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Inhibition by Platelet-activating Factor of Src- and Hepatocyte Growth Factor-dependent Invasiveness of Intestinal and Kidney Epithelial Cells

PHOSPHATIDYLINOSITOL 3′-KINASE IS A CRITICAL MEDIATOR OF TUMOR INVASION*
Open AccessPublished:June 05, 1998DOI:https://doi.org/10.1074/jbc.273.23.14138
      This study was designed to characterize platelet-activating factor receptor (PAF-R) expression and function in normal and cancerous human colonic epithelial cells. PAF-R gene transcripts were analyzed by reverse transcription-polymerase chain reaction and Southern blot, using three sets of primers corresponding either to the coding region of the human PAF-R sequence (polymerase chain reaction product: 682 base pairs (bp)) or to the leukocyte- and tissue-type transcripts of 166 and 252 bp, respectively. An elongated splice variant was identified in the 5′-untranslated region of the tissue-type PAF-R transcript (334 bp) in colonic epithelial crypts and tumors. In human colonic PCmsrc cells transformed by c-src oncogene, the hepatocyte growth factor (HGF)-dependent invasiveness of collagen gels was abolished by 0.1 μm PAF and restored by the PAF-R antagonists WEB2086 and SR27417. PAF blocked HGF-induced tyrosine phosphorylation of p125 focal adhesion kinase. The phosphatidylinositol 3′-kinase (PI3′-K) inhibitors wortmannin and LY294002 totally blocked the HGF-induced invasion. Similar effects were observed in ts-srcMDCK kidney epithelial cells transformed by a v-Src temperature-sensitive mutant: (i) PAF and wortmannin exerted additive inhibitory effects on Src-induced invasion and (ii) activated and dominant negative forms of p110α PI3′-K, respectively, amplified and abrogated the Src- and HGF-dependent invasiveness of parental and ts-srcMDCK cells. We also provided the first evidence for the contribution of rapamycin-insensitive, pertussis toxin-dependent G-protein pathways to the integration of the signals emerging from activated Met and PAF receptors. These results indicate that PI3′-K is a critical transducer of invasiveness and strongly suggest that PAF exerts a negative control on invasion by inhibiting this signaling pathway. A possible beneficial role of PAF analogs on tumor invasion is therefore proposed.
      Platelet-activating factor (PAF),
      The abbreviations used are: PAF, platelet-activating factor; PAF-R, platelet-activating factor receptor; HGF, hepatocyte growth factor; PI3′-K, phosphatidylinositol 3′-kinase; FAK, focal adhesion kinase; PTx, pertussis toxin; RT, reverse transcription; PCR, polymerase chain reaction; bp, base pair(s); TBS, Tris-buffered saline; PBS, phosphate-buffered saline; UTR, untranslated region; mAb, monoclonal antibody; MDCK, Madin-Darby canine kidney; PMSF, phenylmethylsulfonyl fluoride.
      1The abbreviations used are: PAF, platelet-activating factor; PAF-R, platelet-activating factor receptor; HGF, hepatocyte growth factor; PI3′-K, phosphatidylinositol 3′-kinase; FAK, focal adhesion kinase; PTx, pertussis toxin; RT, reverse transcription; PCR, polymerase chain reaction; bp, base pair(s); TBS, Tris-buffered saline; PBS, phosphate-buffered saline; UTR, untranslated region; mAb, monoclonal antibody; MDCK, Madin-Darby canine kidney; PMSF, phenylmethylsulfonyl fluoride.
      1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, is implicated in human inflammatory processes and gastric ulceration disorders (
      • Rosam A.
      • Wallace J.
      • Whittle B.
      ). In the intestine, PAF contributes to diarrhea in ulcerative colitis (
      • Wardle T.D.
      • Hall L.
      • Turnberg L.A.
      ). It is a potent lipid mediator produced by neutrophils, eosinophils, monocytes, macrophages, platelets, and endothelial cells via two main routes of biosynthesis: the remodeling pathway and a second pathway termed de novo. Products of phospholipid catabolism (PAF and eicosanoids) are detected in the gastrointestinal mucosa and epithelial cells during the inflammatory process and neoplastic progression (
      • Langholz E.
      • Munkholm P.
      • Davidsen M.
      • Binder V.
      ,
      • Sobhani I.
      • Denizot Y.
      • Moizo L.
      • Laigneau J.P.
      • Bado A.
      • Laboisse C.
      • Benveniste J.
      • Lewin M.
      ). Colonic epithelium has been reported to synthesize and secrete PAF acetylhydrolases, which constitute a major pathway for PAF degradation (
      • Riehl T.E.
      • Stenson W.F.
      ). Increased levels of PAF in intestinal mucosa have been reported in experimental models of colitis, Crohn's disease, and ulcerative colitis (
      • Wardle T.D.
      • Hall L.
      • Turnberg L.A.
      ,
      • Sobhani I.
      • Hochlaf S.
      • Denizot Y.
      • Vissuzaine C.
      • Rene E.
      • Benveniste J.
      • Lewin M.M.J.
      • Mignon M.
      ,
      • Ferraris L.
      • Karmeli F.
      • Eliakim R.
      • Klein J.
      • Fiocchi C.
      • Rachmilewitz D.
      ). Numerous clinical and experimental observations reveal that colonic cancer is increasingly frequent after inflammatory bowel disease, ulcerative colitis, primary sclerosing cholangitis, and Crohn's disease (
      • Langholz E.
      • Munkholm P.
      • Davidsen M.
      • Binder V.
      ,
      • Choi P.
      • Zelig M.
      ,
      • Brentnall T.
      • Haggitt R.
      • Rabinovitch P.
      • Kimmey M.
      • Bronner M.
      • Levine D.
      • Kowdley K.
      • Stevens A.
      • Crispin D.
      • Emond M.
      • Rubin C.
      ), suggesting that PAF and its receptors initiate the cancerous progression through inflammatory disorders in the gastrointestinal mucosa. On the other hand, anti-inflammatory drugs are associated with a lower risk of colon cancer and reduced mortality due to this disease (
      • Giovannucci E.
      • Egan K.
      • Hunter D.
      • Stamper M.
      • Colditz G.
      • Willett W.
      • Speizer F.
      ).
      In view of the information above, we are now asking whether functional PAF receptors (PAF-R) are present in both normal and transformed human colonic epithelial cells. As a first step toward answering this question, we investigated 1) the expression of PAF-R transcripts by reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot in normal and transformed human colonic epithelial cells, 2) the effects of PAF on invasion by tumor cells, using two models of PCmsrc human colonic epithelial cells and ts-srcMDCK canine kidney epithelial cells transformed by the src oncogene (
      • Empereur S.
      • Djelloul S.
      • Di Gioia Y.
      • Bruyneel E.
      • Mareel M.
      • Van Hengel J.
      • Van Roy F.
      • Comoglio P.
      • Courtneidge S.
      • Paraskeva C.
      • Chastre E.
      • Gespach C.
      ,
      • Behrens J.
      • Vakaet L
      • Friis R.
      • Winterhager E.
      • Van Roy F.
      • Mareel M.
      • Birchmeier W.
      ), and 3) the functional relationships between PAF receptors and the signaling pathways involved in cell adhesion and invasion, namely p125FAK focal adhesion kinase, phosphatidylinositol 3′-kinase (PI3′-K), and the E-cadherin/catenin invasion suppressor system connected to the actin cytoskeleton via α-catenin (
      • Mareel M.
      • Noë V.
      • Bracke M.
      ).
      In the present investigation, we showed the following. (i) Specific tissue-type PAF-R transcripts are clearly identifiable in human intestinal epithelial cells at various stages of cancerous progression and cell differentiation; (ii) upon PAF addition, there was complete reversion of the Src- and HGF-dependent invasiveness of intestinal and kidney epithelial cells; (iii) the PI3′-K inhibitors wortmannin and LY294002 mimic and cooperate with PAF against invasion; (iv) there was phosphorylation of p125FAK by HGF that was completely blocked by PAF; (v) constitutively activated PI3′-K and dominant negative forms of the PI3′-K p110α catalytic subunit respectively induced and abrogated the invasiveness of parental and Src-transformed MDCK cells; and (vi) the HGF- and PAF-dependent invasion pathways were sensitive to pertussis toxin (PTx), suggesting that heterotrimeric G protein components (Gαi-β/γ dimers) are involved in the signaling cascades that affect the invasiveness of Src- and Met-transformed epithelial cells.
      A preliminary report of the results presented here has been published in abstract form (
      • Noë V.
      • Bruyneel E.
      • Kotelevets L.
      • Myssiakine E.
      • Chastre E.
      • Mareel M.
      • Gespach C.
      ).

      DISCUSSION

      The studies described here demonstrate that normal human colonic epithelial crypts, colonic adenoma, and adenocarcinoma cell lines express functional and specific PAF receptors. A noteworthy finding in this work is the identification of a new alternative splice variant in the 5′-UTR of the PAF-R mRNA, in the form of an elongated version of the tissue-type transcript. This variant includes an additional sequence of 82 nucleotides from the 3′ end of the leukocyte-type exon 1 transcript (
      • Izumi T.
      • Kishimoto S.
      • Takano T.
      • Nakamura M.
      • Miyabe Y.
      • Nakata M.
      • Sakanaka C.
      • Shimizu T.
      ). It has been postulated that long 5′-UTR transcripts are involved in the formation of secondary structures that must be eliminated for the efficient translation initiation (
      ). Elongated forms of 5′-UTR have a potential role in the extensive interactions with multiple factors that control the rates of initiation and translation of human PAF-R mRNA in human colonic epithelial cells. Similarly, the tissue-type transcript and this new variant harbor a second ATG in exon 2. This second open reading frame, comprising 44 codons, is unusually long compared with other G-protein coupled transmembrane receptors (
      • Dixon R.
      • Kobilka B.
      • Strader D.
      • Benovic J.
      • Dohlman H.
      • Frielle T.
      • Bolanowski M.
      • Bennett C.
      • Rands E.
      • Diehl R.
      • Mumford R.
      • Slater E.
      • Sigal I.
      • Caron M.
      • Lefkowitz R.
      • Strader C.
      ), as well as to the leukocyte-type PAF-R transcript (
      • Ye R.
      • Prossnitz E.
      • Zou A.
      • Cochrane C.
      ). The human β2 adrenergic receptor has been shown to have a second upstream ATG and an elongated 19- codon open reading frame in the 5′-UTR. When this upstream open reading frame was deleted from the corresponding cRNA, production of the receptor protein rose about 10-fold, when translated in the reticulocyte system (
      • Kobilka B.
      • McGregor C.
      • Daniel K.
      • Kobilka T.
      • Caron M.
      • Lefkowitz R.
      ), suggesting competitive interactions between the ATG signals. A very similar feature characterizes both the cholinergic and estrogen receptors. Precise function of the elongated form of the 5′-UTR in intestinal PAF-R mRNA remains to be elucidated. Because of the large intron separating exons 1 and 2, previous studies failed to establish the organization of the PAF-R gene (
      • Mutoh H.
      • Bito H.
      • Minami M.
      • Nakamura M.
      • Honda Z.
      • Izumi T.
      • Nakata R.
      • Kurachi Y.
      • Terano A.
      • Shimizu T.
      ). The identification of a new tissue-type splice variant in intestinal cells enabled us to establish the physical map of the human PAF-R gene, as exon 2 was upstream of exon 1 (Fig. 1). Our data suggest that PAF can directly amplify the harmful action of inflammatory agents in human colonic epithelial cells, because this potent lipid mediator, which is generated during cell injury, triggered the expression of the inducible prostaglandin synthase gene that leads to the synthesis of inflammatory agents prostaglandins, prostacyclins, and thromboxanes. Another active mediator of the inflammatory response, such as bacterial lipopolysaccharide, induces PAF release, PAF-R expression, and tumor necrosis factor-α (
      • Ishii S.
      • Matsuda Y.
      • Nakamura M.
      • Waga I.
      • Kume K.
      • Izumi T.
      • Shimizu T.
      ,
      • Han J.
      • Lee J.D.
      • Bibbs R.
      • Ulevitch R.
      ,
      • Jiang Y.
      • Chen C.
      • Li Z.
      • Guo W.
      • Gegner J.
      • Lin S.
      • Han J.
      ,
      • Chastre E.
      • Empereur S.
      • Di Gioia Y.
      • El Mahdani N.
      • Mareel M.
      • Vleminckx K.
      • Van Roy F.
      • Bex V.
      • Emami S.
      • Spandidos D.
      • Gespach C.
      ,
      • Chastre E.
      • Empereur S.
      • Di Gioia Y.
      • El Mahdani N.
      • Mareel M.
      • Vleminckx K.
      • Van Roy F.
      • Bex V.
      • Emami S.
      • Spandidos D.
      • Gespach C.
      ,
      • Baron-Delage S.
      • Capeau J.
      • Barbu V.
      • Chastre E.
      • Levy P.
      • Gespach C.
      • Cherqui G.
      ,
      • Schulam P.
      • Kuruvilla A.
      • Putcha G.
      • Mangus L.
      • Franklin-Johnson J.
      • Shearer W.
      ), suggesting that multiple paracrine and autocrine pathways are involved in cell injury in the intestinal mucosa.
      PAF-R activation is connected with changes in cell shape, cytoskeletal reorganization, and expression of urokinase-type plasminogen activator (
      • Irigoyen J.
      • Besser D.
      • Nagamine Y.
      ). This extracellular serine protease is strongly expressed in transformed cell lines and is directly or indirectly involved in degrading matrix components, including fibrin, fibronectin, laminin, or basement membrane proteins like type IV collagen. Recent studies indicate that the complexes formed by FAK with other cellular proteins are important in the determination of its kinase activity and signaling functions in focal contacts. In immediate signal-dependent changes, FAK is directly connected to extracellular and intracellular signals activated by growth factors, integrin receptors, matrix components, and oncogenic pathways, thus providing docking sites for a complex cluster of SH2-containing proteins and promoting functional links between the cytoskeletal protein network, cell adhesion, and migration (
      • Polte T.
      • Hanks S.
      ). Because both PI3′-K and FAK are downstream components of activated Src and HGF/Met, these functional links led us to investigate the role of PAF and its receptors on these two signaling pathways. We found that PAF impaired the HGF-stimulated tyrosine phosphorylation of FAK that was shown to correlate with its increased kinase activity. Interestingly, we also found that the invasiveness of transformed human colonic and canine kidney epithelial cells is inhibited in a cooperative fashion by PAF-R and PI3′-K inhibitors, and is mimicked by a dominant-negative mutant of PI3′-K p110α, thus providing evidence that activation of PI3′-K is an absolute requirement for Src- and HGF- dependent invasion of tumor cells. In agreement, PAF completely abrogated the invasiveness induced by constitutive activation of the PI3′-K p110α catalytic subunit and inhibited the HGF-induced increase in PI3′-K activity in ts-srcMDCK cells. It was therefore evident that PAF exerts a negative regulation on this cellular activity and signaling pathway, including the downstream mediators of PI3′-K, such as the pleckstrin homology domain-dependent effectors and Rho-like small G-proteins. Alternatively, inhibition of invasion by activated PAF-R may occur through the association of PI3′-K with the p85-kDa regulatory subunit(s) or other non-tyrosine-phosphorylated proteins that influence the stability and activity of the lipid kinase at the plasma membrane. Taken together, our data indicate that the inhibitory action of PAF on invasiveness is coordinated with changes in the activities of both FAK and PI3′-K in Src-transformed epithelial cells.
      Because both wortmannin and LY294002 inhibit the PI3′-K/Akt serine/threonine kinase cascade that activates the p70 ribosomal protein S6-kinase (p70s6k), we next examined the effect of rapamycin on our system. This immunosuppressant indirectly blocks the phosphorylation/activation of p70s6k, forming a complex with FK-506-binding protein and TOR, a lipid kinase upstream of p70s6k. This pathway controls the translation of the proteins required for growth factor signaling, progression through the cell cycle, and delivers signals for cell survival and the polarized distribution of the actin cytoskeleton (
      • Schmidt A.
      • Kunz J.
      • Hall M.
      ). We found that rapamycin had no effect on Src- or HGF/PAF-dependent invasiveness in PCmsrc or ts-srcMDCK cells, suggesting that the signaling elements downstream of TOR-p70s6k are not involved in this invasiveness. Moreover, our present observation that HGF-induced tyrosine phosphorylation of β-catenin was not inhibited by PAF in ts-srcMDCK cells rules out the possibility that the E-cadherin/catenin complex has a functional role in PAF action. In agreement, we observed that invasiveness of ts-srcMDCK cells induced by the monoclonal antibody Degma-1 directed against murine E-cadherin is resistant to PAF (data not shown). We therefore conclude that the PAF-dependent invasiveness of transformed epithelial cells involved E-cadherin- and p70s6k-independent pathways.
      We presented evidence that PAF inhibits invasion via PTx-sensitive and -resistant G-proteins. Although the identity of the individual G-protein isotypes activated by human PAF-R is still a matter of conjecture, the PTx-insensitive isoforms Gs/Gα11/Gαq and the PTx-sensitive isoforms Gαo/Gαi1/Gαi2/Gαi3are probably involved (
      • Moscatello D.
      • Holgado-Madruga M.
      • Emlet D.
      • Montgomery B.
      • Wong A.
      ). PTx-insensitive heterotrimeric GTP-binding proteins include (i) the ubiquitously expressed Gαq, Gα11, Gα12, and Gα13isotypes, which activate specific phospholipase C isoforms and cell growth signaling, and (ii) the Gαz isotype involved in adenylate cyclase inhibition. Consistent with our finding, it has been proposed that PAF receptors trigger either PTx-resistant or -sensitive activation of signaling pathways, depending on the G-protein isotype and the effector system concerned (
      • Ferby I.
      • Waga I.
      • Sakanaka C.
      • Kume K.
      • Shimizu T.
      ,
      • Honda Z.
      • Takano T.
      • Gotoh Y.
      • Nishida E.
      • Ito K.
      • Shimizu T.
      ,
      • Kravchenko V.
      • Pan Z.
      • Han J.
      • Herbert J.
      • Ulevitch R.
      • Ye R.
      ,
      • Neurath M.
      • Pettersson S.
      • Meyer zum Büschenfeld K.H.
      • Strober W.
      ).
      Another interesting finding derived from the experiments described herein is that the HGF-dependent invasiveness of PCmsrc and ts-srcMDCK cells is negatively regulated by PTx. Therefore, PTx exerts opposite effects on the PAF- and HGF-dependent invasion of Src-transformed epithelial cells. This is the first evidence that Met receptors activate (or synergize with) a latent signaling pathway that utilizes a member of the G-family of heterotrimeric proteins. The association of single transmembrane protein-tyrosine kinase receptors with G-proteins was recently documented in the case of the direct interaction between the epidermal growth factor receptor juxtamembrane region and the α-subunit of Gs (
      • Sun H.
      • Chen Z.
      • Poppleton H.
      • Scholich K.
      • Mullenix J.
      • Weipz G.
      • Fulgham D.
      • Bertics P.
      • Patel T.
      ). Our future investigations will be designed to identify the PTx-sensitive G-proteins involved in the molecular and functional integration of the positive and negative signals participating in the cross-talk between PAF-R and the tyrosine kinases Src and Met in tumor invasion. In this connection, recent evidence indicates that four classes of PI3′-K catalytic subunit generate the signaling molecule phosphatidyl-inositol-3,4,5-trisphosphate from upstream receptors: the p110α, β, and γ lipid kinases, and the recently discovered AGE kinase involved in regulating longevity in Caenorhabditis elegans. Most interestingly, the wortmannin-sensitive p110γ isoform was strongly activated by both the α and βγ subunits of heterotrimeric G-proteins and induced mitogen-activated protein kinase activation (
      • Stoyanov B.
      • Volinia S.
      • Hanck T.
      • Rubio I.
      • Loubtchenkov M.
      • Malek D.
      • Stoyanova S.
      • Vanhaesebroeck B.
      • Dhand R.
      • Nürnberg B.
      • Gierschik P.
      • Seedorf K.
      • Hsuan J.
      • Warterfield M.
      • Wetzker R.
      ). One function of βγ is to locate p110γ to the plasma membrane, via a tightly associated adaptator, p101 (
      • Stephens L.
      • Eguinoa A.
      • Erdjument-Bromage H.
      • Lui M.
      • Cooke F.
      • Coadwell J.
      • Smrcka A.
      • Thelen M.
      • Cadwallader K.
      • Tempst P.
      • Hawkins P.
      ). This p110γ isoform of PI3′-K is strongly activated by the PTx-dependent Gαi1 subunit (
      • Stoyanov B.
      • Volinia S.
      • Hanck T.
      • Rubio I.
      • Loubtchenkov M.
      • Malek D.
      • Stoyanova S.
      • Vanhaesebroeck B.
      • Dhand R.
      • Nürnberg B.
      • Gierschik P.
      • Seedorf K.
      • Hsuan J.
      • Warterfield M.
      • Wetzker R.
      ), suggesting that the molecular cluster containing Gαi1, p110γ, and βγ-p101 is a candidate effector of the Met pathway in invasion. Consistent with these interactions, it was recently reported that heterodimeric PI3′-K consisting of p110β and p85 is synergistically activated by the βγ subunits of trimeric G proteins (
      • Kurosu H.
      • Maehama T.
      • Okada T.
      • Yamamoto T.
      • Hoshino S.
      • Fukui Y.
      • Ui M.
      • Hazeki O.
      • Katada T.
      ). Release of Gβγ also promotes tyrosine phosphorylation of Shc and its subsequent association with Grb2-SOS. Complexity of the molecular systems involving PI3′-K is further documented by the molecular and functional diversity of the p85 regulatory subunits of the PI3′-K enzyme family (
      • Inukai K.
      • Funaki M.
      • Ogihara T.
      • Katagiri H.
      • Kanda A.
      • Anai M.
      • Fukushima Y.
      • Hosaka T.
      • Suzuki M.
      • Shin B.-C.
      • Takata K.
      • Yazaki Y.
      • Kikuchi M.
      • Oka Y.
      • Asano T.
      ).
      In conclusion, we showed that, in human colonic mucosa, PAF and its membrane receptors may be directly involved, at the epithelial cell level, in inflammatory processes and normal or neoplastic growth. Because activation and amplification of the Src and Met oncogenes are frequent events in human colon cancer and many human tumors (
      • Empereur S.
      • Djelloul S.
      • Di Gioia Y.
      • Bruyneel E.
      • Mareel M.
      • Van Hengel J.
      • Van Roy F.
      • Comoglio P.
      • Courtneidge S.
      • Paraskeva C.
      • Chastre E.
      • Gespach C.
      ), future studies should center on the downstream elements connected with PAF-R and Src/Met-R kinases. These elements include the PI3′-K cascade and the identification of its downstream targets and ultimate effectors, such as focal adhesions, cytoskeleton components, or proteases. These investigations should prove informative, in view of the possible beneficial effects of PAF derivatives against tumor cell invasion and cancer progression toward metastasis.

      Addendum

      Recently Keely et al. (
      • Keely P.
      • Westwick J.
      • Whitehead I.
      • Der C.
      • Parise L.
      ) and Shawet al. (
      • Shaw L.
      • Rabinovitz I.
      • Wang H.
      • Toker A.
      • Mercurio A.
      ) made similar observations that PI3′-K is involved in carcinoma invasion induced by Rac or α6β4 integrins.

      Acknowledgments

      We gratefully acknowledge Dr. P. Comoglio (University of Turin, Turin, Italy) for providing the recombinant human HGF peptide, Dr. J. Downward (Imperial Cancer Research Fund, London, United Kingdom) for providing parental and MDCK p110* cells, Dr. S. Ohno (Yokohama City School of Medicine, Yokohama, Japan) for providing the expression vector encoding the Eco-S dominant-negative form of PI3′-K, and Dr. P. Mayeux (Hôpital Cochin, Paris, France) and Dr. L. Shaw (Beth Israel Deaconess Medical Center, Boston, MA) for advice regarding the PI3′-K assay.

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