Biochin)ica et Bioplzvsica Acta. 1083 (1991) 121-134 ,i 1991 Elsevier Science Publishers B.V. 0005-2760/91/$03.50 A DONIS 000527609100163R

121

BBALIP 53618 Review

Prostaglandin endoperoxide synthase: regulation of enzyme expression David

L. DeWitt

Department of Biochemistry. Mit'higan State Lim'er~iO'. East La~lsing, MI (U.S.A.)

(Received 9 October 1990)

K,,,y words: Cyclooxygcnase; Transcriptional regulation: Dexamethasone: Renal function; Parturition: Ovulation: Inflammation: 13T3 fibrot)last): flqL-60): 1U937): (Endothelial celtl: (MC3T3-EI cell)

Contents I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

121

II.

Regulation of PGH synthase in fibroblasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

123

IlL

Regulation of PGH synthase expression during the reproducti,,'e cycle . . . . . . . . . . . . . . . . . . . . . A. Ovulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Luteolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Parturition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

125 126 126 127

IV.

Regulation of vascular prostaglandin synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Vascular endothelinm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Smooth muscle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

127 128 129

V.

Regulation of prostaglandin synthesis in macrophage/monocytcs . . . . . . . . . . . . . . . . . . . . . . .

129

VI.

Regulation of renal prostaglandin synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

130

VII. Regulation of prostaglandin synthesis in bone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

130

VIII. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

131

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

132

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

132

Abbreviations: PG. prostaglandin; TX. thromboxane; LT, leukotnene; PDGF, platelet-derived growth factor~ ~('~F, epidermal growth factor~ GM-CSF, granulocyte/macrophage-colony stimulating factor; PMA. 12-O-Tetradecanoylphorbo! !3-acetate; LH. luteinizing hormone; FSH, follicle stimulating hormone; hCG, hi.man chorionic gonadotropin; F(:IF, fibroblast growth factor; TGF-fl, transforming growth factor-/]; PKC, protein kinase C; HUVECS, human umbilical vein endothelial cells; BAECS, bovine aortic endothelial cells; LPS, lipopolysaccharide. Correspondence: D. DeWitt, Depaltment of Biochemistrj, Biochemistry Building, Room 510, Michigan State University, East Lansing MI 48824. U.S.A.

i. I n t r o d u c t i o n P r o s t a g l a n d i n synthesis is initiated w h e n agonists s u c h as h i s t a m i n e [1], b r a d y k i n i n [2-4], leukotrienes i5,6i, a n g i o t e n s i n [7], c y t o k i n e s [8-111, t h r o m b i n [41, or g r : ~ t h factors [ 1 2 - 1 4 ] a c t i v a t e specific p h o s p h o l i p a s e s c a u s i n g the release o f a r a c h i d o n a t e f r o m m e m b r a n e s . R e l e a s e d a r a c h i d o n a t e is c o n v e r t e d to P G H 2 b y p r o s t a g l a n d i n H ( P G H ) s y n t h a s e , the central e n z y m e in the p r o s t a g l a n d i n b i o s y n t h e t i c p a t h w a y (see Ref. 15 for a r e v i e w o f the physical a s p e c t s o f this e n z y m e ) , a n d then,

122 TABLE l Factors which affect PGH ~Tnthase expression in vitro

Effector

Cell system

PG Synthesis

PGHS Activity

PGHS protein

PGHS mRNA

Ref.

PDG F/ serum IL-1

3T3 cells mesangial SV-T2 3T3 dermal fibro. HUVECS dermal fibro. amnion HUVECS HL-60 U937 3T3 cells PO follicles uterus amnion MC3T3-E1 bovine smooth muscle cells HUVECS BAECS MC3T3-EI FRTL-5 MC3T3-EI U937 human monocytes human monocytes U937 dermal fibro.

+ + + + + + NE + + NR NR NR NE +

+ + + + NR + + NR + + NR NR + + +

NE~ _.aNR +h + + NR

+ + NR NR + NR NR NR NR NR + + NR NR

22,24 101 11 32.33,40 8.52 33 62 38 22,90 92 24 46,47 53.54 59-61 107

+ + + + + + + + -

+ NR NR + NR + ND +

NR + + + NR + ND ND ND -

NR NR NR NR NR NR ND ND ND ND _a

23 70 70 106 111,112 108 94 40 40 92 33,40

PMA

LH Progesterone EGF

IL-2 Epinephrine TGF-fl LPS Dcxamethasone

+ + NR + + NR +

" NE, has no effect; NR, was ~ot reported; (+). increases synthesis, activity or expression: ( - ), decreases synthesis, activity or expression. b Increased [35S]methioninelabeling, but protein levels were not determined. Decreased [35S]methioninelabeling, bat protein levels were not determined. d Decreased in vitro translatable mRNA levels, total PGH synthase mRNA levels were not determined.

depending on the cell or tissue type, P G H 2 is converted to a combination of the prostanoids-PGF2a, PGE2, P G D 2, prostacyclin, or t h r o m b o x a n e A 2 [16]. While synthesis of prostaglandins is regulated acutely by activation of phospholipases and release of arachidonate, net prostanoid production is d e p e n d e n t on the level of expi-ession of P G H synthase. Control of the P G H synthase concentration is particularly i m p o r t a n t because the enzymc is inactivated physiologically during catalysis [17-21] attd pharmacologically by aspirin [15]. Thus. bt, th the initial concentiat~ul~ of P G H synthasc and the rate at which it can be resynthesized after inactivation deter,nine the rate of conversion of arachidonate to prosta,~,iandins. Many of the agents which stimulate arachidonate release also increase P G H synthase synthesis, probably by stimulating transcription of the P G H synthase gene. This has been demonstrated in studies with aspirintreated fibroblasts. Enzyme replacement in quiescent fibroblasts pretreated with aspirin takes m o r e than 12 h [22.23]. However, agents such as platelet-derived growth factor ( P D G F ) and interleukin-1 (1L-I) shorten this recovery time to as little as 1 - 4 h [11,22-24]. Thus,

P D G F and IL-1 not only a u g m e n t arachidonate release [11,12], but they also stimulate prostaglandin synthesis in fibroblasts by stimulating synthesis of P G H synthase protein. Finally, w h e n a r a c h i d o n a t e levels exceed P G H synthase biosynthetic capacity, fatty acid release m a y even he curtailed. While it is still unclear which phospholipases are involved in prostaglandin synthesis, studies on a candidate phospholipase A 2 from the m a c r o p h a g e cell line P388D3 [25-27] have shown that this e n z y m e is specifically mhibited by a r a c h i d o n a t e at concentrations near the K m of P G H synthase. Thus, arachidonate release m a y be limited, albeit indirectly, by P G H synthase expression. In this review, I will describe several relevant model systems in which fluctuations in P G H synthase levels occur [see T a b l e I), and I will present results from experiments that were conducted to determine the molecular events underlying these changes. T h e model systems I will e x a m i n e here include: (a) fibroblasts, in which growth factor- and cytokine-stimulated prostaglandin synthesis participates in control of mitogenesis involved in w o u n d repair a n d in modulation of in-

123 flammation; (b) female reproductive organs, where prostaglandins regulate ovulation, luteolysis and parturition; (c) the vasculature, where prostaglandins control hemostasis, and where changes in prostaglandin biosynthesis can influence development of cardiovascular disease; (d) the immune system, where prostaglandins help regulate immune cell funct:.vn and mediate inflammation; (e) bone, where prostaglandins regulate bone stasis; and (f) the kidney, where prostaglandins regulate glomerular filtration, water uptake, and response to injury. In these model systems, two general types of regulation of the PGH synthase expression are apparent. The first type, characteristic of fibrohlasts and enduthelial cells, involves regulation of steady state levels of prostaglandin synthesis. In these systems, prostag!andin synthesis typically increases 5- to 20-fold and is accompanied by relatively small (1- to 3-fold) or undetectable increases in PGH synthase protein levels. In these cells. increased prostaglandin synthesis appears to result from increased transcription of the PGH sya&a=c gene, in creased turnover * of PGH synthase protein, and sustained substrate release. The second type of regulation, characteristic of reproductive tissues and promonocyte cell lines such as HL-60 and U937, involves the acute developmentally-induced expression of PGH synthase in cells that initially contain little or no enzyme. In these systems, prostaglandin synthesis can increase 50to 100-fold, and this is accompanied by up to 15-fold increases in PGH synthase protein levels. Synthesis of prostaglandins in these cells is dependent on significantly enhanced PGH synthase expression coupled with increased arachidonate release. !!. Regulation of i ~ t l synthase in fibroblasts Fibroblasts are envisioned as normally dormant interstitial cells, the structural framework of most soft tissues. Aggregating platelets and monocytes and granulocytes recruited to the site of an injury or infection can release mitogens, such as PDGF, and cytokines, such as IL-I [28]. These agents activate fibroblasts, stimulating them to divide and to produce secondary factors such as GM-CSFs and prostaglandins [9,29]. These secondary factors initially promote the inflammatory process, but later appear to moderate inflammation and stimulate wound repair [30]. Fibroblasts are easily cultured, and thus, the regulation of prostaglandin synthesis in response to factors that stimulate inflammation or wound repair can be readily studied in vivo. Much is already known about the sequential gene expression and the physiological

* In this review the word "turnover"is used to mean the rate at which enzymeis degraded and replaced with new enzyme.

changes that accompany mitogenesis in fibroblasts [31]. To study prostaglandin synthesis during mitogenesis. fibroblasts are first forced to enter the G O phase of the cell cycle (quiescence) by incubation in medium depleted of grovqth factors. The cells can then be stimulated to divide in a synchronous manner by addition of serum, purifie:l growth factors, or IL-I. Two key factors. PDGF [12] and IL-1 [32], have been found to be particularly powerful in inducing prostaglandin synthesis and the synthesis of PGH synthase in fibrob!asts. On the other hand, glucocorticoids have been shown to inhibit stimulated PGH synthase expression [33]. No one laboratory has examined all the steps involved in the induction of prostaglandin synthesis in quiescent fibroblasts. This would require measuring changes in PGH synthase m R N A levels and subsequent changes in protein expression and enzyme activity. Differences in the cell lines used. culture conditions, and e:zpe:'imen'.~l protocols have caused some confusion. I hope to reconcile some of the seemingly anomalous results obtained from the diffcrcn': !aboratoriea. Initial studies on fibroblasts were conducted by Habenicht et al. [22]. who showed that PDGF stimulated a biphasic increase in Swiss 3T3 cell prostaglandin synthesis. In these experiments, cells were made quiescent by incubation for several days in medium containing 5% plasma-derived serum, a supplement devoid of growth factors. Addition of PDGF to these fibroblasts stimulated (a) an early acute burst of synthesis that was complete within 10 min and {b) a later chronic phase of synthesis between 2 - 6 h. The initial burst resulted primarily from PDGF-stimulated phospholipid hydrolysis. The second phase of PDGF-stimulated PGE 2 synthesis (2-6 h) was inhibited by cyc~-~beximide, which suggested that PDGF was stimulating de novo synthesis of PGH synthase. Indeed, additional experiments supported this concept. By measuring prostaglandin synthesis from exogenous arachidonate, it was demonstrated that the total capacity of the cells to synthesize PGE z was increased about 2-fold, 4 h after PDGF stimulation; moreover, kinetic assays of microsomal preparations from quiescent and PDGF-stimulated 3T3 cells showed a 10-fold increase in the Vn,~, of microsomal PGH synthase 16 h after PDGF stimulation, with no change in the K m [34]. Furthermore, actinomycin inhibited the second phase PDGF-stimulated prostaglandin formation, suggesting that PDGF-induced changes in PGH synthase involved transcription as well as translation. In later experiments, it was found that the second phgse of PDGF-induced prostaglandin synthesis was dependent upon uptake of LDL from the medium [35]. It was shown that PDGF stimulated the expression of LDL receptors and tile uptake of LDL. Arachidonate obtained from LDL, presumably derived from the hydrolysis of lipoprotein esters, was found to be the sole

12~ source of prostaglandins synthesized during this second phase. A recent report [36] has further confirmed that LDL is an important source of arachidonate for prostaglandin synthesis. From these experiments, it appears that PDGF has three effects on prostaglandin synthesis in 3T3 cells. PDGF initially stimulates prostaglandin synthesis by augmenting arachidonate release; later, PDGF increases synthesis of PGH synthase and stimulates expression of LDL receptors. The mechanisms underlying the biphasic synthesis of prostaglandins described in these early experiments became a point of controversy later, because in similar experiments with NIH 3T3 cells, Lin et al. [24] found that PDGF stimulated only a single early burst of PGE2 formation. These researchers found that unstimulated 3T3 cells contained sufficient PGH synthase to account for both an early and late phase of prostagla~.Sin synthesis. They also found that PGH synthase protein levels, as measured by Western blotting, did not change upon PDGF stimulation. They, therefore, concluded that PDGF does not stimulate de novo synthesis of iJGi-i syntllase. Although at odds with the results of HabcP.icht et al. [22], the findings of Lin et al. [24] are not necessarily irreconcdab!e. Careful examination of the data of Habenicht et al. [22] shows that the quiescent cells used in these experiments ac:ually contain sufficient PGE 2 synthetic activity to account for both the early and late phase of PDGF-induced PGE 2 production. Thus, the initial explanation, that cycloheximide inhibited de novo synthesis of PGH synthase and prevented the second phase of PGE 2 synthesis, may have been incomplete. While cycloheximide undoubtedly inhibits synthesis of PGH synthase, cycloheximide may also inhibit expression of LDL receptors and prevent uptake of the LDL required for later synthesis. In the experiments of Lin et al. [24], 3T3 cells were first incubated for 18 h in medium containing 0.5% fetal calf serum and then stimulated with PDGF. This differs from the treatment (5% plasma-derived serum) used by Habenicht et al. [22]. One possible explanation for the absence of the second burst of prostaglandin synthesis in experiments by Lin et al. [24] may be that after 18 h their medium contained insufficient LDL to provide arachidonate for the second phase of synthesis. Another possibility is that there are intrinsic differences between the Swiss 3T3 cell line used by Habenicht eta!, [22] and the NIH 3T3 cell line used by Lin et al. [24]. In my laboratory, we have also been unable to detect changes in the PGH synthase protein levels following mitogenic stimulation of 3T3 cells (DeWitt, D.L., Kraemer, S. and Meade, E.A, unpublished data), confirming the findings of Lin et al. [24]. However, when we measured microsomal PGH synthase activity, we found a 2-fold increase 3 h after stimulation with medium containing 10% serum. This increase in activity is about the same as that observed by Habenicht et al.

[22]. Thus, serum, and likely PDGF. can stimulate de novo synthesis of PGH synthase in fibroblasts without significantly in.creasing PGH synthase protein levels. Presumably. inactive enzyme is replaced with active enzyme; in other words, in 3T3 cells, turnover of PGH synthase can increase while the absolute levels of the protein remain constant. The turnover rate of PGH synthase does change depending on the growth state of the cell; there is little turnover in quiescent cells and rapid turnover in growing or mitogen-stimulated ceils. Quiescent 3T3 cells treated with aspirin for 30 min are unable to synthesize prostaglandins for up to 12 h [22]. However, when these cells are treated with PDGF. they regain some biosynthetic capacity after an hour lag and have fully recovered by 3 - 4 h. Similar results with aspirin-treated NIH 3T3 cells were obtained by Lin et al. [24], who also showed that PDGF-dependent recovery of PGE 2 synthesis was inhibited by cycloheximide. Additionally, Burch et al. [11] demonstrated that IL-1 could stimulate PGH synthase recovery in aspirin-treated SV2-T2 3T3 cells. The half-life of PGH synthase in cycling 3T3 cells has only been measured indirectly [22]. Cells stimulated for 6 h with P D G F were tested for their continued ability to synthesize PGE 2 from added arachidonate in the presence of cycloheximide. Cycloheximide reduced PGE 2 synthesis by 80% within 4 h, although approx. 20% of the PGE 2 synthetic capacity remained even after 10 h. This suggests that different pools of enzyme exist which turn over at different rates, Similar results were obtained by Tsai et al. [37,38] who measured the rate of degradation of [35S]methionine labeled PGH synthase in cultured endothelial cells. The majority of PGH synthase in h u m a n umbilical endothelial cells turned over rapidly (ill 2 < 10 min), but a smaller pool of enzyme turns over much more slowly (t~/2 > 150 min). The initial observation that actinomycin inhibited the PDGF-stimulated increase in prostaglandin synthesis suggested that transcription of the PGH synthase gene might be required for increased expression of PGH synthase enzyme [22]. This hypothesis has been supported by experiments conducted by both Lin et al. [24], who showed that PDGF stimulated a 2.5-fold increase in PGH synthase m R N A levels which peaked at 4 h, and by DeWitt et al. [39], who also observed similar increases in PGH synthase m R N A levels in 3T3 cells stimulated with serum. That this increase in PGH synthase m R N A levels coincides with or slightly precedes the increase in PGH synthase activity seen in experiments by Habenicht et al. [22] and by. us (DeWitt, Kraemer and Meade, unpublished data), is additional circumstantial evidence that increased transcription of the PGH synthase gene precedes or accompanies increased synthesis of PGH synthase protein. To confirm the role of transcriptional activation in response to

125 PDGF and serum, however, will require measuring changes in the rate of transcription of the PGH synthase gene in response to these factors. IL-1 is the second major effector which can regulate fibroblast PGH synthase levels. Work by Burch et al. [11] showed that a 24 h pretreatment with IL-I increased arach;donate-stimulated PGE~ synthesis i'a SVT2 3T3 cells by 20-fold. PGE~_ production in IL-1treated SV-T2 3T3 cells was also enhanced in response to bradykinin (10-fold), thrombin (3-fold) and bombesin (10-fold). IL-I increased by 40% receptor O-protein coupling, as determined by measuring bradykinin receptor-stimulated GTPase activity; and IL-1 increased phospholipase A , activity by 80%. PGE synthase activity, measured by addition of exogenous P G H , to cells, did not change. Thus, IL-I stimulation of prostaglandin synthesis in these fibroblasts involves several changes, including increases in phospholipase A a and PGH synthase activity and ;increased signal transduction efficiency. IL-t also increases P~,E,_ synthesis in human dermal fibroblasts [28,32,33,40]. PGH synthase activity, as measured by PGE z production, increased 2- to 5-fold 12 h after IL-I addition. Metabolic labeling of PGH synthase with [3SS]methionine increased in concert with PGH synthase activity. While it is clear that IL-I increased PGH synthase synthesis, it was not determined whether the increase in PGH synthase synthesis led to a net increase in PGH synthase mass. lnhibitors of protein kinase C (H-7, staurosporine) inhibited IL-I induction of PGH synthase, suggesting that the protein kinase C (PKC) is involved in mediating the response to IL-I. However, stimulation with PMA, an activator of PKC, was only about a third as effective as IL-I, indicating that PKC activation alone is insufficient to account for the total IL-l-mediated induction. To further determine the mechanism of the IL-I effects, inhibitors of translation and protein synthesis were tested. Addition of actinomycin (1 v,M) or cycloheximide ( > 0 3 ~:M) during the first 4 h of an 8-h IL-I stimulation inhibited the increase in dermal fibroblast PGH synthase activity and [3sS]-methionine labeling. Thus, during the first 4 h of IL-1 stimulation, both transcription and translation are necessary to permit the increase in PGH synthase synthesis and labeling observed 8 h after the start or IL-I treatment. Cycloheximide, but not actinomycin, prevented the increases in PGH synthase activity and labeling when present at hours 4 - 8 of an 8-h IL-I treatment, indicating that translational, but not transcriptional, events are required in the later stages of IL-1 induction. Raz et al. [33] suggested that induction of PGH syntkase may be divided into a 'transcriptional phase' at from 0 - 4 h and a 'translational phase" at from 4 - 8 h. Another distinct possibility is that IL-I works through an intermediate factor which induces the synthesis of

PGH synthase. A candidate for this role is PDGF. In dermal fibroblasts. IL-I transiently stimulates expression of PDGF-A chain mRNA with a maximum :,nduction at 2 h [41]. Release of PDGF into culture medium can be detected within 4 h, Antibodies against PDGF block mitogenesis in IL-I stimulated cells, so it appears that the growth-promoting activity of 1L-I in dermal fibroblasts is due to autocrine stimulation by PDGF. Therefore, actinomycin and cycloheximide, when added during the early stages of IL-I stimulation, may inhibit synthesis of PGH synthase indirectly by inhibiting dermal fibroblast PDGF production. Glucocorticoids also appear to regulate PGH synthase expression in dermal fibroblasts [33,40]. Dexamethasone, when present during the first 4 h of an 8-h IL-I treatment of dermal fibroblasts, inhibited by more than 50% [ ~SS]-methionine incorporation into PGH synthase (labeling was from 8-12 h). Addition of dexamcthasone at hours 4 - 8 of an 8-h IL-I stimulation completely inhibited incorporation of [xSSlmethionine into PGH synthase (labeling was from 8-12 h). While dexamethasone apparently decreases the rate of translation of PGH synthase, it was not determined whether there was a corresponding decrease in PGH synthase protein levels. It is unclear how dexamethasone inhibits translation of PGH synthase mRNA. Glucocorticoids and other steroids typically enhance or inhibit transcription of genes which contain specific steroid receptor responsive elements [42]. Dexamethasone may increase expression of a protein that inhibits PGH synthase translation. This concept is supported by the results of experiments performed with actinomycin [33]. When actinomycin was added together with dexamethasone at hours 3-7 of a 7-h IL-I stimulation, dexametha,one no longer inhibited PGH synthase labeling. Thus, the dexamethasone effect requires transcription, presumably of a protein that inhibits the metabolic labeling of PGH synthase. Several mechanisms by which a dexamethasone-induced protein could inhibit PGH synthase synthesis include: (a) direct inhibition of PGH synthase translation: (b) reduction of enzyme inactivation (e.g., via reduction of arachidonate release), which could reduce PGH syathase replacement; or (c) reduction of PGH synthase m R N A levels. Recent work provides some support for this last possibility [40]. Dexamethasone added to lL-l-stimulated fibroblast at hours 4 - 8 of an 8-h stimulation reduces the amount of translatable PGH synthase mRNA. IlL Regulation of PGH synthase expression during the reproductive cycle Three events in the female reproductive cycle-ovulation, luteolysis, and parturition-are now known to involve developmentally-regulated increases in prostaglandin synthesis. In each case, increased prostaglandin

126 synthesis is accompanied by increased PGH synthase expression. Enhanced PGH synthase expression in reproductive tissues is unique in that it is under both hormonal and growth factor control, it is transient, and it is temporally correlated with specific developmental events. Thus, regulation in reproductive tissues differs, at least superficially, from regulation of steady state PGH synthase levels in cells such as fibroblasts.

II-A. Ovulation In the rabbit, rat, and pig, prostaglandin synthesis is required for the final step in ovulation-rupture of ovarian follicles and release of mature oocytes [43-45]. Luteinizing hormone (LH) in vivo or follicle-stimulating hormone (FSH), cAMP derivatives, and phorbol esters in vitro stimulate ovulation [46-48]. These hormones substantially increase PGE 2 and PGF2, , synthesis by preovu!atory (PO) follicles and by granulosa and thecal cells obtained from preovulatory follicles. Indomethacin blocks follicular rupture, and the effects of indomethacin are reversed by a~tdition of prostaglandins. The mechanism whereby prostaglandin synthesis increases at ovulation in rat PO follicles has been shown using quantitative Western blotting to involve a dramatic increase in PGH synthase protein [47]. In vivo induetion of ovulation with human chorionic gonadotrophic (hCG) increased rat PO follicle PGH synthase expression within 3 h. Enzyme levels !ncreased by 15-fold at 7 h. then returned to pre-stimulatory levels by 12 h. PGH synthase increased specifically in the granulosa cells of PO follicles and significant induction of PGH synthase was observed only at doses of hCG that induced ovulation (2--10 IU). Similar changes were observed in vitro in excised rat follicles [46]. LH, FSH aud forskolin increased PGH synthase expression in a time- and dose-dependent manner. Each of these compounds increases cAMP levels, suggesting that cAMP is the primary second messenger mediating ovulation and PGH synthase induction (although phorbol esters can also increase prostaglandin synthesis in granulosa cells [48]). Increases in PGH synthase protein were blocked by inhibitors of transcription and translation, but not by PGE 2 and PGFz,. Northern blotting analysis indicated that PG[-I synthase m R N A levels actually decreased preceding ovulation, just at the time that PGH synthase levels increased; conversely, in corpus lutea, PGH synthase expression is reduced and PGH synthase m R N A levels returned to ::,~ higher pre-stimulatiou level. The mechanism c ~,lirolling PGH synthase induction in PO follicles is unresolved. Especially perplexing is the finding that PGH synthase m R N A levels decrease at the same time as PGH synthase protein levels increase. Apparently, transcription of the PGH synthase gene is not required for increased expression of the protein in

follicles, but rather increased enzyme expression is achieved by stimulating translation of extant PGH synthase mRNA. If PGH synthase m R N A were co-translationally degraded, this would coincidentally decrease PGH m R N A while increasing PGH synthase protein; moreover, increased degradation of the m R N A with unchanged or lowered transcription of the gene would limit expression of enzyme. Regulation of protein levels for other genes such as c-los [49], histone [50], and fl-tubulin [51] is thought to be regulated by co-translational degradation of their mRNAs. PGH synthase mRNA, like m R N A for c-los, histone and fl-tubulin, can be superinduced in the presence of cycloheximide [39,52]. Degradation of PGH synthase m R N A may, therefore, be another mechanism regulating PGH synthase protein expression. There is one observation that ostensibly contradicts the hypothesis that follicular PGH synthase synthesis is regulated post-transcriptionally. That is that the transcriptional inhibitor a-amanitin (added together with LH, but not 2 - 4 h later) inhibits LH induction of P G H synthase. Thus, transcription is required for increased PGH synthase enzyme expression in this tissue. While one cannot rule out that transcription of the PGH synthase gene increases, no increases in m R N A levels were detected [46]. It is possible that a transient increase in PGH synthase m R N A was missed, or that increased transcription does not increase m R N A levels. However, an equally plausible explanation is that a-amanitin inhibi)s transcription of a gene that stimulates translation of PGH synthase mRNA.

IlI-B. Luteolysis Although its function in humans is unclear, PGF2o is an endogenous luteolysin in most domestic animals and administration of PGF2,, analogs is a reliable method for synchronizing estrous. In ewes, utero-ovarian venous plasma levels of PGF2,, are about 1 n g / m l on days 0-12 of the estrous cycle and increase more than 10-fold on days 13-15, jusi prior to the onset of luteolysis. Huslig et al. [53] measured cyclooxygenase activity of the PGH synthase in uterine mierosomes derived from ewes at from days 3-15 of estrous. By determining immunochemical equivalence points, these authors were able to demonstrate that PGH synthase enzyme levels increased 3-fold in the uterine caruncular tissue on days 13-15, corresponding to the time of increased prostaglandin synthesis. No significant changes in enzyme activity were observed in other uterine tissue. Luteolysis in vivo requires progesterone pri,,n,ing followed by exposure to estradiol. It is not known how these hormones affect PGH synthase expression. Measurements of PGH synthase m R N A in uterine tissue from sheep treated with progesterone to induce precocious luteolysis revealed changes in PGH synthase

127 m R N A expression that do not correlate anatomically with the observed changes in enzyme activity. In situ hybridization of paraformaldehyde fixed uterine sections from control and progesterone-treated ewes with complementary RNA probes [54] showed that PGH synthase m R N A levels increased 8-fold in the endometrial uterine glands of treated animals. These changes in mRNA levels were localized to the epithelial layer and the underlying lamina propria; changes were not observed in corpus lutea, ovarian stroma, or liver. While immunofluorescent staining of uterine tissue by Huslig et al. [53] showed that the uterine gland was a major site of PGH synthase enzyme expression in the uterus, changes in the activity of non-caruncular endometrium were not observed. One explanation for this apparent discrepancy is that changes in PGH synthase m R N A in caruncles may not precede increases in PGH synthase protein. As noted above, this was found to be the case with PGH synthase induction it, PO follicles. Alternatively, induction of luteolysis by progesterone treatment of ewes may cause metabolic changes that do not exactly mimic enzyme changes that occur in normally cycling ewes. III-C. Parturition

Induction of labor is mediated in part by increased synthesis of prostaglandins by uterine and fetal membranes [55]. Both PGE 2 and PGF:,, contract the uterine myometrium during all stages of pregnancy, and substantial increases in these prostaglandins occur in amniotic fluid and plasma during labor. PGH synthase inhibitors delay premature labor, while PGE~ and PGF2, and their analogs are effective in initiating and stimulating labor. The control of prostaglandin production at term is complicated and involves numerous factors. One important component appears to be increased PGH synthase expression. In humans, fetal membranes, especially amniotic membranes, are major producers of prostaglandins [56]. PGH synthase activity in the amnion has been shown to increase with labor when measured with added arachidonate [56]. Additionally, the amnion from women who delivered vaginally was found to produce 2-4-times the level of prostaglandins as the amnion from women who delivered by cesarean section without entering labor [56-58]. PGH synthase levels in cultured amnion cells are increased by epidermal growth factor (EGF) [59-61], phorbol esters, and diacylglycerol [621, but not by PDGF, FGF, or 'IGF-fl. A unique feature of amnion cells is that each of the factors that increase PGH synthase expression fail to elicit an increase in arachidonate release; thus, an additional factor, such as an endogenous activator of phospholipase, must be involved in vivo to increase prostaglandin synthesis during parturition. This contrasts with the

regulation of prostaglandin synthesis in fibroblasts in which PDGF and IL-I stimulate both substrate availability and PGH synthase synthesis. In cultured amnion cells, EGF stimulated a 2- to 5-fold increase in PGH synthase activity that was maximal by 4 h [59-61]. EGF also increased metabolic labeling of PGH synthase [62] and was required for recovery of prostaglandin synthesis after aspirin inhibition, providing evidence that EGF stimulated de novo synthesis of PGH synthase. EGF had no effect on amnion cell arachidonate release. PGE, synthesis in EGF-treated cells required the presence of medium containing serum or arachidonate or stimulation with A23187 [59,60]. Phorbol esters (PMA) and diacylglycerol also induce prostaglandin synthesis in amnion cells, providing evidence that PKC is an essential link in the signal transduction chain [62]. Actinomycin D, added during the first 60 rain, or cycloheximide, added during the entire incubation period, prevented PMA-stimulated increases in PGH synthase activity, suggesting that transcriptional e',ents as weU as translational events were required. PMA stimulated prostaglandin synthesis to the same degree in both aspirin pretreated cells and untreated cells, indicating that de novo synthesis of PGH synthase was responsible. The addition of exogenous arachidonate to PMA-treated cells potentiated PGE, production 5-fold, providing evidence that activation of PKC had comparatively little effect on arachidonate release. It is likely that the effects of EGF on PGH synthase synthesis are mediated, at least in part, through 1,2 diacylglycerol and PKC. EGF induces phospholipase C-mediated hydrolysis to produce 1,2 diacylglycerol, and the time course of induction of PGH synthase with F.GF and PMA is similar. IV. Reglllation of vascular prostaglandin synthesis Thromboxane production by platelets and prostacyclin production by vessels are important in maintaining vascular homeostasis and may also be crucial in promoting or preventing the development of ca-diovascular disease [63,64]. Aspirin is thought to be effective in reducing cardiovascular disease because it selectively decreases synthesis of the pro-aggregatory prostaglandin, thromboxane A : (TXA2) relative to the antiaggregatory prostaglandin, and prostacyclin (PGI2). This selectivity occurs because ves,,els can synthesize new protein relatively quickly to replace aspirin-inactivated PGH synthase, whereas platelets cannot. Thromboxane synthesis begins to recover only with replacement of platelets, usually after a 1-2 day lag [63], while PGH synthase activity and prostacyclin production in vessels recovers within hours. Bradykininstimulated PGI 2 synthesis, in vivo, recovered by 6 11 after bolus ingestion of aspirin (600 mg) [65]. Pros-

128 tacyclin synthesis, measured cx vivo in saphenous veins removed from patients who received 20 mg aspirin a day for 7 days, returned to pretreatment levels within 24 h [66]. Cultured endothelial cells begin to recover from aspirin inhibition in 2-24 h, depending on the culture conditions [66]. Recovery of PGH synthase activity by cultured smooth muscle cells is slower, but is complete within 48 h: this may reflect either a slower turnover of the enzyme or the fact that smooth muscle cells contain only 5% as much PGH synthase level of endothelial cells [67[. The net effect of aspirin is to skew the P G I j T X A 2 balance towards the synthesis of the antiaggregatory and vasodilatory PGI:. The factors that control the turnover and the level of expression of endothelial and smooth muscle PGH synthase are just beginning to be understood.

IV-A. Vascular endothelium A number of effectors, including histamine [68], thrombin [69], bradykinin [2], ionophore A23187 [69], IL-I [52]. IL-2 [70], and phorhol esters [38], stimulate prostaglandin synthesis in endothelial cells. These effectors can be divided into two groups; those that elicit an acute (10-30 min) release of prostanoids (e.g., histamine, thrombin, bradykinin, and ionophore A23187) and those that elicit a sustained (>_>_1 h) release of prostaglandins (e.g., IL-I, IL-2, and phorbol esters). The former group is thought to simply stimulate arachidonate release, whereas the latter group appears both to stimulate the release of fatty acid substrate and to increase the expression of PGH synthase. IL-1, a product of activated monocytes and a potent modulator of endothelial cell growth, stimulates PGI 2 synthesis and increases PGH synthase m R N A and proteie levels in cultured human umbilical vein endothelial cells (HUVECS) [52]. IL-I increased HUVECS PGH synthase mRNA within 2 h of 1L-I addition [52]; PGH synthase mRNA levels were maximal by 4 h and remained elevated for up to 24 h. PGH synthase protein levels, quantified by Western blotting, increased steadily for 15 h after IL-1 addition. Moreover, the increase in PGH synthase protein appears to be roughly proportional to the 5-fold increase in prostaglandin formation. That stimulated PGI 2 synthesis in these experiments was relatively constant for up to 24 h suggests that IL-I increased the rates of transcription of the PGH synthase gone and translation of PGH synthase m R N A to higher steady state levels, lL-l-stimulated prostaglandin synthesis in HUVECS resulting from increased PGH synthase mRNA levels and subsequent de novo synthesis of PGH synthase represents one of the least complex examples of the l::olecular regulation of prostaglandin synthesis described to date. One striking feature of this system is that cycloheximide potentiates the IL-1stimulated increase in mRNA levels [52]. Cyclohexi-

mide-dependent superinduction of PGH synthase m R N A is also seen in serum-stimulated 3T3 cells [39]. Many immediate early genes, such as c-fos, c-jun and c-myc, are also superinduced by cycloheximide. Superinduction is thought to involve an increased stability of m R N A for these genes and prolonged mitogen-induced transcription of these genes [49,71]. c-fos is thought to inhibit transcription of its own gone by a feedback mechanism. Thus. cycloheximide inhibition of c-fos protein synthesis is thought to allow prolonged transcription of the c-fos gene leading to superinduction of c-fos m R N A [72]. The possibility that PGH synthase may also regulate its own transcription needs to be examined. Two other effectors-lL-2 [701 and PMA [381-were found to increase endothelial prostaglandin production in a manner similar to IL-1. PMA increased HUVECS PGF2, , production by 4 h; synthesis was maximal by 12 h and thereafter declined. While PMA increased PGF2, production by approx. 15-fold, PGH synthase protein levels increased only about 2-fold. Both cycloheximide and actinomycin prevented increased PGF2,, synthesis and PGH synthase protein expression, suggesting that both transcription and translation were required for PMA activation; however, PGH synthase m R N A levels were not measured in these experiments. It is possible, by analogy to the IL-1 stimulation of dermal fibroblast prostaglandin synthesis, that induction of PGH synthase in endothelial cells by IL-1 is mediated in part via PKC. Measurement of the rate of degradation of [35S]methionine-labeled PGH synthase showed that the half-life of the cnz~a,e in I-IUVECS was less than 10 min [38]. While PMA increased the absolute level of PGH synthase, it did not change the half-life of the enzyme, indicating that increased PGH synthase levels must have resulted from increased synthesis. Additional experiments [37] which, for theoretical purposes, assumed a constant rate of protein synthesis and a first order rate constant for degradation showed that PMA increased the rate of PGH synthase synthesis by about 3-fold, but had no effect on the rate constant of enzyme degradation. This model predicts that at steady state, PMA would increase enzyme levels and enzyme turnover by about 3-fold, and the observed changes in protein levels were in close agreement with this prediction. Increasing turnover of the enzyme means that inactivated enzyme is replaced more rapidly. Continuous synthesis of new enzyme allows for a relatively large increase in prostaglandin synthesis with only a modest increase in protein levels. IL-2, a product of activated lymphocytes, also stimulates prostaglandin synthesis in HUVECS and in bovine aortic endothelial cells (BAECS) [701. BAECS and HUVECS displayed differential sensitivity to both natural and recombinant IL-2. Recombinant IL-2 pro-

129 parations substituted with alanine or serine at cysteine125 were inactive. IL-2-stimulated PGI: synthesis was maximal at from 12-24 h. depending on the cell type examined and the IL-2 preparation used: and PGI 2 synthase was increased by as much as 30-fold. The increase in PGI 2 production could be attenuated by either cycloheximide or actinomycin, suggesting that both transcription and ~-ansl ~tion were required. PGH synthase message levels were no~ examined, but Western blots of homogenates from IL-2-si:,aulated cells showed an increase in PGH synthase protein levels. This increase, however, was not commensurate with the increase in total PGI2 production suggesting, again, that sustained substrate release in combination with increased turnover of PGH synthase was responsible for the increase in prostaglandin synthesis observed. The physiological importance of IL-2 in augmenting endothelial cell PGI z production is not obvious. I V . B . Smooth rr,uscle

Cultured smooth muscle cells lose their ability to produce PGI 2 when treated with aspirin, and synthesis does not recover if cells are cultured in spent medium or in a medium devoid of serum or growth factors [23]. Addition of serum, EGF or TGF-/L but not PDGF, restores P{312 production within 3 h. Concurrent restoration of PG12, PGE,, PGF2, and PGD2 production with serum or growth factor treatment indicated that PGH synthase protein was synthesized. Apparently, turnover of PGH synthase in rat smooth muscle is similar to that ia fibrc, bla,:~.s; it is low in confluent quiescent cells and stimulated in the presence of specific mitogens.

V. Regulation of prostaglandin synthesis in macrophage / monocytes Prostaglandins produced at the site of tissue injury or infection are pro-inflammatory by virtue of their ability to cause vasodilation and edema [73] and to potentiate pain [74]. Conversely, prostaglandins can also attenuate the immune response. Some of their anti-inflammatory effects include inhibition of B and T cell proliferation and function [75-79] and inhibition of accessory monocyte/macrophage cell action and function [80-82]. Monocyte/macrophages are the primary immune cells producing prostaglandins. When stimulated by lipopolysaccharide (LPS) [40,83], zymosan [84], immune complexes [78], or complement [85], macrophages produce PGE2 and TXA2, as well as LTB4 products which have both autocrine and paracrine effects. Acting as an autocoid, PGE 2 inhibits macrophage proliferation [86], IL-I and cytokine secretion [87] and macrophage !a expression [81]. Thus, PGE z effectively inhib~:~ further

macrophage accessory function, including the ability to stimulate B and T cell proliferation and differentiation. Two macrophage precursor cell lines have been used to study the regulation of PGH synthase-HL-60, a promyelocytic cell [88] and U937, a promonocytic cell [89]. Both lines can be differentiated into macrophagelike cells by treatment with PMA, diacylglycerol, or vitamin Dg; HL-60 cells can also be induced to differentiate into neutrophil-like cells with dimethylsulfoxide [88]. Upon differentiation to macrophage-like cells, both HL-60 and U937 cells increase prostaglandin synthesis as much as 50- to 100-fold [90-92]. Increases in PGH synthase expression in differentiated HL-60 cells have been demonstrated by measuring increased prostaglandin synthesis from exogenously added arachidonate, by immunoquantitation of protein levels, and by direct kinetic measurements [90,911. PGH synthase activity (V,,,,,,) increases about 5-fold after differentiation of HL-60 cells, while the K., value for arachidonate remains constant. PGH synthase activity also increases about 5-fold in PMA differentiated U937 cells: and enzyme protein levels, determined by dot blotting, i.n,cre,'.~se significantly [92]. Thus, developmental regulation of expression of PGH synthase is an important factor regulating prostaglandin synthesis in differentiating promonocytes. Induction of PGH synthase in these systems most closely resembles induction in follicles during ovulation. Both involve a precipitous increase in PGH synthase activity and protein. Because non-steroidal anti-inflammatory drugs reduce inflammation by inhibiting prostaglandin synthesis, there has been a continuing interest in determining whether anti-inflammatory steroids also work by a similar mechanism. It was once thought that steroids induced the synthesis of inhibitors of phospholipase A.,, but this theory has generally been discarded [93-95]. Thus, the mechanism by which glucocoticoids inhibit prostaglandin synthesis in fibroblasts [96], endothelial cells [95], and monocyte/macrophages [401 is still unclear. Regulation of prostaglandin synthesis in macrophages is e,,,pecially interesting because recent work in vivo [97] suggests that, in the absence of inflammation, glucocorticoids inhibit primarily macrophage prostaglandin synthesis. When subjects ingested the glucocorticoid precursor prednisone for 7 days, prostaglandin formation was inhibited only in cells obtained from bronchoalvcolar lavage cells (macrophages). Prostaglandin synthesis by platelets, vascular endothelia, and blood Icukocytes was unaffected. The mechanism whereby glucocorticoids inhibit prostaglandin synthesis has been investigated in two studies using PMA-differentiated U937 cells and in one study using human peripheral blood monocytes [40,94, 98]. In the first study [94], a 2 h pretreatment with dexamethasone caused a dose-dependent decrease in prostaglandin formation in PMA-differentiated U937

130 cells stimulated for 4 h with either LPS or zymosan. At the highest dexamethasone concentrations, prostaglandin synthesis was reduced to the levels preduced by differentiated cells stimulated only with arachidonate or A23187. Since prostaglandin synthesis from cells stimulated with arachidonate was unaffected by dexa~,~.ethasone, these authors concluded that the site of inh!bition of prostaglandin synthesis was proximal to PGII synthase. However, in these experiments, LPS or zymosan alone increased the synthesis of TXA 2 in differentiated U937 cells 2- to 4-foid above the levels seen in cells stimulated with arachidonate or ionophore A23187. Additionally, zymosan and LPS exhibited a slow onset, displayed a 2 h time lag, and reached a maximum only after 4-7 I1. PDGF [22] and serum (DeWitt, Kraemer and Meade, unpublished data), which stimulate PGH synthase synthesis in fibroblasts, display similar time courses of induction. It is likely that zymosan and LPS also stimulate PGH synthase synthesis and that dexamethasone works by inhibiting enzyme synthesis. This theory is supported by the results of experiments using human blood monocytes [40]. In these cells, LPS stimulates synthesis of PGE 2 by increasing PGH synthase activity, most likely by increasing PGH synthase synthesis. Dexamethasone inhibits the LPS-stimulated increase in monocyte PGH synthase activity. That dexamethasone works by inhibiting PGH synthase synthesis is further supported in a second study using the U937 cell line [98]. In these experiments, a 12-h dexamethasone treatment of differentiated U937 cells decreased PGH synthase activity by 60% and PGH synthase protein expression by 50%. The results of the two studies with U937 cells are not necessarily inconsistent. Even if dexamethasone inhibits synthesis of PGH synthase in U937 cells, one might not detect a decrease in basal enzyme levels during short incubations ( < 2 h), as was reported by Bienkowski et al. [94]. Incubation with dexamethasone for periods of time much longer than the probable half-life of the enzyme (12 h) would, however, produce measurable decreases in the PGH synthase protein levels, as was observed by Koehler et al. [98]. It, thus, seems likely that dexamethasone, by an as yet unknown mechanism, inhibits PGH synthase expression in monocyte/macrophages. That dexamethasone inhibited PGH synthase expression in both monocyte/macrophages and IL-1 stimulated dermal fibroblasts [33] suggests that this may be a common effect of anti-inflammatory glucocorticoids. VI. Regulation of renal prostaglandin synthesis Prostaglandins perform diverse functions in the kidney. PGF2,, and PGE 2 produced by mesangial cells and PGI, and TXA 2 produced by renal capillaries and platelets, regulate glomerular hemodynamics, glomerular cell proliferation and ultrafiltration [99]. PGE 2 pro-

duced in the collecting tubule modulates Na ÷ and water resorption. Pathologically. during development of glomerulonephritis, inflammation results in monocyte/ macrophage invasion of the kidney and leads to increased prostaglandin synthesis and reduced renal function [28,100]. At the molecular level, the regulation of renal prostaglandin expression has best ~ e ~ studied in cultured rat mesangial cells [101]. PGH synthase expression, as measured by enzyme activity, is 4-foid higher in growing, dividing mesangial cells than in quiescent cells. Jmmunoblotting and immunocytochemistry have revealed that this increase in activity is due in part to increased PGH synthase protein. When quiescent cells were stimulated with a medium containing 17% fetal calf serum, there was a time-dependent increase in PGH synthase m R N A levels that peaked at about 2-fold the level of quiescent cells 4 h after stimulation. Thus, mesangial cells appear to be similar to fibroblasts which express PGH synthase at steady state levels, and in which PGH synthase synthesis increases during growth or in response to mitogenic stimulation. There has been one additional report describing the signal transduction pathway for regulating expression of PGH synthase in cultured rat mesangial cells [102]. In these experiments, aspirin treatment inhibited quiescent mesangial cell prostaglandin synthesis for up to 5 h. Treatment of aspirin-inhibited mesangial cells with 10% fetal calf serum stimulated full recovery by 5 h; partial recovery resulted from treatment with PMA (58%) or EGF (25%). The PKC inhibitor, staurosporine, inhibited serumstimulated recovery by greater than 80%. Thus, it appears that PKC is one intracellular signalling mechanism which regulates mesangial PGH synthase expression.

VII. Regulation of prostaglandin synthesis in bone Prostaglandins play a complex and, as yet, poorly defined role in regulating bone metabolism. Prostaglandin synthesis in bone can be stimulated by severa~ cytokines, parathyroid hormone, growth factors and simple mechanical stress [103]. PGE2 stimulates both osteoclastic (bone resorbing) and osteoblastic (m~,neralizing) activities [104,105]. Regulation of bz,ne cell prostaglandin synthesis has been studied primarily in the mouse osteosarcoma cell line MC3T3-E1 [106-108]. PGE,_ synthesis in these cells is stimulated in a timeand dose-dependent manner by addition of epinephrine [106], EGF [107], a n d / o r TGF-fl [108]. PGH synthase activity begins to increase after about an hour lag and is maximal at 3 - 4 h, increasing about 8-fold. The activity of PGE synthase does not change, suggesting that regulation involves only PGH synthase. Increases in PGH synthase activity were prevented by cycloheximide and actinomycin, suggesting that enzyme expression requires increased transcription and translation of the PGH

synthase gene. Stimulation of de novo synthesis of PGH synthase by EGF and TGF-/3 was confirmed by Western blotting experiments which demonstrated about a 2-fold increase in enzyme protein. Again, increases in PGH synthesis can only partially be accounted for by changes in enzyme mass, and it must be assumed that the turnover of the enzyme also increased. EGF- and TGF-fl-induced expression of PGH synthase required the presence of 5% newborn calf serum (which is ineffective by itself), although the essential component(s) of serum were not determined. Epinephrine was found to function via /3-adrenergic receptor-stimulated increases in intracellular cAMP. VIII. Conclusions

Two types of regulation appear to be responsible for controlling PGH synthase levels. The first type, found typically in fibroblasts [22,24,28,33,34,401 and endothelial cells [36,38,70], involves modulation of steady state enzyme levels by increasing the rate of de novo synthesis of PGH synthase in cells that already express the enzyme to a significant extent. Unstimulated quiescent fibroblasts contain relatively high levels of the enzyme, but when they are stimulated with PDGF, IL-1, or serum, PGH synthase m R N A levels increase [24,39] and PGH synthase activity increases [32-34]. In 3T3 fibroblasts, there are no obvious changes in protein levels [24]. In endothelial cells stimulated with PMA, IL-1 or IL-2, PGH synthasc mRNA. PGH synthase activity and PGH synthase protein levels all increase [38,70]. ~luwever, the increase in PGH synthase protein is usually not commensurate with the increase in prostaglandin production. An explanation for these results can be inferred from kinetic measurements of the endothelial cell PGH synthase, wb~':h ~.g~e~ the! h.moral agents that increase the synthesis of PGH synthase also increase the rate of degradation o~" the enzyme [37.38]. Similarly, in fibroblasts, PGH synthase turnover is low in quiescent cells [22], but increases upon mitogenic simulation [11,22,24]. Increased turnover means that inactive enzyme is continuously replaced with active enzyme. As would be expected, treatment with mitogenic agents which increase both arachidonate release and PGH synthase turnover lead to sustained prostaglandin synthesis in fibroblasts and endothelial cells. The second general type of regulation of PGH synthase involves de novo synthesis of PGH synthase in cells that initially express little or no enzyme. The best examples of this are in the developing rat follicle [46,47] and the differentiating promonocyte [59,92.109]. In follicles, PGH synthase increases 15-fold preceding ovulation. HL-60 and U937 PGH synthase increases 5-fold after differentiation from promonocytes to monocyte/ macrophage-like cells [34,90,92]. To determine whether

this second general type of induction is mechanistically different from that described in fibrobiasts and endothelial cells, or whether the two types are quantitative extremes of common regulatory processes, will require a more thorough understanding of the factors regulating PGIt synthase enzyme expression. No one signal transduction mechanism is used by agents which modulate the synthesis of PGH synthase. However, activation of PKC leads, in many cases, to changes in PGH synthase expression. PKC is implicated in increases in PGH syntbase stimulated by IL-l-in human dermal fibroblasts [32,33] and endothelial cells [52] and by PMA in endothelial cells [381, fibroblasts [24], mesangial cells [101], amnion [62]. HL-60 [90] and U937 cells [92.94]. In fibroblasts [32,331 and mesangial cells [1021, PMA is able to stimulate only a fraction of the increase in PGH synthase synthesis elicited by either IL-1 or serum. Thus, while PKC is likely important in regulating PGH synthase expression, other intracellular factors are probably also involved. The second most common intraceUular signalling mechanism controlling PGH synthase levels involves adenylate cyclase. Analogues of cAMP and compounds that increase intracellular cAMP or inhibit cAMP-phosphodiesterases stimulate PGH synthase expression in developing follicles [46.47] and in the osteoclastic cell lille MC3T3-E1 [106]. The inlracellular pathways responsible for mediating PDGF- and serum-induced stimulation of de novo PGH synthase synthesis [22,34,39,101] have not been determined, although each of these treatments can stimulate PKC and adenylate cyclase. Indirect evidence obtained with inkihitors of protein synthesis and transt,.t:4~o,-, iad~ca~c:, :.l~ath-,~st of the factors that increase de novo synthesis of PGH synthase also increase transcription of the PGH synthase gene (see Fig. 1). Direct measurement of changes in PGH synthase gone tran~criotion have not been reported, but in most systems where m R N A levels have been measured (e.g.. fibroblasts [24,39], endothelial cells [521, mesangial cells [101]) increases in PGH synthase mRNA precede or accompany increases in PGH synthase activity a n d / o r protein levels. While increasing transcription of a gone is the most commonly described mechanism for elevating m R N A levels, it is also quite possible that increased stability of m R N A contributes to increased PGH synthase m R N A levels. Induction of PGH synthase in developing follicles follows a unique paradigm [46,47]. Upon stimulation with hCG, PGH synthase mRNA in PO follicles decreases while PGH synthase protein levels increase. Thus, in follicles, PGH synthase expression may be regulated at the level of translation. The coincidental decrease in m R N A levels suggests that PGH synthase m R N A may be degraded during translation. Induction of PGH synthase activity and protein by phorbol esters in HL-60 or U937 cells is similar to that in follicles;

132

IL-1

PMA cZA~M P LPS POH S Y N I H A S E OENE

t ranscrlpl,on

LH(cAMP) ~'~ PGH SYNTHASE mRNA

II, translation

PGH SYNTHASE PROTEIN

(degradation)

Fig. 1. Putative schemefor the regulationof PGH synthaseexpression (see text for details). Factors that increase prostaglandin synthesisby increasing PGH synthasesynthesisappear to enhancetranscriptionof the gene. although in someinstancesincreased PGH synthaseexpression may result from increased translation of extant PGH synthase mR.NA. During translation PGH synthascmRNA may be degraded limiting synthesis. PGH synthase protein could attenuate transcription of the geneor translation of the mRNA. Glucocorticoidsappear to decrease PGH synthaseexpression,although whether it is at the level of transcriptionor translationis not known.

however, the pattern of expression of PGH synthase mRNA in these cells has not been reported. It is not known how transcription of the PGH synthase gene is regulated. The structure of the human [110] and mouse PGH synthase gene have now been determined (DeWitt, Kraemer and Meade, unpublished data). Both genes are remarkably similar and contain 11 exons which together span about ,21 Kb. By coupling the sequences from the 5'-end of ~.he PGH synthase gene to a reporter gene and measuring the reporter gene activity in transfected cells treated with agents that stimulate PGH synthase synthesis, it should be possible to define the regulatory sequences that cor~rol PGH synthase transcription. Moreover, by comparing the sequences of the deduced regulatory sequences with known consensus regulatory sequences, it may be possible to determine which signalling pathways are most important for directly controlling PGH synthase gene expression. From the evidence reported to date, it seems likely that glucocorticoids inhibit prostaglandin synthesis in dermal fibroblasts [33,40] and monocyte/macrophages [40,98], in part by inhibiting synthesis of PGH synthase In human dermal fibroblasts, dexamethasone in;tibits both IL-l-stimulated metabolic labeling of PGH synthase and increases in PGH synthase activity. The inhibitory activity of dexamethasone is itself inhibited by actinomycin [33], indicating that glucocorticoid inhibition requires transcription of an as yet unidentified gene. In these cells, dexamethasone alto decreases the levels of in vitro translatable PGH synthase mRNA. Whether this involves lowering total PGH synthase mRNA levels has not been determined.

Clearly, regulation of prostaglandin synthesis is quite complex. In addition to control of PGH synthase, each of the other enzymes in the pathway, as well as the signal transduction events controlling arachidonate release, are potentially subject to regulation. IL-1 treatment potentiates thrombin-, bradykinin- and bombesinstimulated arachidonate release in SV-T2 3T3 cells, in part by increasing the efficiency of G-protein/effector coupling [11]. Phospholipase A 2 activity is also increased in these cells, but too little is known about the enzymology of phospholipases to determine the relevance of this change. PGI synthase activity is increased in PDGF-stimulated 3T3 cells [34], showing that the expression of the enzymes distal to the PGH synthase may also be regulated (although in many cases they are not [11,107,108]). A number of major questions remain unanswered about control of PGH synthase expression. Little is known about the molecular mechanisms controlling PGH synthase gene transcription. At present, it is still uncertain what role gene transcription plays in regulating mRNA levels. Measurement of changes in the transcription of the gene in response to the various agents found to stimulati~ PGH synthase expression and examination of the transcriptional regulatory elements present in the gene should resolve some of these questions. In many of the systems studied, PGH synthase protein levels are tightly controlled-enzyme levels fluctuate only 1- to 2-fold over steady state levels. How these enzyme levels are controlled, and what controls the rate of turnover seen in different growth states will also be important to determine. It will also be important to confirm whether there really is post-tranScriptional control of PGH synthase expression, as there appears to be in developing rat follicles. Additionally, a fuller understanding of the molecular mechanisms for dexamethasone repression of PGH synthase expression would add much to our understanding of the anti-inflammatory properties of glucocorticoids. Because of the availability of cDNA and genomic clones for PGH synthase, most of these questions could be answered in the next few years. Acknowledgements This work was supporled by NIH grant3 GM40713 and by a Grant-in Aid from the American Heart Association of Michigan. I would like to thank Dr. William Smith for his assistance, both editorial and otherwise in the preparation of this document. References 1 Murayama. T.. Kajiyama, Y, and Nomura. Y. (1990) J. Biol. Chen't. 265, 4290-4295.

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