Abstract

Retinoid Regulation of Gene Transcription

Retinoids, i.e., natural and synthetic analogs of vitamin A, are known to affect proliferation and differentiation of a variety of cells.¹ Most biological activities exerted by retinoids are carried out through binding to specific nuclear retinoid receptors, belonging to a superfamily of receptors which includes steroid, vitamin D, thyroid, peroxisome proliferator-activated and a number of other receptors.^2–4 Retinoic acid receptors (RARα, β, γ) are ligand-dependent transcription factors which are activated by all-trans retinoic acid (RA). Another family of receptors, retinoid X receptors (RXRα, β, γ), has 9-cis RA as a natural agonist.² Usually RAR and RXR proteins form heterodimer complexes.² Both RARs and RXRs contain several functional domains of two of which are important to achieve gene transcription: the DNA-binding E-domain which mediates the binding of the receptors to the target-genes and the ligand-binding C-domain.³

In human epidermis, the predominant RAR isoforms are RARγ but low levels of RARα is also present.⁵ However, the protein levels of RXRs are normally higher than those of RARs with RXRα being the most abundant receptor.⁵ The most common heterodimer complex in human epidermis appears to be RARγ/RXRα,⁵ which are localized to the suprabasal layers whereas the RARα/RXRα heterodimer is dominating in the basal layer.^6,7 The RAR/RXR heterodimers are localized to the nucleus where they bind to specific DNA regulatory sequences; retinoic acid response elements (RAREs), which are usually found in the 5′ upstream region of the target genes.³ The heterodimer complexes are in their unliganded form accompanied by certain co-repressor molecules which inhibit transcription of target genes (reviewed in ref. ⁸). When RA binds to RARs a conformational change is induced in the receptor which dissociates the co-repressors and permits the binding of co-activators to the complex.⁸ It also gives RXR the opportunity to bind its ligand, which could further induce the RAR-mediated transcription of the retinoid-regulated gene.^8–11

Another mechanism by which RA and its receptors regulate the differentiation and proliferation of epidermal keratinocytes is by acting as an antagonist of activating protein-1 (AP-1).¹² AP-1 consists of combinations of fos and jun heterodimers, which are well established regulators of keratinocyte differentiation.^13,14 The activity of AP-1 is determined by the composition of the heterodimer complexes¹⁵ and by posttranscriptional modifications of jun and fos proteins, of which phosphorylation is most important.^16,17

Also, reciprocal interactions between RARs and nuclear factor kappaB (NFκB) has been shown, which can be of relevance when designing retinoids for chemo-preventive and anti-inflammatory interventions.^18,19 NFκB transactivation of target genes requires that heterodimers are formed between class I NFκB proteins (p105/p50 and p100/p52) and class II proteins (RelA, RelB and c-Rel), although some homodimer combinations also are reported to activate gene transcription.²⁰ It is important to note that the antagonistic effects performed by retinoids on AP-1 and NFκB activity do not require RAREs in the target genes.

Keratin Expression in Normal Human Skin and Cultured Keratinocytes

Recent reports have described the tissue-specific expression of type I (KRT 9–20) and type II (KRT 1–8) keratin genes, the formation of keratin filaments and the role of other proteins in keratin filament assembly.^21–26 In normal human epidermis, heterodimers of keratin 5 (K5) and K14 form the cytoskeleton of undifferentiated cells in the basal layer, which is replaced in suprabasal cells by K1 and K10, accompanied by K2 in the granular layer (Fig. 1).²¹ In palms and soles, the type I keratin K9 is expressed in suprabasal layers.²¹

In cultured keratinocytes, the keratin expression profile differs from normal epidermis. Furthermore, the profile also depends on the culture conditions used. Keratinocytes grown in serum-containing medium and in the presence of feeder cells, express K5, K6, K14, K16, K17 and low levels of K13, K15 and K19,²⁷ whereas in serum-free low-calcium medium containing epidermal growth factor (EGF) they show a more restricted mRNA expression, including KRT5, KRT13, KRT14, KRT6 and KRT17.^28–30 Upon differentiation of keratinocytes, e.g., by raising the calcium level with or without the addition of growth factors (EGF and fibroblast growth factor-10) or adding the EGF-receptor inhibitor PD153035, expression of the differentiation-related keratins K1 and K10 are induced in a subpopulation of cells.^28,30–35

Retinoid Regulation of Keratins in Cultured Keratinocytes and Normal Skin

Several keratins have been reported to be retinoid regulated in cultured human epidermal keratinocytes and normal human skin. As shown in Table 1, the effect of retinoids on keratins in vitro and in vivo is paradoxical. In vitro, retinoids reduces the expression of KRT5, KRT14, KRT6, KRT1 and KRT10 in cultured keratinocytes,^36–40 whereas KRT7, KRT13, KRT15 and KRT19 are induced.^27,38,40

By contrast, a different keratin profile is observed when retinoic acid is added to reconstructed human skin in vitro or topically to human skin in vivo. Almost similar effects are generated in the two multilayered tissues including increased expression of K4, K6, K13, K16, K17 and K19 whereas no effects are observed on K5, K14, K1 and K10 proteins.^41–47 On the other hand KRT1, KRT2, KRT10 and KRT14 mRNA's, are downregulated by retinoid treatment.^{42,43,45–47} In fact, a number of these keratin genes, e.g., KRT5, KRT6, KRT14 and KRT17, have been reported to carry positive or negative RAREs,^48–50 whereas in KRT2 and KRT4 genes no RAREs have been identified so far. Thus, it is likely that these latter two retinoid-regulated keratins are among the many genes that are indirectly influenced by retinoids through mechanisms that do not require RAREs.⁵¹

Clearly, keratin gene expression is affected by retinoids in many complex ways, and it is obvious that classical RARE-activation by RAR/RXR heterodimers is not always involved.^48,50–55 Among the indirect effects associated with the regulation of several keratin genes are interactions with AP-1 and NFκB signaling pathways (see above). Recently it was shown in mouse skin that topical application of the natural compound sulforaphane induced K16 and K17 via AP-1 activation.⁵⁶ Furthermore, using a co-transfection strategy others reported that the upstream regulatory regions of KRT5, KRT6 and KRT14 genes are activated by AP-1 (c-Fos and c-Jun).⁵⁷ On the other hand, certain NFκB proteins, suppressed the KRT5 and KRT14 promoters while the KRT6 promoter was activated by the p65 NFκB protein.⁵⁷ These results suggest that keratin gene expression might depend on interplay between retinoid signaling with that of AP-1 and/or NFκB proteins, an interplay which is unique to each affected keratin. In the case of KRT4, KRT13 or KRT2 genes no retinoid response element has been reported yet. However, in the case KRT2, an anti-AP-1 mechanism was ruled out since CD2409, an anti-AP1 retinoid without affinity for RARs, did not alter the mRNA expression of this gene (Virtanen M, et al. unpublished observation).

Another possibility, as in the case of regulation of KRT4 mRNA, is that AP-2 activation is involved. An AP-2 site in the KRT4 gene was found that increased the activity of a reporter gene in the HaCaT cell line, although an indirect effect was not completely ruled out.⁵⁸ It is also important to keep in mind that retinoid regulation of this and other keratin mRNAs could also involve post-transcriptional changes in mRNA stability as previously noted for KRT19.⁴⁰

Results from a recent study also add retinoylation (acylation by RA) to the list of putative mechanisms regulating keratin expression. In this study, retinoic acid was reported to bind to a number of proteins, including K10 and K16.⁵⁹ However, the physiological importance of these direct interactions needs still to be further explored.

Keratins are Regulated by Ligands with RAR-Specificity

The pronounced upregulation of KRT4 and KRT13 mRNA and downregulation of KRT2 by retinoic acid in vivo^42,45,47 has also been verified in organotypic skin models.^60,61 Since the effects in vivo and in reconstructed skin in vitro are almost similar, the latter model can be used to explore the role of the various isoforms of RARs in retinoid regulation of keratins. A pre-requisite is that the expressions of RARα, RARγ and RXRα in organotypic skin are similar to human skin which was recently shown.⁶¹ This suggests that RARα and RARγ, but not RABβ mediate the effects of retinoids on keratin expression.⁶¹

Over the last 20 years a number of synthetic retinoids that are highly selective for a single RAR-isoform or two or more isoforms have been developed. In a recent study, it was found that the most potent compounds affecting keratin gene expression were two RARα agonists and a pan-RAR agonist.⁶¹ One of the two RARα agonists, Am580/CD336, has previously been shown to downregulate VEGF and NOS-2 with similar efficiency as a pan-RAR agonist.^62,63 However, the role of RARα agonists as potent modulators of gene transcription in keratinocytes is obviously not a general phenomenon, since Am580/CD336 was less effective than retinoic acid in increasing the expression of other retinoid-regulated genes in cultured keratinocytes.⁶⁴

Keratin Mutations in Epidermolysis Bullosa Simplex (EBS) and Epidermolytic Ichthyosis (EI)

Almost two decades ago it was disclosed by several groups that EBS and EI are caused by mutations in KRT5 or KRT14 and KRT1 or KRT10 genes, respectively.^65,66 The disease-causing mutations are point and dinucleotide mutations and deletions.^67,68 The mutations are usually located in the conserved domains of the α-helix (1A and 2B),²¹ but can also be found in the non-helical linker regions.⁶⁹ These dominant-negative mutations cause destabilization of the cytoskeleton since the mutant keratin forms a defective heterodimer with its keratin partner. This may cause cells to become less resistant to mechanical trauma and other types of stress, and result in epidermolysis in the basal or suprabasal parts of epidermis in EBS and EI patients, respectively.^21,70

Retinoid Therapy for Keratinization Disorders of the Skin

The use of retinoids in keratinization disorders depends on the severity of skin manifestations and the patients' general health, sex, age, etc.⁷¹ Mild forms of ichthyosis can be treated with topical retinoids such as RA (tretinoin) and tazarotene whereas severe cases respond better to oral therapy with synthetic retinoids (acitretin or isotretinoin).⁷¹ Despite being in use for a long time the exact mechanism of action in these disorders is not completely understood. The general hypothesis is that retinoids normalize keratinocyte differentiation. Other possible mechanisms of retinoids could include downregulation of desmosomal proteins, an anti-proliferative effect or regulation of lipid synthesis, growth factors and cytokines.^72–74

In EI, an alternative hypothesis is that retinoids act through the regulation of suprabasal keratins. This is supported by the fact that EI-patients with KRT10 mutations respond to retinoid treatment, whereas patients with KRT1 mutations do not respond of unknown reasons.⁷⁵ One explanation might be that patients with KRT10-mutations are not as sensitive to the negative effect of retinoid-induced downregulation of both type II keratins (K1 and K2) in the suprabasal compartment as patients with KRT1-mutations⁷⁵ or that the mechanism involve the recently reported interaction between retinoids and K10.⁵⁹ However, it would be tempting to suggest that RARα ligands would be suitable as intervention for epidermolytic keratinopathies due to either dominant negative K2 mutations (i.e., Ichthyosis bullosa of Siemens; IBS) or K1/10 mutations (epidermolytic ichthyosis) where upregulation of K4/13 might compensate for the retinoid-induced reduction in K1/K10. In the case of IBS, pharmacological silencing of the mutated KRT2 gene by retinoids or Retinoic acid metabolism blocking agents (RAMBAs⁷⁶) should hypothetically be the perfect treatment and IBS-patients actually respond well to low-dosage of synthetic retinoids.⁷⁸ However, in healthy control skin it has been reported that retinoids or RAMBAs reduces the KRT2 mRNA levels, but less effect was observed at the protein level, thus suggesting involvement of other mechanisms than solely downregulation of KRT2.^47,77

It is well established that a common adverse effect during retinoid treatment is skin irritation. Interestingly, the retinoid-induced skin irritation has been shown to be a receptor-mediated process involving activation of RARβ and RARγ, but not RARα,^79,80 which suggests that RARα agonists could be a better therapeutic option than RARβ/γ agonists from a clinical perspective with respect to both efficacy and tolerance.

In conclusion, the regulation of keratins by retinoids in human keratinocytes is complex and putatively a RARα-mediated event. An improved knowledge of keratin regulation may be of considerable significance under both physiologic and pathological conditions as well as during retinoid therapy of disorders of keratinization.

Acknowledgments

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