Prostaglandin F2alpha amplifies tumor necrosis factor-alpha promoter activity by the FPB prostanoid receptor

This study examines the regulation of tumor necrosis factor-alpha (TNF-alpha) promoter activity by prostaglandin F2alpha ( PGF2alpha ) in HEK cells stably expressing either the FPA or FPB prostanoid receptors. Cells were transiently transfected with a luciferase reporter plasmid under the control of a TNF-alpha promoter and luciferase activity was measured. In the absence of PGF2alpha basal TNF-alpha reporter gene activity is elevated in FPB cells as compared with FPA cells. This elevated basal activity is blocked by pretreatment with a Rho inhibitor, but not by pretreatment with an inhibitor of protein kinase C (PKC). TNF-alpha reporter activity in FPB cells is stimulated by PGF2alpha and this is decreased by pretreatment with a chelator of intracellular calcium or by a gap junction inhibitor. In FPB cells pretreatment with a Rho inhibitor combined with either a calcium chelator or a gap junction inhibitor decreases both basal and PGF2alpha stimulated TNF-alpha reporter activity. Interestingly post-treatment of FPB cells with an inhibitor of PKC decreased PGF2alpha stimulated TNF-alpha reporter gene activity even though pretreatment did not. It, therefore, appears that PGF2alpha stimulated TNF-alpha reporter activity in FPB cells is amplified by a Rho-dependent mechanism involving calcium, gap junctions, and PKC. These findings may help in understanding the function of the FPB isoform in the corpus luteum.     

Delayed reversal of shape change in cells expressing FP(B) prostanoid receptors. Possible role of receptor resensitization

Prostaglandin F(2 alpha) (PGF(2 alpha)) receptors are G-protein-coupled receptors consisting of two alternative mRNA splice variants, named FP(A) and FP(B). As compared with the FP(A) isoform, the FP(B) isoform lacks the last 46 amino acids of the carboxyl terminus and, therefore, represents a truncated version of the FP(A). We recently found (Pierce, K. L., Fujino, H., Srinivasan, D., and Regan, J. W. (1999) J. Biol. Chem. 274, 35944-35949) that stimulation of both isoforms with PGF(2 alpha) leads to activation of a Rho signaling pathway, resulting in tyrosine phosphorylation of p125 focal adhesion kinase, formation of actin stress fibers, and cell rounding. Although the activation of Rho and subsequent cell rounding occur at a similar rate for both isoforms, we now report that following the removal of PGF(2 alpha) the reversal of cell rounding is much slower for cells expressing the FP(B) isoform as compared with the FP(A) isoform. Thus, in HEK-293 cells that stably express the FP(A) isoform, the reversal of cell rounding appears to be complete after 1 h, whereas for FP(B)-expressing cells there is essentially no reversal even after 2 h. Similarly, the disappearance of stress fibers and dephosphorylation of p125 focal adhesion kinase following removal of agonist are much slower in FP(B)-expressing cells than in FP(A)-expressing cells. The mechanism of this differential reversal appears to involve a difference in receptor resensitization following the removal of agonist. Based upon whole cell radioligand binding, agonist-induced stimulation of inositol phosphate formation, and mobilization of intracellular Ca(2+), the FP(B) isoform resensitizes more slowly than the FP(A) isoform. These findings suggest that the carboxyl terminus of the FP(A) is critical for resensitization and that the slower resensitization of the FP(B) isoform leads to prolonged signaling. This differential signaling distinguishes the FP(A) and FP(B) receptor isoforms and could be important toward understanding the physiological actions of PGF(2 alpha).     

Sphingosine 1-phosphate amplifies phosphoinositide hydrolysis stimulated by prostaglandin f2 alpha in osteoblasts: involvement of p38MAP kinase

We previously showed that sphingosine 1-phosphate phosphorylates p42/p44 mitogen-activated protein (MAP) kinase and p38 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of sphingosine 1-phosphate on phospholipase C-catalyzing phosphoinositide hydrolysis induced by prostaglandin F2alpha (PGF2 alpha) in these cells. Sphingosine 1-phosphate significantly amplified the inositol phosphates formation by PGF2 alpha. Sphingosine 1-phosphate did not enhance the formation induced by NaF, a direct activator of heterotrimeric GTP-binding proteins. PD98059, an inhibitor of the kinase that activates p42/p44 MAP kinase, had little effect on the amplification by sphingosine 1-phosphate. SB203580, an inhibitor of p38 MAP kinase, reduced the effect of sphingosine 1-phosphate on the formation of inositol phosphates by PGF2 alpha. The phosphorylation of p42/p44 MAP kinase by PGF alpha was attenuated by PD98059. SB203580 suppressed the phosphorylation of p38 MAP kinase by PGF2 alpha. Tumor necrosis factor-alpha enhanced the PGF2 alpha-stimulated formation of inositol phosphates. These results strongly suggest that sphingosine 1-phosphate amplifies PGF2 alpha-induced phosphoinositide hydrolysis by phospholipase C through p38 MAP kinase in osteoblasts.     

Glycation of the human erythrocyte glucose transporter in vitro and its functional consequences.

Stimulation of NIH-3T3 cells with prostaglandin F2 alpha (PGF2 alpha) caused a dose- and time-dependent generation of inositol phosphates. The first detectable changes were in the levels of Ins(1,4,5)P3 and Ins(1,3,4,5)P4. Increases in Ins(1,3,4)P3, InsP2 and InsP were detected later, and only minor changes were observed in putative InsP5 or InsP6. The accumulation of inositol phosphates was synergistically increased by the addition of calf serum, whereas PGF2 alpha had no effects on cell proliferation in either the presence or the absence of calf serum. Stimulation of a different clone of NIH-3T3 cells (AmNIH-3T3) or Swiss 3T3 cells with PGF2 alpha resulted in both inositol phospholipid breakdown and cell proliferation. No differences were found in the characteristics of PGF2 alpha-stimulated inositol phosphate generation between the two clones of NIH-3T3 cells, nor was there any difference in receptor number of Kd. These results question the role of inositol phospholipid breakdown in mitogenesis and demonstrate significant differences in the biochemical properties of apparently the 'same' cells.     

Effect of phorbol ester on prostaglandin regulation of proliferation in rabbit endometrial cells

We have proposed that two of the endogenously synthesized endometrial prostaglandins, prostaglandin F2 alpha (PGF2 alpha) and prostaglandin E1 (PGE1), play a regulatory role in growth control of the endometrium. PGF2 alpha increases DNA synthesis and PGE1 inhibits that effect. Primary cultures of rabbit endometrial cells were used here to examine the effects of the tumor-promoting, diacylglycerol mimicking, phorbol ester, 12-O-tetradecanoyl phorbol-13-acetate (TPA), on the prostaglandin control of cell proliferation. TPA treatment of these cultures results in: a decrease in control levels of proliferation and complete inhibition by TPA of PGF2 alpha stimulated DNA synthesis; a reduction in [3H]PGF2 alpha binding with short term treatment but an increase to above control binding level with long term treatment; an inhibition of the normal PGF2 alpha stimulated inositol polyphosphate synthesis; and a small increase in accumulation of PGF2 alpha in the culture media. Furthermore, in this culture system, TPA does not down regulate [3H]PGE1 binding; it does not alter the normal PGE1 stimulation of cAMP synthesis; and it has no effect on the normal endogenous PGE1 synthesis by these cultures. The above results are consistent with our previous observations that PGF2 alpha works through the intracellular messengers inositol polyphosphate/diacylglycerol whereas PGE1 works through cAMP.     

Zinc suppresses IL-6 synthesis by prostaglandin F2alpha in osteoblasts: inhibition of phospholipase C and phospholipase D

We previously reported that prostaglandin F2alpha (PGF2alpha) induces phosphoinositide hydrolysis by phospholipase C and phosphatidylcholine hydrolysis by phospholipase D through heterotrimeric GTP-binding protein, resulting in the activation of protein kinase C (PKC) in osteoblast-like MC3T3-E1 cells and that PGF2alpha stimulates the synthesis of interleukin-6 (IL-6) via PKC-dependent p44/p42 mitogen-activated protein (MAP) kinase activation. In the present study, we investigated whether zinc affects the PGF2alpha-induced IL-6 synthesis in these cells. Zinc complex of l-carnosine (l-CAZ) dose-dependently suppressed the PGF2alpha-stimulated IL-6 synthesis. In addition, zinc alone reduced the IL-6 synthesis. L-CAZ suppressed the PGF2alpha-induced p44/p42 MAP kinase phosphorylation. However, the p44/p42 MAP kinase phosphorylation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), a direct activator of PKC, or NaF, a direct activator of GTP-binding protein, was not affected by l-CAZ. l-CAZ reduced the PGF2alpha-stimulated formation of inositol phosphates and choline. However, l-CAZ did not affect the formation of inositol phosphates or choline induced by NaF. These results strongly suggest that zinc reduces PGF2alpha-induced IL-6 synthesis via suppression of phosphoinositide-hydrolyzing phospholipase C and phosphatidylcholine-hydrolyzing phospholipase D in osteoblasts.     

Endothelin-1 stimulates phospholipid hydrolysis and prostaglandin F2α production in primary human decidua cell cultures

The regulation of prostaglandin (PG) production by the uterine decidua may be an important mechanism controlling the onset and maintenance of human parturition. The present in vitro study has evaluated the potential for endothelin-1 (ET-1) to activate cell signalling and PGE2 alpha production in human primary decidua cell cultures. ET-1 stimulated a dose-dependent increase in inositol phospholipid hydrolysis and PG precursor release as evidenced by respective increases in [3H] inositol monophosphate accumulation and [14C] arachidonate release from radiolabelled decidua cells. PGF2 alpha production was increased in some but not all cell preparations in response to ET-1 alone. Pretreatment of decidua cells with interleukin-1 beta (IL-1 beta) enhanced PGF2 alpha production but not arachidonate release in response to ET-1. These in vitro observations support a possible role for ET-1 in the regulation of decidual PG production during parturition.     

Astrocytes possess prostaglandin F2 alpha receptors coupled to phospholipase C.

We examined the effect of prostaglandin (PG) F2 alpha on phosphoinositide (PI) hydrolysis in rat cultured astrocytes. PGF2 alpha stimulated the formation of [3H]inositol phosphates in [3H]inositol-labeled astrocytes with the ED50 value of 23 nM, whereas PGD2 and PGE2 were much less effective than PGF2 alpha. Transformation of astrocytes was accompanied by an increase in the stimulatory response of PGF2 alpha. Pretreatment of the astrocytes with pertussis toxin and cholera toxin did not affect the PGF2 alpha-evoked PI hydrolysis. In the digitonin-permeabilized astrocytes, PGF2 alpha significantly enhanced the GTP gamma S-evoked PI hydrolysis in the presence of Ca2+. These results indicate that rat cultured astrocytes possess PGF2 alpha receptors coupled to phospholipase C.     

p38 MAP kinase is involved in the signalling of sphingosine in osteoblasts: sphingosine inhibits prostaglandin F(2alpha)-induced phosphoinositide hydrolysis

We previously showed that sphingosine inhibits prostaglandin F(2alpha) (PGF(2alpha))-stimulated interleukin-6 synthesis in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of sphingosine on phospholipase C-catalyzing phosphoinositide hydrolysis induced by PGF(2alpha) in these cells. Sphingosine inhibited the inositol phosphates formation by PGF(2alpha) or NaF, a GTP-binding protein activator. Sphingosine induced the phosphorylation of p38 mitogen-activated protein (MAP) kinase but did not affect the phosphorylation of p42/p44 MAP kinase. SB203580 and PD169316, inhibitors of p38 MAP kinase, rescued the inhibitory effect of sphingosine on the formation of inositol phosphates by PGF(2alpha) or NaF. These results indicate that sphingosine inhibits PGF(2alpha)-induced phosphoinositide hydrolysis by phospholipase C via p38 MAP kinase in osteoblasts.     

Proliferative effect of PGD2 on osteoblast-like cells; independent activation of pertussis toxin-sensitive GTP-binding protein from PGE2 or PGF2 alpha.

PGD2 stimulated DNA synthesis and decreased alkaline phosphatase activity dose-dependently between 10 nM and 10 microM in osteoblast-like MC3T3-E1 cells. PGD2 had little effect on cAMP production, but caused very rapid enhancement of phosphoinositide (PI) hydrolysis dose-dependently between 10 nM and 10 microM. The formation of inositol trisphosphate (IP3) induced by PGD2 reached the peak within 1 min and decreased thereafter, which is more rapid than that induced by PGE2 or PGF2 alpha and both PGE2 and PGF2 alpha affected PGD2-induced IP3 formation additively. Pertussis toxin (PTX) inhibited both PGD2-induced formation of inositol phosphates and DNA synthesis. The degree of these PTX (1 micrograms/ml)-induced inhibitions was similar. In addition, neomycin, a phospholipase C inhibitor, inhibited PGD2-induced DNA synthesis as well as the formation of IP3, and the patterns of both inhibitions were similar. In the cell membranes, PTX-catalyzed ADP-ribosylation of a 40-kDa protein was significantly attenuated by pretreatment of PGD2. Time course of the attenuation of PTX-catalyzed ADP-ribosylation by PGD2 was apparently different from that by PGE2 or PGF2 alpha. These results indicate that PGD2 activates PTX-sensitive GTP-binding protein independently from PGE2 or PGF2 alpha and stimulates PI hydrolysis resulting in proliferation of osteoblast-like cells.     

Effects of prostaglandin E2 and F2 alpha on cytoplasmic pH in a clonal osteoblast-like cell line, MOB 3-4.

Prostaglandin E2 (PGE2, 5 ng/ml to 5 micrograms/ml) induced a dose-dependent increase in cAMP accumulation, inositol phosphates (IPs) accumulation, and cytoplasmic free Ca2+ ([Ca2+]i) in a clonal osteoblast-like cell line, MOB 3-4. In contrast, prostaglandin F2 alpha (PGF2 alpha, 5 ng/ml to 5 micrograms/ml) stimulated increases in IPs accumulation and [Ca2+]i without stimulating an increase in cAMP accumulation. Both PGE2 (greater than 0.5 micrograms/ml) and PGF2 alpha (greater than or equal to 5 micrograms/ml) increased cytoplasmic pH (pHi) from approximately 7.15 to 7.35 in BCECF-loaded cells. A tumor promotor, phorbol 12-myristate 13-acetate (PMA, 0.1-100 nM) also increased pHi without effect on phosphoinositide hydrolysis. Both PGE2-(5 micrograms/ml) and PMA- (100 nM) induced cytoplasmic alkalinization was inhibited by removal of extracellular Na+, or by pretreatment of the cells with amiloride (0.5 mM), an inhibitor of Na+/H+ exchange, or H-7 (100 microM), a nonspecific inhibitor of protein kinase C. Thus, MOB 3-4 cells appeared to possess PGE2 receptors and PGF2 alpha receptors: the former are coupled to adenylate cyclase and phospholipase C, and the latter are predominantly coupled to phospholipase C. Also the cells appeared to possess an amiloride-sensitive Na+/H+ exchange activity, which increases pHi in response to PGE2 and PGF2 alpha, as well as to PMA. Long-term (48 hr) exposure of the cells to PGE2 at a high concentration (5 micrograms/ml), but not to PGF2 alpha and PMA, decreased DNA synthesis in the serum-deficient medium. Thus, cytoplasmic alkalinization appeared insufficient for cell replication. At least in MOB 3-4 cells, the inhibitory effect of PGE2 on DNA synthesis may be due to the cAMP messenger system.     

Parathyroid hormone and prostaglandin E2 stimulate both inositol phosphates and cyclic AMP accumulation in mouse osteoblast cultures.

Parathyroid hormone (PTH) and prostaglandin E2 (PGE2) are physiological agonists which stimulate bone cells to resorb bone, a process by which the mineralized extracellular bone matrix is dissolved. Bone resorption has a key role in the maintenance of plasma calcium levels. It has been established that both PTH and PGE2 activate adenylate cyclase in osteoblasts, but it is apparent that (1) the two agents have qualitatively different effects on osteoblasts, and (2) the generation of cyclic AMP cannot account for all the effects of PTH on bone cell metabolism. Others have demonstrated that PTH and PGE2 may also elevate intracellular calcium levels, but the mechanism by which this is achieved has not been fully defined. Here we have investigated the effects of PTH on neonatal mouse osteoblasts in culture and shown that physiological concentrations of the hormone (50 nM) caused a small increase (22%) in total inositol phosphates accumulation, with a larger increase (40%) in inositol trisphosphate. We found that this activation occurred at lower concentration than was necessary to activate adenylate cyclase. PGE2 was a more effective activator of inositol phosphates accumulation than PTH, causing up to 300% increase in the total inositol phosphates after 30 min. Both PTH and PGE2 stimulated cyclic AMP accumulation, but the activation of adenylate cyclase by forskolin did not enhance inositol phosphates production. We conclude that both PTH and PGE2 stimulate phosphoinositide turnover in mouse osteoblasts and suggest that this mechanism may contribute to their elevation of intracellular calcium in bone cells.     

Prostaglandin E2 and F2 alpha inhibit growth of human gastric carcinoma cell line KATO III with simultaneous stimulation of cyclic AMP production

The effects of prostaglandins (PGs) on the growth of human gastric carcinoma cell line KATO III were investigated. PGE2 as well as PGF2 alpha significantly and dose-dependently inhibited the growth of this gastric carcinoma cell line (PGE2 greater than PGF2 alpha). This inhibition of cell growth by the PGs was associated with the increase in cyclic AMP production (PGE2 greater than PGF2 alpha), whereas inositol-phospholipid turnover was not affected by either PGE2 or PGF2 alpha as assessed by the formation of 3H-inositol phosphates. Furthermore, the proliferation of these gastric carcinoma cells was also suppressed by the administration of forskolin as well as of dibutyryl cyclic AMP. These results suggest that PGE2 and PGF2 alpha inhibit the growth of cultured human gastric carcinoma cells KATO III via stimulation of cyclic AMP production.     

Effects of prostaglandins on the production of collagenase by rabbit uterine cervical fibroblasts

Effects of prostaglandins on the production of collagenase by rabbit uterine cervical fibroblasts were investigated. Exogenous prostaglandin E2 (PGE2) and PGF2 alpha significantly stimulated the production of collagenase in a dose dependent manner, whereas PGI2 did not. Addition of arachidonic acid in the presence of absence of indomethacin to the cell culture did not show any increase in collagenase production. Recombinant human interleukin-1 (rhIL-1) also promoted the production of cervical collagenase independently of endogenous prostaglandin(s). Furthermore both exogenous PGE2 and PGF2 alpha enhanced the rhIL-1-induced collagenase production whereas PGI2 and/or indomethacin did not. These results suggested that exogenous PGE2 and PGF2 alpha but not endogenous prostaglandin(s) participate in cervical ripening and dilation by enhancing collagenase production by rabbit uterine cervical cells.     

Protein kinase C activation amplifies prostaglandin F2 alpha-induced prostaglandin E2 synthesis in osteoblast-like cells.

In cloned osteoblast-like cells, MC3T3-E1, prostaglandin F2 alpha (PGF2 alpha) stimulated arachidonic acid (AA) release in a dose-dependent manner in the range between 1 nM and 10 microM. 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC) activator, which by itself had little effect on AA release, markedly amplified the release of AA stimulated by PGF2 alpha in a dose-dependent manner. 4 alpha-phorbol 12,13-didecanoate, a phorbol ester which is inactive for PKC, showed little effect on the PGF2 alpha-induced AA release. 1-oleoyl-2-acetylglycerol (OAG), a specific activator for PKC, mimicked TPA by enhancement of the AA release induced by PGF2 alpha. H-7, a PKC inhibitor, markedly suppressed the effect of OAG on PGF2 alpha-induced AA release. Quinacrine, a phospholipase A2 inhibitor, showed partial inhibitory effect on PGF2 alpha-induced AA release, while it suppressed the amplification by OAG of PGF2 alpha-induced AA release almost to the control level. Furthermore, TPA enhanced the AA release induced by melittin, known as a phospholipase A2 activator. On the other hand, TPA inhibited the formation of inositol trisphosphate stimulated by PGF2 alpha. Under the same condition, PGF2 alpha indeed stimulated prostaglandin E2 (PGE2) synthesis and TPA markedly amplified the PGF2 alpha-induced PGE2 synthesis as well as AA release. These results indicate that the activation of PKC amplifies PGF2 alpha-induced both AA release and PGE2 synthesis through the potentiation of phospholipase A2 activity in osteoblast-like cells.     

Prostaglandin E receptor enhancement of catecholamine release may be mediated by phosphoinositide metabolism in bovine adrenal chromaffin cells

In the presence of ouabain, prostaglandin (PG) E2 stimulated a gradual secretion of catecholamines from cultured bovine adrenal chromaffin cells. PGE2 or ouabain alone evoked a marginal secretory response. The synergism of ouabain was also observed with muscarine. PGE2, like muscarine, induced a concentration-dependent formation of inositol phosphates: rapid rises in inositol trisphosphate and inositol bisphosphate followed by a slower accumulation of inositol monophosphate. This effect on phosphoinositide metabolism was accompanied by an increase in cytosolic free Ca2+. The potency of PGs (PGE2 greater than PGF2 alpha greater than PGD2) to stimulate catecholamine release was well correlated with that to affect phosphoinositide metabolism and that to increase the level of intracellular Ca2+. PGE2 did not stimulate cAMP generation significantly in bovine chromaffin cells. The effect of PGE2 on catecholamine release was mimicked by 12-O-tetradecanoylphorbol 13-acetate and A23187, but not by the cAMP analogue dibutyryl cAMP nor by forskolin. These results indicate that PGE2 may enhance catecholamine release from chromaffin cells by activating protein kinase C in concert with the increment of intracellular Ca2+.     

PGE2 is a more potent vasodilator of the lamb ductus arteriosus than is either PGI2 or 6 keto PGF1alpha.

It has been shown in vitro that the lamb ductus arteriosus forms prostaglandins PGE2, PGF2alpha, 6 keto PGF1alpha (and its unstable precursor PGI2). In this study the relative potencies of these endogenous prostaglandins were investigated on isolated lamb ductus arteriosus preparations contracted by exposure to elevated PO2 and indomethacin. All the prostaglandins (except PGF2alpha) relaxed the vessel. This is consistent with the hypothesis that endogenous prostaglandins inhibit the tendency of the vessel to contract in response to oxygen. Only PGE2, however, relaxed the vessel at concentrations below 10(-8)M. PGI2 and 6 keto PGF1alpha had approximately 0.001 and 0.0001 times the activity of PGE2. Although PGE2 has been observed to be a minor product of prostaglandin production in the lamb ductus arteriosus, the tissue's marked sensitivity to PGE2 might make it the most significant prostaglandin in regulating the patency of the vessel.