Signalling Transduction And Function Analysis Of Dmrt1

Sex determinaton/differentiation is a key process in research work of developmental biology, which is focused on by many biologists. Dmrt1 (doublesex-mab3-related transcription factor 1), is an only important functional gene which found to plays key roles in different phyla by now. In all animals, different Dmrtl proteins have a common DNA binding domain, which is conserved from Drosphila to mammalian animals.Dsx and mab-3, which come from fly and worm respectively, is homologue genes of Dmrt1. These two genes are found to be involved in sex determination and gonadal development of both fly and worm. In medaka, a kind of teleost fish, Dmrtlb(Dmy) is a key gene to control sex determination. When Dmy is mutant, male medaka develops to female phenotype. In amphibian frogs, Dmrtl and its homologue gene Dm-w control sex determination and gonadal development of male and female, respectively. In mammalian, such as mouse, homozygous Dmrt1 gene mutations result in infertile male phenotype. In the seminiferous tubes of mutant mouse, germ cells death and Sertoli cells are immature and overproliferate. The mutant of 9 chromosome fragment, containing Dmrt1 gene and others in human being induces totally infertile phenotype, and even sex reversal takes place in some patients. To look for the factors which control and regulate the expression of Dmrtl and how Dmrtl gene play its role in the process of gonadal development are so necessary and significative because of the important status of Dmrtl gene. In this paper, we focus on the post-transcriptional and post-translational modification of Dmrtl, including trans-splicing of Dmrtl mRNA and phosphorylation and ubiquitination of Dmrtl protein.We found a novel signal transduction pathway involved isulin, Dmrt1, Mdm2 and p53, which may participate in apoptosis and male sexual development. These results in the work make us to further understand the status of Dmrtl gene itself and the network involved in the sex determination and differentiation.1. Insulin is involved in post-translational modification of DmrtlWe focus on the modication of Dmrtl protein in this part. First, we found phosphtyrosine of Dmrtl protein. We identified two putative phosphotyrosine sites in Dmrtl protein, Y31 and Y82. The Y82 site is conserved from chicken to human being. Then we found the growth factor, insulin and igf-1 can up-regulate the protein level of Dmrtl. By RT-PCR and Western Blotting, we confirmed the insulin can not activate the transcription of Dmrt1. When cells were treated with insulin, Dmrt1 protein was more stable because of the lower ubiquitination level. We found the treatment with insulin effectively down-regulated the ubiquitination of Dmrtl protein through MEK/ERK pathway. By in vivo labelling with 32P, we found the insulin can not influence the phosphotyrosine level of Dmrtl, but at the same time, we also found the phosphorylation of Dmrtl protein without two putative phostyrosine sites was sensitive with cAMP/PKA pathway, which means Sersine or Throsine phosphrylation. These results showed that there are multiple modifications in Dmrtl protein and these phosphorylation sites make the Dmrtl protein to keep a right structure to avoid of degradation.In diabetic mouse, we found higher level of ubiquitination of Dmrtl and lower protein level, which reconfirmed the results got from primary sertoli cells. We also found disordered seminiferous tubes in diabetic mouse. This phenotype is similar with the Dmrtl mutant mouse model, which may according to the reduced Dmrtl protein level.2. Dmrtl-Mdm2 complex upregulates p53 to induce apoptosisCoimmunoprecipitation confirmed interaction between Mdm2 and Dmrtl. Immuofluorecence indicated nuclear localization of both endogenous Mdm2 and Dmrtl in new dissected mouse primary Sertoli cells. Mdm2 was observed in the cytoplasm after in cuture for 10 days when endogenous Dmrtl was not expressed. Interestingly, relocalization of endogenous Mdm2 to the nucleus was observed following ectopic expression of Dmrtl-gfp again in the cells. These results suggested interaction betweeen Mdm2 and Dmrtl. Mdm2-Dmrtl interaction leads to stabilization of Dmrtl but not degradation, which indicated that Mdm2 is not an E3 ubiquitin ligase of Dmrtl although it really binds to. Binding of both proteins looks to stabilize each other.We further examined whether Mdm2 affects function of Dmrtl as a transcription factor using cis-element Dmrtl binding site linked with luciferase reporter. Co-transfection of Dmrtl, Dmrtl-luciferase reporter and an increased Mdm2 showed that Mdm2 inhibited transcriptional activation of Dmrtl in a dose dependent manner.Because Mdm2 and p53 are linked to each other through an autoregulatory negative feedback loop, we challenged functional significance of Dmrtl, Mdm2 and p53. We found Mdm2 and p53 proteins are up-regulated by Dmrtl expression in TM4 cells. Furthure, we found Dmrtl stablized p53 by inhibiting polyubiquitination of p53. These data suggested that Dmrt1, as a tumor supressor, can induce apoptosis through stabilizing p53.3. Insulin-Dmrt1-Mdm2-p53 pathway is involved in dysmorphologic seminiferous tubules in STZ and db/db testisAn open question is whether Mdm2, p53 and Dmrt1 synergetically regulate in vivo. We investigated these protein expression and Sertoli cell proliferation in both STZ and leprdb/db diabetic mouse models. Western blot analysis showed that decreased expression of the three proteins in the STZ diabetic testis. Immunofluorecance staining indicated that similar expression pattern of the three proteins in normal testis, including Sertoli cells and pre-meiosis germ cells. In the STZ diabetic testis, Sertoli cell marker Gata4 showed Sertoli cell spread from the center to the periphery in the seminiferous tubules, in contrast to only in the periphery of the tubules in normal testis.In leprdb/db diabetic mice, we also observed decreased protein levels of Dmrt1, Mdm2 and p53, which is similar to those in STZ diabetic testis. leprdb/db testis showed more severely dysmorphologic seminiferous tubules than the STZ testis and nearly devoid of spermatogenesis. Over proliferation of Sertoli cells was further confirmed by immunofluorecance staining of Gata4. These results indicated dysmorphologic seminiferous tubules, over proliferation of Sertoli cells, associated with decreased Mdm2, p53 and Dmrt1 protein levels in both STZ and leprdb/db diabetic testis.