Interaction Of MAD2B And TCF4and Its Effects On The Function Of Dermal Papilla Cells

Backgrounds and ObjectivesHair follicle is an appendant organ of the skin, as well as the subject of the hair growth.As the only human organ that can be completely recycled, the studies on hair follicles haveimportant biological significance. Moreover, hair follicle-related diseases, since it mayaffect the patient’s appearance, would have great psychological pressure on patients,thereby affecting the physical and mental health. Therefore, there is a growing emphasis onhair follicles research.Dermal papilla (DP) is located at the bottom of hair follicles, which can induce thereconstruction of hair follicles in culture in vitro, and plays a key role in morphogenesis andperiodic regeneration of hair follicle. Dermal papilla cell (DPC) is the only cell constitutingthe DP. It not only induces formation of hair follicles of epithelial cells in embryonic period,but also regulates the periodic regeneration of mature hair follicles after birth. DPC acts onits own and neighboring cells via autocrine and paracrine, which involves multiplesignaling pathways and regulatory factors, including mainly the proteins and cytokines ofWnt, Notch protein, bone morphogenetic protein (BMP), fibroblast growth factor (FGF)and vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) andinsulin-like growth factor (IGF-1). Therefore, DPC is currently one of the hot cells in hairfollicles research. Aggregative growth is the key biological characteristic of DPC, which isrelated to the functional status of the DP. And now, the aggregative growth and mechanismof inducting of hair follicle reconstructure in vitro remains unclear, while further researchon the regulation effect of a variety of signaling pathways on DPC is required.The study demonstrated that the Wnt pathway plays an important role in hair growthand cycle regulation, as wells as in maintaining the agglutination growth ability of DPC.Our previous experiments confirmed that the T cell factor4(TCF4), the key regulatoryfactor of Wnt signaling pathway is the DPC-specific expression of the aggregation growth,hence, the enhancement of TCF4expression would promote the DPC cultured in vitro, facilitate the expressions of secreted factors of DPC，SCF and HGF, while regulate the hairfollicle development via autocrine and paracrine mechanisms. MAD2B protein is identifiedby the yeast two-hybrid and can be bound with TCF4, playing an important role in the cellregulation of the mitosis. This subject intends to study whether the DPC growing understate of aggregation shows the interaction of MAD2B protein and TCF4, whether it hasregulation effect on the Wnt pathway activity regulated by the TCF4as well as theexpression of downstream cytokines, and affecting the biological function of DPC.Experimental methods and results1.DPC was isolated from human hair follicle of scalp and cultured for observation ofthe primary and passage DPC growth characteristics. The total RNA was then extracted forfollow-up testing.2. Co-immunoprecipitation was used to determine the whether the interaction existsbetween MAD2B protein and TCF4protein in DPC.3. pIRES2-MAD2B and pIRES2-TCF4plasmids were constructed, which were thentransfected to DPC with LipofectamineTM2000liposome transfection method afterrestriction enzyme digestion; the inverted fluorescence microscope and Western blot werethen adopted to observe whether it had overexpression in DPC. The plasmid constructionand transfection were successful.4. Test was randomized as pIRES2-MAD2B/pIRES2-TCF4co-transfection group,pIRES2-TCF4transfection group and normal DPC control group. As confirmed by theresults, comparing with the pIRES2-TCF4transfection group, the Wnt/β-catenin signalswere significantly reduced after co-transfection of pIRES2-MAD2B and pIRES2-TCF4(p＜0.05), whereas, and yet the co-transfection of siMAD2B and pIRES2-TCF4may correctsuch reduction. The Wnt/β-catenin signals of pIRES2-TCF4transfection group andpIRES2-MAD2B/pIRES2-TCF4co-transfection group showed significant increasecomparing with the control group (p＜0.05). CCK-8was used to determine its effect on cellproliferation activity, where the D450mean of pIRES2-MAD2B/pIRES2-TCF4co-transfection group was0.49±0.18, pIRES2-TCF4transfection group was0.79±0.15, andnormal DP control group was0.57±0.15, among which, the pIRES2-TCF4transfectiongroup and pIRES2-MAD2B/pIRES2-TCF4co-transfection group showed significantdifference with normal DP control group (p＜0.05); there was no significant difference between pIRES2-MAD2B/pIRES2-TCF4co-transfection group and normal DP controlgroup.5. Through the fluorescent quantitative PCR (QF-PCR), it then found that the mRNAexpressions of IGF-1, VEGF and HGF secreted by the DPC with overexpressed MAD2Bwere significantly reduced comparing with the normal control group.Conclusion1. Co-immunoprecipitation further corroborates the interaction between MAD2B andTCF4in DPC.2. The overexpression of MAD2B may inhibit the DPC proliferation via Wnt signalingpathway.3. The overexpressed MAD2B may inhibit the synthesis of paracrine factors of IGF-1,VEGF and HGF.Above experiment establishes the foundation for further understanding of DPCbiological functions, especially the regulatory mechanism of the Wnt pathway in the DPC.