J Am Acad Dermatol

J Am Acad Dermatol. localized around these ARE sites, we used ChIP DNA immunoprecipitated by anti-DHT/testosterone and anti-IgG antibody. for semi-quantitative RT-PCR analysis. The ChIP specific primers covering promoter regions R1-R10 are listed in Stable 1. The fold enrichment of androgen conversation with different regions (R1-R10) in the promoter was quantified (Ct value Chebulinic acid of Input DNA/Ct value of androgen ChIP DNA) and represented graphically. The unfavorable control anti-IgG ChIP DNA did not show amplification with R1-R10 primers. TRPM8 expression is usually regulated by androgens [11]. In this study we investigated if the gene is usually regulated by androgens, which depends on actions of androgens to bind AR and activate it. The androgen impartial pathways do not require androgens, but can be activated by growth factors acting through kinase pathways, such as the MAPK pathway or the PI3K pathway, which phosphorylate and activate AR in the absence of androgens [22]. Our aim was to study the androgen-dependent regulation of and several putative ARE have been indicated at the 5 flank region of gene [20, 21]. To investigate whether androgen-AR complex binding to the promoter is usually localized around these ARE sites, we performed chromatin anti-DHT/testosterone immunoprecipitation (ChIP) using DNA isolated from LNCaP, PC3 and HEK-TRPM8 control, and testosterone-induced cells which were then cloned, sequenced and analyzed. The ChIP analysis identified a number of short individual DNA fragments (Supplementary Physique 1A), consisting of sequences lying between putative ARE I and II elements in the gene promoter (Physique ?(Figure1B).1B). To further confirm the androgen binding to ARE I and II elements, we used ChIP DNA immunoprecipitated by anti-DHT/testosterone and anti-IgG antibodies. The semi-quantitative RT-PCR was carried out using primers for regions (R) named 1C10 by scanning the first 2064 bp 5-flanking region of the human gene (“type”:”entrez-nucleotide”,”attrs”:”text”:”NW_004929306.1″,”term_id”:”528475943″,”term_text”:”NW_004929306.1″NW_004929306.1) identified by ChIP analysis (Physique ?(Figure1B).1B). Androgen enrichment at R10, which includes putative ARE I Chebulinic acid site, was greater than at other regions made up of ARE II (R2, R3) or no ARE (R4, R5, R6, R7, R8 and R9) sites (Physique 1B and 1C). The coefficient of androgen conversation indicated that androgens/AR bind to promoter in a region detected by R10 primers (Figure ?(Figure1C).1C). Interestingly, when compared to Chebulinic acid testosterone-induced cells, LNCaP and PC3 control cells showed increased androgen enrichment on the promoter. These contradictory observations in the androgen-unresponsive PC3 cells may be due to the relatively low but detectable levels of AR mRNA [23, 24]. Whereas in HEK-TRPM8, testosterone-induced cells showed prominent androgen/AR binding of the promoter when compared to control cells (Figure ?(Figure1C).1C). Although, we did not detect the IgG2b Isotype Control antibody (FITC) AR protein in PC3 cells, we observed the AR expression in HEK-293 cells by immunoblot analysis (Supplementary Figure 1B). Furthermore, these results demonstrated inverse correlation of androgen-mediated promoter regulation with androgen response of cells (LNCaP PC3 HEK-TRPM8). Role of androgens in TRPM8-mediated Ca2+ uptake Previous studies showed that TRPM8 acts as a Ca2+-permeable channel in androgen-responsive LNCaP cells [21]. To test whether androgen regulates TRPM8-mediated Ca2+ uptake, LNCaP, PC3 and HEK-TRPM8 control, 1 M – DHT (o/n) and testosterone (3 Chebulinic acid h) -induced cells were analyzed using Ca2+ imaging (Figure 2A and 2B). The time- and dose-dependent effects of androgens were standardized initially to induce the highest TRPM8 protein expression. The standardization of conditions for TRPM8 activation was done using HEK-TRPM8 cells as described previously [25]. In these experiments TRPM8 was activated using its agonist, menthol, and resulted Ca2+-uptake was compared among the cell lines (Figure ?(Figure2A).2A). We found that menthol did not induce any noticeable Ca2+ uptake in LNCaP control or Chebulinic acid DHT-induced cells. However, testosterone-induced LNCaP cells demonstrated elevated basal Ca2+ levels and also responded to 50 M menthol (Figure ?(Figure2A),2A), indicating enhanced TRPM8 activity induced by testosterone. PC3 cells showed small menthol-induced TRPM8 responses (Figure 2A and 2B). Open in a separate window Figure 2 TRPM8 activity by intracellular Ca2+-measurementsA. Fluorescence measurements of intracellular Ca2+ concentration were performed on PC cells upon DHT (o/n) and testosterone (3 h) induction by calcium-imaging. B. The summary of 50 M and 500 M menthol-induced TRPM8 responses are represented graphically. Role of androgens, AR.