Gu Yangguang, Wang Yu, Lan Yinghua, Feng Jianglong, Zeng Wen, Zhang Wei, Lu Hongguang
Department of Dermatology, Affiliated Hospital of Guizhou Medical University, Guiyang, China.
Department of Dermatology and Venereology, Clinical College of Medicine, Guizhou Medical University, Guiyang, China.
Front Cell Dev Biol. 2022 Apr 4;10:787730. doi: 10.3389/fcell.2022.787730. eCollection 2022.
Photoreceptive proteins play critical physiological roles in human skin cells. The retinal G protein-coupled receptor (RGR) is a photoisomerase in the human retina, but its expression and cellular functions in human skin cells have not been reported. We aimed to detect RGR expression in various skin cells and evaluate its regulation of the cellular functions of keratinocytes. The expression, distribution, and subcellular location of the RGR in normal human epidermal keratinocytes and cells with pathological conditions including psoriasis, seborrheic keratosis, and squamous cell carcinoma were determined using microscopic tools (immunohistochemical staining, immunofluorescence staining, and immunoelectron microscopy) and Western blotting (WB). The protein levels of the RGR in primary human melanocytes, keratinocytes, and fibroblasts isolated from the neonatal foreskin were measured by WB. The expression and subcellular localization of the RGR in these cells were detected by immunofluorescence staining under a fluorescence microscope and laser scanning confocal microscope. Additionally, the levels of RGR expression in normal keratinocytes exposed to ultraviolet (UV)-A or total ultraviolet radiation (UVR) in the presence or absence of all-trans-retinal were measured by WB. Furthermore, the effects of the RGR on human keratinocyte functions including proliferation, migration, and apoptosis were evaluated using the Cell Counting Kit 8, wound healing, and Transwell assays after reducing the RGR mRNA level in keratinocytes using small interfering RNA technology. The RGR was primarily located in the epidermal basal and spinous layers and skin appendages. Its expression increased in psoriatic lesions, seborrheic keratosis, and squamous cell carcinoma. Confocal microscopy showed that the RGR was located in the cell membrane and nucleus of keratinocytes, melanocytes, and fibroblasts. Keratinocytes had a higher expression of the RGR than melanocytes and fibroblasts, as well as nuclear expression, according to nuclear/cytoplasmic fractionation. Colloidal gold immunoelectron microscopy technology further confirmed that the RGR is mainly located in the nucleoplasm and mitochondria and is scattered in the cytoplasm and other organelles in the epidermal keratinocytes. Notably, RGR knockdown in keratinocytes led to the inhibition of cell proliferation and migration, augmenting cell apoptosis. This study is the first to demonstrate the presence of RGR in the human skin. Our findings indicate that the RGR may play a critical role in the physiological function of epidermal keratinocytes.
光感受蛋白在人类皮肤细胞中发挥着关键的生理作用。视网膜G蛋白偶联受体（RGR）是人类视网膜中的一种光异构酶，但其在人类皮肤细胞中的表达及细胞功能尚未见报道。我们旨在检测RGR在各种皮肤细胞中的表达，并评估其对角质形成细胞功能的调节作用。使用显微镜工具（免疫组织化学染色、免疫荧光染色和免疫电子显微镜）及蛋白质免疫印迹法（WB），确定RGR在正常人类表皮角质形成细胞以及患有银屑病、脂溢性角化病和鳞状细胞癌等病理状况的细胞中的表达、分布及亚细胞定位。通过WB测定从新生儿包皮分离的原代人黑素细胞、角质形成细胞和成纤维细胞中RGR的蛋白水平。在荧光显微镜和激光扫描共聚焦显微镜下，通过免疫荧光染色检测这些细胞中RGR的表达及亚细胞定位。此外，通过WB测定在存在或不存在全反式视黄醛的情况下，暴露于紫外线A（UV-A）或总紫外线辐射（UVR）的正常角质形成细胞中RGR的表达水平。此外，在使用小干扰RNA技术降低角质形成细胞中RGR mRNA水平后，使用细胞计数试剂盒8、伤口愈合和Transwell实验评估RGR对人类角质形成细胞功能（包括增殖、迁移和凋亡）的影响。RGR主要位于表皮基底层、棘层和皮肤附属器。其表达在银屑病皮损、脂溢性角化病和鳞状细胞癌中增加。共聚焦显微镜显示，RGR位于角质形成细胞、黑素细胞和成纤维细胞的细胞膜和细胞核中。根据细胞核/细胞质分级分离，角质形成细胞中RGR的表达高于黑素细胞和成纤维细胞，且有核表达。胶体金免疫电子显微镜技术进一步证实，RGR主要位于核质和线粒体中，在表皮角质形成细胞的细胞质和其他细胞器中呈散在分布。值得注意的是，角质形成细胞中RGR的敲低导致细胞增殖和迁移受到抑制，并加剧细胞凋亡。本研究首次证明RGR在人类皮肤中的存在。我们的研究结果表明，RGR可能在表皮角质形成细胞的生理功能中起关键作用。
