Isotretinoin Temporally Regulates Distinct Sets of Genes in Patient Skin

TO THE EDITOR The goal of this study is to gain insight into putative pathways by which 13-cis retinoic acid (13-cisRA) improves acne by comparing the temporal changes in gene expression in the skin of acne patients. Gene array analysis and immunohistochemistry were performed on skin biopsies from patients at baseline and after 8 weeks of isotretinoin therapy and compared to data obtained at 1 week (Nelson et al., 2008). As 13-cisRA drastically decreases sebaceous gland size after 16 weeks (Goldstein et al., 1982); we chose an 8-week time point to examine changes in skin histology and gene expression. All protocols were approved by the Institutional Review Board of The Pennsylvania State University College of Medicine and were conducted according to the principles outlined in the Declaration of Helsinki. Eight patients who were prescribed isotretinoin for their severe acne enrolled in the study after giving informed consent and 5-mm punch biopsies of uninvolved skin were taken from their upper backs at baseline and 8 weeks of treatment. Demographic data are presented in Figure 1a. After 8 weeks, sebaceous gland size was reduced by 76% (4.17-fold) compared to baseline (P=0.009) (Figure 1b and d). At 1 week, glands were decreased by approximately 49% (not significant; Figure 1c and d). Gene array expression analysis was performed on biopsies at baseline and after 8 weeks of therapy. Using a false discovery rate of 0.05 corresponding to a 5% chance of false positives, 197 genes were significantly upregulated and 587 genes were significantly downregulated. Select genes are listed in Table 1 (complete list, S1). The preponderance of genes that were downregulated at 8 weeks involve the metabolism of steroids, cholesterol, and fatty acids, consistent with the known decreases in sebum production induced by 13-cisRA. Upregulated genes at 8 weeks encode structural proteins such as collagens and fibronectin, consistent with the known effects of retinoids in rebuilding extracellular matrix (Weiss et al., 1988; Griffiths et al., 1993; Kafi et al., 2007). In comparison, at 1 week, significant increases in expression were noted in genes broadly categorized as tumor suppressors, protein processors, and genes involved in transfer or binding of ions and small molecules (Nelson et al., 2008). Only three genes were found in common between the 1-week and 8-week gene arrays illustrating that isotretinoin regulates the temporal expression of separate and distinct groups of genes. These three genes were downregulated 2- to 5-fold and include solute carrier family 26, member 3 (SLC26A3); phospholipase A2, group VII (PLA2G7); and phosphodiesterase 6A (PDE6A). PLA2 family members hydrolyze membrane phospholipids to generate arachidonic acid, a known inflammatory mediator (Yedgar et al., 2006). Retinoids, including 13-cisRA, inhibit PLA2 activity and prevent arachidonic acid release in human synovial fluid which may explain its anti-inflammatory properties (Hope et al., 1990). PDEA6 encodes the cyclic-GMP-specific PDE6A α-subunit expressed in cells of the retinal rod outer segment. Mutations in PDE6A occur in autosomal recessive retinitis pigmentosa, which is associated with night blindness (Wang et al., 2001). Although night blindness is a known potential side effect of isotretinoin treatment, it has not been linked with changes in retinal expression of PDE6A. To determine if 13-cisRA preferentially influences a particular subset of genes, genes were categorized according to their “gene ontology”, “protein domains”, “chromosomal location”, and “pathway” annotations using dChip software. For the 8-week data, 21 protein domains and 10 pathways were significantly enriched (P<0.001). Protein domains for collagen and fibronection were significantly increased. Domains for steroid metabolizing enzymes were decreased. Ten pathways were downregulated; each is linked to lipid metabolism, further supporting 13-cisRA's role in sebum suppression (Table 1). At 1 week, five gene ontology terms, three protein domains (lipocalin related, calcium binding, and latexin), and one chromosomal location (1q21) were enriched. The 1q21 locus is the site of the epidermal differentiation complex. Five genes on this locus are affected by isotretinoin: cellular retinoic acid binding protein 2 (CRABP2), S100A2, S100A7, S100A9, and involucrin (IVL). Increased CRABP2 expression supports a previous study demonstrating increased CRABP2 levels in the patients' sebaceous glands following 3–16 weeks of isotretinoin therapy (Sitzmann et al., 1995). The specific S100 proteins whose genes are upregulated are induced by oxidative or inflammatory stress. S100A7 (psoriasin) has antimicrobial effects against Proprionibacterium acnes and chemoattractant activity (Eckert et al., 2004; Lee et al., 2008). Initial upregulation of S100 proteins by 13-cisRA could relate to the “acne-flare” observed in some patients receiving retinoids. The clustering of retinoic acid responsive genes on 1q21 raises the question of whether each gene is individually regulated or whether retinoic acid affects epigenetic regulation of the epidermal differentiation complex through a locus control region, similar to the β-globin gene locus (Mahajan et al., 2007). The promoter regions of all genes whose expression changed significantly at 1 week or 8 weeks were analyzed for retinoid receptor consensus sequences using Transcriptional Element Search System and TRANSFAC databases (Wingender et al., 1996; Schug and Overton, 1997). Approximately half of the genes in both data sets contain retinoic acid receptor response elements within their promoters, suggesting possible direct gene regulation through classical nuclear retinoid receptors. Although 13-cisRA itself does not activate RAR or RXR, it is isomerized to all-trans retinoic acid, which can directly activate these receptors (Allenby et al., 1993; Tsukada et al., 2000). However, not all gene promoters contained retinoid receptor consensus sequences, indicating that indirect regulation is possible. This study demonstrates that 13-cisRA markedly decreases sebaceous gland size by 8 weeks with a trend noted at 1 week. These data support the observation that sebum secretion is markedly reduced by 13-cisRA as early as 2 weeks (Stewart et al., 1983; Hughes and Cunliffe, 1994). The temporal changes in gene expression within patient skin induced by isotretinoin substantiate many clinical, histological and biochemical effects of isotretinoin noted in previous studies. These data combined with recent findings regarding isotretinoin's effects on apoptosis, cell cycle arrest, and induction of lipocalin 2 expression suggest that after the initial induction of apoptosis and cell cycle arrest within the sebaceous gland; the skin adopts a wound healing-like pattern of gene expression and subsequently undergoes substantial repair and remodeling as evidenced by increased expression of extracellular matrix proteins (Nelson et al., 2006, 2008). The mechanism by which isotretinoin induces long-term or permanent remission of acne however remains to be determined. The authors state no conflict of interest. We thank Amy Longenecker and Silvia Gosik for coordination of patient procedures. This study was supported by NIH NIAMS R01 AR047820 to DMT and NIH General Clinical Research Center Grants M01RR010732 and C06RR016499 to the Pennsylvania State University College of Medicine and by the Jake Gittlen Cancer Research Foundation and the Department of Dermatology at the Pennsylvania State University College of Medicine. SUPPLEMENTARY MATERIAL Table S1. Significantly changed genes in patients after 8 weeks isotretinoin treatment.