Ultraviolet responses of Gorlin syndrome primary skin cells.

BACKGROUND: Gorlin syndrome, or naevoid basal cell carcinoma syndrome (NBCCS), is an autosomal dominant disorder associated with mutations in the PTCH1 gene, which encodes the receptor of SONIC HEDGEHOG. In addition to developmental abnormalities, patients with NBCCS are prone to basal cell carcinoma (BCC), the most frequent type of nonmelanoma skin cancer in humans. OBJECTIVES: As ultraviolet (UV) exposure plays a prominent role in the development of sporadic BCC, we aimed to determine whether primary NBCCS skin cells exhibit differential responses to UV exposure compared with wild-type (WT) skin cells. METHODS: Primary fibroblast and keratinocyte strains were isolated from nonlesional skin biopsies of 10 patients with characteristic NBCCS traits. After identification of PTCH1 mutations, capacities of NBCCS cells to repair UV-induced DNA lesions and to survive after UV irradiation, as well as p53 responses, were compared with those of WT skin cells. RESULTS: The c1763insG PTCH1 mutation is described for the first time. DNA repair and cell survival analyses following UV irradiation revealed no obvious differences between responses of NBCCS and WT fibroblasts and keratinocytes. However, p53 accumulation after UV irradiation was abnormally persistent in all NBCCS primary keratinocyte strains compared with WT keratinocytes. CONCLUSIONS: Our observations that NBCCS cells harbour normal DNA repair and survival capacities following UV irradiation better explain that BCC proneness of patients with NBCCS does not solely concern body areas exposed to sunlight and suggest rather that it might be due to cell cycle alterations.

[Clinical and genetic study in 22 patients with basal cell nevus syndrome]. / Etude clinique et recherche de mutations germinales du gène PTCH 1 dans le syndrome des hamartomes basocellulaires.

BACKGROUND: Nevoid basal cell carcinoma syndrome is an autosomal dominant disorder characterized by developmental abnormalities and cancer predisposition. The PTCH 1 gene, the human homolog of the Drosophila segment polarity gene patched, has been shown to be involved in the development of nevoid basal cell carcinoma syndrome. PTCH 1 is mapped to chromosome 9q22.3. The aim of the present study was to report on clinical and genetic characteristics in patients followed for nevoid basal cell carcinoma syndrome and to compare them to the data in the literature. PATIENTS AND METHODS: Screening for PTCH 1 mutations was done in 22 patients followed between 1981 and 2003 for clinical suspicion of nevoid basal cell carcinoma syndrome. Clinical and radiological data were reviewed retrospectively from records. Genetic analysis was performed using blood samples after patient informed consent was obtained. When possible, DNA was also analyzed from the parents of patients in whom PTCH 1 mutations were found. RESULTS: All patients had developed basal cell carcinomas: 45% palmar and plantar pitting, 62% jaw cysts and 66% calcification of falx cerebri. Medulloblastomas and meningiomas were the most common associated tumors. PTCH 1 mutations were identified in 13 patients: 6 familial cases, 3 sporadic cases and for 4 patients, it was not possible to conclude. Nine different new germ-line mutations were identified. DISCUSSION: Genetic analysis allows molecular confirmation of diagnosis in about half of all patients. Early diagnosis is essential for detection of clinical and radiological manifestations in young patients and for provision of advice concerning protection of the skin from the sunlight.

Differential behaviors toward ultraviolet A and B radiation of fibroblasts and keratinocytes from normal and DNA-repair-deficient patients.

Xeroderma pigmentosum (XP) and trichothiodystrophy (TTD) are rare genodermatoses transmitted as recessive and autosomal traits that result in reduced capacity to repair UV-induced DNA lesions. Although XP, but not TTD, patients are prone to basal and squamous cell carcinomas, to date no comparative studies of the XP and TTD phenotypes have included epidermal keratinocytes. We compared the DNA repair capacity (by unscheduled DNA synthesis) and cell survival (by clonal analysis) of epidermal keratinocytes and dermal fibroblasts grown from normal individuals and patients with xeroderma pigmentosum and trichothiodystrophy following UVA and UVB irradiation. The same dose of UVB (1000 J/m2) induced twice as many DNA lesions in normal fibroblasts as in normal keratinocytes. UV survival rates were always higher in keratinocytes than in fibroblasts. Normal and TTD keratinocytes survived better following UVA and UVB irradiation than XP-C and XP-D keratinocytes. XP-C keratinocytes exhibited exacerbated sensitivity toward UVA radiation. Unscheduled DNA synthesis at UV doses leading to 50% cell survival indicated that the ratio of DNA repair capacity to cell survival is higher in keratinocytes than in fibroblasts. In addition, UVA and UVB irradiation induced a transition from proliferative to abortive keratinocyte colonies. This transition varied between donors and was in part correlated with their cancer susceptibility. Altogether these data provide the first evidence of the differential behaviors of normal, XP, and TTD keratinocytes toward UV radiation.

Upregulation of galectin-7 in murine lymphoma cells is associated with progression toward an aggressive phenotype.

We have previously shown that ICAM-1-deficient mice were resistant to lymphoma dissemination of intravenously injected 164T2 lymphoma cells. Highly aggressive variants of this cell line, however, could overcome this resistance. To discern the complex pattern of gene expression involved in the evolution of aggressiveness in lymphoma cells, we compared the transcriptome of 164T2 cells with that of their aggressive variants using cDNA arrays. We identified several genes that were differentially expressed in nonmetastatic lymphoma cells and their metastatic variants. Galectin-7, associated with the development of chemically induced mammary carcinoma, was one such gene whose expression was significantly upregulated. We showed that it was constitutively expressed in aggressive variants, at both mRNA and protein levels. Galectin-7 expression in aggressive lymphoma cells was induced upon in vivo selection in several organs, including the thymus, the spleen and kidneys. We also showed that treatment of nonaggressive lymphoma cells with 5-aza-2'-deoxycytidine was sufficient to induce galectin-7 gene expression. This report is the first to show that galectin-7 is expressed in aggressive lymphoma.

Expression of galectin-7 during epithelial development coincides with the onset of stratification.

Galectin-7 is a 14 KDa member of the galectin family that we have cloned from human, rat and mouse. Our previous studies have shown that in the adult, galectin-7 is expressed in all cell layers of epidermis and of other stratified epithelia such asthe cornea and the lining of the oesophagus. This suggested that galectin-7 expression might be induced at a particular stage in the embryonic development of stratified epithelia. In the present study we have investigated this hypothesis by in situ hybridization of galectin-7 mRNA in mouse embryos. Starting from E13.5, weak expression of galectin-7 was detected in bilayered ectoderm, and stronger expression was found in areas of embryonic epidermis where stratification was more advanced. Galectin-7 expression was maintained in all living layers after epidermal development was completed. Galectin-7 was also strongly and specifically expressed in stratified regions of ectodermally-derived non-epidermal epithelia such as the lining of the buccal cavity, the oesophagus and the ano-rectal region. In contrast, no expression of galectin-7 was found in epithelia derived from endoderm, such as lining of the intestine, kidney and lung. Our results demonstrate that galectin-7 is expressed in all stratified epithelia examined so far, and that the onset of its expression coincides with the first visible signs of stratification. These results establish galectin-7 as the first region-independent marker of epithelial stratification.

Delayed onset of epidermal differentiation in psoriasis.

In normal epidermis, as previously reported, the first signs of differentiation occur within the basal layer in a subpopulation of keratinocytes that start to express K1 and K10 "supra-basal" keratin transcripts (20-30% of the basal cells) and proteins (5-10% of the basal cells). We found that in psoriatic lesions, the basal layer was devoid of cells expressing these early differentiation markers. This was already the case at the periphery of the lesions, where epidermis, although slightly acanthotic, still completes the keratinization process. In the center of the lesions, not only the basal layer, but also several rows of suprabasal cells, were negative for keratin K10 transcripts or protein. Moreover, the upper nucleated layers of involved epidermis were also devoid of K10 keratin transcripts or proteins. In normal epidermis, as previously reported, transcripts for the "basal" K5 keratin were mainly restricted to the basal layer, whereas the protein persisted in a few suprabasal layers. We found that in psoriatic epidermis, K5 keratin transcripts persisted in several suprabasal layers up to the level where K10 keratin transcripts appeared. These data, although not contradictory with previous reports showing a reduction of K1-K10 keratins and other differentiation markers in psoriasis, demonstrate that these quantitative changes are in fact the result of major qualitative differences in the distribution of these markers in psoriatic versus normal skin. Our results indicating that the onset of differentiation is delayed in psoriasis show that, contrary to conclusions accepted so far, not only the suprabasal compartment, but also the basal one, is abnormal in psoriatic epidermis.

Germline and somatic mutations of the INK4a-ARF gene in a xeroderma pigmentosum group C patient.

Xeroderma pigmentosum is an inheritable autosomal recessive DNA repair deficient syndrome characterized by a high predisposition to skin cancers. An elevated proportion of tumors from xeroderma pigmentosum patients harbor ultraviolet-induced mutations (CC:GG > TT:AA tandem transitions) of the p53 and/or the INK4a-ARF genes. Here, we report the clinical and molecular features of a 12 y old xeroderma pigmentosum patient who, in addition to severe cutaneous clinical symptoms, also had three unusual tumors, a mediastinal lymphoblastic lymphoma, an atypical fibroxanthoma, and an epithelioid hemangioma. Single strand conformation polymorphism and sequencing analysis of the p53 and INK4a-ARF genes were carried out in DNA from normal skin and different tumors (four actinic keratosis, two microinvasive squamous cell carcinomas, one basal cell carcinoma, and one atypical fibroxanthoma) from the patient. After characterization of the xeroderma pigmentosum C complementation group, we found unexpectedly that this patient also carried a germline mutation of the INK4a-ARF locus affecting the p16INK4A reading frame. Three different somatic mutations that all harbor the signature of ultraviolet light (two of p16INK4A and one of p53) were also detected in the basal cell carcinoma. We hypothesize that the germline mutation of p16INK4A, in association with the nucleotide excision repair defect, could explain the patient's unusual phenotype. Furthermore, this study confirms that concomitant somatic mutations of INK4a-ARF and p53 occur in some xeroderma pigmentosum associated tumors, and seem to accumulate during tumor progression rather than the initiation step.

Transcriptional regulators of expression of K#16, the disease-associated keratin.

In most malignant and benign skin diseases, the normal pattern of keratin expression is altered. Among other phenotypic changes, the expression of hyperproliferation- and activation-associated keratins K#16 and K#6 is induced. Because the molecular mechanisms and the nuclear regulators involved in this induction are unknown, we have characterized the transcriptional regulators of expression of the keratin K#16 promoter. Our previous studies have shown that the transcription of K#16 is strongly and specifically induced in epidermal keratinocytes by epidermal growth factor (EGF), through the EGF-responsive element (RE). In the present work, using an electrophoretic mobility-shift assay, we have found several nuclear protein binding sites that have been identified as an Sp1 site, an AP2 site, the EGF-RE, and an enhancer element. The function of each site was assessed in transfection assays using specific deletions. Both the Sp1 and EGF-RE sites are essential for K#16 promoter activity. The site that functions as an independent enhancer, E, was found adjacent to and interacting with a sequence recognized by the AP2 transcription factor. This knowledge of the nuclear regulators of expression of the disease-associated K#16 keratin provides insight into the molecular parameters that might be important in skin diseases.

Expression of the carcinoma-associated keratin K6 and the role of AP-1 proto-oncoproteins.

The normal pattern of keratin expression in epidermis is altered in carcinomas as well as in nonmalignant diseases such as psoriasis and wound healing. Under these circumstances, the transcription of differentiation-specific keratins K1 and K10 is suppressed, whereas the activation- and hyperproliferation-associated keratins K6 and K16 are induced. Very little is known regarding transcriptional regulators involved in this switch. To investigate the nuclear factors that participate in regulation of expression of the K6 gene, we have characterized the binding sites for nuclear proteins on the promoter DNA of the K6 gene by gel retardation assays and site-specific deletion mutagenesis. We found four nuclear protein binding sites in the K6 gene promoter. Two are near the TATA box, but their ability to bind HeLa or keratinocyte nuclear extracts is independent of the TATA box-binding protein complex. The third binding site is a large palindrome. The sequences of these three sites do not correspond to any described target sequences for characterized transcriptional factors. The fourth is an AP-1 site, the target sequence for the proto-oncoproteins fos and jun. All four sites are independent of the previously characterized epidermal growth factor-responsive element, EGF-RE. These findings suggest that there may be two parallel pathways of induction of K6 transcription. One proceeds through the EGF-RE, which may be involved in nonmalignant hyperproliferation processes; the other, through the AP-1 site and the fos-jun proto-oncoproteins, may be related to induction in malignant processes.

On the role of AP2 in epithelial-specific gene expression.

Transcription factor AP2 plays an important role in transcription of keratin genes, and it has been suggested that AP2 confers epithelial specificity. Promoters of keratin genes contain AP2 sites, usually within tight clusters of binding sites for other nuclear transcription factors. The role of AP2 was examined by in vitro gel shift analysis, AP2 binding site mutagenesis, and stable and transient transfection experiments. Nonepithelial cells, such as GM10 fibroblasts and melanocytes, neither express keratin nor become phenotypically epithelial when transfected with an AP2-expressing vector. However, in 3T3 and HeLa cells, co-transfection of an AP2-expressing vector increases the level of transcription from keratin gene promoters. This increase requires an intact AP2 binding site. Thus, the role of AP2 in keratin gene expression is quantitative rather than qualitative. AP2 interacts with other transcription factors and may convey extracellular regulatory signals to the transcription complex in the promoters of keratin genes.

Overexpression of matrix metalloproteinase 1 in dermal fibroblasts from DNA repair-deficient/cancer-prone xeroderma pigmentosum group C patients.

Xeroderma pigmentosum (XP) is a rare, recessively inherited genetic disease characterized by skin cancer proneness and premature aging in photoexposed area. The disease results from defective nucleotide excision repair of ultraviolet (UV)-induced DNA lesions. Reconstruction of group C (XP-C) skin in vitro previously suggested that patients' dermal fibroblasts might be involved in promoting skin cancer development, as they elicited microinvasions of both control and XP-C keratinocytes within dermal equivalents. Here we show that in the absence of UV exposure XP-C fibroblasts exhibit aged-like features such as an elongated and dendritic shape. We analysed the repertoire of expression of matrix metalloproteinases (MMPs) involved in skin aging and cancer. All XP-C fibroblasts tested in this study overexpressed specifically and significantly MMP1. MMP1 expression was also found increased in the dermis of XP-C skin sections suggesting the active contribution of XP-C mesenchymal cells to skin aging and exacerbated carcinogenesis. Increased MMP1 expression in cultured XP-C fibroblasts resulted from MMP1 mRNA accumulation and enhanced transcriptional activity of the MMP1 gene promoter. Deletion analysis revealed the essential role of AP-1 activation in constitutive MMP1 overexpression in XP-C primary fibroblasts. In parallel, levels of reactive oxygen species and FOSB DNA-binding activity were found increased in XP-C fibroblasts. Altogether, these observations suggest that beyond its role in nucleotide excision repair the XPC protein may be important in cell metabolism and fate in the absence of UV.

Heterozygous mutations in the tumor suppressor gene PATCHED provoke basal cell carcinoma-like features in human organotypic skin cultures.

Basal cell carcinoma of the skin is the most common type of cancer in humans. The majority of these tumors displays aberrant activation of the SONIC HEDGEHOG (SHH)/PATCHED pathway, triggered by mutations in the PATCHED tumor suppressor gene, which encodes a transmembrane receptor of SHH. In this study, we took advantage of the natural genotype (PATCHED(+/-)) of healthy keratinocytes expanded from patients with the nevoid basal cell carcinoma or Gorlin syndrome to mimic heterozygous somatic mutations thought to occur in the PATCHED gene early upon basal cell carcinoma development in the general population. PATCHED(+/-) epidermis developed on a dermal equivalent containing wild-type (WT) PATCHED(+/+) fibroblasts exhibited striking invasiveness and hyperproliferation, as well as marked differentiation impairment. Deciphering the phenotype of PATCHED(+/-) keratinocytes revealed slight increases of the transcriptional activators GLI1 and GLI2-the latter known to provoke basal cell carcinoma-like tumors when overexpressed in transgenic mice. PATCHED(+/-) keratinocytes also showed a substantial increase of the cell cycle regulator cyclin D1. These data show for the first time the physiological impact of constitutive heterozygous PATCHED mutations in primary human keratinocytes and strongly argue for a yet elusive mechanism of haploinsufficiency leading to cancer proneness.

Nucleotide excision repair and related human diseases.

Nucleotide excision repair (NER) of DNA-lesions is the most versatile DNA repair mechanism involved in genome maintenance, cell and organismal preservation. Deciphering the stepwise mechanism of NER has mostly relied on cells from rare patients presenting photosensitive, recessively inherited genetic disorders such as xeroderma pigmentosum (XP), trichothiodystrophy (TTD) and Cockayne (CS) syndromes. Cells from these patients share various extents of impaired capacity of repairing UV-induced DNA lesions (cyclobutane pyrimidine dimers, 6-4 pyrimidine-pyrimidone photo products) located either in transcribed DNA strands or in inactive DNA. We review here the essentials of NER actors and how impairment of their activity may lead to distinct and characteristic human disorders whose presentation may be limited to developmental trait (TTD; CS), or cumulate with cancer susceptibility toward genotoxic aggressions, most notably short wavelength ultraviolets.

CD24 (heat stable antigen, nectadrin), a novel keratinocyte differentiation marker, is preferentially expressed in areas of the hair follicle containing the colony-forming cells.

We have isolated, by subtractive and differential hybridization from a library constructed from keratinocyte colony-forming cells (K-CFCs), a cDNA coding for the rat CD24 (nectadrin, heat stable antigen). CD24, a glycoprotein thought to be involved in cell-cell adhesion and signalling, is highly expressed in keratinocytes located in the bulge area of the rat vibrissa which contains the most K-CFCs. CD24 is also expressed in the outer epithelial sheath of human hair follicles and in glabrous epidermis. However, its expression is not restricted to K-CFCs as demonstrated by cell sorting experiments, and it is thus not a specific marker of clonogenic keratinocytes. Rather, its preferential distribution in keratinocytes located in the most innervated area of the rat vibrissal follicle, i.e., the bulge, suggests that is function could be related to the tactile role of the hair follicle.

Galectin-7 overexpression is associated with the apoptotic process in UVB-induced sunburn keratinocytes.

Galectin-7 is a beta-galactoside binding protein specifically expressed in stratified epithelia and notably in epidermis, but barely detectable in epidermal tumors and absent from squamous carcinoma cell lines. Galectin-7 gene is an early transcriptional target of the tumor suppressor protein P53 [Polyak, K., Xia, Y., Zweier, J., Kinzler, K. & Vogelstein, B. (1997) Nature (London) 389, 300-305]. Because p53 transcriptional activity is increased by genotoxic stresses we have examined the possible effects of ultraviolet radiations (UVB) on galectin-7 expression in epidermal keratinocytes. The amounts of galectin-7 mRNA and protein are increased rapidly after UVB irradiation of epidermal keratinocytes. The increase of galectin-7 is parallel to P53 stabilization. UVB irradiation of skin reconstructed in vitro and of human skin ex vivo demonstrates that galectin-7 overexpression is associated with sunburn/apoptotic keratinocytes. Transfection of a galectin-7 expression vector results in a significant increase in terminal deoxynucleotidyltransferase-mediated UTP end labeling-positive keratinocytes. The present findings demonstrate a keratinocyte-specific protein involved in the UV-induced apoptosis, an essential process in the maintenance of epidermal homeostasis.

Galectin-7, a marker of all types of stratified epithelia.

Galectin-7 is a 14-kDA member of the lectin family we have previously cloned in the human. Its expression was found at all stages of differentiation of the human epidermis and was reduced but not suppressed when oral metaplasia of reconstructed epidermis was induced by retinoic acid. This suggested that galectin-7 could be a marker of both keratinized and non-keratinized stratified epithelia. To ascertain this hypothesis, we cloned the rat and the mouse cDNAs and produced a specific antiserum raised against a synthetic peptide. The distribution of galectin-7 mRNAs and protein was studied by in situ hybridization and immunolabelling of various human, rat and mouse epithelia. Galectin-7 was found to be expressed in interfollicular epidermis and in the outer root sheath of the hair follicle, but not in the hair matrix, nor in the sebaceous glands. It was present in esophagus and oral epithelia, cornea, Hassal's corpuscles of the thymus, but not in simple and transitional epithelia. Galectin-7 can thus be considered as a marker of all subtypes of keratinocytes. In that respect it differs from both "basal-specific" keratins K5-K14 and from "suprabasal-specific" markers such as keratins K1-K10 and involucrin. Galectin-7 also differs from some desmosomal proteins, which are present in all types of epithelia and in myocardium. Galectin-7 was absent from cultured carcinoma cell lines and was reduced both in human carcinomas and in murine tumors produced with the two-stage carcinogenesis protocol.

Galectin-7, a human 14-kDa S-lectin, specifically expressed in keratinocytes and sensitive to retinoic acid.

A cDNA encoding a novel member of the S-lectin family has been cloned from human epidermis. The 14-kDa protein of pI7 predicted by the 136-amino-acid open reading frame of the sequence was called galectin-7 according to the presently accepted nomenclature. A GST fusion protein authentified the lactose-binding properties expected for a member of this lectin subfamily. Galectin-7 was identified on two-dimensional gels of keratinocyte protein extracts. Differential and in situ hybridizations indicate that this lectin is specifically expressed in keratinocytes. It is expressed at all stages of epidermal differentiation (i.e., in basal and suprabasal layers). It is moderately repressed by retinoic acid, a behavior contrasting with those of other keratinocyte markers sensitive to this agent, which, either basal, are induced, or suprabasal, are repressed. This effect of retinoic acid on a keratinocyte cell type marker such as galectin-7 is more reminiscent of its metaplasiogenic effect in vivo than of its inhibitory effect on terminal epidermal differentiation in vitro. This interpretation is supported by the fact that in chick epidermis a 14-kDa S-lectin is suppressed during retinoic acid-induced mucous metaplasia.

Isolation of a GC-rich cDNA identifying mRNA present in human epidermis and modulated by calcium and retinoic acid in cultured keratinocytes. Homology with murine loricrin mRNA.

Differential screening of a human epidermal cDNA library led to the isolation of cDNA clones homologous to mRNAs specifically expressed in epidermis but weakly or not expressed in the undifferentiated squamous carcinoma cell line TR146. One of these 'differentiation-specific' cDNA clones, A8, hybridized with a 1.7 kb transcript among RNAs isolated from normal human epidermis, but with several transcripts ranging from 1.4 to 2.1 kb when mRNAs were isolated from cultured keratinocytes. We examined the effects of modulators of epidermal differentiation such as calcium and retinoic acid on the production of these transcripts. Their amount was found to increase in the presence of high calcium concentration, but to decrease in the presence of retinoic acid. These results strongly suggest that A8 messages are up-regulated during epidermal differentiation. The sequence of the 1371 bp of A8 cDNA shows a very high GC content. Because of its homology with the murine loricrin mRNA, A8 is likely to correspond either to the human loricrin or to a related protein.

Expression of loricrin is negatively controlled by retinoic acid in human epidermis reconstructed in vitro.

In epidermis, the last steps of keratinocyte differentiation are characterized by the covalent cross-linking of cornified envelope precursors such as involucrin and loricrin, a hydrophobic protein recently described in mouse and human epidermis. In situ hybridization of normal human skin sections with a human loricrin cRNA probe and immunolabeling with an antiserum directed against a synthetic peptide corresponding to the carboxyterminus of human loricrin revealed the presence of loricrin transcripts and protein in the granular layers of epidermis. In human epidermis reconstructed in vitro by growing keratinocytes on dermal equivalents, loricrin and loricrin mRNAs were also restricted to granular cells, but their amounts seemed higher than in epidermis from skin biopsies. The reactivities for both loricrin and loricrin mRNAs were abolished by a treatment of the cultures with a retinoic acid concentration (10(-6) M) provoking a complete inhibition of terminal epidermal differentiation (parakeratosis). Thus, the regulation of loricrin synthesis is different from that of another envelope precursor, involucrin, which does not seem to be significantly modulated by retinoic acid. Together with the well-documented inhibition of epidermal transglutaminase by retinoic acid, our results provide a molecular basis for the inhibition of cornified envelope formation by retinoic acid.

Epidermal growth factor and transforming growth factor alpha specifically induce the activation- and hyperproliferation-associated keratins 6 and 16.

Epidermal injury results in activation of keratinocytes which produce and respond to growth factors and cytokines and become migratory. Activated keratinocytes express a specific pair of keratin proteins, K6 and K16, distinct from the keratins in the healthy epidermis. Keratinocytes can be activated, for example, by binding of the appropriate ligands to the epidermal growth factor receptor (EGFR). We have analyzed the effects of EGFR activation on keratin gene transcription by transfecting DNAs containing keratin promoters linked to a reporter gene into primary cultures of human epidermal keratinocytes in the presence or absence of EGF or transforming growth factor alpha (TGF alpha), two growth factors that activate EGFR. The activation of EGFR had no effect on the promoters of simple epithelial, basal-layer-specific, or differentiation-specific keratins. In contrast, the expression of K6 and K16 was strongly and specifically induced. A 20-bp DNA segment of the K16 gene promoter conveyed the EGF regulation, functioned in a heterologous construct, and therefore constituted an EGF-responsive element. A nuclear protein specifically bound to this element and to the analogous sequence of the K6 promoter. Thus, EGF specifically induces K6 and K16, markers of activated keratinocytes, via nuclear proteins that bind to EGF-responsive elements in the promoters of these keratin genes.