of sPLA2 on the one hand, but attenuating how that cytokine up-regulates PGHS-2 on the other. Thus, our observations suggest that Jak2 functions as a pacemaker for prostanoid biosynthesis. They are consonant with the generally accepted view that Jak2, through its coupling with other components of the Jak/STAT pathway, participates in the expression/induction of multiple enzymes in this pathway.

We have previously reported that the induction by IL-1β of PGHS-2 expression in orbital fibroblasts is mediated through multiple mechanisms (16). The cytokine leads to a time-dependent albeit modest and transient up-regulation of PGHS-2 gene promoter activity but a more substantial enhancement of PGHS-2 mRNA stability. It appears that Jak2 modulates turnover of this transcript, an influence that can be partially overcome through its inhibition (Fig. 3B). The basis for the inherent instability of PGHS-2 relates to multiple AUUUA elements, of which 22 can be identified in the human 3'-UTR (34, 45). The impact of Jak2 on sPLA2 mRNA stability is very different. AG490 shortens the survival of the hybridizable transcript and IL-1β is only modestly effective in its rescue (Fig. 6C). The sPLA2 3'-UTR is relatively short (~260 bp) and includes few recognizable regulatory elements. Two 15-lipoxygenase DICE-like sequences, spanning 73–89 and 765–784 nt, can be identified in the 5'-UTR and 3'-UTR, respectively. There are no instability motifs analogous to those in the PGHS-2 3'-UTR. Thus, the very different impact of Jak2 on PGHS-2 and sPLA2 transcript stability can be reconciled by differences in the primary structure of these 3'-UTRs.

The generation of PGE2 results from a complex interplay between several enzymes and the signaling events influencing their expression. For instance, cPLA2 appears to be required for the induction of sPLA2 and PGHS-2 by IL-1β and TNF-{alpha} (46). Moreover, functional coupling between PLA2 and PGHS exhibits isoform specificity (47). Considering the remarkable fidelity with which PGHS and PGES seem to interact (10, 48), it is not surprising that the signaling pathways used in their regulation by cytokines also cross-talk. Thus, a complex pattern of functional coupling between these enzymes tightly regulates PGE2 production under physiological and pathological circumstances. Our finding that sPLA2 overexpression could enhance IL-1β induction of PGHS-2 and mPGES (Fig. 8) is consistent with the relationship shared by these enzymes.

The impact of high PGE2 concentrations on immune responses and tissue remodeling in orbital connective tissue can be substantial. In TAO and other inflammatory diseases, cytokines emanating from recruited or residential cells drive PGE2 production and therefore bias the profile and behavior of B and T lymphocytes and mast cells (49, 50, 51). A very recent study using microarray gene analysis of affected orbital adipose tissue has identified PGHS-2 as an overexpressed gene in active TAO (52). Jak2 signaling may prove an attractive therapeutic target in diseases where PGHS-2 plays a prominent role. Moreover, it might represent a determinant of the inflammatory phenotype exhibited by orbital fibroblasts. An inventory of additional IL-1β-activated genes affected by Jak2 might prove interesting.


   Acknowledgments
 
We are grateful to Debbie Hanaya for her expert secretarial assistance in the preparation of this manuscript. We thank Dr. Raymond Douglas for his help in procuring the fibroblast strains used in these studies.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by National Institutes of Health Grants EY008976, EY011708, DK063121, and RR00425. We gratefully acknowledge generous support from the Bell Charitable Foundation. Back

2 R.H. and B.C. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Terry J. Smith, Division of Molecular Medicine, Building C-2, Harbor-University of California Los Angeles Medical Center, 1124 West Carson Street, Torrance, CA 90502. E-mail address: tjsmith{at}ucla.edu Back

4 Abbreviations used in this paper: sPLA2, secreted phospholipase A2; cPLA2, cytoplasmic PLA2; mPGES, microsomal PLA2 synthase; PGHS, PG endoperoxide H synthase; DN, dominant negative; DRB, 5,6-dichlorobenzimidazole; TAO, thyroid-associated ophthalmopathy; EIA, enzyme immunoassay; UTR, untranslated region; AU, arbitrary unit. Back

Received for publication May 4, 2007. Accepted for publication September 1, 2007.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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