Knockout of p16INK4a promotes aggregative growth of dermal papilla cells

ORIGINAL ARTICLE Knockout of p16INK4a promotes aggregative growth of dermal papilla cells Knockout of p16INK4a promotes aggregative growth of dermal papilla cells Yi Cheng1, Yang Gao2, Lu Zhao1*, Shunqiang Gao1, Guoqiang Zhang1, Yan Zhang1 Department of Dermatology, the Fourth Hospital of Hebei Medical University, Shi Jiazhuang, Hebei Province, China 1 2 Department of Interventional Radiology, Hebei Children’s Hospital, Shi Jiazhuang, Hebei Province, China Summary Study conducted at Fourth Hospital of Hebei Medical University and Hebei Children’s Hospital, Shi Jiazhuang, Hebei Province, China Article received: 3/10/2017 Accepted for publication: 3/11/2017 *Correspondence: Department of Dermatology Fourth Hospital of Hebei Medical University Address: No.12 Jiankang Road Shi Jiazhuang, Hebei Province – China Postal code: 050011 http://dx.doi.org/10.1590/1806-9282.63.10.883 Objective: Dermal papilla cells (DPCs) are located in the hair follicles and play an important role in hair growth. These cells have the ability to induce hair follicle formation when they display aggregative behavior. DPCs derived from the androgenetic alopecia (AGA) area undergo premature senescence in vitro, associated with p16INK4a expression. The aim of the current study was to investigate the expression of p16INK4a in aggregative and non-aggregative DPCs and the effect of p16INK4a downregulation in these cells by adenovirus-mediated RNA interference (RNAi). Method: DPCs were isolated and cultured from healthy human scalp. p16INK4a gene and protein were detected in aggregative and non-aggregative cells. Expression of p16INK4a in DPCs was silenced by infection with rAd5-CDKN1A-1p2shRNA. Cell fate was monitored after infection. The growth of cells was measured by MTT assay. Cell cycle was evaluated by flow cytometry (FCM). Results: DPCs were isolated by digestion and showed aggregative behavior for six passages. The expression of p16INK4a showed a clear upward trend in non-aggregative cells when compared with aggregative group. p16INK4a expression was silenced by rAd5-CDKN1A-1p2shRNA (p<0.05). The p16INK4a-silenced cells grew more rapidly and exhibited a trend towards aggregative growth. There was an increase in the proportion of cells in G1 phase, while those in S phase were reduced after p16INK4a gene silencing (p<0.05). Conclusion: Our results suggest that p16INK4a plays an important role in the premature senescence and aggregative behavior of DPCs. These observations can lead to novel therapeutic strategies for treatment of AGA. Keywords: hair follicle, transfection, hair/growth and development. Introduction Dermal papilla cells (DPCs) are the main mesenchymal cells located at the bottom of the hair follicle (HF) and compose the dermal papilla. The biological characteristic of these cells is the ability to induce HF formation, both in vivo as well as in vitro. DPCs are a cluster of specialized fibroblasts and occur at the central link in the morphology of the HF and its cyclic growth regulation.1,2 During the initial stage of HF morphogenesis in the course of embryonic development, the ectodermal epithelial cells proliferate and differentiate downward continuously after being stimulated by dermis signaling molecules to form the hair peg. The hair peg provides feedback to the dermis and induces the formation of dermal concentrate Rev Assoc Med Bras 2017; 63(10):883-889 by dermal fibroblast in the dermis, to give rise to the dermal papilla precursor. Some of the aggregative dermal fibroblast cells then differentiate into dermal papilla cells while others turn into dermal sheath cells surrounding the columnar hair follicle epithelial cells.3-5 In other words, HF formation is directed by an aggregation of dermal mesenchymal cells, which form the origin of DPCs in the embryonic skin. Furthermore, cultured DPCs also have hair-forming activity if they are in an aggregative mode. Aggregative growth is a crucial feature of DPCs to induce hair follicle formation and is characterized by radial growth that occurs prior to fusion of DPCs.3 DPCs that grow in a radial pattern in an aggregative manner are spindle-shaped with abundant cytoplasm. But the characteristic 883 Cheng Y et al. of aggregative growth gradually disappears in vitro, eventually ceasing entirely. It was reported that when DPCs of low passage were inoculated into a small incision on the mouse auricle, with high-passage cells and fibroblasts as control, clusters of hair fibers grew on the incision containing low passage DPCs. These hair fibers were thicker and longer than naturally occurring hair on the ear, and were similar to the tentacles from where the DPCs were taken. On the other hand, high-passage DPCs and fibroblasts did not show this phenomenon.6 These observations confirm that low passages of cultured DPCs with aggregative growth not only have the ability to induce complete regeneration of hair follicles, but also carry the information needed to determine the nature of the hair, as corroborated by subsequent research.7,8 It should be emphasized that the ability of DPCs to induce HF formation is dependent on their aggregative growth. But the mechanism by which the aggregative behavior disappears is not yet clear. p16INK4a is a cyclin-dependent kinase (CDK) inhibitor that slows down cell cycle by inhibiting transition from G1 to S phase. Normally, CDK4/6 binds cyclin D to form an active protein complex that phosphorylates retinoblastoma protein (pRB). Once phosphorylated, pRB disassociates from the transcription factor E2F1, thus liberating E2F1 from its cytoplasm bound state, thereby allowing it to enter the nucleus. In the nucleus, E2F1 promotes transcription of target genes that are essential for transition from G1 to S phase.9,10 Tissue ageing causes p16INK4a concentration to increase dramatically.11 p16INK4a has also been used as a target to delay certain changes related to ageing in mice.12 Recent reports have demonstrated that DPCs taken from male androgenetic alopecia (AGA) patients undergo premature senescence in vitro associated with the expression of p16INK4a. We hypothesized that non-aggregative growth was a feature of ageing and that aggregative growth characteristics were correlated with p16INK4a. We studied p16INK4a expression in DPCs and inhibited p16INK4a expression in DPCs by adenovirus-mediated RNA interference to explore the possible mechanisms of cultured human DPCs losing aggregative growth characteristics. Method Ethics statements The Fourth Hospital of Hebei Medical University institutional review board approved all described studies. The study was conducted according to the Declaration of Helsinki Principles. Informed written consent was obtained from all patients. Isolation and culture of dermal papilla cells Specimens were taken from the occipital scalp of six male individuals undergoing surgical excision of benign cutaneous tumors. The patients were not using any hair loss medications wh (...truncated)