Up-regulation of steroid biosynthesis by retinoid signaling: Implications for aging

Abstract

Retinoids (vitamin A and its derivatives) are critical for a spectrum of developmental and physiological processes, in which steroid hormones also play indispensable roles. The StAR protein predominantly regulates steroid biosynthesis in steroidogenic tissues. We have reported that regulation of retinoid, especially atRA and 9-cis RA, responsive StAR transcription is largely mediated by an LXR-RXR/RAR heterodimeric motif in the mouse StAR promoter. Herein we demonstrate that retinoids are capable of enhancing StAR protein, P-StAR, and steroid production in granulosa, adrenocortical, glial, and epidermal cells. Whereas transient expression of RARα and RXRα enhanced 9-cis RA induced StAR gene transcription, silencing of RXRα with siRNA, decreased StAR and steroid levels. An oligonucleotide probe encompassing an LXR-RXR/RAR motif bound to adrenocortical and epidermal keratinocyte nuclear proteins in EMSAs. ChIP studies revealed association of RARα and RXRα with the StAR proximal promoter. Further studies demonstrated that StAR mRNA levels decreased in diseased and elderly men and women skin tissues and that atRA could restore steroidogenesis in epidermal keratinocytes of aged individuals. These findings provide novel insights into the relevance of retinoid signaling in the up-regulation of steroid biosynthesis in various target tissues, and indicate that retinoid therapy may have important implications in age-related complications and diseases.

Keywords: RAR; RXR; Retinoids; StAR expression; Steroid biosynthesis; Steroidogenic cells; cAMP/PKA.

Fig. 1

Effect of atRA on (Bu)₂cAMP stimulated StAR, P-StAR, CYP11A1, and steroid levels in KK-1, H295R, and A172 cells, and in HaCaT keratinocytes. These cells were treated without (Basal) or with atRA (10 μM), (Bu)₂cAMP (0.1 mM), or their combination, for either 6h (A and B) or 24h (C and D), as indicated, and subjected to cellular protein preparation. A172 and HaCaT cells were treated in the presence of SU-10603 (20 μM) and cyanoketone (5 μM). Representative immunoblots illustrate StAR, P-StAR, and CYP11A1 levels using 20-30 μg (A and B) or 60-70 μg (C and D) of total protein. Immunoblots shown are representative of 4-6 independent experiments. β-actin expression was assessed for loading controls. Accumulation of progesterone (A and B; bottom panels) and pregnenolone (C and D; bottom panels) in the media was determined from different groups and expressed as ng/mg protein. Data represent the mean ± SE of 4 independent experiments. Different letters above the bars indicate that these groups differ significantly from each other at least at p < 0.05. Note the different scales on the graphs.

Fig. 2

Overexpression of RARα and RXRα in 9-cis RA induced StAR promoter activity. KK-1 (A), H295R (B), A172 (C), and HaCaT (C) cells were transfected with pCMX, pCMX-RARα (RARα), and pCMX-RXRα (RXRα), within the context of either wild type −254/−1 bp StAR-Luc (Wt-LXR-RXR/RAR) or mutant (Mut-LXR-RXR/RAR) plasmid, as indicated, in the presence of pRL-SV40. Following 36h of transfection, cells were treated without (Basal) or with 9-cis RA (10 μM), for either an additional 6h (A and B) or 24h (C and D). Luciferase activity in the cell lysates was determined and expressed as StAR promoter activity, RLU (luciferase/Renilla). Data represent the mean ± SE of 3-5 independent experiments.

Fig. 3

Silencing of RXRα in 9-cis RA induced StAR protein and steroid levels. KK-1 (A), H295R (B), A172 (C), and HaCaT (D) cells were transfected with either 100 nM of negative control (scrambled) or specific human RXRα (RXRα) siRNA, as indicated. Following 36-48h of transfection, cells were treated without or with 9-cis RA (10 μM) for either an additional 6h (A and B) or 24h (C and D), and subjected to cellular protein preparation. Representative immunoblots illustrate expression of RXRα, StAR, and CYP11A1 in different treatment groups. Immunoblots are representative of 4-6 independent experiments. Accumulation of progesterone (A and B; bottom panels) and pregnenolone (C and D; bottom panels) in the media was determined and expressed as ng/mg protein (n = 4, ± SE). Different letters above the bars indicate that these groups differ significantly from each other at least at p < 0.05. Note the different scales on the graphs.

Fig. 4

Binding of the LXR-RXR/RAR motif in the StAR promoter to H295R and HaCaT NE in EMSAs, and association of RARα and RXRα with the StAR promoter by ChIP analyses. NE (10-15 μg) obtained from control, atRA (10 μM), (Bu)₂cAMP (0.1 mM), and atRA plus (Bu)₂cAMP treated H295R (A) and HaCaT (B) cells were incubated with the ³²P-labeled LXR-RXR/RAR probe. A major protein:DNA complex was observed with the LXR-RXR/RAR probe and H295R (A) and HaCaT (B) NE (lanes 1-7 in both cases). Protein:DNA binding was challenged with unlabeled DR5 RARE consensus sequence (lane 3) and, RARα (lane 6) and RXRα (lane 7) Abs (A and B). Cold competitor was used at 100-fold molar excess. Migration of free probes is shown in panels A and B. Data are representative of 3-4 independent experiments. ChIP assays were carried out as described under Materials and Methods (C and D). Crosslinked sheared chromatin obtained from control (None) and 9-cis RA treated groups was immunoprecipitated (IP) without or with anti-RARα and anti-RXRα Abs. Recovered chromatin was subjected to PCR analysis using primers specific to the proximal −255/−39 bp region of the StAR promoter, as specified under Materials and Methods. Representative autoradiograms illustrate the association of RARα and RXRα with the StAR promoter. Data shown are representative of 3 independent experiments.

Fig. 5

Expression of StAR mRNA in men and women skin tissues. De-identified, formalin fixed, paraffin-embedded skin specimens of various ages (14-86 years) and body locations were obtained from the Dermatology clinic at TTUHSC. Total RNA from these skin specimens was extracted and purified using the procedures described under Materials and Methods. StAR mRNA levels in different samples were determined by a semi-quantitative RT-PCR. A total of 106 skin specimens were analyzed and were made up of the following samples: elderly (37-86 years; 25 men and 24 women) and young (14-35 years; 26 men and 31 women).

Fig. 6

Relative levels of StAR mRNA in various inflammatory skin diseases. De-identified, formalin fixed, paraffin-embedded diseased and non-diseased men and women skin specimens were obtained from the Dermatology clinic at TTUHSC. Total RNA from these skin tissues was extracted and purified using procedures as described in the legend of Fig. 5 and under Materials and Methods. StAR mRNA levels in these skin specimens (a total of 87 samples were analyzed; 46 men and 41 women) were determined by a semi-quantitative RT-PCR.

Fig. 7

Effect of 9-cis RA on (Bu)₂cAMP-stimulated StAR mRNA, StAR protein, P-StAR, and pregnenolone levels in isolated epidermal keratinocytes. De-identified fresh skin tissues from elderly men (64-83 years; 5 different specimens) were obtained from the Dermatology clinic at TTUHSC upon surgery. Epidermal keratinocytes were isolated from these aged skin tissues following the procedures described under Materials and Methods. Primary cultures of isolated keratinocytes were treated without (Basal) or with 9-cis RA (10 μM), (Bu)₂cAMP (0.1 mM), or their combination, for 24h, as indicated, in the presence of SU-10603 (20 μM) and cyanoketone (5 μM). Following treatments, cells were processed for either total RNA extraction or cellular protein preparation. A representative autoradiogram illustrates expression of StAR mRNA in different treatment groups using a semi-quantitative RT-PCR (A). Integrated optical density (IOD) values of each StAR band was quantified and normalized with the corresponding L19 bands and presented below the autoradiogram. Representative immunoblots illustrate StAR, P-StAR, and CYP11A1 levels in different treatment groups using 60-70 μg of total protein in each lane (B). Autoradiogram and immunoblots are representative of 3-5 independent experiments. β-actin expression was assessed as a loading control. Accumulation of pregnenolone in the media was determined from different treatment groups and expressed as ng/mg protein C). Data represent the mean ± SE of 4 independent experiments. *, p < 0.05; ***, p < 0.01; vs. basal.