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Retinoid-responsive transcriptional changes in epidermal keratinocytes.

Retinoid-responsive transcriptional changes in epidermal keratinocytes

Lee DD, Stojadinovic O, Krzyzanowska A, Vouthounis C, Blumenberg M, Tomic-Canic M.

J Cell Physiol, 2009, 220, 427-439

Retinoids play a major role in regulating growth, differentiation and apoptosis of many cell types, including keratinocytes, fibroblasts and sebocytes. The molecular mechanisms mediating these effects involve binding of the retinoid to ligand-activated nuclear hormone receptors forming dimers and subsequent binding to specific DNA sequences called retinoic acid response elements (RARFs) to regulate target genes [11. Therapeutic uses of retinoids include treatment of psoriasis, acne and some preneoplastic lesions and cancers [21. These benefits are limited by the side effects of scaling, erythema and dryness. The most efficacious acne treatment, 13-cis retinoic acid (RA) or Roaccutane[R], is well known for causing considerable improvement in very severe acne conditions (for example acne conglobata) but usage is also limited by its potential teratogenic effects. Therefore, it would be advantageous to be able to design retinoids which have the profound beneficial effects of retinoid treatment, while designing out the negative effects. Extensive work on identifying such retinoid compounds has not been successful to date. Understanding the downstream effects transduced by retinoids activating their receptors might help to provide insights into how better to develop such compounds and this study was designed to comprehensively define the transcriptional targets regulated by retinoids in human epidermal keratinocytes.

mRNA was obtained from cultured keratinocytes at 1, 4, 24, 48 and 72h of culture in the presence and absence of 1uM RA. mRNA was hybridised to Affymetrix gene chips HGU133A 2.0 arrays and genes were considered regulated if the expression levels differed by more than 1.62-fold relative to untreated controls at any time point. Regulated genes were clustered to look for specific pathways affected by RA according to the Gene Ontology (GO) pathways.

The Affymetrix gene microarrays used in this study allow for the analysis of 22,200 mRNAs simultaneously. Of these, approximately 12,900 genes were found expressed in keratinocytes and 3346 genes were regulated by RA. Keratinocytes responded to RA very rapidly with 134 genes induced and 181 suppressed at 1h, and maximum number of induced genes reaching 768 at 48h. The total number of suppressed genes continued to increase at each time point and at 72h (the final time point) had reached a peak of 979 genes. Two hundred and fifty-three genes showed a temporal regulation, being induced at some time points and suppressed at others; an example was the expression of cellular retinoic acid binding protein II (CRABP2), which was induced at 4h and suppressed after 24h.

Cluster analysis of the data showed that RA suppressed protein markers of cornification, biosynthesis of epidermal lipids, long chain fatty acids, cholesterol and sphingolipids. The pathways of RA synthesis, esterification and metabolism are activated, thus indicating that RA regulates its own bioavailability. Additionally, genes associated with cell cycle and apoptosis were also regulated as might be expected considering the influence of RA on keratinocyte differentiation and cell death. A comparison of the data with an analysis of genes regulated in psoriasis showed that RA influences many of the psoriasis-related genes.

This paper significantly extends our understanding of RA effects on human epidermal keratinocytes and shows the power of advances in gene technology when applied to skin research. The ability to investigate the genes expressed in the entire human genome in one experiment allows for a global investigation of all pathways modulated in this case through RA signal transduction pathways.

Another study investigating the effect of 13-cis RA on acneic skin was recently published as a Letter to the Editor in Journal of Investigative Dermatology [3]. These authors obtained punch biopsies from patients undergoing Roaccutane[R] treatment for acne. The methodology, similar to the study described above, used gene array analysis and found that 197 genes were significantly upregulated and 587 genes were significantly down regulated by in vivo administration of retinoid treatment at 8 weeks compared to baseline. The latter group of genes included those genes regulating steroid metabolism, cholesterol and fatty acids. Upregulated genes included those responsible for remodelling skin, such as structural proteins collagen and fibronectin. A temporal response was observed when gene array data from 1 week of treatment was compared to 8 weeks. Only three genes were found to be common to both data sets. The temporal observation is consistent with the in vitro data for the human epidermal kcratinocyte study discussed above.

These two studies show the power of applying gene chip technology to skin research to take a look at the entire pattern of gene expression--both upregulation and downregulation--effected by an ingredient at different time points. To date, experimental work has had to proceed by investigating one or just a few proteins or genes. The current advances in experimentation provide a huge amount of data, requiring detailed analysis, and eventually providing insights not thought to be possible, into how skin functions.


[1.] Chambon P. A decade of molecular biology of retinoic acid receptors. FASEB J, 1996, 10. 940-954.

[2.] Rook AH, Jaworsky C. Nguyen T et al. Beneficial effect of low-dose systemic retinoid in combination with topical tretinoin for the treatment and prophylaxis of premalignant and malignant skin lesions in renal transplant recipients. Transplantation, 1995, 59. 714-719.

[3.] Nelson AM. Zhao W, Gilliland KL, Zaenglein AL, Liu W, Thiboutot DM. Isotretinoin temporally regulates distinct sets of genes In patient skin. J Invest Dermatol, 2009, 129. 1038-1042.
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Author:Knaggs, Helen
Publication:Clinical Dermatology
Date:Jun 1, 2009
Previous Article:Topical tretinoin therapy and all-cause mortality.

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