Synergism of calcium sensing receptor and vitamin D receptor mediated signaling in epidermal differentiation [CaSR KO]

Calcium and 1,25-dihydroxyvitamin D3 (1,25D3), through the actions of their respective receptors, the Ca2+-sensing receptor (CaSR) and the vitamin D receptor (VDR), potentiate keratinocyte differentiation. VDR regulates epidermal keratinocyte proliferation and differentiation by modulating gene transcription, whereas the CaSR, a member of the family C G-protein coupled receptor, calcium mobilizes intracellular calcium and induces the formation of cell-cell junctions. 1,25D3 augments the sensitivity of the prodifferentiating actions of calcium by increasing the expression of CaSR. CaSR- and VDR-deficient keratinocytes share common characteristics such as abnormal intercellular adhesion and sphigolipid metabolism. Both CaSR and VDR are abundantly expressed in epidermal stem cell populations including CD34 expressing bulge keratinocytes in hair follicles and basal cells in interfollicular epidermis. To delineate the role of CaSR- and VDR-dependent pathways in regulating epidermal development and functions in physiological state, we generated conditional CaSR-null and VDR-null mice, where Casr and VDR gene was removed from keratinocytes. Keratinocyte-specific CaSR-null and VDR-null mice manifest distinct phenotypes. CaSR-null mice display defective epidermal permeability barrier function due to aberrant keratinocyte differentiation. VDR-null mice develop alopecia after completing the first hair follicle cycling. Concurrent ablation of CaSR and VDR genes in keratinocytes delays rate of wound repair and increases incidence of skin tumor formation to a greater extent than deletion of the CaSR or VDR alone, indicative of synergistic effects of calcium and 1,25D3 signaling. Gene expression profiles and subsequent pathway analysis on the epidermis derived from 5-day-old neonates revealed that ablation of CaSR or VDR increased expression of genes associated with cancer progression and metastasis. Deletion of VDR markedly inhibited signaling pathways that regulate hair development. Furthermore, concurrent ablation of CaSR and VDR significantly suppressed calcium, VDR, Wnt/b-catenin signaling, as well as epithelial adherence junction signaling to maintain appropriate keratinocyte adhesion. These results indicated the interplay of calcium/CaSR and 1,25D3/VDR signaling in keratinocyte proliferation and differentiation, and their importance in maintaining normal epidermal adhesion and functions. n=3 CON and KO (each sample contain RNA isolated from neonatal epidermis separated from 3 mice)

钙（calcium）与1,25-二羟维生素D3（1,25-dihydroxyvitamin D3, 1,25D3）可分别通过其受体——钙敏感受体（Ca2+-sensing receptor, CaSR）与维生素D受体（vitamin D receptor, VDR）的介导，增强角质形成细胞（keratinocyte）的分化过程。VDR通过调控基因转录，调节表皮角质形成细胞的增殖与分化；而作为C家族G蛋白偶联受体（G protein-coupled receptor, GPCR）成员的CaSR，则可动员细胞内钙并诱导细胞间连接的形成。1,25D3可通过上调CaSR的表达，增强钙的促分化作用敏感性。CaSR与VDR缺陷的角质形成细胞具有共同的异常特征，包括细胞间黏附异常与鞘脂代谢紊乱。CaSR与VDR均在表皮干细胞群中高表达，包括毛囊中表达CD34的隆突角质形成细胞以及毛囊间表皮的基底细胞。为阐明CaSR与VDR依赖通路在生理状态下调控表皮发育与功能的作用，我们构建了角质形成细胞特异性条件性敲除Casr与VDR基因的小鼠模型，即Casr与VDR基因可在角质形成细胞中被特异性敲除。角质形成细胞特异性CaSR敲除与VDR敲除小鼠呈现出不同的表型：CaSR敲除小鼠因角质形成细胞分化异常，出现表皮通透屏障功能缺陷；VDR敲除小鼠在完成首次毛囊周期后会发生脱发。与单独敲除CaSR或VDR相比，同时敲除角质形成细胞中的CaSR与VDR基因会更显著地延缓伤口修复速率并增加皮肤肿瘤形成的发生率，这表明钙与1,25D3信号通路存在协同效应。对5日龄新生小鼠表皮组织进行基因表达谱分析及后续通路分析发现，敲除CaSR或VDR会上调与肿瘤进展及转移相关的基因表达；敲除VDR则会显著抑制调控毛发发育的信号通路。此外，同时敲除CaSR与VDR会显著抑制钙信号、VDR信号、Wnt/β-连环蛋白（Wnt/β-catenin）信号以及上皮黏附连接信号通路，以维持角质形成细胞的正常黏附功能。上述结果表明，钙/CaSR与1,25D3/VDR信号通路在角质形成细胞增殖与分化过程中存在相互作用，且二者在维持正常表皮黏附与功能中发挥重要作用。样本设置：对照组（CON）与敲除组（KO）各3份，每份样本的RNA均提取自3只新生小鼠分离的表皮组织。