The Mechanism Of Saposin C Upregulating Expression And Function Of AR, And Stimulating Proliferation Of PCa Cells

Prosaposin is a highly conserved glycoprotein(65-72kDa,527 amino acids),the gene was localized to the long arm of chromosome 10(q21-22)and encompasses 17 kb of genomic sequence with 14 exons.Prosaposin is the precursor of four small heat-stable sphingolipid activator proteins(saposins A,B,C and D,which are required for the enzymatic hydrolysis of sphingolipids in lysosomes).In addition to its intracellular presence and function,Prosaposin is also expressed as a secretory protein and also a well-known neurotrophic factor in vivo and in vitro.As a secretory protein, Prosaposin could promote survival,prevent apoptosis and stimulate synapse growth of neuroglial-derived cells.Prosaposin functional sequence is localized to a 12-amiono acid stretch at NH₂-terminal end of the saposin C domain (LIDNNRTEELLY).Several synthetic peptides(14-22 residues,e.g.TX14A)derived from this region are equally as bioactive as Prosaposin.Prosaposin,saposin C,and prosaptides exert their neurotrophic effects by binding to a putative high affinity G protein-coupled receptor(GPCR)which could be blocked by Pertussis Toxin.Some reports demonstrated that inactivation of prosaposin gene affected the development of the prostate gland.Koochekpour S et al affirmed that prosaposin gene amplified and overexpressed in prostate cancer cells,and also demonstrated that exogenous saposin C and Prosaptide TX14A stimulated growth,metastasis and invasion via activated MAPK,PI3K/Akt et al singling pathways in prostate cancer.Prostate cancer is the highly strong lethal disease,once relapsing most of the patients will develop into hormone-resistant or hormone-independent prostrate cancer. At present,this is no effective treatment options for the patients.Although the mechanisms involved in the progression of prostate cancer are not entirely understood, androgen receptor(AR)has been shown to play a critical role because AR was the only overexpressed persistently molecular during the development and progression of the AIPC.The AR is a ligand-dependent transcription factor of the nuclear steroid hormone receptor superfamily.Binding androgen,AR can be activated by phosphorylation.The active AR enters into the nuclear and binds to the androgen-responsive element(ARE)of the target DNA in order to activate the target gene transcription and promote cells growth.During the development of AIPC the singling pathways AR mediating involved in AR gene mutation,AR gene amplification,the aberrant regulation of AR by growth factors and cytokines,and AR cofactors change.Evidences have been shown that ligand-independent activation of AR is concerned with the development of AIPC.The present study is aimed to determine effect of Saposin C and its function domain neurotrophic peptide(NP)on androgen receptor(AR)expression and transcriptional activity and cells proliferation in prostate cancer.Then research deeply on molecular mechanism.We constructed DNA vectors that can express Saposin C/NP or a chimeric peptide of a viral TAT transduction domain and Saposin C/NP by gene cloning technology.The effect of ectopic expression of Saposin C/NP with or without the TAT transduction domain on cell growth was examined by MTT assay. Then reverse transcriptase-polymerase chain reaction(RT-PCR),Western blot analysis, transient transfection experiments and confocal laser microscopy were used to determine the effect of Saposin C/NP on AR expression and activation.To further approach the mechanism of Saposin C/NP on AR,we used pertussis toxin and inhibitor of MAPK or PI3K/Akt by Western Blot analysis,transient transfection experiments,confocal laser microscopy and co-immunoprecipitation.Our research showed that the eukaryotic expression vectors pcDNA-NP/His, pcDNA-TAT-Saposin C/pcDNA-TAT-NP and pcDNA-Saposin C/pcDNA-NP were constructed and confirmed by sequencing,and they can normally express in prostate cancer cells.Immunofluorescence staining demonstrated that TAT-Saposin C located on both cell membrane and interior of cell,Saposin C and NP located only in cell.Flow cytometry analysis showed that expressions of TAT-NP and NP had a stimulating effect on prostate cancer cell proliferation,and promoted the cells to enter S and G₂/M phase.MTT analysis showed that expressions of TAT-Saposin C/TAT-NP and Saposin C/NP had a stimulating effect on prostate cancer cells proliferation.The RT-PCR and Western blot analyses displayed that TAT-Saposin C/TAT-NP and Saposin C/NP induced AR gene expression.Dual-luciferace experiment results demonstrated that TAT-Saposin C/TAT-NP and Saposin C/NP activated transactivation of the AR.Immunofluorescence staining showed that TAT-Saposin C,Saposin C and NP could activate AR and locate at cellular nucleus in the absence of androgens.The effect of intracellular Saposin C/NP on the AR function and the proliferation of prostate cancer cells was not through the G-protein receptor pathway,but with Src and AR interaction,to form a complex,thereby activating non-receptor tyrosine protein kinase Src.The activated Src further activated MAPK and PI3K/Akt pathway. AR activation may also be directly activated Src.Collectively,our study showed that Saposin C,as a growth stimulating factor to PCa cells,exerted the activity by increasing the expression and activation of the AR in a ligand-independent manner.It is reasonably believed that Saposin C could stimulate proliferation of PCa by non-GPCR pathway,but interact with Src and AR form a complex.The Src and AR were activated in turn.It is certainly that the MAPK or PI3K/AKT pathway participate the course of AR activation.Furthermore,the synergistic effect of Saposin C and androgen on the expression and transactivation of AR was observed.