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Transcriptional Regulation of Keratin Gene Expression

*.

* Corresponding Author: The Departments of Dermatology and Biochemistry and The Cancer Institute, NYU School of Medicine, 550 First Avenue, New York, New York, U.S.A. Email: blumem01@med.nyu.edu

Keratin synthesis is regulated at the level of transcription. Each keratin genes appears to be regulated by a characteristic constellation of transcription factors and DNA binding sites. Often these occur in clusters and complexes, providing a mechanism for fine-tuning the expression levels. Most commonly, the important regulatory sites are found in the promoter regions, infrequently coding and downstream sequences also play a role. Transcription factors Sp1, AP1 and AP2 are important components in regulation of many keratin genes, and the nuclear receptors for retinoic acid and thyroid hormone also regulate majority of keratins. In addition, the expression of most keratin genes can be modulated by extracellular signals, such as growth factors. Universal, or general regulators for all keratin genes have not been found; apparently each keratin protein has its own, characteristic circuits and machinery for regulation of expression.

Introduction

Since the first cloning of a keratin gene, just over 20 years ago,1,2 we have come a long way toward understanding their structure, function and regulation. The control of keratin expression occurs primarily, perhaps exclusively, at the transcriptional level. Therefore, several groups have worked very hard on elucidating the transcriptional regulation of keratin gene expression. Keratins K5, K6, K16 and K18 have been particularly well explored, others, e.g., K7 and hair keratins, await their turn. A common thread that connects the transcriptional regulation of keratin genes is that their promoters contain complex sites that simultaneously bind a multitude of transcription factors (Table 1). Many transcriptional factor binding sites in keratin gene promoters have been mapped, and their functional significance ascertained. However, the overall picture of the mechanisms and circuits that regulate keratin gene expression is not known. At present, we see many trees, but not yet the forest. Still, many details are known, which combined with systematic genomic studies, promises that a comprehensive picture will appear before long.

Table 1. Transcription factors shown to regulate keratin genes.

Table 1

Transcription factors shown to regulate keratin genes.

Transcriptional Regulation of Keratin K5 Expression

A common feature of all epithelial cells is the presence of keratin proteins that assemble into an intermediate filament cytoskeletal network. Whereas other cell types often use a specific master transcription factor to coordinate cell type-specific transcription, analysis of transcriptional regulation of keratin genes suggests that specific groupings of widely expressed transcription factors, acting on clusters of recognition elements in the promoter regions, confer epithelia-specific transcription. Keratins K5 and K14 form the cytoskeletal intermediate filament network in mitotically competent basal cells in all stratified epithelia, and therefore have received extensive attention focused on the mechanisms of their regulation.

To initiate analysis of the protein factors that interact with the human K5 keratin gene upstream region, we have used gel-retardation and DNA-mediated cell-transfection assays. A cluster of three sites that binds five transcription factors was found in the promoter of the human K5 keratin gene.3,4 Within this cluster, an unusual Sp1 site binds the Sp1 transcription factor and two additional proteins. Flanking the Sp1 site are an AP2 site and another sequence that binds a transcription factor. Similar clusters of recognition sites for the same transcription factors have been also identified in other keratin genes. DNA-protein interactions at two of the sites apparently increase transcription levels, at one decrease it, while the importance of the remaining two sites is, at present, unknown. In addition, the location of the retinoic acid and thyroid hormone nuclear receptor action site has been determined, and it involves a cluster of five sites similar to the consensus recognition elements. The complex constellation of protein binding sites upstream from the K5 gene probably reflects the complex regulatory circuits that govern the expression of the K5 keratin in mammalian tissues.5 Such clusters may play a role in epithelia-specific expression of keratins.

In transgenic mice, as few as 90 bp of the human K5 promoter still directed expression to stratified epithelia.6 However, the truncated K5 promoter expression was not limited to the basal layer. A 6Kb segment of the 5' upstream K5 gene directed proper basal cell-specific expression in all stratified epithelia. An open chromatin region containing a DNase I-hypersensitive site within the 6 kb of 5' upstream regulatory sequence acted independently to drive abundant and keratinocyte-specific reporter gene activity in culture and in transgenic mice. A 125-bp segment of this element is an independent strong enhancer element with keratinocyte-specific activity in vivo. Its activity is restricted to a subset of cells located within the sebaceous gland. The adjacent segment can suppress the sebocyte-specific expression and induce expression in the inner root sheath. Thus, the K5 gene expression is determined by multiple regulatory modules, which may contain AP-2 and/or Sp1/Sp3 binding sites as well as additional sites that determine cell type specificity.7

Similar regulation mechanism regulate K5 expression in other organism; for example, the 5' upstream region located between the cap-site and nucleotide -605 of the bovine K5 gene was found to contain the cis-regulatory DNA elements involved in the cell-type-specific expression. These elements enhanced the specific expression in the epithelial cells that express the endogenous gene, but not in cells that do not.8 Analogous epithelium-specific expression was also observed in murine keratinocytes, suggesting that similar regulatory mechanisms have been conserved through evolution. The 5.2 kilobases preceding the gene contain the regulatory sites responsible for the cell type-specific expression of bovine K5. A strong enhancer is located between positions -762 and —1009.9 The only regulatory element found in this enhancer by electrophoretic mobility shift, competition, and footprinting experiments is a consensus AP-1 site. Mutation of this site abolishes the activity of the enhancer and reduces to 25% the activity of the 5.2-kilobase upstream promoter region. Surprisingly, although the AP-1 sequence presents indistinguishable footprints in all cell types tested, the enhancer is active only in some of them. Furthermore, an oligonucleotide containing the AP-1 region is active in epithelial cells lines but not in fibroblasts, suggesting that this region could constitute an epithelium-specific AP-1 element. Thus the regulation of K5 keratin gene by AP-1 must be complex and different from other suprabasal, AP-1-regulated cellular and viral genes.

The tissue specificity of the bovine K5 expression was used ingeniously to produce strict conditional expression of genes in the mouse epidermis. Transgenic mouse lines were generated in which the tetracycline-regulated transcriptional transactivators, tTA and rTA, are linked to the bovine keratin 5 promoter.10 When crossed with the tetOlacZ indicator line, the K5/tTA line induced beta-galactosidase enzyme activity in the epidermis at a level 500-fold higher than controls, and oral and topical administration of doxycycline caused a dose- and time-dependent suppression of beta-galactosidase mRNA levels and enzyme activity. Histochemical analyses localized the beta-galactosidase expression to K5 positive tissues, i.e., the basal layer of the epidermis and the outer root sheath of the hair follicle. The K5-dependent tetracycline regulatory system produces effective conditional gene expression in the mouse epidermis, and has been used to suppress and activate foreign genes specifically in the basal layer of the epidermis.

Regulation of K14

A construct containing 300 base pair segment corresponding to the promoter region of a human K14 gene was introduced into various mammalian cell lines and primary cultures.11 The 300 base pair segment was active in all epithelial cells, including transformed simple epithelial cells, cell lines derived from stratified epithelia as well as primary cultures of epithelial cells, but it was inactive in all nonepithelial cells tested including fibroblasts and melanocytes. Using a series of deletions, an essential function was localized within a 40 bp sequence, thus identifying the keratin gene promoter that is sufficient to confer epithelial-specific expression.11

A much larger construct containing approximately 2.500 kb of the human K14 keratin gene generated tissue-specific and differentiation-specific expression in transgenic mice.12 Four DNase I-hypersensitive sites are present in the 5' regulatory sequences of the K14 gene in cells where the gene is actively expressed.13 Two of these sites are conserved in position and sequence within the human and mouse K14 genes. A novel 700-bp regulatory domain encompassing these sites is sufficient to confer epidermis-specific activity to a heterologous promoter. A 125-bp DNA fragment encompassing DNase I-hypersensitive site-II harbors the majority of the activity in vitro, and AP-1, ets, and AP-2 proteins orchestrate the keratinocyte-preferred expression.

The suprabasal suppression of the K5 and K14 gene expression may depend on the POU homeodomain factors Skn-1a and Tst-1, because K14 mRNA expression persists in suprabasal cells in Skn-1/Tst-1 double knockout mice. Both Skn-1a and Tst-1 repress the K14 promoter, with the POU domain being sufficient for repression.14 DNA-binding defective mutants of Skn-1a and Tst-1 are as effective at mediating repression as the wild-type proteins. A 100-base pair sequence, lacking POU-binding sites, adjacent to the transcription start site of the K14 gene is sufficient and required for repression by Skn-1a, suggesting that protein-protein interactions rather than direct DNA binding effect the repression. (CBP)/p300 coactivators activate K14 gene expression and interact directly with the POU domain of Skn-1a, suggesting that POU domain factors repress K14 gene expression by interfering with CBP/p300.

When we compared the functions of epidermal keratin genes, K5, K6, K10 and K14 by transfection into nonepithelial and transformed epithelial cell lines, as well as in primary cultures of cells derived from simple and stratified epithelia, we found that the four promoters were functional only in epithelial cells.15 While the promoter for the K14 gene was active in all epithelial cells tested, its basic-type partner, K5, and the promoter for the hyper-proliferation-associated K6 were active only in primary cultures of stratified epithelia. The promoter for the epidermal differentiation-specific K10 keratin gene was active at a low level in primary cultures of stratified epithelial cells on nonepidermal origin. Thus, the K14 gene promoter is functional in all epithelial cells, but the upstream regions of the K5 and K6 keratin genes restrict their expression to stratified epithelia, whereas the epidermal determinants of the K10 gene are not in the proximal upstream sequences.

We studied the effects of transcription factors of the AP-1 and NF-κB families on the expression of those four keratin genes using gene transfection.16 AP-1 and NFκB are activated by many extracellular signals, including those in hyperproliferative and inflammatory processes. K5 and K14 promoters, which are coexpressed in vivo, are regulated in parallel: both were activated by the c-Fos and c-Jun components of AP-1, but not by Fra1. On the other hand, the NFκB proteins, especially p65, suppressed these two promoters. The K17 promoter was specifically activated by c-Jun, whereas the other transcription factors tested had no significant effect. In contrast, the K6 promoter was very strongly activated by all AP-1 proteins, especially by the c-Fos + c-Jun and Fra1 + c-Jun combinations. It was also strongly activated by the NFκB p65 protein. AP-1 and NFκB synergistically activated the K6 promoter, although the AP-1 and the NF-κB responsive sites could be separated physically. These results suggest that the interplay of AP-1 and NFκB proteins regulates epidermal gene expression and that the activation of these transcription factors by extracellular signaling molecules brings about the differential expression of keratin genes in epidermal differentiation, cutaneous diseases, and wound healing.

The transcription of K14 and some other epidermal marker genes is regulated by AP1 interactions at their promoters. c-Jun and JunD activate and JunB downregulates the transcription of both basal and suprabasal genes.17 The effect of c-Jun is exerted through interactions with c-Fos at the AP1 motifs in the target promoters, whereas both JunB and JunD act independently of the binding at the AP1 sites. The differentiation specificity of the AP1 regulation seems determined by interactions involving other transcriptional regulators and transcription factor AP2 plays a role in K14 keratin gene expression. Functional AP2 binding sites were found upstream from several epidermal genes, suggesting that AP2 may be generally involved in epidermal gene regulation.18 The role of AP2 was examined by in vitro gel shift analysis, AP2 binding site mutagenesis, and stable and transient transfection experiments.19 Nonepithelial cells, such as fibroblasts and melanocytes, neither express keratin nor become phenotypically epithelial when transfected with an AP2-expressing vector. However, cotransfection of an AP2-expressing vector increases the level of transcription from keratin gene promoters. Thus, the role of AP2 in keratin gene expression seems to be quantitative rather than qualitative.

Regulation of K15

Basal layers of stratified squamous epithelia express keratins K15 in addition to K5 and K14, although at lower levels. Nonkeratinizing stratified epithelia, e.g., esophagus, produce more keratin K15 than epidermis. We cloned the promoter of the K15 gene and examined its regulation. Using cotransfection, gel mobility shift assays and DNAse I footprinting, we have identified the regulators of the K15 promoter activity and their binding sites.20 We focused on those that can be manipulated with extracellular agents, transcription factors C/EBP, AP-1, NF-κB, nuclear receptors for thyroid hormone, retinoic acid and glucocorticoids, as well as the cytokine IFNγ. We found that C/EBPβ and AP1 induced, while retinoic acid and glucocorticoid receptors and NFκB suppressed the K15 promoter, along with other keratin gene promoters. However, the thyroid hormone and IFNγ uniquely and potently induced the K15 promoter.20

Regulation of K1 and K10

The process of epidermal differentiation is profoundly influenced by the level of intracellular calcium within keratinocytes, which, in turn, regulates the expression of the differentiation-specific keratin genes K1 and K10. A 249-bp region in the 3'-flanking region of the human K1 gene, located 7.9 kb downstream from the promoter, can activate a minimal promoter construct in transfected keratinocytes.21 Importantly, this activity was enhanced by increased levels of calcium. The 249-bp fragment demonstrated a marked specificity for epidermal keratinocytes and was not active in other cell types. Moreover, this fragment could activate CAT expression in a construct driven by the K1 promoter, which alone had no intrinsic CAT activity. An AP-1 site is implicated in mediating the calcium response. These data identified and characterized a calcium-responsive regulatory element of the K1 gene.21

Forced expression of C/EBPβ in BALB/MK2 keratinocytes inhibited growth, induced morphological changes consistent with a more differentiated phenotype, and induced expression of two early markers of differentiation, K1 and K10 but had a minimal effect on the expression of late-stage markers, loricrin and involucrin.22 Conversely, C/EBPβ-deficient mice revealed decreased expression of K1 and K10 but not of involucrin and loricrin. C/ EBPβ-deficient primary keratinocytes were partially resistant to calcium-induced regulation. Thus, C/EBPβ modulates the early events of keratinocyte differentiation including K1 and K10 expression.

Similarly, overexpression of Whn (Hfh11, Foxn1), a winged-helix/forkhead transcription factor that, when mutated causes the nude phenotype, stimulates the expression of K1, while suppressing later markers, profilaggrin, loricrin, and involucrin.23 This suggests a role for Whn, the nude gene, in the earliest stages of epithelial terminal differentiation, the stages when the expression of keratins K1 and K10 commences.

Differentiation-associated transcription factors C/EBPα, C/EBPβ, and AP-2 regulate K10 gene expression.24 In cultured cells, C/EBPα and C/EBPβ can activate the K10 promoter using three binding sites. The selection of C/EBPα vs. C/EBPβ for K10 regulation is determined through a third transcription factor, AP-2 (see below). Unique gradients of expression exist for each transcription factor, i.e., C/EBPβ and AP-2 are most abundant in the lower epidermis, C/EBPα in the upper layers. In response to differentiation signals, loss of AP-2 expression leads to induction of C/EBPα and activation of the K10 promoter.

Regulation of K4 and K13

The esophageal stratified epithelium, like the epidermis, comprises an actively proliferating basal layer that, unlike the epidermis, undergoes a differentiation program not involving keratinization. Given its localization, K4 is a marker of the early differentiated suprabasal compartment of nonkeratinizing stratified epithelia. The transcriptional regulatory signals that orchestrate the switch from proliferation to differentiation also regulate the human K4 promoter.25 A critical cis-regulatory element contains an inverted CACACCT motif that binds esophageal-specific zinc-dependent transcriptional factors. Importantly, the interaction between Sp1 and cell cycle regulatory proteins is important in regulating K4 expression.26 Sp1 activation of the K4 promoter was reduced in cyclin D1-overexpressing cells, which is possibly mediated through direct interaction between Sp1 and cyclin D1; the reduction is opposed by a complex between Sp1 and pRB.

AP-2 also transactivates K4 transcription. The promoter region of K4 contains a functional AP-2 binding site in the vicinity of the transcriptional start.27 Various constructs, which did or did not contain the K4 promoter AP-2 site, were ballistically transfected into differentiating HaCaT keratinocytes. The results revealed that the AP-2 site is functional, although additional regulatory elements were found to be necessary for the full transcription of K4. These include the Kruppel-like transcriptional factors KLF6 and KLF4, which physically interact and coactivate the K4 promoter.28 KLF6 is a widely expressed member of the Kruppel-like family found in the esophagus. Using transient transfection, KLF6 was found to transactivate the human gene K4 promoter; cotransfection of KLF6 and KLF4, another member of the Kruppel-like factors and expressed in the esophageal squamous epithelium, leads to additive activation. The promoter contains a CACCC-like motif previously shown to bind KLF4. In a transient transfection system, KLF4 increased the activity of K4 promoter >25-fold, which depended on the CACCC-like element.29

The ets transcription factors contribute to diverse cellular functions and include a novel epithelial-specific member of the ets family ELF3 (a.k.a. ESE-1, ERT, jen, ESX). Interestingly, ELF3 suppressed the basal expression of K4 promoter in both esophageal and cervical epithelial cancer cell lines, while simultaneously activating the SPRR2A promoter, linked to late-differentiation.30 ELF3 may have dual functions in the transcriptional regulation of squamous epithelial differentiation, including transcriptional suppression of the K4 promoter.

K13, together with K4, its basic partner, is expressed in the suprabasal layers of noncornified stratified epithelia. Sequence analysis has revealed that two transcription-start sites were utilized, the major being at 61 and the minor at 63 nucleotides upstream of ATG.31 The promoter contains a TATA box and several other putative transcription factor binding sites. K13 is aberrantly expressed in murine epidermal tumors and constitutes an early marker of malignant progression.32 In vitro, expression of K13 in transformed epidermal cell lines can be induced either by Ca2+ or, indirectly, by retinoic acid.

Regulation of K3 and K12

Corneal epithelial cells initially express K5 and K14 keratins, characteristic of basal keratinocytes, and then undergo biochemical differentiation, as evidenced by the subsequent expression of K3 and K12 keratin markers of corneal epithelial differentiation. Using rabbit corneal epithelial cells, the promoter region of the rabbit K3 promoter was analyzed in transfection experiments. Serial deletion experiments narrowed this keratinocyte-specific promoter to within -300 bp upstream of the transcription initiation site. Its activity was not regulated by the coding or 3'-noncoding sequences. This 300 bp sequence can function in vitro as a keratinocyte-specific promoter and contains two clusters of partially overlapping motifs, one with an NFκB consensus sequence and another with a GC box.33 The combinatorial effects of these multiple motifs and their cognate binding proteins may play an important role in regulating the expression of this tissue-restricted and differentiation-dependent keratin gene. Electrophoretic mobility shift assays established that corneal keratinocyte nuclear proteins bind in vitro to the two sites. Immunosupershift and UV cross-linking show that NFκB, consisting of the p65 and p50 subunits, bind to the sequence GGGGCTTTCC, -262 to -253 bp. The second site contain unusual overlapping Sp1 and AP-2 GC-rich motif, CCGCCCCCTG, at -203 to -194 bp. This site bound an Sp1-related keratinocyte nuclear protein. Mutagenesis of the NFκB site, GC motif, and both abolished 20, 50, and 75% of the promoter activity, respectively, in transfected keratinocytes. These results indicate that NFκB is present in significant quantities in keratinocyte nuclei and that the tissue restriction of the NFκB- and Sp1-related proteins, in combination with other factors, may contribute to the keratinocyte specificity of rabbit K3 promoter. Furthermore, Sp1 activates, while AP-2 represses the K3 promoter.34 Although corneal basal cells express approximately equal amounts of Sp1 and AP-2 DNA-binding activities, the differentiated cells drastically down-regulate their AP-2 activity, which results in a six to sevenfold increase of the Sp1/AP-2 ratio. This change coincides with the activation of the differentiation-related K3 gene and suppression of the K14 keratin gene. In addition, polyamines, which are present in a high concentration in proliferating basal keratinocytes, inhibit the binding of Sp1 to its binding motif, but do not inhibit the binding of AP-2. These results suggest that the low Sp1/AP-2 ratio and the polyamine-mediated inhibition of Sp1 may account for the suppression of the K3 gene expression in the corneal basal cells, while the elevated Sp1/AP-2 ratio may be activating the K3 gene in the differentiated corneal epithelial cells. The switch of the Sp1/AP-2 ratio during corneal epithelial differentiation apparently plays a role in the reciprocal expression of the K3 and K14 genes during corneal differentiation.

K12 is the acidic type pair of K3 in differentiated corneal epithelial cells. The 2.5-kb DNA fragment 5'-flanking K12 contains corneal epithelial cell-specific regulatory cis-DNA elements. Pax-6 is a positive transcription factor essential for K12 expression. Three 5' truncated fragments of the keratin K12 promoter (1.03, 0.71 and 0.25 Kb) showed higher functional and tissue-specific promoter activity in a human corneal epithelial cell line than other cell lines.35 The 250 bp K12 promoter fragment was active in cultured rabbit corneal epithelial cells, suggesting that the tissue-specific expression in corneal epithelial cells extends across species lines. The paired box homeotic gene 6 (PAX-6), which plays a role in controlling eye development, stimulates the activity of keratin K12 promoter.36 The cis-regulatory elements located 600 bp upstream of the murine K12 gene were analyzed in rabbit corneas using particle-mediated gene transfer “Gene Gun” technology, while DNA foot-printing and electrophoresis mobility shift assay were performed to identify the cis-regulatory elements using bovine corneal epithelial cell nuclear extracts. The sequences between -181 to -111 and -256 to -193 bound to nuclear proteins; these two regions were potential binding sites for many transcription factors, such as AP1, c/EBP, and KLF6.37

Regulation of Hair Keratins

During hair growth, cortical cells differentiate and synthesize large amounts of hair keratin proteins. Hair keratin gene regulation has been studied in mouse and humans, as well as in sheep, where wool production has major economic consequences. The organogenesis of hair follicles is a very complex process, regulated both positively and negatively by several members of the Delta/Notch signaling system.38,39

HOXC13 when mutated or overexpressed in mice produces a fragile hair phenotype. HOXC13, but not a homeobox-deleted HOXC13 mutant, strongly activated the promoters of coexpressed human hair keratin genes.40 The hair keratin promoters contained numerous putative Hox binding core motifs TAAT, TTAT, and TTAC. Electrophoretic mobility shift assays identified the core motifs concentrated in the proximal promoter regions and allowed the deduction of an HOXC13 consensus binding sequence TT(A/T)ATNPuPu. Thus, there seems to be a direct involvement of HOXC13 in the control of hair keratin expression during early trichocyte differentiation.

Minimal promoter of the wool keratin gene K2.10 spanning nucleotides -350 to +53 was sufficient to direct expression of the lacZ gene to the hair follicle cortex of transgenic mice.41 Mutation introduced into the binding site for lymphoid enhancer factor 1 (LEF-1) decreased promoter activity, without affecting specificity. The constituent proteins of the LEF/TCF transcription complexes change during hair follicle differentiation; the LEF/TCF complexes seem to regulate directly the expression of hair keratin genes.42 DNase I footprinting analyses and electrophoretic mobility shift assays identified LEF-1, Sp1, AP2-like and NF1-like proteins bound to the promoter. The LEF-1 binding site is an enhancer element of the K2.10 promoter in the hair follicle cortex and additional factors may regulate the tissue- and differentiation-specificity of the promoter.

Regulation of Keratin Gene Expression by Hormones and Vitamins

Nuclear receptors for retinoic acid and thyroid hormone regulate transcription of keratin genes.43 By cotransfecting the vectors expressing nuclear receptors for retinoic acid, RAR, and thyroid hormone, T3R, along with the constructs that contain K5, K6, K10 and K14 gene promoters into epithelial cells, we have demonstrated that the receptors can suppress the promoters of keratin genes. The suppression is ligand dependent. The regulation of keratin gene expression by RAR and T3R occurs through direct binding of these receptors to the receptor response elements of the keratin gene promoters.44 The DNA binding domains of the receptors are essential for regulation, but the NH2-terminal “A/B” domains are not required. These findings indicate that the mechanism of regulation of keratin genes by RAR and T3R differs significantly from the mechanisms described for other genes modulated by these receptors. The RAR and T3R binding sites in the K5 promoter are adjacent to each other whereas in the K15 and K17 promoters they overlap.20

Similar and confirming results were obtained with squamous carcinoma cells of head and neck, where receptor-specific ligands implicated RARβ in inhibition of keratinization and suppression of K1 keratin expression.45 Curiously, although vitamin D3 acts through a nuclear receptor, VDR, a member of the RAR/T3R family, and regulates keratinocyte differentiation, VDR does not directly interact with the keratin genes.46 The keratin promoters shown not to be regulated by vitamin D3 include K3, K5, K10, K14, and K16.

We identified the RAR- and T3R-responsive site in the K14 gene using site-specific mutagenesis and found that the site consists of a cluster of consensus palindrome half-sites in various orientations, relatively close to the TATA box.47 Similar clusters of half-sites that share structural organization with the K14 regulatory site were found in the K5, K6, K10, K15, K16, and K17 keratin gene promoters and are responsible for retinoic acid-mediated transcription regulation of keratin synthesis in the epidermis.48 This means that the clustered structure of the RAR/T3R responsive elements is a common characteristic for keratin genes. Furthermore, in the absence of the ligand, T3R activates keratin gene expression, and the heterodimerization with the retinoid X receptor is not essential for activation by the unliganded T3R. Unlike other nuclear receptor binding sites, the response elements in keratin genes bind RAR, T3R and the glucocorticoid receptor, GR, as monomers or homodimers.49 Interestingly, addition of ligand to the receptor changes the binding pattern of the T3R from homodimer to monomer, reflecting the change in regulation from induction to inhibition. Such specific DNA-receptor interactions are crucial for the repression signal of transcription. Thus, they not only provide a docking platform for the receptors, but also directing the receptors to bind in a particular configuration and coordinate the interactions among different receptors. Furthermore, the response elements allow simultaneous binding of multiple receptors, thus providing fine-tuning of transcriptional regulation.

Glucocorticoids are important regulators of epidermal growth, differentiation, and homeostasis, and are used extensively in the treatment of skin diseases. GC action is mediated via GR. Transgenic mice overexpressing GR in epidermis have altered skin development and impaired proliferative and inflammatory responses.50 The developmental and proliferative phenotype includes lesions that range from epidermal hypoplasia to a complete absence of epidermis. Additional abnormalities resemble the clinical findings in patients with ectodermal dysplasia, including aplasia cutis congenitalis. The anti-inflammatory role of glucocorticoids is also evident in the transgenic animals, and is partly due to interference with other transcription factors, such as NFκB.50 We have described a novel mechanism of keratin gene regulation in skin through glucocorticoid receptor monomers.51 Glucocorticoids repress the expression of a subset of keratin genes, the basal-cell-specific keratins K5 and K14 and disease-associated keratins K6, K16, and K17, but not the differentiation-specific keratins K3 and K10 or the simple epithelium-specific keratins K8, K18, and K19. The regulated keratins are all associated with diseases, and in this way glucocorticoids differ from retinoic acid and thyroid hormone, which regulate all keratin genes tested. Detailed footprinting analysis revealed that the GR binds as four monomers to adjacent sites in keratin gene promoters.51 Using cotransfection and antisense technology we have found that coregulators SRC-1 and GRIP-1 are not involved in the suppression of keratin genes, while histone acetyltransferase and CBP are, which is another unusual and keratin gene-specific aspect of regulation by GR. In addition, GR blocks the induction of keratin gene expression by AP1 independently from direct binding of AP1 proteins to their responsive elements. Therefore GR suppresses keratin gene expression through two independent mechanisms: directly, through interactions of four GR monomers with responsive elements in keratin genes, as well as indirectly, by blocking the AP1-mediated induction of keratin gene expression.

A large variety of signals modulates epidermal keratin gene expression. Hormones and vitamins, which act via nuclear receptors, affect the differentiation process, whereas growth factors and cytokines, which act via cell surface receptors (see below), affect keratinocyte activation and related events. We examined the interaction between the nuclear receptor and cell surface receptor pathways in regulating the expression of keratin genes.52 Expecting to find dominance of one of the pathways, we were surprised to find that the two pathways are codominant. While EGF induces and retinoic acid suppresses expression of K6 and K16 keratin genes, when both EGF and retinoic acid are present simultaneously, the level of expression is intermediate. Similar codominant effects were found on other keratin genes with IFNγ, TGFβ, and thyroid hormone signaling. A judicious combination of hormones, vitamins, growth factors, and cytokines may be used to target specific expression of appropriate genes in the treatment of human epidermal diseases.20

Regulation of Keratin Gene Expression by Growth Factors and Cytokines

Keratin K17, the myoepithelial keratin, while not present in healthy skin, is expressed under various pathological conditions. Psoriasis is associated with production of IFNγ. The primary molecular effect of IFNγ is activation of specific transcription factors, such as STAT1, which regulate gene expression in target cells. Induction of cutaneous delayed-type hypersensitivity reactions resulted in activation and nuclear translocation of STAT1 in keratinocytes in vivo and subsequent induction of transcription of keratin K17. Within the promoter of the K17 keratin gene, we have identified the site that confers the responsiveness to IFNγ and that binds the transcription factor STAT1, and thus characterized at the molecular level the signaling pathway produced by the infiltration of lymphocytes in skin and resulting in the specific induction of K17 keratin gene expression in keratinocytes.53 The induction of K17 is specific for the inflammatory reactions associated with high levels of IFNγ and activation of STAT1, such as psoriasis and dermatitis caused by delayed type hypersensitivity, but not in samples of atopic dermatitis, which is not.54 Two cytokines, interleukin-6 and leukemia inhibitory factor, which can induce phosphorylation of STAT1, can also induce K17 expression, whereas interleukin-3, interleukin-4, interleukin-10, and granulocyte macrophage colony stimulating factor have no effect on K17 expression. Therefore, in inflammatory skin diseases, lymphocytes, through the cytokines they produce, differently regulate not only each other, but also keratin gene expression in epidermis one of their target tissues.

In the promoter region of the K17 gene we identified eight protein binding sites.55 Five of them bind the known transcription factors NF1, AP2, and Sp1 and three bind still unidentified proteins. Using site-directed mutagenesis, we have demonstrated the importance of the protein binding sites for the promoter function involved in both constitutive and IFNγ-induced expression of the K17 keratin gene. Interestingly, UVA irradiation (320-400 nm), but not UVB (290-320 nm), induced an increase in K17, showing a differential gene regulation between these two ultraviolet ranges.56 Importantly, UVB, a common damaging agent in the epidermis, increased the transcription of K19 gene and to a lesser extent the K6, K5, and K14 genes.56

In contrast, TGFβ causes transcriptional induction of K5 and K14 keratin genes.57 No other keratin gene promoters were induced. TGFβ is an important regulator of epidermal keratinocyte function because it suppresses cell proliferation. The effect of TGFβ is concentration-dependent, can be demonstrated in HeLa cells, does not depend on keratinocyte growth conditions and can be elicited by both TGFβ1 and TGFβ2. These results suggest that TGFβ promotes the basal cell phenotype in stratified epithelia such as the epidermis, and that the effects of TGFβ are not anti-proliferative, but merely anti-hyperproliferative.

We will not go into details of the analysis of the activation-specific keratin genes K6 and K16, which have been studied by many investigators,58,59,60,61,62,63,64 and which are described elsewhere in this issue (see chapter by Navarro et al). We will briefly focus on the effects of proliferative and proinflammatory cytokines on K6 and K16 expression. For example, interleukin-1 (IL-1) and tumor necrosis factor-α (TNFα) induce K6b keratin synthesis through a transcriptional complex containing NFκB and C/EBPβ,65,66 while the epidermal growth factor (EGF) and transforming growth factor-α (TGFα) specifically induce K6 and K16, apparently through AP1 transcription elements.67

Regulation of Small Keratins, K7, K8, K18 and K19

So far, very little is known about the transcriptional regulation of K7, although a systematic exploration of its expression in several organisms has begun and, hopefully, will soon yield much more data.68 K7 is expressed observed in lung, bladder, mesothelium, hair follicle, filiform papillae of the tongue and in a range of “hard” epithelial tissues.

The human keratins K8 and K18 genes are expressed in diverse simple epithelial tissues and in various carcinomas. Relatively little is known about the regulation of the basic type simple epithelial keratin K8, originally known as EndoA.69,70 Its expression is regulated by p53 through a binding site in the 5' untranslated region of the gene.71 In addition, an enhancer at the 3'- end of the gene contains seven Ets binding sites that bind ETS1, ETS2 and ERGB/FLI-1 transcription factors and regulate transcription in combination with the K8 promoter.71,72

In contrast, the regulation of expression of the K18 gene has received much attention because of several very exciting features unique to this gene. In transgenic mice, a 10-kilobase DNA segment of the human K18 gene contains all the necessary information for proper tissue-specific expression. Furthermore, this expression is copy number-dependent and integration site-independent. The 10 Kb sequence contains several interdependent regulatory sites, including a promoter, an intronic enhancer, a regulatory site in the exon, and the locus controlling regions, LCRs, bracketing the gene.

The promoter of the K18 gene is regulated in a cell-type specific manner. The “minimal promoter” contains the TATA box and an initiation site, and the TATA box is the only essential element of the minimal promoter.73 The K18 expression is different in tumorigenic and nontumorigenic cell lines. The differential expression depends on an Sp1 site close upstream from the TATA box. Three different proteins bind to the Sp1 site, one of them is Sp1 itself. Importantly, the acetylation state of the proteins bound to DNA in the K18 promoter greatly affect the promoter activity: histone deacetylase inhibitors stimulate the activity of the K18 promoter.74 CBP/p300 coactivator protein seems involved in the regulation of K18 promoter activity, at least in nontumorigenic cell lines.75

The promoter activity is increased by an enhancer element in intron 1 (see below). The intronic enhancer can also stimulate transcription from a cryptic promoter, apparent in the absence of the minimal promoter. Furthermore, this cryptic promoter sequence is AP-1-dependent.76 Apparently, the minimal promoter, the cryptic promoter/enhancer, and the intron-1 enhancer interact in a very complex way, involving AP1, Sp1, CBP/p300 and protein acetylation to regulate the levels of activity of the K18 promoter.

This complexity, however, can be exploited to deliver transgenes to the airway epithelia, which is potentially very useful for development of gene therapy approaches.77 A helper-dependent adenoviral vectors utilizing the K18 sequence as a tissue-specific promoter, directed expression of linked genes in airway epithelial and submucosal cells, apparently circumventing the side effects such as acute toxicity and inflammation, and could be important in the development of gene therapy for cystic fibrosis.

The induction of K18 in embryonal carcinoma (EC) and embryonic stem (ES) cells can be triggered in culture by exposure to retinoic acid. The indiction depends, in part, on the complex enhancer element located within the first intron of K18.78 ETS-2 and AP-1 transcription factor c-Jun, and JunB also mediate the induction of K18. These transcription factors act by opposing three silencer elements also located within the first intron of the K18 gene. Therefore, the induction of K18 is due to a combination of relief from negative regulation and direct positive activation by the ETS and AP-1 transcription factors. The methylation of the ETS binding site causes a repression of the K18 gene expression, because ETS proteins do not bind to methylated sites in DNA. Such methylation adds another layer of complexity to the regulation of K18 keratin gene expression.79

DNase-hypersensitive sites often correlate with regulatory regions of genes. Four such sites are found in the promoter region and the first intron of K18. Two DNase-hypersensitive sites were found in an Alu repetitive sequence immediately upstream of the promoter.80 The final hypersensitive site was mapped to exon 6 of K18. This novel regulatory element in exon 6 modulates K18 gene expression in transgenic mice. While the exon 6 site can bind c-Jun and c-Fos, it can stimulate transcription independent of AP-1 proteins.81 Therefore, the protein-coding sequences of the K18 gene have a regulatory function as well.

Arguably the most unusual and least understood DNA regulatory sites are the locus control regions, LCRs.82 These sequences, first described bracketing the hemoglobin genes, confer integration site-independent transcription in transgenic mice. LCR serves to insulate the region from transcriptional influences of surrounding DNA. Neither read-through, nor enhancers nor silencers have any effect in the segments protected by LCRs. This means that the level of expression of a transgene depends only on the copy number, and not on the site of integration.

The group of Robert Oshima identified and characterized the LCR elements of the human K18 keratin gene. Transgenic animals express the human K18 gene at levels proportional to the copy number and independently of the integration sites.83,84 Integration site-independent expression of K18 depends on a 323 bp sequence from the 5' flanking sequence and 3.5 kb of the 3' flanking sequence. The LCR activity is orientation-dependent. Interestingly, the integration site-independent expression and copy number-dependent expression can be separated. The 323 bp sequence can confer LCR function even on heterologous transgenes.85 This sequence comprises an Alu repetitive element and its LCR activity correlates with its RNA polymerase III promoter activity.

Keratin K19 is expressed in simple and nonkeratinizing stratified epithelia. Its expression seems associated with malignant transformation in esophageal and pancreatic cancers. This expression depends on a sequence containing binding sites for KLF4 and Sp1. Furthermore, overexpression of KLF4 and Sp1 induces K19 production.86 An enhancer sequence containing an AP1 site has been identified in the K19 3'-flanking region.87 K19 is specifically and directly induced by retinoic acid88 and by estrogen.89 The estrogen regulation depends on a complex enhancer region in the first intron, which contains several estrogen receptor binding sites and AP1 sites.

The K19 upstream sequences contain transcription regulatory elements in -2249 to -2050 bp and -732 bp to the first ATG. Six protein-binding sites, including an Sp1 site, a CCAAT box and a TATA box were detected in the segment from -732 bp to the first ATG by the DNA footprinting technique.90 The K19 promoter has been used for targeting and identification of K19-producing cells in transgenic mice.91 Expression was found in ductal epithelia of the pancreas, small intestinal villi, in surface colonocytes, and gastric isthmus cells. The expression of K19 correlated with and overlapped that of KLF4 protein expression.

Specific Transcription Factors Implicated in Regulation of Transcription of Keratin Genes

The transcription factors AP-2 comprise three isoforms, AP-2-α, -β and -γ, similar in structure. AP-2α is present in basal keratinocytes, but is significantly induced in proliferating diseases; AP-2β is present only in sweat glands, whereas AP-2γ is present throughout the epidermis in normal and psoriatic skin as well as in the SCC lesions. The K14 promoter binds to AP-2α and AP-2γ, whereas the K1 promoter predominantly binds to AP-2γ. In contrast, AP-2β does not bind to either keratin DNA.92 The AP2 proteins may be general regulators of keratin gene expression,18,19 but their precise role remains to be elucidated.

Ectopic expression of c-Myc,93 KLF4,94 Gli295 or Ras and Raf96 in the epidermis alters cell phenotype and keratin expression. However, the effects of these transcription factors on keratin gene expression may be indirect. Particularly interesting are the roles of the Kruppel-like transcription factors.94 KLF4, besides affecting epidermal barrier function in development, directly regulates K4 and K19 (see above).28,86 On the other hand, K12, the corneal keratin gene is regulated by KLF6, presumably through the KLF6 binding site identified in the human K12 promoter.97

Conclusions and Future Directions

The common characteristic of all keratin gene promoters analyzed so far indicates that each individual gene is regulated by numerous transcription factors that bind nearby DNA elements and assemble into multi-protein complexes. These transcription factors respond to extracellular influences, such as hormones, vitamins and growth factors. Therefore, the level of expression of each keratin gene seems to be affected by the signals from the cellular milieu.

Conspicuously absent is the “epithelia-specific master regulator”, a transcription factor common to all epithelial cells and specific only for epithelial cells, necessary for expression of all keratin genes. Hope for finding such a protein derives from the axiomatic association of keratins and epithelia. Its absence means that every epithelial cell type has elaborated a set of transcription factors that, in combinations, are responsible for expression of keratins. Furthermore, we did not find “epidermal”, “simple epithelial” or even “differentiation-specific” master regulators that are either permissive or necessary for expression of a subclass of keratins.

Similarly, while keratin proteins tend to be expressed in specific pairs that consist of a basic and an acidic keratin, the pair-wise coexpression regulators have not been identified. Again, we are left with the notion that each keratin gene is regulated by its own regulatory circuits and expressed independently from other keratins. The pair-wise coexpression, then, is a fortuitous result of multiple independent regulatory mechanisms.

On the other hand, the frequent encounter of a small number of transcription factors in the regulation of many keratin genes indicates that same regulatory motifs play a role in the expression of keratin genes. The recently elucidated sequences of several mammalian species will soon lead to new genomics breakthroughs. Before long, algorithms for large throughput sequence analysis of promoters will be available. Hopefully, many more discoveries and surprises are in store for keratinologists in the near future.

Acknowledgments

Our experiments have been supported by grant AR41850 from the National Institutes of Health. I want to thank my collaborators over the past years, especially Marjana Tomic-Canic, Alix Gazel, Chun-Kui Jiang, Tomohiro Banno and Mayumi Komine, among others, for joining me on different legs of this exciting and fun journey.

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