VDR Regulated The Proliferation And Differentiation Of Murine Dpidermal And Hair Follicle Keratinocytes

[Objective] Vitamin D receptor (VDR), a nucleus receptor, after binding with its ligand,1,25(OH)2D3, recruits RXR to form a VDR-1,25(OH)2D3 polymer, then combines with Vitamin D response elements (VDRE) of target genes to activate the gene transcriptions. This project aims at uncovering the underneath mechanisms of how VDR regulates the development of epidermis and hair follicle in mice, so as to afford some basal data for the further researches of the human diseases of epidermis and hair follicle.[Methods] 1. VDR gene knockout mice (VDR KO) were constructed to observe the effects of VDR gene on the growth and development of mouse epidermis and hair follicle, and re-transfection of human VDR (hVDR) gene sequence was used to verify its affection. Routine HE staining was performed to check the histological features, and RT-PCR was utilized to detect the genes transcription regarding cell proliferation and differentiation before and after VDR knockout, such as FLQ LOR, K10, K14, Ki-67. Behaviors of proliferation and differentiation in cultured keratinocytes of WT and KO mice were examined in vitro. After making wounds in skin, layers of proliferative cell, numbers of BrdU-positive cells and expressions of involucrin, an indicative protein of keratinocyte differentiation, were observed during wound healing. Finally, the positioning feature of hair follicle stem cells was detected by CD34 immunofluorescence, and quantitative comparison was analysed by flow cytometry with CD34 and CD49f double positive label in WT and KO mice afterbirth 18 days.2. Hair follicle anagen was induced by depilation, and target gene expressions of cWnt and Shh signaling pathway were evaluated by RT-PCR in WT and KO mice, including Shh, Gli1, Ptc2, Msx2 and wif1. Then Shh agonist (SAG) was topically applicated to trigger the hair regrowth and the activations of Shh and cWnt pathway were investigated. Finally, CHIP-RT-PCR was performed to investigate the physical bindings between VDR and DNA regulatory region of cWnt and Shh pathway target genes. 3. Immunofluorescence stainings were performed to detect the dynamic expression of DDIT4 at different ages and hair follicle cycles. Then RT-PCR was used to detect the DDIT4 gene transcriptions before and after VDR knockout in cultured WT and KO keratinocytes and hair follicle stem cells, as well as verified by hVDR retransfection. DDIT4 expression was also investigated in the periphery tissue after wounds by immunofluorescence staining. Different concentrations of 1,25(OH)2D3 were added to stimulate the cultured keratinocytes of WT and KO mouse in vitro to investigate its inductive effect on DDIT4 expressions. Finally, physical binding of VDR and DDIT4 gene promoter region was clarified by CHIP-RT-PCR to decide how VDR participates in the complex networks of "DDIT4-cWnt-Shh" pathways.[Results] 1. After constructing VDR KO mice by specificly knockout the amino acid residues of second zinc finger on VDR protein, it was found that the mice, upon loss of functional VDR, presented skin and hair follicle dysplasia and even alopecia, which, in turn, could be reversed by re-transfected with the human VDR (hVDR) gene. Moreover, in KO mice, the expression of FLG, LOR, K10, but K14 were significantly reduced, while Ki-67 increased. During skin wound healing, KO mice recovered much faster than WT mice in the mid-phrase after wounded, and the layers of proliferating epidermal cell and positive staining cells of BrdU among wound edges increased significantly, but the cell differentiation marker, involucrin, was much lower than WT. These results indicated that VDR played an important role in epidermal cell proliferation and differentiation. In addition, flow cytometry was used to sort CD34 and CD49f double positive hair follicle stem cells (HSC) to evaluate HSC number, and found a significant reduction in KO mice on postnatal 18 day, compared to the WT, which infered KO mice suffer a progressive reduction of HSC after the hair follicle morphogenesis, which might partially contribute to the postnatal hair loss in KO mice.2. To further clarify the underneath mechanism of how VDR regulates the epidermal and follicle development, the crosstalk between VDR, cWnt and Shh signaling pathway was examined. Hair anagen initiation was found to be impaired by VDR ablation after depilation. PCR results revealed the impaired induction of cWnt and Shh targeted gene expression in KO mice, including Shh, Gli1, Ptc2, Msx2 and wif1. SAG treatment could not initiate the hair regrowth in both 16-day and 7-week old KO mice. After stimulation of SAG, Wifl, Gli1, Shh, Ptc2 gene were typically increased, with much more BrdU-positive keratinocytes obversed in WT mice; while attenuated response of these gene expression with only a few BrdU-positive cells was found in KO. CHIP-RT-PCR analysis showed that VDR protein potentially bind to the DNA regulatory region of Glil, Axin2, cMYC as well as OC gene in WT primary keratinocytes, but no enrichment at any site in KO. It indicated VDR might be involved in the regulation of expression of cWnt and Shh target genes by binding to their DNA regulatory regions.3. DDIT4 expressions were dynamically involved in murine epidermal growth and hair follicle cycles. At catagen, DDIT4 appeared high expression in WT mice, along with the proliferation of hair follicle cells and decrease of protein synthesis; but in anagen, DDIT4 expression decreased; then raised again in the following telogen. Those dynamic features of DDIT4 expression are coincident with the physiological characteristics of hair follicle cycle. But conversely, loss of VDR in KO mice impaired this dynamic characteristics of DDIT4. It was inferred low expression of DDIT4 in KO epidermis partially attributed to its excessive epidermal proliferation, while continuing high expression during hair follicle cycle produced persistent inhibition of hair regrowth, and finally led to progressive hair loss after hair follicle morphogenesis. RT-PCR and Western blot results also confirmed the lower expression of DDIT4 in KO primary keratinocytes in vitro than that of WT, and which could be reversed by transfection of hVDR. However, HSC sorted by flow cytometry showed the level of DDIT4 in KO was higher than that of WT. DDIT4 expression was also ligand-dependent in vitro cultured keratinocyte.1,25(OH)2D3 could increase the DDIT4 expression in a concentration-dependent manner. During skin wound healing, DDIT4 was mainly expressed in the basal layer of epidermis after wounded in WT mice, and its intensity was higher than that of KO, which were consistent with the result in the first part of the keratinocyte in KO mice proliferated faster than WT mice at the mid phase after wounded. CHIP-RT-PCR analysis showed that VDR bind to the promoter region of DDIT4 DNA, wherein the strongest binding site was at-963～1154bp, and also was enhanced by 1,25(OH)2D3. These results manifested that VDR may regulate the expression of DDIT4 by binding to its regulatory sequence of DNA, and thus participate in the regulation of epidermis and hair follicle.[Conclusion] 1. VDR is involved in the regulation of mouse epidermis and hair follicles regrowth and development. After VDR ablation, KO mice appear excessive proliferation of epidermis and formation of cyst, as well as progressive hair loss in a ligand and calcium or phosphorus metabolism independent manner, which may result from the the abnormal gene expressions induced by VDR abliation and contribute to the impaired cell proliferation and differentiation in skin. KO mice hair follicle stem cells begin to decrease after birth 18 days.2. In keratinocytes, VDR can bind to the DNA regulatory regions of cWnt and Shh pathway target genes and then initiate their transcriptions. A crosstalk among VDR-cWnt-Shh pathway was thereby formed to regulate epidermal and hair follicle keratinocytes proliferation and differentiation.3. DDIT4 is dynamically involved in the murine epidermal growth and hair follicle cycles. DDIT4 is continuously sustained at high expression in hair follicle bugle region, which is functionally inferred to maintain the status of hair follicle stem cells. DDIT4 expression in KO hair follicle is higher than that of WT may contribute to the postnatal hair follicle recycle defects. VDR can bind to DDIT4 DNA promoter region at -963～1154bp site, and 1,25(OH)2D3 is able to enhance this process to promote DDIT4 transcription. For cWnt and Shh is also reported at downstream of DDIT4 pathway, so it may further form a greater network between "VDR-DDIT4-cWnt-Shh" signaling pathways.