The Signal Transduction Pathway Of PGE₂Promoting Liver Cancer Cell Growth

Background:Prostaglandin E₂（PGE₂） is predominant metabolic product of arachidonic acidafter Cyclooxygenase-2（COX-2） catalytic effect. It can obviously promote theproliferation, invasion and metastasis of multiple tumors. But the exact mechanismof the regulation of PGE₂has not been illustrated.PGE₂mediates its effects by binding and activating four differentG-protein-coupled EP receptors （EP1, EP2, EP3and EP4receptor）. The transfectionof EP receptor in the mouse and human HEK293cell indicates that EP1receptormainly couples Gq protein, which induces the increase of Ca2+in the cell andactivates PKC. EP2and EP4receptors mainly couple Gs protein, which activatesadenylate cyclase （AC）, then increases the level of cAMP and induces PKAactivation. EP3receptor couples Gi protein, which inactivates adenylate cyclase（AC）, then decreases the level of cAMP. The specific target of PGE₂regulating thegrowth of cancer cell through EP receptors has not been reported.Far upstream element binding protein （FBP） family comprise three proteins.They are FBP1, FBP2and FBP3. FUSE binding protein can predominantly bindsingle strain DNA and RNA, as a transcription factor, regulates the stability of genetranscription and promotes the expression of c-myc. On the contrary, thedown-regulation of FBP1can inhibit the expression of c-myc. It is reported that theincrease of FBP1can induce the proliferation of liver cancer cell.Our previous study showed that PGE₂and the agonist of EP3receptorup-regulated the level of FBP1protein in liver cancer cell line CCLP1and promoted cell growth. The results indicate that PGE₂might up-regulate the level of FBP1protein through EP3receptor and induce the increase of liver cancer cell growth.Moreover, recently, it has been reported that EP3receptor also couples Gs protein,which activates adenylate cyclase （AC）, then increases the level of cAMP andfacilitates the gowth, blood vessel formation and metastasis of tumors.cAMP can activate PKA. The activated PKA maybe inhibit the activity ofTGF-β₁TGF-β₁/Smad can inhibit the gowth and metastasis of tumors. So theinhibited TGF-β₁/Smad may promote the growth of cancer cell.Meanwhile, the decreased growth of can cell induced by TGF-β₁is related withJTV1protein. JTV1is also called p38or AIMP2. It is first identified as a factorassociated with a macromolecular protein complex consisting of several differentaminoacyl-tRNA synthetases. The increase of JTV1inhibits the proliferation ofcancer cell. TGF-β₁induces the increase of JTV1protein.JTV1regulates the ubiquitination of FBP1in nucleus. The overexpression ofJTV1increases the ubiquitination of FBP1. JTV1binding FBP1is necessary for theubiquitination of FBP1. Therefore, TGF-maybe up-regulate the level of JTV1protein, then enhance the binding of JTV1and FBP1and induce the degradation ofFBP1.According to the reports and our findings, the presumption is proposed: PGE₂couples Gs protein by EP3receptor and activates cAMP-PKA. cAMP-PKA caninhibit the activity of TGF-β₁Moreover, the suppressed TGF-β₁down-regulates thelevel of JTV1protein and inhibits the ubiquitination of FBP1, which inceases FBP1protein and promotes liver cancer cell growth.This presumption will be observed by some methods, such as stimulation andinhibition of EP3receptor, overexpression and RNA interfering of EP3receptor andtargeting the signals of transduction pathway. This research has not been reported in the world. It can further illustrate the mechanism of PGE₂promoting liver cancercell.Aims:1. To clarify the expression of EP3receptor in liver cancer cell.2. To explore the role of EP3receptor in PGE₂-induced liver cancer cell growth.3. To investigate that PGE₂promotes liver cancer cell through EP3receptor byup-regulating FBP1protein.4. To investigate that PGE₂couples Gs protein by EP3receptor and activates cAMP-PKA. cAMP-PKA can inhibit the activity of TGF-Moreover, the suppressedTGF-down-regulates the level of JTV1protein and inhibits the ubiquitination ofFBP1, which inceases FBP1protein and promotes liver cancer cell growth.Methods:1. Hepatocellular carcinoma cells HUH-7, Hep3B, HepG2and cholangiocarcinomacells HuCCT1, CCLP1, SG231were all cultured in conventional conditions.2. Real-Time PCR was used to detect the expression of EP3receptor splicingvariants in liver cancer cell.3. WST assay was used to detect the growth of liver cancer cell induced by EP3receptor agonist sulprostone, Gi protein inhibitor PTX, AC inhibitor SQ22536andPKA inhibitor H89.4.2-D DIGE was used to detect the downstream proteins, such as FBP1after EP3receptor stimulation.5. The cells were treated with EP3receptor inhibitor L-798106, EP3receptor siRNA,PGE₂and EP3receptor agonist sulprostone. Western blot was used to detect the levelof FBP1protein.6. The ELISA kit was used to detect the level of cAMP after PGE₂and EP3receptoragonist sulprostone stimulation. 7. The cells were treated with PKA inhibitor H89, AC agonist Forskolin, cAMPanalogy db-cAMP, PGE₂and EP3receptor agonist sulprostone. Western blot wasused to detect the level of phosphorylated Smad2protein.8. The cells were treated with TGF-β₁1, AC agonist Forskolin, cAMP analogydb-cAMP, PGE₂and EP3receptor agonist sulprostone. Western blot was used todetect the level of phosphorylated Smad2protein.9. The cells were treated with TGF-β₁1, AC agonist Forskolin, cAMP analogydb-cAMP, PGE₂and EP3receptor agonist sulprostone. Western blot andimmunoprecipitation were used to detect the levels of FBP1protein, JTV1protein,the binding of JTV1and FBP1and the ubiquitination of FBP1.10. The cells were treated with PGE₂,EP3receptor agonist sulprostone, AC inhibitorSQ22536and PKA inhibitor H89. Western blot and immunoprecipitation were usedto detect the levels of FBP1protein, JTV1protein, the binding of JTV1and FBP andthe ubiquitination of FBP1.Results:1. EP3-4，EP3-5，EP3-6and EP3-7splicing variants were observed in HUH-7,Hep3B, HepG2, HuCCT1and CCLP1cells, while EP3-4，EP3-5，EP3-7plicingvariants were observed in SG231cell.2. EP3-4stable cell line of CCLP1and control cell were treated with10μMSulprostone and50nM Gi protein inhibitor PTX for24h. In EP3-4stable cell line,Sulprostone significantly increased the growth rate by35.17%（P <0.01）, whilecompared with the treatment of “Sulprostone”, the treatment of“Sulprostone+PTX” had no effect on the cell growth induced by Sulprostone. Thecontrol cell had no effect. EP3-4stable cell line of CCLP1and control cell weretreated with10μM Sulprostone,50μM SQ22536and10μM H89for24h. In EP3-4stable cell line, Sulprostone significantly increased the growth rate by35.17%（P <0.01）, while compared with the treatment of “Sulprostone”, the treatment of“Sulprostone+H89” dereased the growth rate from135.17%to99.4%（P <0.01）and the treatment of “Sulprostone+SQ22536” dereased the growth rate from135.17%to116.9%（P <0.01）. The control cell had no effect.3. EP3-4stable cell line of CCLP1and the empty pcDNA-transfected cell weretreated with10μMPGE₂and10μM Sulprostone. In EP3-4stable cell line, the levelof FBP1protein of “PGE₂”gruoup was1.43fold to solvent control and the level ofFBP1protein of “Sulprostone” gruoup was1.72fold to solvent control at the timeof1h（P <0.05）. The empty pcDNA-transfected cell had no effect at this time.EP3-4stable cell line of CCLP1and control cell were treated with10μMPGE₂and10μM EP3receptor inhibitor L-798106. In EP3-4stable cell line, the level ofFBP1protein of “PGE₂”gruoup was1.68fold to solvent control（P <0.05）, whilecompared with “PGE₂”group, the level of FBP1protein of “PGE₂+L-798106”group was0.57fold to “PGE₂”gruoup（P <0.05）. The empty pcDNA-transfectedcell had no effect. EP3-4stable cell line of CCLP1was treated with10μMPGE₂and10μM Sulprostone after transfection by EP3-4siRNA and the negative RNA.PGE₂and Sulprostone had no effect on the the level of FBP1protein in EP3-4siRNA transfected cell. The levels of FBP1protein of “PGE₂”and “Sulprostone”groups were respectively1.46and2.02fold to control in negative RNAtransfected cell （P <0.05）.4. EP3-4stable cell line of CCLP1and the empty pcDNA-transfected cell cell weretreated with10μMPGE₂and10μM Sulprostone. In EP3-4stable cell line, the levelof cAMP induced by PGE₂and Sulprostone relatively increased by248.56%and99.42%（P <0.01）. The empty pcDNA-transfected cell had no effect.5. EP3-4stable cell line of CCLP1and the empty pcDNA-transfected cell cell weretreated with10μMH89,10μM Forskolin,100μM db-cAMP,10μM Sulprostone and 10μMPGE₂. In EP3-4stable cell line, the level of phosphorylated Smad2proteininduced by H89was1.49fold to solvent control（P <0.05）. The levels ofphosphorylated Smad2protein of “H89+Forskolin”,“H89+db-cAMP”,“H89+Sulprostone” and “H89+PGE₂” groups were respectively0.51fold,0.74fold,0.68fold and0.62fold（P <0.05） to “H89” group. The empty pcDNA-transfected cellhad no effect.6. EP3-4stable cell line of CCLP1and the empty pcDNA-transfected cell cell weretreated with2ng/ml TGF-,10μM Forskolin,100μM db-cAMP,10μM Sulprostoneand10μMPGE₂. In EP3-4stable cell line, the level of phosphorylated Smad2proteininduced by TGF-β₁was2.26fold to solvent control（P <0.05）. The levels ofphosphorylated Smad2protein of “TGF-+Forskolin”,“TGF-β₁+db-cAMP”,“TGF-β₁+Sulprostone” and “TGF-β₁+PGE₂” groups were respectively0.76fold,0.60fold,0.60fold and0.70fold（P <0.05） to “TGF-β₁” group. The emptypcDNA-transfected cell had no effect.7. EP3-4stable cell line of CCLP1and the empty pcDNA-transfected cell cell weretreated with2ng/ml TGF-β₁,10μM Forskolin,100μM db-cAMP,10μM Sulprostoneand10μMPGE₂. In EP3-4stable cell line, the level of FBP1protein induced byTGF-β₁was0.63fold to solvent control（P <0.05）. The levels of FBP1protein of“TGF-β₁+Forskolin”,“TGF-+db-cAMP”,“TGF-β₁+Sulprostone” and “TGF-β₁+PGE₂” groups were respectively1.90fold,1.56fold,1.84fold and1.62fold to“TGF-β₁” group（P <0.05）. The level of JTV1protein induced by TGF-β₁was1.48fold to solvent control（P<0.05）. The levels of JTV1protein of “TGF-β₁+Forskolin”,“TGF-β₁+db-cAMP”,“TGF-β₁+Sulprostone” and “TGF-+PGE₂” groups wererespectively0.46fold,0.55fold,0.56fold and0.55fold to “TGF-β₁” group（P<0.05）. The empty pcDNA-transfected cell had no effect.8. EP3-4stable cell line of CCLP1and the empty pcDNA-transfected cell were treated with2ng/ml TGF-β₁,10μM Forskolin,100μM db-cAMP,10μM Sulprostoneand10μMPGE₂. In EP3-4stable cell line, the binding of FBP1and JTV1induced byTGF-was1.47fold to solvent control（P <0.05）. The bindings of FBP1and JTV1of “TGF-+Forskolin”,“TGF-+db-cAMP”,“TGF-β₁+Sulprostone” and “TGF-β₁+PGE₂” groups were respectively0.41fold,0.52fold,0.41fold and0.37fold to“TGF-β₁” group（P <0.05）. The ubiquitination of FBP1induced by TGF-β₁was1.76fold to solvent control（P <0.05）. The ubiquitinations of FBP1of“TGF-β₁+Forskolin”,“TGF-+db-cAMP”,“TGF-β₁+Sulprostone” and “TGF-β₁+PGE₂” groups were respectively0.59fold,0.64fold,0.43fold and0.67fold to“TGF-β₁” group（P <0.05）. The empty pcDNA-transfected cell had no effect.9. EP3-4stable cell line of CCLP1and the empty pcDNA-transfected cell weretreated with10μM Sulprostone,50μM SQ22536and10μM H89. In EP3-4stablecell line, the level of FBP1protein induced by Sulprostone was1.82fold to solventcontrol（P <0.05）. The level of FBP1protein of “Sulprostone+H89”group was0.58fold to “Sulprostone”group and the level of FBP1protein of“Sulprostone+SQ22536”group was0.53fold to “Sulprostone”group（P <0.05）. Thelevel of JTV1protein induced by Sulprostone was0.60fold to solvent control（P<0.05）. The level of JTV1protein of “Sulprostone+H89”group was1.36fold to“Sulprostone”group and the level of JTV1protein of “Sulprostone+SQ22536”groupwas1.66fold to “Sulprostone”group（P <0.05）. The empty pcDNA-transfected cellhad no effect.10. EP3-4stable cell line of CCLP1and the empty pcDNA-transfected cell weretreated with10μM PGE₂,50μM SQ22536and10μM H89. In EP3-4stable cell line,the level of FBP1protein induced by PGE₂was1.73fold to solvent control（P <0.05）.The level of FBP1protein of “PGE₂+H89”group was0.78fold to “PGE₂”group andthe level of FBP1protein of “PGE₂+SQ22536”group was0.73fold to “PGE₂”group（P <0.05）. The level of JTV1protein induced by PGE₂was0.80fold tosolvent control（P <0.05）. The level of JTV1protein of “PGE₂+H89”group was1.61fold to “PGE₂”group and the level of JTV1protein of “PGE₂+SQ22536”group was1.94fold to “PGE₂”group（P <0.05）. The empty pcDNA-transfected cell had noeffect.11. EP3-4stable cell line of CCLP1and the empty pcDNA-transfected cell weretreated with10μM Sulprostone,50μM SQ22536and10μM H89. In EP3-4stablecell line, the level of binding of FBP1and JTV1induced by Sulprostone was0.36fold to solvent control（P<0.05）. The level of binding of FBP1and JTV1of“Sulprostone+H89”group was3.31fold to “Sulprostone”group and the level ofbinding of FBP1and JTV1of “Sulprostone+SQ22536”group was3.39fold to“Sulprostone”group（P <0.05）. The level of ubiquitination of FBP1induced bySulprostone was0.69fold to solvent control（P <0.05）. The level of ubiquitination ofFBP1of “Sulprostone+H89”group was1.77fold to “Sulprostone”group and thelevel of ubiquitination of FBP1of “Sulprostone+SQ22536”group was1.79fold to“Sulprostone”group（P <0.05）. The empty pcDNA-transfected cell had no effect.12. EP3-4stable cell line of CCLP1and the empty pcDNA-transfected cell weretreated with10μM PGE₂,50μM SQ22536and10μM H89. In EP3-4stable cell line,the level of binding of FBP1and JTV1induced by PGE₂was0.56fold to solventcontrol（P <0.05）. The level of binding of FBP1and JTV1of “PGE₂+H89”group was2.21fold to “PGE₂”group and the level of binding of FBP1and JTV1of“PGE₂+SQ22536”group was2.34fold to “PGE₂” group（P <0.05）. The level ofubiquitination of FBP1induced by PGE₂was0.70fold to solvent control（P <0.05）.The level of ubiquitination of FBP1of “PGE₂+H89”group was1.53fold to“PGE₂”group and the level of ubiquitination of FBP1of “PGE₂+SQ22536”groupwas1.46fold to “Sulprostone”group（P <0.05）. The empty pcDNA-transfected cell had no effect.Conclusions:PGE₂activates EP3receptor. EP3receptor couples Gs protein and activatescAMP-PKA. cAMP-PKA can inhibit the activity of TGF-Moreover, thesuppressed TGF-down-regulates the level of JTV1protein and inhibits theubiquitination of FBP1, which inceases FBP1protein and promotes liver cancer cellgrowth.