Functional Interaction Between Mouse Rhox5 And Prosaposin

Mouse reproductive homeobox on the X chromosome (Rhox) is a novel homeobox gene cluster. Rhox5, also called Pem, belongs to theβsubcluster of Rhox. More and more studies implied an important role for Rhox5 during embryogenesis and reproductive development. But till now, little information is available regarding how the Rhox5 protein exerts its function. Prosaposin is a multiple-functional protein. Despite of its function during sphingolipid catabolism, prosaposin also plays an important role in reproductive system,nervous system and the development of prostate cancer. In our prior research, we found Rhox5 could interact with prosaposin in yeast, which gave some clues to their function study. Previous study suggested prosaposin could promote survival and prevent apoptosis via PI3K/Akt-dependent pathway in prostate cancer cells. Due to their specific expression pattern in mouse reproductive system and the functional study about prosaposin, we postulate that Rhox5/prosaposin interaction may also regulated the reproductive development via PI3K/Akt signal pathway. Advanced research about Rhox5/prosaposin functional interaction in vivo, the critical structure for its interaction and its putative interaction model may help us to elucidate the possible mechanism of the male reproductive organs development and maintenance.The main results are as follows:1. High-level expression, polyclonal antibody preparation and sub-cellular localization analysis of mouse Rhox5 proteinThe prokaryotic expression vector pET22b-Rhox5 was constructed successfully. To achieve high-level expression ofRhox5, Rosetta^TM2 (DE3) cells, expressing 7 tRNA genes of rare codons, were used to overcome problems associated with codon bias in Rhox5 cDNA squence. The 6His-tagged Rhox5 was expressed efficiently in Rosetta^TM2 (DE3), compared with marginal expression in BL21 (DE3). The fusion protein amounted to 16% of the total bacterial protein after induction with 0.4 mM IPTG for 1.5 h at 37℃. The protein Rhox5-6His was purified with Ni²⁺ affinity chromatography and the purified product was ultrafiltered to desalt and then lyophilized. After purification, Rhox5-6His was used to immunize New Zealand white rabbits following standard protocol. The polyclonal antiserum was got and the ELISA assay indicated its titer was 1:16 000. The homemade antiserum could detect both endogenous Rhox5 protein expressed in eukaryotic cells (Cos-7) and exogenous GFP-Rhox5 protein. Furthermore, the antiserum was used to determine the localization of Rhox5 in NIH3T3 cells using an immunofluorescence technique. The results demonstrated that Rhox5 was localized predominantly in the nucleus. Preparation of an anti-Rhox5 polyclonal antibody will facilitate further functional study of Rhox5. 2. Rhox5 could interact with prosaposin directly.In yeast two-hybrid screening, we obtained a novel interaction partner of Rhox5: No. 1-14.Two-ways yeast two-hybrid assay indicated that Rhox5 could interact with No. 1-14 in yeast. GST-pull assay confirmed its physical interaction in vitro. The sequence of No. 1-14 was blasted in NCBI. Through comparing and searching, a sequence containing a whole open reading frame that absolutely matched with No. 1-14 (100%) was obtained. The sequence was prosapsin (Psap) gene, encoding prosaposin protein. No. 1-14 was located in the C-terminal of the Psap gene. Then the full-lenth of prosaposin gene was cloned by PCR from the 7-day mouse embryo library. The prosaposin we amplified was one of the prosaposin splicing transcripts without exon8. Two-ways yeast two-hybrid assay indicated Rhox5 could also interact with prosapsoin (without exon8) in yeast. GST-pull assay also confirmed its physical interaction. Indirect immunofluorescence assay suggested that both proteins colocalised predominatly in the nucleus. Furthermore, coimmunoprecipation experiments confirmed that prosaposin could interact not only with exogenous Rhox5 but with endogenous Rhox5.3. The critical structure for Rhox5/prosaposin interaction.In order to study the interaction between Rhox5 and prosaposin protein (with exon8), overlap extension PCR was used to amply the full length of prosaposin cDNA with exon8. Two-ways yeast two-hybrid assay and GST-pull down assay indicated that Rhox5 could interact with prosaposin (with exon8) in yeast and in vitro. This impled that the interaction between Rhox5 and prosaposin (with or without exon8) might both have the same physiologica functions.In order to determine the minimal domain of Rhox5 for Rhox5/prosaposin interaction, we constructed two truncate fragments: Rhox5 A and Rhox5 B. Rhox5 A, without homeobox domain, included the 104 amino acids in the N terminal of Rhox5 protein. Two-ways yeast two-hybrid assay and GST-pull down assay suggested this truncated mutants couldn't interact with prosaposin. Rhox5 B, including the 105 amino acids in the C terminal of Rhox5 protein, contained the conservative homeobox domainin. Two-ways yeast two-hybrid assay and GST-pull down assay indicated that it could interact with prosaposin. This suggested the homeodomain of Rhox5 protein was critical for its interaction with prosaposin.In order to determine the minimal domain ofprosaposin for Rhox5/prosaposin interaction, four truncate fragments were constructed: Psap, including the amino acids besides No.1-14; No.1-14, saposin C, including the amino acids from 313 to 392; saposin D, including the amino acids from 438 to 517. Two-ways yeast two-hybrid assay and GST-pull down assay indicated that all the truncated mutants of prosaposin with disulfide linkage could bind with Rhox5. With the decrease of the number of the amino acid residues and the disulfide linkage, the affinity with Rhox5 also decreased.But there was a variance between its bonding force: Prosaposin＞Psap A, No. 1-14＞saposin C, D. Using bioinformatical method to analyze their three-dimensional models, it suggested all the truncated mutants had the similar 3D model.Furthermore, two saposin D domain mutants (saposin D M3C and saposin D M6C) were constructed. For saposin D M3C and saposin D M6C, three or six cysteine residues were mutated into serine in order to destroy the conservative disulfide bond, decrease the ratio of the hydrophobic amino acid and destroy the hydrophobic core. Two-ways yeast two-hybrid assay and GST-pull down indicated that saposin D M3C could interact with Rhox5 as the wild saposin D due to its similar 3D model. However, Due to its disulfide linkage removal and the alteration of its 3D model, saposin D M6C mutants loosed the ability of interacting with Rhox5.4. Rhox5/prosaposin interaction and PI3K/Akt signal pathwayTo study the physiological function of Rhox5, prosaposin alone and its interaction, pcDNA-Rhox5-HA and pcDNA-Psap-Myc plasmids were transfected alone or cotransfected into NIH3T3 cells. Through G418 and hygromycin B screening, RT-PCR and western blotting verification, we abtained the four cells: pcDNA3.1 in NIH3T3, Rhox5-HA in NIH3T3, Psap-Myc in NIH3T3 and Rhox5/Psap in NIH3T3. The proliferation of these four cell lines was detected by MTT assay and the result indicated that both Rhox5 and prosaposin overexpression could promote cell proliferation, while cooverexpression of Rhox5 and prosaposin resulted in decreased proliferation comparing with the effect of prosaposin alone. The PI3K-specific inhibitor, LY294002, could inhibit the proliferation promoted by prosaposin and its cooperation with Rhox5 significantly.Under serum-starvation stress, we decteced the apoptosis of this four cell line. The results suggested: prosaposin could prevent apoptosis significantly; Rhox5 could also inhibit the apoptosis slightly; while the cooverexpression of Rhox5 and prosaposin resulted in increased cell apoptosis comparing with the effect of prosaposin alone.To elucidate the possible mechanism for their effect on cell proliferation and cell apoptosis, Western blotting was used to detect the phosphorylative level of PI3K/Akt pathway. Direct immunoblotting of these four cell lines was showed that Rhox5 overexpression could upregulate phosphorylative activity of PDK1 at serine 241, Akt at Serine 473 and GSK3βat serine 9, prosaposin upregulated these proteins' phosphorylative antivity significantly, while there is a decrease on the proteins' phosphorylative activity when Rhox5 and prosaposin were coexpressed. Pretreatment of cells with LY294002 substantially reduced phosphorylation levels of these proteins. Results of Realtime PCR suggested that both Rhox5 and prosaposin downregulated the expression of P27^Kipl genes and upregulated the expression of CyclinD 1 and survival genes.While coexpression of Rhox5 and prosaposin resulted in upregulating the expression of P27^Kipl genes and downregulating the expression of CyclinDl and survival genes to some extant comparing with the effect of prosaposin alone.In this study, we developed a method for the efficient expression and purification of the Rhox5-6His fusion protein using a modified Rosetta^TM2 (DE3) strain. In addition, we reported the first production of highly specific antiserum against Rhox5 and the nuclear localization of endogenous Rhox5. We discovered the interaction between Rhox5 and prosaposin in vitro and in vivo and difined the critical structure for its interaction. Most importantly, we postulate: Under pathological conditions (neoplastic growth of the prostate or disorder of Androgen-regulated genes), Rhox5 and/or prosaposin, two androgen-regulated genes, would be overexpressed. The overexpressed prosaposin protein, as a nutrition factor or cell factor, will promote cell survival and prevent apoptosis via PI3K/Akt pathway. The overexpressed Rhox5 protein would activate the PI3K/Akt pathway by phosphorylating its serine and threonine residues or via its targeted molecules, and then promote cell survival and prevent apoptosis. However, when prosaposin and Rhox5 were cooverexpressed, the overexpressed Rhox5, as a suppressor, may bind to prosaposin, intercept the secretion of prosaposin and alter the phisilogical effect caused by prosaposin via PI3K/Akt pathway.This work of ours provided a solid basis for further study the fuctional role of Rhox5, prosaposin and its interaction during the maintance of prostate and the development and invasion of prostate cancer.