Regulation And Mechanisms Of The Intracellular Cross-talk Between CAMP And ERK1/2 Pathway In The Cell Proliferation And Growth Hormone Secretion Of Pituitary Somatotropinomas

Part ?.The existence of cross-talk between cAMP and ERK1/2 pathway in human pituitary somatotropinomasObjective To identify the existence of cross-talk between cAMP and ERK1/2 pathway in human pituitary somatotropinomas,and to investigate the effect of gsp mutations on the tumor biological characteristics.Methods The gsp mutations in 36 cases of human pituitary somatotropinomas were detected by PCR-DNA direct sequencing analusis.Ki67 expression was detected by immunohistochemical method and western blot.GH mRNA expression was determined by SYBR green real-time fluorescent quantitative PCR.The protein expression levels of phosphorylation of ERK1/2,Ki67 tohether with growth hormone were determined by western blot.In vitro,human sommatotroph cells harboring gsp oncogenes were treated with or without the adenylate cyclase inhibitor Bithionol,and the cells without gsp mutations were treated with or without phosphodiesterase enzymes inhibitor Rolipram,then the phosphorylation levels of ERK1/2 were detected by western blot.Results Gsp mutations were observed in 13 cases.The expression levels of GH,Ki67 together with ERK1/2 phosphorylation were significantly higher in gsp-positive adenomas than in negative adenomas(P<0.05).Bithionol significantly reduced the phosphorylation levels of ERK1/2 in cells harboring gsp oncogenes,yet Rolipram stimulated ERKl/2 phosphorylation(P<0.05).Conclusion Results supplied evidence of the existence of cross-talk between cAMP and ERK1/2 pathway in pituitary somatotropinomas.Gsp oncogenes could affect the proliferative activity and the regulation of growth hormone in pituitary somatotropinomas.Part ?.The intracellular cross-talk between cAMP and ERK1/2 pathway regulates proliferation of somatotropinomas in a B-Raf and C-Raf dependent manner Objective To figure out the effect of the cross-talk between cAMP and ERK1/2 on the proliferation of somatotropinomas and the role of B-Raf and C-Raf in the effect mediated by the cross-talk.Methods By employing human sommatotroph cells and a rat cell line(GH3),selective inhibitors of B-and C-Raf and knockdown with siRNAs were used in conjunction with forskolin to investigate effects on cell proliferation assayed using CCK-8 and EdU.In addition,effects on phosphorylation of ERK1/2,levels of cyclins D1 and D3,together with B-Raf and C-Raf activity were determined.Reslts Elevation of cAMP by exogenously added forskolin exerts significant stimulatory effects on somatotroph proliferation.Moreover,forskolin induced phosphorylation of ERX1/2?and increased levels of cyclins D1 and D3(P<0.01).These effects were attenuated by B-Raf and C-Raf inhibitors and by specific knockdown with siRNAs.Forskolin elevated B-Raf and C-Raf activity within a short time(P<0.01).Conclusion Results indicate that cAMP promotes proliferation of tumorous somatotrophs via cross-talk with the ERK1/2 pathway in a B-Raf and C-Raf dependent manner.Part ?.The cross-talk between cAMP and ERK1/2 pathway stimulates growth hormone secretion of somatotropinomas via CREB phosphorylation Objective To investigate the effect of the cross-talk between cAMP and ERK1/2 on the growth hormone secretion of somatotropinomas and the role of CREB in the effect mediated by the cross-talk.Methods By employing human sommatotroph cells and a rat cell line(GH3),selective inhibitors of B-and C-Raf and knockdown with siRNAs were used in conjunction with forskolin to investigate effects on growth hormone secretion.In addition,effects on phosphorylation of CREB together with B-Raf/C-Raf binding were determined.Reslts Elevation of cAMP by exogenously added forskolin exerts significant stimulatory effects on growth hormone secretion in time dependent manner.Moreover,forskolin induced phosphorylation CREB(P<0.01).These effects were attenuated by B-Raf and C-Raf inhibitors and by specific knockdown with siRNAs(P<0.01).B-Raf and C-Raf are not heterodimerized in somatotrophs.Conclusion Results indicate that the functional cross-talk between cAMP and ERK1/2 pathway stimulates growth hormone via CREB phosphorylation in pituitary somatotropinomas.In particular,heterodimerization of B-Raf and C-Raf does not appear to be necessary in this mechanism in somatotrophs.