Stimulatory Effect Of PGE2 Research Articles

PGE2 upregulates the Na+/K+ ATPase in HepG2 cells via EP4 receptors and intracellular calcium.

The Na+/K+ ATPase is a key regulator of the hepatocytes ionic homeostasis, which when altered may lead to many liver disorders. We demonstrated recently, a significant stimulation of the Na+/K+ ATPase in HepG2 cells treated with the S1P analogue FTY 720P, that was mediated through PGE2. The mechanism by which the prostaglandin exerts its effect was not investigated, and is the focus of this work. The type of receptors involved was determined using pharmacological inhibitors, while western blot analysis, fluorescence imaging of GFP-tagged Na+/K+ ATPase, and time-lapse imaging on live cells were used to detect changes in membrane abundance of the Na+/K+ ATPase. The activity of the ATPase was assayed by measuring the amount of inorganic phosphate liberated in the presence and absence of ouabain. The enhanced activity of the ATPase was not observed when EP4 receptors were blocked but still appeared in presence inhibitors of EP1, EP2 and EP3 receptors. The involvement of EP4 was confirmed by the stimulation observed with EP4 agonist. The stimulatory effect of PGE2 did not appear in presence of Rp-cAMP, an inhibitor of PKA, and was imitated by db-cAMP, a PKA activator. Chelating intracellular calcium with BAPTA-AM abrogated the effect of db-cAMP as well as that of PGE2, but PGE2 treatment in a calcium-free PBS medium did not, suggesting an involvement of intracellular calcium, that was confirmed by the results obtained with 2-APB treatment. Live cell imaging showed movement of GFP–Na+/K+ ATPase-positive vesicles to the membrane and increased abundance of the ATPase at the membrane after PGE2 treatment. It was concluded that PGE2 acts via EP4, PKA, and intracellular calcium.

Abstract 4080: TGF-β Effects on prostate cancer cell migration are mediated by PGE2 through activation of PI3K/AKT/mTOR pathway.

Abstract Transforming growth factor-beta (TGF-β) plays an important role in the progression of prostate cancer. It exhibits both tumor suppressor and tumor promoting activities. Correlations between COX-2 overexpression and enhanced production of PGE2 have been implicated in cancer progression; however, there are no studies indicating that TGF-β effects in prostate cancer cells involve PGE2 synthesis. In this study, we investigated TGF-β regulation of COX-1 and COX-2 expression in prostate cancer cells and whether the effects of TGF-β on cell proliferation and migration are mediated by PGE2. COX-1 protein was ubiquitously expressed in prostate cells; however, COX-2 protein levels were only detected in prostate cancer cells. TGF-β treatment increased COX-2 protein levels and PGE2 secretion in PC3 cells. Exogenous PGE2 and PGF2α had no effects on cell proliferation in LNCaP, DU145, and PC3 cells while PGE2 and TGF-β induced migratory behavior only in PC3 cells. Only EP2 and EP4 receptors were detected at mRNA levels in prostate cells. The EP4-targeting siRNA inhibited PGE2 and TGF-β-induced migration of PC3 cells. PGE2 and TGF-β induced phosphorylation of AKT which was blocked by antagonists of PGE2 (EP4) receptors (L161982, AH23848) and PI3-kinase inhibitor (LY294002) in PC3 cells. Pretreatment with L161982 or AH23848 blocked the stimulatory effects of PGE2 and TGF-β on cell migration, while LY294002 or rapamycin completely eliminated PGE2, TGF-β, and EGF-induced migration PC3 cells. We conclude that TGF-β increases COX-2 levels and PGE2 secretion in prostate cancer cells which, in turn, mediates TGF-β effects on cell migration through the activation of PI3K/AKT/mTOR pathway. Acknowledgements: These studies were supported by the NIH/NIMHD/RCMI grant #G12MD007590, NIH/NIMHD #5P20MD002285, and DOD grant # W8I-08-1-0077. Citation Format: Baohan T. Vo, Derrick Morton, Shravan Komaragiri, Ana C. Millena, Shafiq A. Khan. TGF-β Effects on prostate cancer cell migration are mediated by PGE2 through activation of PI3K/AKT/mTOR pathway. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4080. doi:10.1158/1538-7445.AM2013-4080

Prostaglandin EP Receptor Subtypes Involved in Regulating HCO3- Secretion from Gastroduodenal Mucosa

Gastroduodenal HCO(3)(-) secretion is a key process that aids in preventing acid-peptic injury. The HCO(3)(-) secretion in rats and mice was increased in response to PGE(2) as well as mucosal acidification, the latter response occurring with a concomitant enhancement of mucosal PG production. The duodenal responses to PGE(2) and acid were decreased in mice lacking EP3 receptors and reduced by coadministration of an EP3 or EP4 antagonist in rats, complete inhibition being observed when the EP3 and EP4 antagonists were given together. By contrast, the gastric responses disappeared in EP1-knockout mice and were prevented by an EP1 antagonist but not other EP antagonists. Furthermore, duodenal HCO(3)(-) secretion was stimulated by the EP3 and EP4 agonists, whereas gastric HCO(3)(-) secretion was increased only by the EP1 agonist. In addition, the HCO(3)(-) stimulatory effect of sulprostone (an EP1/EP3 agonist) in the duodenum was inhibited by verapamil, a Ca(2+) antagonist, and enhanced by isobutyl- methylxanthine, a phosphodiesterase (PDE) inhibitor, but the response in the stomach was inhibited by verapamil and not affected by isobutylmethylxanthine. In the mouse duodenum but not stomach, the response to PGE(2) was potentiated by both vinpocetine (a PDE1 inhibitor) and cilostamide (a PDE3 inhibitor). These results suggest that the HCO(3)(-) stimulatory effect of PGE(2) in the duodenum is mediated by both EP3 and EP4 receptors, being coupled intracellularly with Ca(2+) and cAMP, while that in the stomach is mediated by EP1 receptors, coupled with Ca(2+). In addition, both PDE1 and PDE3 are involved in the regulation of duodenal HCO(3)(-) secretion.

PGE 2 in Synergy with IL-23 Drives Robust IL-17 Production and Th17 Differentiation by Inhibiting the STAT6 Signaling Pathway

RATIONALE: IL-17-producing Th17 cells are a newly established subset of differentiated CD4 T cells. Although the lipid molecule PGE2 has been shown to increase IL-17 production by human and murine T cells, the mechanism for the pro-Th17 function of PGE2 is not well understood. We investigated the role of STAT6 signaling pathway in PGE2-augmented IL-17 production and Th17 differentiation. METHODS: We activated splenic CD4 T cells of STAT6 knockout (KO) mice and control BALB/c mice with anti-CD3, anti-CD28 and IL-23 in the presence or absence of PGE2 for 5 days. The levels of IL-17, IL-17A and IL-22 in the culture supernatant were determined by ELISA and Luminex multiple cytokine bead-array assay. The levels of IL-23R and RORγt mRNAs were determined by real-time PCR. RESULTS: PGE2 increased IL-17 production and Th17 differentiation of the wild type CD4 T cells. PGE2 also enhanced the production of other Th17 cytokines including IL-17A and IL-22, but inhibited Th1 and Th2 cytokine production. The pro-Th17 effect was correlated with PGE2-mediated upregulation of IL-23R and RORγt mRNA expression. In contrast, the stimulatory effect of PGE2 on IL-17, IL-17A and IL-22 production was abrogated in STAT6 KO T cells. These results indicate that PGE2 increases Th17 differentiation by suppressing STAT6-signaling pathway. CONCLUSIONS: The lipid product PGE2 in the arachidonic acid metabolic pathway is a potent Th17-driving factor. Frequent use of cyclooxygenase-inhibiting and PGE2-suppressing drugs may disturb the balance of Th lineages in the immune system and affect the host defense and disease development.

PGE2 potentiates tonicity-induced COX-2 expression in renal medullary cells in a positive feedback loop involving EP2-cAMP-PKA signaling

Cyooxygenase-2 (COX-2)-derived PGE2 is critical for the integrity and function of renal medullary cells during antidiuresis. The present study extended our previous finding that tonicity-induced COX-2 expression is further stimulated by the major COX-2 product PGE2 and investigated the underlying signaling pathways and the functional relevance of this phenomenon. Hyperosmolality stimulated COX-2 expression and activity in Madin-Darby canine kidney (MDCK) cells, a response that was further increased by PGE2-cAMP signaling, suggesting the existence of a positive feedback loop. This effect was diminished by AH-6809, an EP2 antagonist, and by the PKA inhibitor H-89, but not by AH-23848, an EP4 antagonist. The effect of PGE2 was mimicked by forskolin and dibutyryl-cAMP, suggesting that the stimulatory effect of PGE2 on COX-2 is mediated by a cAMP-PKA-dependent mechanism. Accordingly, cAMP-responsive element (CRE)-driven reporter activity paralleled the effects of PGE2, AH-6809, AH-23848, H-89, forskolin, and dibutyryl-cAMP on COX-2 expression. In addition, the stimulatory effect of PGE2 on tonicity-induced COX-2 expression was blunted in cells transfected with dominant-negative CRE binding (CREB) protein, as was the case in a COX-2 promoter reporter construct in which a putative CRE was deleted. Furthermore, PGE2 resulted in PKA-dependent phosphorylation of the pro-apoptotic protein Bad at Ser155, a mechanism that is known to inactivate Bad, which coincided with reduced caspase-3 activity during osmotic stress. Conversely, pharmacological interruption of the PGE2-EP2-cAMP-PKA pathway abolished Ser155 phosphorylation of Bad and blunted the protective effect of PGE2 on cell survival during osmotic stress. These observations indicate the existence of a positive feedback loop of PGE2 on COX-2 expression during osmotic stress, an effect that apparently is mediated by EP2-cAMP-PKA signaling, and that contributes to cell survival under hypertonic conditions.

PGE2 inhibits apical K channels in the CCD through activation of the MAPK pathway

We used the patch-clamp technique and Western blot analysis to explore the effect of PGE(2) on ROMK-like small-conductance K (SK) channels and Ca(2+)-activated big-conductance K channels (BK) in the cortical collecting duct (CCD). Application of 10 microM PGE(2) inhibited SK and BK channels in the CCD. Moreover, either inhibition of PKC or blocking mitogen-activated protein kinase (MAPK), P38 and ERK, abolished the effect of PGE(2) on SK channels in the CCD. The effect of PGE(2) on SK channels was completely blocked in the presence of SC-51089, a specific EP1 receptor antagonist, and mimicked by application of sulprostone, an agonist for EP1 and EP3 receptors. To determine whether PGE(2) stimulates the phosphorylation of P38 and ERK, we treated mouse CCD cells (M-1) with PGE(2). Application of PGE(2) significantly stimulated the phosphorylation of P38 and ERK within 5 min. The dose-response curve of PGE(2) effect shows that 1, 5, and 10 microM PGE(2) increased the phosphorylation of P38 and ERK by 20-21, 50-80, and 80-100%, respectively. The stimulatory effect of PGE(2) on MAPK phosphorylation was not affected by indomethacin but abolished by inhibition of PKC. This suggests that the effect of PGE(2) on MAPK phosphorylation is PKC dependent. Also, the expression of cyclooxygenase II and PGE(2) concentration in renal cortex and outer medulla was significantly higher in rats fed a K-deficient diet than those on a normal-K diet. We conclude that PGE(2) inhibits SK and BK channels and that there is an effect of PGE(2) on SK channels in the CCD through activation of EP1 receptor and MAPK pathways. Also, high concentrations of PGE(2) induced by K restriction may be partially responsible for increasing MAPK activity during K restriction.

Prostaglandin E2 Increases Transforming Growth Factor-β Type III Receptor Expression through CCAAT Enhancer-Binding Protein δ in Osteoblasts

Variations in individual TGF-beta receptors (TbetaRs) may modify TGF-beta activity and significantly alter its effects on connective tissue growth or repair. Differences in the amount of TbetaR type III (TbetaRIII) relative to signal transducing TbetaRI occur on bone cells during differentiation or in response to other growth regulators. Here we investigated prostaglandin (PG) E2, a potent effector during trauma, inflammation, or mechanical load, on TbetaR expression in primary osteoblast-enriched cultures. PGE2 rapidly increased TbetaRIII mRNA and protein expression and enhanced TbetaRIII gene promoter activity through a discrete region within 0.4 kb of the transcription start site. PGE2 alters osteoblast function through multiple signal-inducing pathways. In this regard, protein kinase A (PKA) activators, PGE1 and forskolin, also enhanced gene expression through the TbetaRIII gene promoter, whereas protein kinase C activators, PGF2alpha and phorbol myristate acetate, did not. The stimulatory effect of PGE2 on TbetaRIII promoter activity was suppressed by a dominant negative PKA-regulatory subunit, but not by dominant negative protein kinase C. PGE2 specifically increased nuclear factor CCAAT enhancer-binding protein delta (C/EBPdelta) binding to a half-binding site upstream of the basal TbetaRIII promoter region, and promoter activity was sensitive to C/EBPdelta overexpression and to dominant-negative C/EBPdelta competition. In parallel with their effect on TbetaRIII expression, activators of PKA decreased TGF-beta-induced activity. In summary, high levels of PGE2 that occur with inflammation or trauma may, through PKA-activated C/EBPdelta, preferentially increase TbetaRIII expression and in this way delay TGF-beta-dependent activation of osteoblasts during the early stabilization phase of bone repair.

PGE2 inhibits ROMK channels in the CCD through activation of MAPK pathway

We have used the patch clamp technique and Western blot analysis to explore the effect of PGE2 on ROMK-like small conductance K (SK) channels in the CCD. Application of 10 uM PGE2 inhibited SK channels in the CCD. Moreover, inhibition of P38 and ERK MAPK not only increased SK channel activity but also abolished the effect of PGE2 on SK in the CCD. To determine whether PGE2 stimulates the phosphorylation of P38 and ERK, we treated mouse CCD cells (M-1) with PGE2. Application of PGE2 significantly stimulated the phosphorylation of P38 and ERK within 5 min. The dose response curve of PGE2 effect shows that 1, 5 and 10 uM PGE2 increased the phosphorylation of ERK and P38 by 25, 50 and 80%, respectively. The stimulatory effect of PGE2 on MAPK phosphorylation was not affected by indomethacin but abolished by inhibition of PKC. This suggests that the effect of PGE2 on MAPK phosphorylation requires PKC. Also, we examined the COX2 expression in the rat kidney. The expression of COX2 and PGE2 concentration in renal cortex and outer medulla were significantly higher in rats fed on K-depleted diet (KD) than that on a normal K. We conclude that PGE2 inhibits SK channels in the CCD through MAPK pathways and that high concentrations of PGE2 induced by K restriction may be partially responsible for increasing MAPK activity during K restriction.

Prostaglandin E2 increases cyp19 expression in rat granulosa cells: Implication of GATA-4

The expression of Cyp19, the key gene of estrogen biosynthesis, in granulosa cells (GC) is essential for follicular growth and coordination of the ovulatory process. The goal of this study was to examine the effect of PGE2 and PGF2α on Cyp19 expression in undifferentiated and luteinized GC (UGC and LGC). In UGC, PGE2 increased Cyp19 mRNA and Cyp19 protein levels whereas PGF2α had no effect. In LGC, PGF2α decreased Cyp19 expression whereas PGE2 had no effect. Gene-reporter experiments demonstrated that PGE2 increases Cyp19 transcription in UGC. A protein kinase A inhibitor blocked PGE2-induced increase in Cyp19 promoter activity. PGE2 increased GATA-4 binding to the Cyp19 promoter. Mutation of the GATA binding site resulted in the loss of PGE2 stimulation. This study demonstrates that PGE2 stimulates Cyp19 expression in rat GC and suggests that GATA-4 may mediate (at least in part) the stimulatory effect of PGE2.

Evidence for post-transcriptional regulation of Na,K-ATPase by prostaglandin E 1

The stimulatory effect of PGE 1 on the activity of the Na,K-ATPase in MDCK cells is associated with an increase in the rate of transcription of the Na,K-ATPase β1 subunit gene and an increase in the rate of biosynthesis of the Na,K-ATPase [M.L. Taub, Y. Wang, I.S. Yang, P. Fiorella, S.M. Lee, Regulation of the Na,K-ATPase activity of Madin-Darby canine kidney cells in defined medium by prostaglandin E1 and 8-bromocyclic AMP, J. Cell. Physiol. 151 (1992) 337–346]. In order to further define the molecular mechanisms, transient transfection and biosynthesis studies were conducted with dibutyryl cAMP resistant (DB r) MDCK cells, defective in cAMP dependent protein kinase, and PGE 1 independent (PGE 1 Ind) MDCK cells with elevated intracellular cAMP. Transient transfection studies with the human Na,K-ATPase β1 promoter/luciferase construct, pHβ1–1141 Luc [J. Feng, J. Orlowski, J.B. Lingrel, Identification of a functional thyroid hormone response element in the upstream flanking region of the human Na,K-ATPase beta 1 gene, Nucleic Acids Res. 21 (1993) 2619–2626], showed that the stimulatory effect of PGE 1 and 8Br-cAMP on β1 subunit gene transcription is retained in the DB r and PGE 1 independent variants. However, the stimulatory effect of PGE 1 and 8Br-cAMP on Na,K-ATPase biosynthesis was lost in DB r (unlike PGE 1 Ind) variants. These results can be explained by a defect in post-transcriptional regulation.

Relationship between prostaglandin E 2 and vascular endothelial growth factor (VEGF) in angiogenesis in human vascular endothelial cells

To address the role of prostaglandin E 2 (PGE 2) in tube formation of endothelial cells and the relationships between the action of PGE 2 and vascular endothelial growth factor (VEGF), cultured human umbilical vein endothelial cells (HUVECs) were used to evaluate tube formation on Matrigel and the expression of angiogenesis-related genes. PGE 2 treatment stimulated the tube-like formation of HUVECs. Whereas VEGF-induced tube formation was significantly suppressed by ETYA, an inhibitor of arachidonic acid metabolism, or SU5614, an inhibitor of VEGF-receptor tyrosine kinase, the stimulatory effect of PGE 2 was observed in the presence of ETYA or SU5614. Thus, PGE 2 counteracted both ETYA- and SU5614-induced blockage of angiogenesis in the presence of VEGF. VEGF induced cyclooxygenase (COX) -2 mRNA expression in HUVECs and increased the PGE 2 concentration in the medium. PGE 2 treatment enhanced the expression of VEGF mRNA. These findings suggest that PGE 2 directly stimulates angiogenesis, apart from VEGF signaling, and further induces VEGF expression in HUVECs. In addition, the effect of VEGF on angiogenesis may be mediated, in part, by PGE 2 secretion.

Prostaglandin E2 activates outwardly rectifying Cl(-) channels via a cAMP-dependent pathway and reduces cell motility in rat osteoclasts.

We examined changes in electrical and morphological properties of rat osteoclasts in response to prostaglandin (PG)E(2). PGE(2) (>10 nM) stimulated an outwardly rectifying Cl(-) current in a concentration-dependent manner and caused a long-lasting depolarization of cell membrane. This PGE(2)-induced Cl(-) current was reversibly inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), and tamoxifen. The anion permeability sequence of this current was I(-) > Br(-) approximately Cl(-) > gluconate(-). When outwardly rectifying Cl(-) current was induced by hyposmotic extracellular solution, no further stimulatory effect of PGE(2) was seen. Forskolin and dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) mimicked the effect of PGE(2). The PGE(2)-induced Cl(-) current was inhibited by pretreatment with guanosine 5'-O-2-(thiodiphosphate) (GDPbetaS), Rp-adenosine 3',5'-cyclic monophosphorothioate (Rp-cAMPS), N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide dihydrochloride (H-89), and protein kinase A inhibitors. Even in the absence of nonosteoclastic cells, PGE(2) (1 microM) reduced cell surface area and suppressed motility of osteoclasts, and these effects were abolished by Rp-cAMPS or H-89. PGE(2) is known to exert its effects through four subtypes of PGE receptors (EP1-EP4). EP2 and EP4 agonists (ONO-AE1-259 and ONO-AE1-329, respectively), but not EP1 and EP3 agonists (ONO-DI-004 and ONO-AE-248, respectively), mimicked the electrical and morphological actions of PGE(2) on osteoclasts. Our results show that PGE(2) stimulates rat osteoclast Cl(-) current by activation of a cAMP-dependent pathway through EP2 and, to a lesser degree, EP4 receptors and reduces osteoclast motility. This effect is likely to reduce bone resorption.

Histamine and prostaglandin interaction in regulation of oxytocin and vasopressin secretion.

Prostaglandins and histamine in the hypothalamus are involved in the regulation of oxytocin and vasopressin secretion, and appear to be involved in the mediation of pituitary hormone responses to immunochallenges. Therefore, we investigated in conscious male rats: (i) whether blockade of H1 or H2 receptors affected the oxytocin and vasopressin responses to prostaglandins and (ii) whether blockade of prostaglandin synthesis affected the oxytocin and vasopressin responses to histamine or to Escherichia coli lipopolysaccharide (LPS), in order to determine any interaction between prostaglandins and histamine in the hypothalamus. Oxytocin secretion was dose-dependently stimulated by intracerebroventricular infusion of 1 or 5 microg of PGE1, PGE2 or PGF2alpha, with PGE2 being the most potent of the compounds used. Prior central infusion of the H1 receptor antagonist mepyramine or the H2 receptor antagonist cimetidine significantly inhibited the oxytocin response to all three prostaglandins by approximately 50%. Vasopressin secretion was increased by PGE1 but not by PGE2 or PGF2alpha. The stimulatory effect of PGE1 was almost annihilated by prior administration of mepyramine or cimetidine. Central infusion of histamine or immunochallenge with LPS administered intraperitoneally increased oxytocin and vasopressin secretion four- and two-fold, respectively. Pretreatment with systemic injection of the prostaglandin synthesis inhibitor indomethacin dose-dependently reduced the oxytocin response and prevented the vasopressin response to histamine or LPS. We conclude that histamine and PGE1, PGE2 or PGF2alpha interact in the regulation of oxytocin secretion, whereas histamine and only PGE1 interact in the regulation of vasopressin secretion. Furthermore, histamine as well as LPS may affect oxytocin and vasopressin neurones via activation of prostaglandins, probably in the hypothalamic supraoptic nucleus.

Effects of progesterone on prostaglandin E(2)-induced changes in glycosaminoglycan synthesis by human cervical fibroblasts in culture.

Prostaglandins are known to induce cervical ripening and this effect may be mediated by an increase in glycosaminoglycan (GAG) concentration. The aim of this study was to assess the effects of progesterone on prostaglandin E(2) (PGE(2))-induced changes in GAG synthesis by human cervical cells in culture. Human cervical fibroblasts were obtained by cervical biopsies in hormonally active women and cultured. Cells were submitted to an incubation with progesterone or control medium. A second incubation was then performed with increasing concentrations of PGE(2). GAG synthesis by the cervical cells was assayed after extraction, by incorporation of [(3)H]-glucosamine and [(35)S]-sulphate into GAGs. It was found that progesterone alone induced a dose-dependent increase in GAG synthesis. After pre-incubation with progesterone, PGE(2) further increased [(3)H]-glucosamine and [(35)S]-sulphate uptake. However, when expressed as percentage of stimulation, the stimulatory effect of PGE(2) on GAG synthesis was inhibited at high progesterone concentrations. Therefore we concluded that, although high concentrations of progesterone increase the overall synthesis of GAG, they may also play a preventative role against PGE(2)-induced changes in GAG production during pregnancy.

Activation of the insulin-like growth factor-binding protein-5 promoter in osteoblasts by cooperative E box, CCAAT enhancer-binding protein, and nuclear factor-1 deoxyribonucleic acid-binding sequences.

Insulin-like growth factor (IGF)-binding protein-5 (IGFBP-5) has IGF-dependent and -independent actions. PGE2 rapidly increases IGFBP-5 expression by osteoblasts through cAMP-dependent processes. A minimal DNA sequence required for basal and PGE2-stimulated IGFBP-5 promoter activity spans -69 to -35 bp. This region adjoins a functional TATA box and contains E box, CCAAT enhancer-binding protein (C/EBP), nuclear factor-1 (NF-1), and activator protein-2 (AP-2) transcription factor related binding motifs. In this study we compared minimal promoter sequences of -74 to +120 bp, without or with mutations in each potential regulatory element, by reporter gene expression and electrophoretic mobility shift assays. Mutation of the E box-related element reduced basal promoter activity by 50% and eliminated the 2-fold stimulatory effect of PGE2. In contrast, mutations in the C/EBP- or NF-1-related elements also reduced basal promoter activity without fully eliminating the PGE2 effect. Overexpression of C/EBPdelta stimulated basal IGFBP-5 promoter activity, and this effect was eliminated by mutating the C/EBP-binding site. However, mutation of the AP-2-binding site or overexpression of AP-2 did not correlate with basal or PGE2-induced promoter activation. By electrophoretic mobility shift assay, prominent gel shift complexes occurred with osteoblast nuclear extracts and 32P-labeled probes spanning the E box-, C/EBP-, and NF-1-related motifs. These gel shift complexes were depleted by specific binding site mutations and were enhanced by PGE2. Increased binding by extracts from PGE2-treated cultures was blocked by cycloheximide treatment. These results identify several elements as integral binding sequences for both basal and PGE2-stimulated IGFBP-5 promoter activity. They further reveal that multiple sequences within this cluster form a basic transcription unit where nuclear factors can accumulate in a protein synthesis-dependent way and enhance IGFBP-5 expression by osteoblasts in response to PGE2.

Endogenously produced prostanoids stimulate calcium reabsorption in the rabbit cortical collecting system.

1. The influence of endogenously produced prostanoids on active transepithelial Ca2+ transport and cAMP formation was investigated in immunodissected rabbit kidney connecting and cortical collecting tubule cells grown to confluency on permeable supports. 2. The cyclo-oxygenase inhibitor indomethacin dose-dependently (IC50 = 18 nM) reduced the net apical-to-basolateral Ca2+ transport by 57%. Inhibition was reversed in medium obtained from monolayers incubated in the absence of indomethacin. 3. HPLC analysis following incubation with 14C-labelled arachidonic acid revealed the presence of a wide variety of radiolabelled prostanoids in both the apical and basolateral media. These findings are compatible with the endogenous production and subsequent release of stimulatory prostanoids. 4. The inhibitory action of indomethacin was reversed by the addition of the prostanoids PGE1, PGE2 and PGA2, but not PGD2, PGF2 alpha, the stable PGI2 analogue cicaprost or the thromboxane A2 mimetic U-46619. PGE2 stimulated transepithelial Ca2+ transport dose dependently (EC50 = 3 nM), irrespective of the compartment of which it was added. The stimulatory effect of PGE2 was paralleled by increased cAMP formation, suggesting the apical and basolateral presence of stimulatory prostanoid receptors EP2 and/or EP4. 5. Sulprostone, an analogue selective for EP1 and EP3 receptors, inhibited transepithelial Ca2+ transport in indomethacin-treated monolayers only when applied basolaterally, suggesting the exclusive presence of inhibitory EP receptors on the basolateral membrane. 6. The percentage by which parathyroid hormone and arginine vasopressin increased both transepithelial Ca2+ transport and cAMP formation was dramatically increased in indomethacin-inhibited cells as compared with control cells, demonstrating that indomethacin unmasks the actions of these hormones to their full extent.

Effects of prostaglandins on deoxyribonucleic acid and aggrecan synthesis in the RCJ 3.1C5.18 chondrocyte cell line: role of second messengers.

PGs play an important role in regulating articular chondrocyte function in both normal and pathological states. However, the mechanisms of the effects of PG on chondrocyte function remain undefined. We, therefore, examined the effects of PGE1, PGE2, and PGE2 alpha on second messenger generation in relation to DNA and aggrecan synthesis in the nontransformed rat RCJ 3.1C5.18 (RCJ) chondrocyte cell line. RCJ cells were grown under minimal attachment conditions on a composite collagen-agarose (0.15%/0.8%) gel to maintain a differentiated phenotype. PGE1 and PGE2 (0.001-100 microM) produced a similar dose-related increase in cAMP accumulation, with a maximal 8-fold increase over basal values, whereas PGF2 alpha produced a minimal 1.3-fold increase in cAMP levels only at 100 microM. On the other hand, both PGE2 and PGE2 alpha raised the intracellular free calcium ([Ca2+]i) concentration, derived primarily from extracellular sources, whereas PGE1 was without effect on [Ca2+]i. These three PGs also had divergent effects on DNA synthesis, as measured by [3H]thymidine ([3H]TdR) incorporation. PGF2 alpha (0.001-5 microM) produced a dose-related increase in [3H]TdR incorporation, with a maximal 1.6-fold increase over baseline values at 5 microM and a slight decline to below maximal levels at 10 microM. PGE2 exhibited a contrasting inverse biphasic response, with an initial small suppressive effect that was maximal at 0.1 microM and a secondary stimulatory phase producing a small increase over control values at 5 microM. PGE1 had a uniformly suppressive effect, producing a 30% decrease at 10 microM. Despite the divergent effects of PGE1, PGE2, and PGE2 alpha on second messenger generation and DNA synthesis, all three PGs produced a dose-related stimulation of aggrecan synthesis. PGF2 alpha was the most potent, producing significant stimulation at 0.001 microM and a maximal 104% increase at 5 microM. PGE1 and PGE2 were approximately equipotent and approximately 60% as effective as PGF2 alpha in stimulating aggrecan synthesis. Northern analysis demonstrated that the effects of PG on aggrecan synthesis were not accompanied by changes in aggrecan core protein steady state messenger RNA levels. Thus, the effects of PG on aggrecan production in RCJ cells appear to be regulated at the posttranscriptional level. Forskolin and (Bu)2cAMP mimicked the suppressive effects of PGE1 on [3H]TdR incorporation, as well as the stimulatory effect of PGE1 on aggrecan synthesis. In addition, the phorbol ester 12-O-tetradecanoyl phorbol acetate mimicked PGF2 alpha stimulation of [3H]TdR incorporation and aggrecan synthesis, and the effects of PGE2 alpha on these processes were blocked by protein kinase C inhibitors. Therefore, it appears that in mammalian chondrocytes, PGE1 primarily activates the cAMP-protein kinase A second messenger system, PGE2 alpha affects primarily the Ca2(+)-protein kinase C system, and PGE2 activates both pathways. Moreover, PG posttranscriptional regulation of aggrecan synthesis in chondrocytes involves both the cAMP-protein kinase A and Ca2(+)-protein kinase C second messenger systems.

Effects of prostaglandins on deoxyribonucleic acid and aggrecan synthesis in the RCJ 3.1C5.18 chondrocyte cell line: role of second messengers

PGs play an important role in regulating articular chondrocyte function in both normal and pathological states. However, the mechanisms of the effects of PG on chondrocyte function remain undefined. We, therefore, examined the effects of PGE1, PGE2, and PGE2 alpha on second messenger generation in relation to DNA and aggrecan synthesis in the nontransformed rat RCJ 3.1C5.18 (RCJ) chondrocyte cell line. RCJ cells were grown under minimal attachment conditions on a composite collagen-agarose (0.15%/0.8%) gel to maintain a differentiated phenotype. PGE1 and PGE2 (0.001-100 microM) produced a similar dose-related increase in cAMP accumulation, with a maximal 8-fold increase over basal values, whereas PGF2 alpha produced a minimal 1.3-fold increase in cAMP levels only at 100 microM. On the other hand, both PGE2 and PGE2 alpha raised the intracellular free calcium ([Ca2+]i) concentration, derived primarily from extracellular sources, whereas PGE1 was without effect on [Ca2+]i. These three PGs also had divergent effects on DNA synthesis, as measured by [3H]thymidine ([3H]TdR) incorporation. PGF2 alpha (0.001-5 microM) produced a dose-related increase in [3H]TdR incorporation, with a maximal 1.6-fold increase over baseline values at 5 microM and a slight decline to below maximal levels at 10 microM. PGE2 exhibited a contrasting inverse biphasic response, with an initial small suppressive effect that was maximal at 0.1 microM and a secondary stimulatory phase producing a small increase over control values at 5 microM. PGE1 had a uniformly suppressive effect, producing a 30% decrease at 10 microM. Despite the divergent effects of PGE1, PGE2, and PGE2 alpha on second messenger generation and DNA synthesis, all three PGs produced a dose-related stimulation of aggrecan synthesis. PGF2 alpha was the most potent, producing significant stimulation at 0.001 microM and a maximal 104% increase at 5 microM. PGE1 and PGE2 were approximately equipotent and approximately 60% as effective as PGF2 alpha in stimulating aggrecan synthesis. Northern analysis demonstrated that the effects of PG on aggrecan synthesis were not accompanied by changes in aggrecan core protein steady state messenger RNA levels. Thus, the effects of PG on aggrecan production in RCJ cells appear to be regulated at the posttranscriptional level. Forskolin and (Bu)2cAMP mimicked the suppressive effects of PGE1 on [3H]TdR incorporation, as well as the stimulatory effect of PGE1 on aggrecan synthesis. In addition, the phorbol ester 12-O-tetradecanoyl phorbol acetate mimicked PGF2 alpha stimulation of [3H]TdR incorporation and aggrecan synthesis, and the effects of PGE2 alpha on these processes were blocked by protein kinase C inhibitors. Therefore, it appears that in mammalian chondrocytes, PGE1 primarily activates the cAMP-protein kinase A second messenger system, PGE2 alpha affects primarily the Ca2(+)-protein kinase C system, and PGE2 activates both pathways. Moreover, PG posttranscriptional regulation of aggrecan synthesis in chondrocytes involves both the cAMP-protein kinase A and Ca2(+)-protein kinase C second messenger systems.

Differential effects of LH and PGE2 on progesterone secretion by small and large porcine luteal cells

This study examined the effects of LH and PGE2 on progesterone secretion by small and large porcine luteal cells with or without low-density lipoproteins. Corpora lutea were isolated from gilts 13-14 days after administering gonadotrophins; enzymatically dissociated and small and large cells were isolated by elutriation. Culture plates, 24-well, were then seeded with 150,000 small or 30,000 large luteal cells suspended in 1 ml M199 medium supplemented with 5 micrograms insulin ml-1, 40 ng hydrocortisone ml-1 and with or without low-density lipoproteins (50 micrograms cholesterol ml-1) or PGE2. Cells were cultured for up to 24 h in a humidified incubator at 37 degrees C under 5% CO2 in air. The low-density lipoproteins stimulated (P < 0.05) progesterone secretion by large, but not small, luteal cells. Prostaglandin E2 stimulated (P < 0.05) progesterone production by large luteal cells in a dose-dependent manner, and the stimulatory effects of PGE2 were greater (P < 0.05) in the presence than in the absence of low-density lipoproteins. Progesterone secretion by small luteal cells was not significantly affected by PGE2. Progesterone production by small luteal cells was enhanced (P < 0.05) by LH, and the stimulatory effects of LH were greater (P < 0.05) in the presence than in the absence of low-density lipoproteins. In the absence of these lipoproteins, LH had no effect on progesterone secretion by large luteal cells; however, in the presence of low-density lipoproteins, LH increased (P < 0.05) progesterone secretion by large cells, though to a lesser (P < 0.05) extent than the effect of LH on small cells. These data demonstrate that progesterone secretion by porcine luteal cells is stimulated differentially by LH and PGE2 and that small luteal cells are more responsive to LH and PGE2 acts primarily on large luteal cells.

In vitro effect of hormones and growth factors on the incorporation of [ 3H]leucine, [ 35S]sulfate and [ 3H]proline by chondrocytes of aging mice

The ability of parathyroid hormone (PTH 1–84), dexamethasone, prostaglandin E 1 (PGE 1), prostaglandin E 2 (PGE 2) and human transforming growth factor beta (hTGF-β) to stimulate the synthesis of matrical components in articular cartilage of aging mice, was studied in an organ culture system. A marked age-dependent decrease was observed in the synthesis of sulfated glycosaminoglycans (GAGs), protein, collagen disgestible protein (CDP) and non-collagen protein (NCP) between 1 and 18 months of age. The addition of hTGF-β (1 ng/ml) into the culture medium resulted in a significant ( P < 0.01) increase of both protein and sulfated GAGs in condylar cartilage from animals aged 1, 3, 6 and 12 months. PGE 2 (10 μg/ml) induced [ 3H]leucine and [ 35S]SO 4 incorporation into condylar cartilage from 1, 3 and 6 months old animals. A stimulatory effect of PGE 1 (10 μg/ml) on [ 3H]leucine incorporation was noted at 1 and 3 months of age. The effect of PTH appeared to be stimulatory only for protein synthesis in young (1 and 3 months old) animals, whereas it had no effect at 6, 12 and 18 months of age. In contrast, dexamethasone exerted a stimulatory effect on young adults (6 months old) and in matured (12 months old) animals, respectively and a slight inhibitory effect on young (1 and 3 months old) animals. [ 3H]Proline incorporation was enhanced by all the factors tested in 1-month-old animals. In cultures from 6- and 18-months old animals only PGE 1 and PGE 2 appeared to be stimulatory. It is concluded that synthesis of protein, sulfated GAGs and collagen by chondrocytes from maturing and osteoarthritic senescent animals can be stimulated by hormones and growth factors. The efficiency of this response, however, varied according to the animal's age and the factor studied.