Somatic gene therapy offers, ultimately, a promise to treat many inherited disorders; however, not all genetic diseases are amenable to this approach. Replacing a deficient enzyme or a signaling protein in a cell type that can easily repopulate the affected organ is conceptually straightforward (14
). It is much more difficult to treat a structural protein mutation in a tissue where the stem cells are concealed, such as the epidermis. While skin can be used to deliver a missing protein systemically, treating inherited skin diseases with replacement gene therapy, targeting the entire organ, is a much more difficult task and calls for alternative approaches (13
). At the same time, in gene families, one member can often substitute for another mutated or missing one, which commonly and surprisingly leads to a lack of a phenotype in knockouts of genes thought to be essential. Indeed, a whole section in this journal, “Mammalian genetic models with minimal or complex phenotypes,” is devoted largely to such phenomena. These considerations led us to hypothesize that increasing the expression of one structural protein, K15, could compensate for another, mutated protein of the same family, K14.
Keratins are a family of about 30 proteins that form intermediate filament in epithelial cells. Obligate heteropolymers of a type I and a type II subunit, they contribute to the structure and strength of the cytoskeleton (49
). Mutations in keratin genes cause keratinopathies, inherited diseases of the skin and its appendages (39
). For example, mutations in K5 or K14 genes give rise to epidermolysis bullosa simplex (EBS), those in K1, K2e, or K10 give rise to epidermolytic hyperkeratosis, those in K6, K16, or K17 give rise to pachyonychias, etc. Gene therapy approaches that replace the mutant gene are not feasible for treatment of keratinopathies because the entire integument needs to be treated. Therefore, searching for an alternative to gene therapy for EBS, we hypothesized that boosting the expression of K15 may compensate for the disrupted K14 and restore the mechanical strength of the epidermal basal layer. Increasing the expression of a related protein from the same gene family to compensate for the mutated one is a novel paradigm in gene therapy for inherited disorders and could offer real promise in the case of EBS.
The mitotically active keratinocytes of all stratified squamous epithelia are characterized by their contact with the basement membrane and expression of keratins K5 and K14 and, less abundantly, keratin K15 (8
). Keratin K15 belongs to the “acidic” or type I keratin family and assembles into a keratin filament network paired with K5, its “basic,” type II keratin expression partner. In response to unknown stimuli, the basal cells are triggered to detach from the basement membrane, initiate their migration through the suprabasal layers, and terminally differentiate, ending transcription of K5, K14, and K15 while inducing a new sets of differentiation-specific keratins (18
). The differentiation of basal keratinocytes is also accompanied by changes in expression of several transcription factors, such as C/EBP, AP-1, and NF-κB (34
Studies with mice with a deletion of the K14 gene have shown that K15 can be a major component of the basal keratin network of stratified, nonkeratinizing epithelia, protecting them from mechanical damage in the absence of K14 (30
). Consequently, the blistering in patients with EBS due to a mutation in K14 is less severe in internal stratified epithelia, e.g., the esophagus, than in cornified epithelia, e.g., the epidermis. Such differences are not present in EBS patients with mutations in K5 (11
). Elegant transgenic experiments have shown that decreased expression of the mutant keratin 14 relative to the healthy allele results in normal morphology and function of the skin (9
). The mechanisms of regulation of K15 expression thus become of major importance because increasing the content of K15 could significantly alleviate the symptoms in EBS patients with a mutation in K14.
The molecular mechanisms that control the expression of the K15 keratin gene are still unknown. Human K15 is encoded on chromosome 17 (3
). The K14/K15 ratio can change dramatically during postnatal development (30
). Under hyperproliferating conditions, in which keratinocytes are activated, the K15 protein and its mRNA are suppressed, suggesting that K15 expression may not be compatible with the activated phenotype (16
). Transcription of K15 appears to be suppressed by transforming growth factor beta, tumor necrosis factor alpha (TNF-α), epidermal growth factor, and keratinocyte growth factor in HaCaT cells (59
With this in mind, we set out to clone the promoter of the K15 gene and to determine the molecular regulators of its expression. We were particularly interested in those regulators that can be affected by extracellular stimuli. These include nuclear receptors for retinoic acid, thyroid hormone, and glucocorticoids. We have shown previously that nuclear receptors for retinoic acid, thyroid hormone, and glucocorticoids and their ligands play an important role in regulating keratin synthesis (40, 41
). Specifically, the receptors and their ligands suppress K5 and K14, the basal keratins, and K6, K16, and K17, the inflammation- and wound healing-associated keratins. Therefore, we hypothesized that retinoic acid, thyroid hormone, and glucocorticoids are involved in the regulation of K15 gene expression in the epidermis.
We also examined the regulation of K15 expression by C/EBP, AP-1, NF-κB, and STAT transcription factors because these also respond to extracellular stimuli (1
). C/EBP-α and C/EBP-β are found in the suprabasal layers (34
), the AP-1 proteins, JunB, JunD, and c-Fos, are found in the basal and granular layers, while Fra-1 and Fra-2 are in basal cells (44
). NF-κB, an activation-associated transcription factor, has antiproliferative effects in the skin (12
), while STAT-1 is activated by gamma interferon (IFN-γ) and induces transcription of keratin K17 and other genes (21
). We have found that the C/EBP, AP-1, NF-κB, and STAT proteins regulate expression of several epidermal keratin genes, including K5, K6, K14, and K17 (21
). Taken as a whole, these results led us to hypothesize that the C/EBP, AP-1, NF-κB, and STAT-1 proteins might be good candidates for regulation of K15 transcription.
To explore regulation of K15 transcription, we have used DNA-mediated cell transfection, electrophoretic mobility shift assays (EMSA), and DNase I footprinting of the K15 promoter. We have shown that the transcription of the K15 keratin gene is regulated by the receptors for thyroid hormone, retinoic acid, and glucocorticoids. C/EBPβ, AP-1, and IFN-γ are activators of K15 expression, while NF-κB is a suppressor. Thus, K15 promoter activity is, uniquely among keratin genes, increased by both IFN-γ and thyroid hormones. Furthermore, IFN-γ and thyroid hormones increase the amount of K15 protein produced in an ex vivo skin model. We expect that our findings regarding regulation of K15 expression may lead to a better understanding of the role of K15 keratin in normal and pathological conditions and perhaps to treatments of diseases such as EBS based on modulating keratin gene expression.