Temporal changes in gene expression in the skin of patients treated with isotretinoin provide insight into its mechanism of action

Abstract

Isotretinoin (13-cis RA) is the most potent agent in the treatment of acne. Insights into its mechanism of action can lead to drug discovery of alternative compounds with comparable efficacy but improved safety. The goal of this study is to compare the temporal changes in gene expression in the skin of acne patients after 1 week and 8 weeks of treatment with isotretinoin. Microarray analysis was performed on skin biopsies taken from eight acne patients prior to and at 8 weeks of treatment with isotretinoin. Results were compared with data obtained from seven acne patients biopsied at one week of treatment in a prior study. Distinctly different patterns of gene expression were noted. At 8 weeks, genes encoding extracellular matrix proteins were upregulated and numerous genes encoding lipid metabolizing enzymes were downregulated. At 1 week, genes encoding differentiation markers, tumor suppressors and serine proteases were upregulated. Only three genes were commonly downregulated. The temporal changes in gene expression in patient skin noted with isotretinoin substantiate many previously reported effects of isotretinoin and other retinoids, suggesting a model wherein isotretinoin induces apoptosis leading to reduced sebaceous gland size, decreased expression of lipid metabolizing enzymes and increased matrix remodeling during acne resolution.

Keywords: 13-cis retinoic acid; acne; sebaceous gland.

Figure 1

Sebaceous gland size is significantly reduced by isotretinoin at 8 weeks of treatment compared to one week. Hematoxylin and eosin sections of skin biopsies were taken from patients before and at approximately 8 weeks or 1 week of isotretinoin treatment. (A) A total of 16 sections at baseline and 12 sections at 8 weeks of treatment were taken from 8 patients and analyzed using image analysis software. The mean area (±SEM) of sebaceous glands in the baseline samples was 0.17 ± 0.04 mm² compared to 0.04 ± 0.07 mm² in the 8-week samples; a four-fold reduction in size (p = 0.009). (B) A total of 17 sections at baseline and 19 sections at 1 week of treatment were taken from six patients and analyzed using image analysis software. In contrast to 8 weeks of isotretinoin treatment, the mean area (±SEM) of sebaceous glands in the 1-week baseline samples was 0.23 ± 0.09 mm2 compared to 0.12 ± 0.02 mm2 after 1 week of treatment, which was not significant using a paired t-test (p = 0.16). Representative images from three patients at each time point are shown at original magnification of 100x. Magnification bars = 250 µm. *Initial publication: Journal of Investigative Dermatology advance online publication, 6 November 2008; doi:10.1038/jid.2008.338.

Figure 2

QPCR verifies the magnitude and direction of gene array changes. In order to verify the direction and magnitude of the changes in gene expression induced by isotretinoin in the gene array expression analysis, QPCR was performed using primers to select genes whose expression was significantly changed by 13-cis RA in the array analysis. Data represent the mean ± SEM of the fold-change in gene expression as determined by QPCR in four subjects compared to array analysis performed in eight subjects: 3β-hydroxysteroid dehydrogenase (3βHSD1), HMG CoA reductase (HMGCR), phospholipase A2 group 7(PLAG7), insulin induced gene 1 (INSIG), carnitine acyltransferase (CRAT) and zinc finger binding protein 145 (ZBTB16). Significant changes in gene expression with the microarray were identified using Significance Analysis of Microarrays (SAM) computer software and QPCR results were analyzed by the REST-XL software program; *p < 0.05 was considered significant for both analyses.

Figure 3

One week Clustering Diagram. Hierarchical clustering was used to compute a dendogram that assembled all samples and genes into a single tree. Normalized gene array expression data from each patient’s baseline and treatment biopsy was imported into dChip software version 1.3. Each row represents a single gene and each column represents a patient sample. B = baseline and A = after treatment. The color reflects the level of expression when compared to the mean level of expression for the entire 1 week biopsy set.

Figure 4

Temporal effects of isotretinoin.