Comparative study of the effects of polyunsaturated fatty acids and their metabolites on cell growth and tyrosine kinase activity in oesophageal carcinoma cells

The effects of exogenous gamma-linolenic acid (GLA), arachidonic acid (AA), prostaglandin E2 (PGE2) and prostaglandin A2 (PGA2) were evaluated on cell growth in two squamous oesophageal carcinoma cell lines, WHCO1 and WHCO3 and normal monkey kidney (NMK) cells. In both cancer cell lines all four compounds inhibited cell growth significantly. Indomethacin (I) alone, or in combination with either GLA or AA, caused marked inhibition of cell growth in WHCO3. Total tyrosine kinase (TK) activity was determined after exposure of all three cell types to the lipid compounds. Negligible differences were observed in TK activity between treated and untreated NMK cells. Small increases were noticed in WHCO1. Marked TK stimulation was observed in WHCO3. Addition of indomethacin to WHCO3 also increased TK activity above control value. Tyrosine phosphorylation status of exposed cells indicated that a band of approximately 55 kDa (approximately 55 kDa) was primarily influenced in both WHCO3 and WHCO1. PGA2 caused a decrease in tyrosine phosphorylation of the approximately 55 kDa protein in all three cell types. Negligible differences were observed in the tyrosine phosphorylation status of the approximately 55 kDa in NMK cells exposed to GLA, AA and PGE2 respectively. However, tyrosine phosphorylation of a number of other proteins (21.5-97.4 kDa) was observed in NMK cells. Flow cytometry studies showed an increase in S phase and decrease in G1 phase in WHCO3 exposed to PGE2 and PGA2. Indomethacin alone, or in combination with GLA and AA, respectively, lead to an increase in G1 and a decrease in S phase. Induction of p53 levels was observed in WHCO3 cells exposed to GLA, AA, PGA2, indomethacin and the combination of indomethacin and GLA or AA.     

Identification of a tyrosine kinase-phosphorylated protein in arachidonic acid- and prostaglandin A(2)-treated cells in vitro.

The effects of 20 microg/ml exogenous arachidonic acid (AA) and prostaglandin A(2) (PGA(2)) were evaluated on total tyrosine kinase (TK) activity and tyrosine phosphorylation status in HeLa and MCF-7 cells. AA and PGA(2) increased TK activity in both HeLa and MCF-7 cells. Western blotting employing an anti-phosphotyrosine antibody showed only one protein of approximately 55 kDa (approximately 55 kDa) to be phosphorylated in the MCF-7 cells, while a variety of proteins were phosphorylated in the HeLa cells, including the approximately 55 kDa protein. Amino acid analyses as well as Matrix Assisted Laser Desorption Ionization were conducted on this protein from different cell lines and it was shown to be similar. Comparison to p53 did not show similarities. The identity of this protein needs to be further characterized to help elucidate the signal transduction pathways of AA and PGA(2).     

Transduction of a signal for arachidonic acid metabolism by untriggered CSF-1 receptor induces an opposite effect to that induced by CSF-1 receptor and its ligand: separate regulation of phospholipase A2 and cyclooxygenase by CSF-1 receptor/CSF-1.

The mouse hematopoietic cell line, 32D, was transfected with c-fms, which encodes for the CSF-1 receptor, a tyrosine kinase (TK). In the absence of CSF-1, transfected cells show moderate levels of arachidonic acid (AA) release and produce a substantial amount of prostaglandin E2 (PGE2) in comparison with the original cell line. Exposure of transfected cells to CSF-1, while inducing a substantial increase in arachidonate release, nevertheless resulted in inhibition of PGE2 production. Addition of ST638, a tyrosine kinase inhibitor, to cells transfected with c-fms in the absence of CSF-1 inhibited PGE2 production within 10-60 min. Its addition to the same cells in the presence of CSF-1 induced an opposite effect, but required longer treatment (24 h). In either cell type, AA release was not affected by this agent. These data indicate that CSF-1 may regulate cyclooxygenase activity. The different effect of CSF-1 receptor on PGE2 production in the presence or absence of CSF-1 and the opposite effect of a tyrosine kinase inhibitor on PGE2 suggest that both the receptor alone or the receptor-ligand complex may transduce an active, but different, signal through tyrosine phosphorylation. CSF-1 receptor and CSF-1 may exert separate, but related, effects on phospholipase A2 and cyclooxygenase activity which, in concert, or along with other tyrosine kinases, regulate prostaglandin production.     

Wild-type TP53 inhibits G(2)-phase checkpoint abrogation and radiosensitization induced by PD0166285, a WEE1 kinase inhibitor

The WEE1 protein kinase carries out the inhibitory phosphorylation of CDC2 on tyrosine 15 (Tyr15), which is required for activation of the G(2)-phase checkpoint in response to DNA damage. PD0166285 is a newly identified WEE1 inhibitor and is a potential selective G(2)-phase checkpoint abrogator. To determine the role of TP53 in PD0166285-induced G(2)-phase checkpoint abrogation, human H1299 lung carcinoma cells expressing a temperature-sensitive TP53 were used. Upon exposure to gamma radiation, cells cultured under nonpermissive conditions (TP53 mutant conformation) underwent G(2)-phase arrest. However, under permissive conditions (TP53 wild-type conformation), PD0166285 greatly inhibited the accumulation of cells in G(2) phase. This abrogation was accompanied by a nearly complete blockage of Tyr15 phosphorylation of CDC2, an increased activity of CDC2 kinase, and an enhanced sensitivity to radiation. However, under permissive conditions (TP53 wild-type conformation), PD0166285 neither disrupted the G(2)-phase arrest nor increased cell death. The compound inhibited Tyr15 phosphorylation only partially and did not activate CDC2 kinase activity. To understand the potential mechanism(s) by which TP53 inhibits PD0166285-induced G(2)-phase checkpoint abrogation, two TP53 target proteins, 14-3-3rho and CDKN1A (also known as p21), that are known to be involved in G(2)-phase checkpoint control in other cell models were examined. It was found that 14-3-3rho was not expressed in H1299 cells, and that although CDKN1A did associate with CDC2 to form a complex, the level of CDKN1A associated with CDC2 was not increased in response to radiation or to PD0166285. The level of cyclin B1, required for CDC2 activity, was decreased in the presence of functional TP53. Thus inhibition of PD0166285-induced G(2)-phase checkpoint abrogation by TP53 was achieved at least in part through partial blockage of CDC2 dephosphorylation of Tyr15 and inhibition of cyclin B1 expression.     

Isoproterenol induces actin depolymerization in human airway smooth muscle cells via activation of an Src kinase and GS

In a previous study, we showed that isoproterenol induced actin depolymerization in human airway smooth muscle cells by both protein kinase A (PKA)-dependent and -independent signaling pathways. We now investigate the signaling pathway of PKA-independent actin depolymerization induced by isoproterenol in these cells. Cells were briefly exposed to isoproterenol or PGE(1) in the presence and absence of specific inhibitors of Src-family tyrosine kinases, phosphatidylinositol-3-kinase (PI3 kinase), or MAP kinase, and actin depolymerization was measured by concomitant staining of filamentous actin with FITC-phalloidin and globular actin with Texas red DNase I. Isoproterenol, cholera toxin, and PGE(1) induced actin depolymerization, indicated by a decrease in the intensity of filamentous/globular fluorescent staining. Pretreatment with the Src kinase inhibitors 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyriimidine (PP2) or geldanamycin or the PKA inhibitor Rp-cAMPS only partly inhibited isoproterenol- or PGE(1)-induced actin depolymerization. In contrast, PP2 and geldanamycin did not inhibit forskolin-induced actin depolymerization, and AG-213 (an EGF receptor tyrosine kinase inhibitor) did not inhibit isoproterenol- or PGE(1)-induced actin depolymerization. PI3 kinase or MAP kinase inhibition did not inhibit isoproterenol-induced actin depolymerization. Moreover, isoproterenol but not forskolin induced tyrosine phosphorylation of an Src family member at position 416. These results further confirm that both PKA-dependent and PKA-independent pathways mediate actin depolymerization in human airway smooth muscle cells and that the PKA-independent pathway by which isoproterenol induces actin depolymerization in human airway smooth muscle cells involves Src protein tyrosine kinases and the G(s) protein.     

Induction of cytosolic phospholipase A2 by lipopolysaccharide in canine tracheal smooth muscle cells: involvement of MAPKs and NF-kappaB pathways

Cytosolic phospholipase A2 (cPLA2) plays a pivotal role in mediating agonist-induced arachidonic acid (AA) release for prostaglandins (PG) synthesis induced by bacterial lipopolysaccharide (LPS) and cytokines. However, the intracellular signaling pathways mediating LPS-induced cPLA2 expression and PGE2 synthesis in canine tracheal smooth muscle cells (TSMCs) remains unknown. LPS-induced expression of cPLA2 and release of PGE2 was attenuated by inhibitors of tyrosine kinase (genistein), phosphatidylcholine-phospholipase C (D609), phosphatidylinositol-phospholipase C (U73122), PKC (GF109203X and staurosporine), removal of Ca2+ by BAPTA/AM plus EDTA, MEK1/2 (PD98059), p38 (SB202190), JNK (SP600125), and phosphatidylinositol 3-kinase (PI3-K; LY294002 and wortmannin). The involvement of MPAKs in LPS-induced responses was further confirmed by transfection of TSMCs with dominant negative mutants of ERK2 and p38. LPS-induced cPLA2 expression and PGE2 synthesis was inhibited by a selective NF-kappaB inhibitor (helenalin) and transfection with dominant negative mutants of NF-kappaB inducing kinase (NIK), IkappaB kinase (IKK)-alpha, and IKK-beta, consistent with that LPS-stimulated both IkappaB-alpha degradation and NF-kappaB translocation into nucleus in these cells. LPS-stimulated cPLA2 phosphorylation was inhibited by PD98059, GF109203X, and staurosporine, indicating the regulation by p42/p44 MAPK and PKC. Moreover, LPS-induced up-regulation of cPLA2 and COX-2 linked to PGE2 synthesis was inhibited by AACOCF3 (a selective cPLA2 inhibitor), implying the involvement of cPLA2 in these responses. These findings suggest that phosphorylation and expression of cPLA2 correlates with the release of PGE2 from LPS-challenged TSMCs, at least in part, mediated through MAPKs and NF-kappaB signaling pathways. LPS-mediated responses were modulated by PLC, Ca2+, PKC, tyrosine kinase, and PI3-K in TSMCs.     

Stimulation of CFU-f formation by prostaglandin E2 is mediated in part by its degradation product, prostaglandin A2

Prostaglandins (PG) of the E series are known to rapidly undergo non-enzymatic dehydration in culture medium containing serum albumin to produce the cyclopentenone PGs of the A series. We investigated the actions of PGA1 and A2 in the in vitro calcifying fibroblastic-colony forming unit assay which can partially mimic the in vivo anabolic effects of PGE2. It was found that PGA1 and A2 both stimulated colony formation in a dose-dependent manner with a maximum at 10(-6) M and to a similar degree to PGE2. In contrast to PGE2, PGA1 and PGA2 both caused an inhibition of cAMP accumulation. Furthermore, the addition of protein kinase A inhibitors, H8 and H89, had no significant effect on the stimulation of colony number by PGE2. These data suggest that (a) the bone anabolic effects of PGE1 and E2 are, in part at least, mediated by their dehydration products PGA1 and A2 and (b) that they are mediated via pathways not necessarily involving the cAMP/protein kinase A cascade.     

Metformin‐induced alterations in nucleotide metabolism cause 5‐fluorouracil resistance but gemcitabine susceptibility in oesophageal squamous cell carcinoma

5‐Fluorouracil (5‐FU) is a chemotherapeutic agent used to treat a variety of gastric cancers including oesophageal squamous cell carcinoma (OSCC), for which the 5‐year mortality rate exceeds 85%. Our study investigated the effects of metformin, an antidiabetic drug with established anti‐cancer activity, in combination with 5‐FU as a novel chemotherapy strategy, using the OSCC cell lines, WHCO1 and WHCO5. Our results indicate that metformin treatment induces significant resistance to 5‐FU in WHCO1 and WHCO5 cells, by more than five‐ and sixfolds, respectively, as assessed by MTT assay. We show that this is due to global alterations in nucleotide metabolism, including elevated expression of thymidylate synthase and thymidine kinase 1 (established 5‐FU resistance mechanisms), which likely result in an increase in intracellular dTTP pools and a “dilution” of 5‐FU anabolites. Metformin treatment also increases deoxycytidine kinase (dCK) expression and, as the chemotherapeutic agent gemcitabine relies on dCK for its efficient activity, we speculated that metformin would enhance the sensitivity of OSCC cells to gemcitabine. Indeed we show that metformin pre‐treatment greatly increases gemcitabine toxicity and DNA fragmentation in comparison to gemcitabine alone. Taken together, our findings show that metformin alters nucleotide metabolism in OSCC cells and while responsible for inducing resistance to 5‐FU, it conversely increases sensitivity to gemcitabine, thereby highlighting metformin and gemcitabine as a potentially novel combination therapy for OSCC.     

Stimulation of CFU-f formation by prostaglandin E(2) is mediated in part by its degradation product, prostaglandin A(2)

Prostaglandins (PG) of the E series are known to rapidly undergo non-enzymatic dehydration in culture medium containing serum albumin to produce the cyclopentenone PGs of the A series. We investigated the actions of PGA(1) and A(2) in the in vitro calcifying fibroblastic-colony forming unit assay which can partially mimic the in vivo anabolic effects of PGE(2). It was found that PGA(1) and A(2) both stimulated colony formation in a dose-dependent manner with a maximum at 10(-6) M and to a similar degree to PGE(2). In contrast to PGE(2), PGA(1) and PGA(2) both caused an inhibition of cAMP accumulation. Furthermore, the addition of protein kinase A inhibitors, H8 and H89, had no significant effect on the stimulation of colony number by PGE(2). These data suggest that (a) the bone anabolic effects of PGE(1) and E(2) are, in part at least, mediated by their dehydration products PGA(1) and A(2) and (b) that they are mediated via pathways not necessarily involving the cAMP/protein kinase A cascade.     

Insulin stimulates spreading of skeletal muscle cells involving the activation of focal adhesion kinase,phosphatidylinositol 3-kinase and extracellular signal regulated kinases

Insulin plays an important role in muscle cell survival and proliferation. However, there is no report showing the role of insulin in spreading of muscle cells. In the present report, we showed that insulin enhances muscle cell spreading concomitant with enhanced tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin. Moreover, insulin can stimulate the cell spreading even in presence of integrin alpha5 blockers although to a lesser extent as compared to control. Cell adhesion was not dependent on insulin and serum, and decreased in presence of integrin blockers. We found direct association of FAK with affinity purified insulin receptors using in vitro kinase assay. The increase in FAK tyrosine phosphorylation was associated with increase in its kinase activity and further supported by increased phosphotyrosine accumulation on focal adhesions and increased membrane localization of FAK after stimulation by insulin. Moreover, insulin-mediated muscle cell spreading was dependent upon phosphatidylinositol 3-kinase (PI 3-kinase) activity. PI 3-kinase activity was found to be associated with FAK and the FAK associated PI 3-kinase activity enhanced when cells were plated in presence of insulin. We also observed activation of MAP kinases, i.e., ERK-1/-2 during insulin mediated muscle cell spreading. In conclusion, FAK, PI 3-kinase, and MAP kinase are important components of pathway(s) that regulate insulin stimulated muscle cell spreading.     

Inhibition of G1 cyclin-dependent kinase activity during growth arrest of human breast carcinoma cells by prostaglandin A2.

Prostaglandin A2 (PGA2) potently inhibits cell proliferation and suppresses tumor growth in vivo, but little is known regarding the molecular mechanisms mediating these effects. Here we demonstrate that treatment of breast carcinoma MCF-7 cells with PGA2 leads to G1 arrest associated with a dramatic decrease in the levels of cyclin D1 and cyclin-dependent kinase 4 (cdk4) and accompanied by an increase in the expression of p21. We further show that these effects occur independent of cellular p53 status. The decline in cyclin D and cdk4 protein levels is correlated with loss in cdk4 kinase activity, cdk2 activity is also significantly inhibited in PGA2-treated cells, an effect closely associated with the upregulation of p21. Immunoprecipitation experiments verified that p21 was indeed complexed with cdk2 in PGA2-treated cells. Additional experiments with synchronized MCF-7 cultures stimulated with serum revealed that treatment with PGA2 prevents the progression of cells from G1 to S. Accordingly, the kinase activity associated with cdk4, cyclin E, and cdk2 immunocomplexes, which normally increases following serum addition, was unchanged in PGA2-treated cells. Furthermore, the retinoblastoma protein (Rb), a substrate of cdk4 and cdk2 whose phosphorylation is necessary for cell cycle progression, remains underphosphorylated in PGA2-treated serum-stimulated cells. These findings indicate that PGA2 exerts its growth-inhibitory effects through modulation of the expression and/or activity of several key G1 regulatory proteins. Our results highlight the chemotherapeutic potential of PGA2, particularly for suppressing growth of tumors lacking p53 function.     

Involvement of the Src kinase Lyn in phospholipase C-gamma 2 phosphorylation and phosphatidylinositol 3-kinase activation in Epo signalling

We examined the role of the Src kinase Lyn in phospholipase C-gamma 2 (PLC-gamma 2) and phosphatidylinositol (PI) 3-kinase activation in erythropoietin (Epo)-stimulated FDC-P1 cells transfected with a wild type (WT) Epo-receptor (Epo-R). We showed that two inhibitors of Src kinases, PP1 and PP2, abolish both PLC-gamma 2 tyrosine phosphorylation and PI 3-kinase activity in WT Epo-R FDC-P1 cells. We also demonstrated that Epo-phosphorylated Lyn is associated with tyrosine phosphorylated PLC-gamma 2 and PI 3-kinase in WT Epo-R FDC-P1-stimulated cells. Moreover Epo-activated Lyn phosphorylates in vitro PLC-gamma 2 immunoprecipitated from unstimulated cells. Our results suggest that the Src kinase Lyn is involved in PLC-gamma 2 phosphorylation and PI 3-kinase activation induced by Epo.     

Interleukin-1beta-induced cyclooxygenase-2 expression is mediated through activation of p42/44 and p38 MAPKS,and NF-kappaB pathways in canine tracheal smooth muscle cells

Interleukin-beta (IL-1beta) was found to induce inflammatory responses in the airways, which exerted a potent stimulus for PG synthesis. This study was to determine the mechanisms of IL-1beta-enhanced cyclooxygenase (COX)-2 expression associated with PGE(2) synthesis in tracheal smooth muscle cells (TSMCs). IL-1beta markedly increased COX-2 expression and PGE(2) formation in a time- and concentration-dependent manner in TSMCs. Both COX-2 expression and PGE(2) formation in response to IL-1beta were attenuated by a tyrosine kinase inhibitor, genistein, a phosphatidylcholine-phospholipase C inhibitor, D609, a phosphatidylinositol-phospholipase C inhibitor, U73122, protein kinase C inhibitors, GF109203X and staurosporine, removal of Ca(2+) by addition of BAPTA/AM plus EGTA, and phosphatidylinositol 3-kinase (PI3-K) inhibitors, LY294002 and wortmannin. IL-1beta-induced activation of NF-kappaB correlated with the degradation of IkappaB-alpha in TSMCs. IL-1beta-induced NF-kappaB activation, COX-2 expression, and PGE(2) synthesis were inhibited by the dominant negative mutants of NIK and IKK-alpha, but not by IKK-beta. IL-1beta-induced COX-2 expression and PGE(2) synthesis were completely inhibited by PD98059 (an inhibitor of MEK1/2) and SB203580 (an inhibitor of p38 inhibitor), but these two inhibitors had no effect on IL-1beta-induced NF-kappaB activation, indicating that activation of p42/44 and p38 MAPK and NF-kappaB signalling pathways were independently required for these responses. These findings suggest that the increased expression of COX-2 correlates with the release of PGE(2) from IL-1beta-challenged TSMCs, at least in part, independently mediated through MAPKs and NF-kappaB signalling pathways in canine TSMCs. IL-1beta-mediated responses were modulated by PLC, Ca(2+), PKC, tyrosine kinase, and PI3-K in these cells.     

Effects of TGF-beta, TNF-alpha, IL-beta and IL-6 alone or in combination, and tyrosine kinase inhibitor on cyclooxygenase expression, prostaglandin E2 production and bone resorption in mouse calvarial bone cells

Cyclooxygenase-2 (COX-2) and tyrosine kinase, which are involved in the biosynthesis of prostaglandin E(2) (PGE(2)) in mouse calvarial osteoblasts, are stimulated by cytokine interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha) and/or interleukin-6 (IL-6). IL-1beta and IL-6 and, to a lesser extent, TNF-alpha, enhances COX-2 mRNA levels in calvarial osteoblasts. Simultaneous treatment with IL-6 and IL-1beta and TNF-alpha resulted in enhanced COX-2 mRNA levels accompanied by the cooperative stimulation of PGE(2) biosynthesis compared to cells treated with IL-1beta or TNF-alpha or IL-6 alone. In contrast, the presence of TGF-beta reduced COX-2 mRNA level, PGE(2) biosynthesis and bone resorption induced by IL-1beta, TNF-alpha, IL-6 or a combination thereof. However, neither IL-1beta, TNF-alpha, IL-6 nor a combination of IL-1beta, TNF-alpha, IL-6 enhanced COX-1 mRNA levels in calvarial osteoblasts. A novel Src tyrosine kinase inhibitor, Herbimycin A (HERB), reduced COX-2 mRNA levels as well as PGE(2) production induced by IL-1beta, TNF-alpha and IL-6 or a combination of IL-1beta, TNF-alpha, IL-6, whereas COX-1 mRNA levels remained unaffected. Finally, HERB was found to inhibit in vitro bone resorption. These results indicate that the cooperative effects of IL-beta, TNF-alpha, IL-6 on PGE(2) production are due to the enhanced expression of the COX-2 gene and that tyrosine kinase(s) are involved in COX-2 signal transduction in mouse calvarial osteoblasts. Thus, the Src family of kinase inhibitors may be useful in treating diseases associated with elevated bone loss.     

Cyclooxygenases-1 and 2 couple to cytosolic but not group IIA phospholipase A2 in COS-1 cells

Phospholipases A2 (PLA2) and cyclooxygenases (COX) are important enzymes responsible for production of potent lipid mediators, including prostaglandins (PG) and thromboxane A2. We investigated coupling between PLA2 and COX isoforms by using transient transfection in COS-1 cells. Untransfected cells, incubated with or without phorbol ester + the Ca2+ ionophore ionomycin, generated trivial amounts of PGE2. In cells co-transfected with cytosolic PLA2 (cPLA2) and COX-1 or COX-2, phorbol ester + ionomycin markedly stimulated PGE2 production. There was no preferential coupling of cPLA2 to either of the COX isoforms. In contrast, group IIA secretory PLA2 (sPLA2) co-transfected with COX-1 or COX-2 did not lead to an increase in PGE2 production, despite high levels of sPLA2 enzymatic activity. Transfection of cPLA2 did not affect basal free arachidonic acid (AA) levels. Phorbol ester + ionomycin stimulated release of AA in cPLA2-transfected COS-1 cells, but not in untransfected cells, whereas sPLA2 transfection (without stimulation) led to high basal free AA. Thus, AA released by cPLA2 is accessible to both COX isoforms for metabolism to PG, whereas AA released by sPLA2 is not metabolized by COX.     

IL-2 binding activates a tyrosine-phosphorylated phosphatidylinositol-3-kinase.

IL-2 was shown to induce phosphatidylinositol-3-kinase activity in the CTLL-2 murine lymphocyte line. Tyrosine phosphorylation of phosphatidylinositol-3-kinase was demonstrated by immunoabsorption with antiphosphotyrosine antibody and was coincident with activation of an IL-2R-associated tyrosine kinase. Half-maximal activation of this enzyme, and of cell proliferation, occurred at 30 pM IL-2. Incubation of cells with genistein, a selective tyrosine kinase inhibitor, blocked IL-2-dependent activation of receptor-associated tyrosine kinase and phosphatidylinositol-3-kinase activities, suggesting that tyrosine phosphorylation-dependent activation of phosphatidylinositol-3-kinase is a component of the IL-2R signal transduction process.     

Prostaglandin E2 upregulates EGF-stimulated signaling in mitogenic pathways involving Akt and ERK in hepatocytes

Prostaglandins (PGs) such as PGE2 enhance proliferation in many cells, apparently through several distinct mechanisms, including transactivation of the epidermal growth factor (EGF) receptor (EGFR) as well as EGFR-independent pathways. In this study we found that in primary cultures of rat hepatocytes PGE2 did not induce phosphorylation of the EGFR, and the EGFR tyrosine kinase blockers gefitinib and AG1478 did not affect PGE2-stimulated phosphorylation of ERK1/2. In contrast, PGE2 elicited EGFR phosphorylation and EGFR tyrosine kinase inhibitor-sensitive ERK phosphorylation in MH1C1 hepatoma cells. These findings suggest that PGE2 elicits EGFR transactivation in MH1C1 cells but not in hepatocytes. Treatment of the hepatocytes with PGE2 at 3 h after plating amplified the stimulatory effect on DNA synthesis of EGF administered at 24 h and advanced and augmented the cyclin D1 expression in response to EGF in hepatocytes. The pretreatment of the hepatocytes with PGE2 resulted in an increase in the magnitude of EGF-stimulated Akt phosphorylation and ERK1/2 phosphorylation and kinase activity, including an extended duration of the responses, particularly of ERK, to EGF in PGE2-treated cells. Pertussis toxin abolished the ability of PGE2 to enhance the Akt and ERK responses to EGF. The results suggest that in hepatocytes, unlike MH1C1 hepatoma cells, PGE2 does not transactivate the EGFR, but instead acts in synergism with EGF by modulating mitogenic mechanisms downstream of the EGFR. These effects seem to be at least in part G(i) protein-mediated and include upregulation of signaling in the PI3K/Akt and the Ras/ERK pathways.     

Inhibition of phospholipase A2-mediated arachidonic acid release by cyclic AMP defines a negative feedback loop for P2Y receptor activation in Madin-Darby canine kidney D1 cells

In Madin-Darby canine kidney D1 cells extracellular nucleotides activate P2Y receptors that couple to several signal transduction pathways, including stimulation of multiple phospholipases and adenylyl cyclase. For one class of P2Y receptors, P2Y2 receptors, this stimulation of adenylyl cyclase and increase in cAMP occurs via the conversion of phospholipase A2 (PLA2)-generated arachidonic acid (AA) to prostaglandins (e.g. PGE2). These prostaglandins then stimulate adenylyl cyclase activity, presumably via activation of prostanoid receptors. In the current study we show that agents that increase cellular cAMP levels (including PGE2, forskolin, and the beta-adrenergic agonist isoproterenol) can inhibit P2Y receptor-promoted AA release. The protein kinase A (PKA) inhibitor H89 blocks this effect, suggesting that this feedback inhibition occurs via activation of PKA. Studies with PGE2 indicate that inhibition of AA release is attributable to inhibition of mitogen-activated protein kinase activity and in turn of P2Y receptor stimulated PLA2 activity. Although cAMP/PKA-mediated inhibition occurs for P2Y receptor-promoted AA release, we did not find such inhibition for epinephrine (alpha1-adrenergic) or bradykinin-mediated AA release. Taken together, these results indicate that negative feedback regulation via cAMP/PKA-mediated inhibition of mitogen-activated protein kinase occurs for some, but not all, classes of receptors that promote PLA2 activation and AA release. We speculate that receptor-selective feedback inhibition occurs because PLA2 activation by different receptors in Madin-Darby canine kidney D1 cells involves the utilization of different signaling components that are differentially sensitive to increases in cAMP or, alternatively, because of compartmentation of signaling components.     

TNF-alpha-induced cyclooxygenase-2 expression in human lung epithelial cells: involvement of the phospholipase C-gamma 2, protein kinase C-alpha, tyrosine kinase, NF-kappa B-inducing kinase, and I-kappa B kinase 1/2 pathway

TNF-alpha induced a dose- and time-dependent increase in cyclooxygenase-2 (COX-2) expression and PGE2 formation in human NCI-H292 epithelial cells. Immunofluorescence staining demonstrated that COX-2 was expressed in cytosol and nuclear envelope. Tyrosine kinase inhibitors (genistein or herbimycin) or phosphoinositide-specific phospholipase C inhibitor (U73122) blocked TNF-alpha-induced COX-2 expression. TNF-alpha also stimulated phosphatidylinositol hydrolysis and protein kinase C (PKC) activity, and both were abolished by genistein or U73122. The PKC inhibitor, staurosporine, also inhibited TNF-alpha-induced response. The 12-O-tetradecanoylphorbol 13-acetate (TPA), a PKC activator, also stimulated COX-2 expression, this effect being inhibited by genistein or herbimycin. NF-kappaB DNA-protein binding and COX-2 promoter activity were enhanced by TNF-alpha, and these effects were inhibited by genistein, U73122, staurosporine, or pyrolidine dithiocarbamate. TPA stimulated both NF-kappaB DNA-protein binding and COX-2 promoter activity, these effects being inhibited by genistein, herbimycin, or pyrolidine dithiocarbamate. The TNF-alpha-induced, but not the TPA-induced, COX-2 promoter activity was inhibited by phospholipase C-gamma2 mutants, and the COX-2 promoter activity induced by either agent was attenuated by dominant-negative mutants of PKC-alpha, NF-kappaB-inducing kinase, or I-kappaB (inhibitory protein that dissociates from NF-kappaB) kinase (IKK)1 or 2. IKK activity was stimulated by both TNF-alpha and TPA, and these effects were inhibited by staurosporine or herbimycin. These results suggest that, in NCI-H292 epithelial cells, TNF-alpha might activate phospholipase C-gamma2 via an upstream tyrosine kinase to induce activation of PKC-alpha and protein tyrosine kinase, resulting in the activation of NF-kappaB-inducing kinase and IKK1/2, and NF-kappaB in the COX-2 promoter, then initiation of COX-2 expression and PGE2 release.