The possible role of yes-associated protein (YAP) on IGF-1-induced sebum production

Acne is a chronic inflammatory disorder of the pilosebaceous unit. Increased sebum excretion and alterations in certain lipid components are major features of acne pathogenesis.1 Human sebum is composed mainly of triglycerides, squalene and wax esters. Free fatty acids broken down from triglyceride by C. acnes and increased amount of squalene and their peroxidants were reported as contributing factor of acne.2 Recent research suggested that a diet with a high glycemic load may influence the development of acne through insulin-like growth factor (IGF)-1-induced sebum production.3 In vitro, IGF-1 upregulated the expression of sterol response element binding protein-1 (SREBP-1) through the PI3K/Akt signalling pathway, resulting in increased lipogenesis in SEB-1 sebocytes.4 Yes-associated protein (YAP) is an effector of the Hippo pathway, which plays an important role in regulating epidermal and sebaceous gland proliferation. Recent studies have revealed that YAP stimulates de novo lipogenesis via mTORC1 signalling and subsequent activation of SREBP.5 In cancer background, several reports revealed that the IGF-1-YAP signalling loop regulates cancer progression.6 However, there is no investigation into the role of IGF-1-YAP signalling in the sebum production of sebocytes. To investigate the effect of IGF-1 on YAP activity in sebocytes, we treated them with 50 ng/mL IGF-1 in a time-dependent manner. We used immortalized sebocytes which were transduced with a recombinant retrovirus expressing simian virus 40 T antigen (SV40Tag).7 Interestingly, the nuclear localization of YAP was significantly increased after 30 min of IGF-1 treatment in sebocytes (Figure 1A). To validate the result obtained from immunofluorescence staining, we performed Western blot with nuclear and cytoplasmic fractionation. Similarly, the abundance of YAP was increased in nuclear fraction and decreased in cytoplasmic fraction after IGF-1 treatment (Figure 1B). To evaluate the transcriptional activity of YAP after treatment of IGF-1 to sebocytes, we performed RT-qPCR to observe the expression of two well-known YAP target genes, CTGF and CYR61. Expression of these two genes was significantly upregulated after IGF-1 treatment (Figure 1C). These results indicate that IGF-1 increases YAP activity by inducing nuclear translocalization in sebocytes. To further examine the effect of YAP on IGF-1-induced lipogenesis, we used verteporfin which is a chemical inhibitor of the interaction of YAP with TEAD and blocks transcriptional activation of targets downstream of YAP. We examined that verteporfin decreased the expression of CTGF and CYR61 in sebocytes (Figure S1). This suggests that verteporfin can inhibit YAP activity in sebocytes. Next, we performed thin layer chromatography (TLC) to investigate the change of lipid profiles of sebum secreted by sebocytes after YAP inhibition. IGF-1 treatment increased the quantity of squalene and triglycerides in sebocytes. Interestingly, pretreatment with verteporfin inhibited IGF-1-induced increase of these two lipids in sebocytes (Figure 1D). To investigate the mechanism of YAP inhibition on IGF-1-induced lipogenesis, we evaluated the protein expression of important transcription factors related to lipid production. IGF-1 treatment increased the levels of transcription factors including the mature form of SREBP-1 and SREBP-2. Pretreatment with verteporfin inhibited IGF-1-induced mature SREBP-1 and SREBP-2 expression (Figure 1E). We next examined the effect of YAP inhibition on the expression of lipogenic enzymes, such as FDFT1 and SCD which are involved in the production of squalene and triglyceride, respectively. Pretreatment of verteporfin markedly decreased IGF-1-induced FDFT1 and SCD expression (Figure 1F). To evaluate whether the inhibitory effect of verteporfin is dependent on IGFR-mTOR signalling, we checked the phosphorylation levels of IGFR and mTOR after treatment of IGF-1 and verteporfin. We found that verteporfin significantly inhibited IGF-1-induced these signalling in sebocytes (Figure 1G). From these results, we determined that IGF-1 increased YAP activity in sebocytes and YAP inhibition attenuated IGF-1-induced lipid production by preventing IGFR-mTOR signalling pathway. We speculated that the IGF-1-YAP axis may affect the pathogenesis of acne by increased sebum production, especially, squalene and triglyerides. A more elaborate study will be needed to verify these result and the possibility of YAP as a therapeutic target in acne. S.P. conceived the study. J.M.S and S.P. designed the experiments. J.M.S. and K.M.K. performed the experiments. J.M.S. and S.P. wrote the manuscript, and Y.L. and C.D.K. reviewed and edited the manuscript. All authors reviewed the manuscript. This research was supported by a grant from the National Research Foundation of Korea (NRF-2021R1I1A1A01061447). This research was supported by a grant from the National Research Foundation of Korea (NRF-2021R1I1A1A01061447). None declared. The data that support the findings of this study are available from the corresponding author upon reasonable request. Figure S1: Effect of verteporfin on cell viability and expression of YAP downstream genes in sebocytes. Appendix S1: Primary culture and immortalization. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.