Human Derived Immortalized Dermal Papilla Cells With a Constant Expression of Testosterone Receptor

Abstract

Androgenetic alopecia (AGA) is the most common type of hair loss, and is mainly caused by the biological effects of testosterone on dermal papilla cells (DPCs). In vitro culturing of DPCs might be a useful tool for the screening of target molecule of AGA. However, primary DPCs cannot continuously proliferate owing to cellular senescence and cell culture stress. In this study, we introduced mutant cyclin-dependent kinase 4 (CDK4), Cyclin D1, and telomerase reverse transcriptase (TERT) into DPCs. We confirmed protein expression of CDK4 and Cyclin D1, and enzymatic activity of TERT. Furthermore, we found the established cell line was free from cellular senescence. We also introduced the androgen receptor gene using a recombinant retrovirus, to compensate the transcriptional suppressed endogenous androgen receptor in the process of cell proliferation. Furthermore, we detected the efficient nuclear translocation of androgen receptor into the nucleus after the treatment of dihydrotestosterone, indicating the functionality of our introduced receptor. Our established cell line is a useful tool to identify the downstream signaling pathway, which activated by the testosterone.

Combinatorial expression of cell cycle regulators is more suitable for immortalization than oncogenic methods in dermal papilla cells

“…Although the exact mechanism is unclear, the endogenous AR expression level became lower after several passages of DPCs (Kwack et al, 2008). In agreement with previous reports of AR suppression, we also detected downregulation of AR in DPCs immortalized using the K4DT method (Fukuda et al, 2020).…”

“…As shown in Figure S1, although the efficiency of gene introduction was only around a few percent, by selecting the successfully introduced cells using antibiotics (G418 for SV40, hygromycin for E6/E7), we successfully established human DPCs that expressed SV40 or E6/E7. We previously reported the establishment of immortalized DPCs with combinatorial expression of R24C mutant cyclin-dependent kinase 4 (CDK4), cyclin D1, and telomerase reverse transcriptase (TERT) (Fukuda et al, 2020). Hereafter, we will refer to immortalized cells with the combinatorial expression of R24C mutant CDK4, cyclin D1, and TERT as K4DT.…”

“…iScience Article Tani, T., Eitsuka, T., Katayama, M., Nagamine, T., Nakaya, Y., Suzuki, H., Kiyono, T., Nakagawa, K., Inoue-Murayama, M., Onuma, M., et al (2019) The detailed method for establishing immortalized DPCs using the K4DT method was described in our previous publication (Fukuda et al, 2020). and analysis was performed according to the manufacturer's instructions.…”

“…However, expression of R24C mutant CDK4 and cyclin D1 induces constitutively elevated CDK4-cyclinD1 complex activity since p16 protein cannot bind to mutant CDK4 due to this point mutation (Shiomi et al, 2011). The expression of R25C mutant CDK4 and cyclin D1 constitutively enhances cell proliferation in various types of human cells, such as myogenic cells, fibroblasts, corneal epithelial cells (Fukuda et al, 2019a), and dermal papilla cells (DPCs) (Fukuda et al, 2020). An important characteristic of immortalization using R25C mutant CDK4 and cyclin D1 is the intact condition of the p53 tumor suppressor gene, which enables cell expansion without genomic instability.…”

Comprehensive transcriptome data to identify downstream genes of testosterone signalling in dermal papilla cells

“…However, the gene networks controlled by testosterone, especially in dermal papilla cells (DPCs) are not fully understood. Interestingly, we accidentally found that the expression of AR is strongly suppressed even in the early passage of human DPCs 3 . The suppression of AR gene expression under the cultured cell conditions of DPCs has been previously reported in rat-derived DPCs, which is consistent with our finding 4 .…”