The Expression And Function Of SIRP α And Gankyrin In Neural Development And Differentiation

SIRP a and Gankyrin are two genes highly expressed in nervous system. SIRP a is transmembrane molecule with three extracellular immunoglobulin superfamily domains. The expression of SIRP a in neuronal tissue is unique for it is the only known inhibitory receptor expressed in neuron. Gankyrin is one cytoplasmic protein consisting of seven ankyrin repeat domains. It has been demonstrated that Gankyrin is involved in core cell cycle apparatus regulation and can interact with 19S proteasome non-ATPase subunit. The neuronal function of SIRP a and Gankyrin still remain unresolved. In this study, we established the model of mouse brain development and maturation and investigated the expression pattern of SIRP a and Gankyrin genes in this model. In addition, using the widely used in vitro model of NGF stimulated sympathetic like neuronal differentiation of PC 12 cell, we studied the regulation and biological and neuronal functions of these two genes during neural differentiation.Western Blot analysis with specific rabbit anti-SIRP α polyclonal antibody showed that the expression of SIRP a protein increases with the development and maturation of the mouse nervous system. Our results showed that there is maximum SIRP a protein expression in mouse brain tissue 40 days after birth.In situ hybridization(ISH) and immunohistochemistry (IH)analysis showed that there is no SIRP α mRNA and protein expression in embryonic brain tissue, but postnatal brain tissue exhibits apparent staining signal in both ISH and IH analysis. In both cerebellum and cerebral cortex, SIRP a protein expression signal mainly appears in the molecular layer, however, SIRP a is also widely expressed in other regions of the cortex.In the model of NGF induced neuronal differentiation of PC 12, SIRP a protein expression is elevated after 9 days of NGF stimulation. Although MTT assaydemonstrated that transfection of SIRP a enhanced the proliferation of PC 12 in the absence of NGF stimulation, SIRP a have no effect on the proliferation halt response of PC 12 to NGF stimulation. Comparing to the parental PC 12 or the empty vector transfected control cell, SIRP a transfectant cell exhibits poor morphological differentiation response to NGF stimulation(P<0.001).Biochemically, Western Blotting with anti-phospho JNK antibody showed that SIRP a expression reduced the activating phosphorylation of MAP kinase JNK. In contrast, luciferase reporter gene analysis showed that transcriptional activity of NF-* B is significantly increased by SIRP a expression in PC 12 cell( (P<0.01) .Western Blotting results with anti-Gankyrin antibody showed that Gankyrin is also induced by NGF stimulation in PC 12 cell. The expression pattern of Gankyrin and SIRP a after NGF stimulation is different. The induction of Gankyrin gene expression by NGF is enhanced by SIRP a expression. In embryonic brain tissue, there is no detectable Gankyrin protein expression. After birth, Gankyrin expresses in many regions of mouse brain. The most prominent expression of Gankyrin appears in the hippocampal pyramidal cell neucleus.Kinase specific inhibitor treament experiments showed that the induction of Gankyrin protein expression by NGF in PC 12 cell in correlated with PI3K, NF- K B and MAPK signaling pathways.Our results showed that both SIRP and Gankyrin are induced during neuronal differentiation and maturation, the expression pattern and functione of SIRP a and Gankyrin suggest that SIRP a maybe involved in refining the neuronal process growth programe, while Gankyrin maybe involved in neuronal survival regulation.