Functional analysis of vitamin D receptor (VDR) using adenovirus vector
Functional analysis of vitamin D receptor (VDR) using adenovirus vector
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DOI：
10.1016/j.jsbmb.2023.106275
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发表时间：
2023-03-06
影响因子：
4.1
通讯作者：
Sakaki, Toshiyuki
中科院分区：
文献类型：
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作者：
Kise, Satoko;Iijima, Ayano;Sakaki, Toshiyuki
最近，我们通过基因组编辑获得了包括VDR(R270L)、VDR(H301Q)、VDR(R270L/H301Q)和VDR基因敲除(KO)在内的II型软骨病模型大鼠。所有产生的动物都表现出软骨病的症状，包括生长迟缓和异常的骨形成。其中，只有VDR-KO大鼠表现出皮肤形成异常和脱发。为了阐明VDR功能与软骨病症状的关系，用腺病毒载体在人HaCaT-VDR-KO细胞中表达了每种VDR。我们还构建了与引起脱发的人VDR(V346M)相对应的表达VDR(V342M)的腺病毒载体。我们比较了分别在10和100 nM的最终浓度下加入1α，25-二羟基维生素D3(1，25D3)或25-羟基维生素D3(25D3)后VDR的核明显易位。25D3和1，25D3均可诱导野生型VDR和VDR(V342M)的核易位。相反，VDR(R270L)易位在100 nM 25D3存在下观察到，而在10 nM 1，25D3处理后几乎没有易位。VDR(R270L/H301Q)核易位失败。这些结果与它们对每个配体的亲和力是一致的。值得注意的是，VDR(R270L/H301Q)在生理条件下可能以未连接的形式存在，与VDR(R270L/H301Q)相互作用的因子可能参与毛发生长周期。因此，这个使用腺病毒载体的新系统在阐明维生素D受体功能方面可能有价值。
Recently, we generated type II rickets model rats, including Vdr(R270L), Vdr(H301Q), Vdr(R270L/H301Q), and Vdr-knockout (KO), by genome editing. All generated animals showed symptoms of rickets, including growth retardation and abnormal bone formation. Among these, only Vdr-KO rats exhibited abnormal skin formation and alopecia. To elucidate the relationship between VDR function and rickets symptoms, each VDR was expressed in human HaCaT-VDR-KO cells using an adenovirus vector. We also constructed an adenovirus vector expressing VDR(V342M) corresponding to human VDR(V346M) which causes alopecia. We compared the nu-clear translocation of VDRs after adding 1 alpha,25-dihydroxyvitamin D3 (1,25D3) or 25-hydroxyvitamin D3 (25D3) at final concentrations of 10 and 100 nM, respectively. Both 25D3 and 1,25D3 induced the nuclear translocation of wild type VDR and VDR(V342M). Conversely, VDR(R270L) translocation was observed in the presence of 100 nM 25D3, with almost no translocation following treatment with 10 nM 1,25D3. VDR(R270L/H301Q) failed to undergo nuclear translocation. These results were consistent with their affinity for each ligand. Notably, VDR (R270L/H301Q) may exist in an unliganded form under physiological conditions, and factors interacting with VDR(R270L/H301Q) may be involved in the hair growth cycle. Thus, this novel system using an adenovirus vector could be valuable in elucidating vitamin D receptor functions.