Regulation of K3 keratin gene transcription by Sp1 and AP-2 in differentiating rabbit corneal epithelial cells

Rabbit corneal epithelial cells cultured in the presence of 3T3 feeder cells undergo biochemical differentiation, as evidenced by their initial expression of K5 and K14 keratins characteristic of basal keratinocytes, followed by the subsequent expression of K3 and K12 keratin markers of corneal epithelial differentiation. Previous data established that mutations of an Sp1 site in a DNA element, E, that contains overlapping Sp1 and AP-2 motifs reduce K3 gene promoter activity by 70% in transfection assays. We show here that Sp1 activates while AP-2 represses the K3 promoter. Although undifferentiated corneal epithelial basal cells express equal amounts of Sp1 and AP-2 DNA-binding activities, the differentiated cells down-regulate their Sp1 activity slightly but their AP-2 activity drastically, thus resulting in a six- to sevenfold increase in the Sp1/AP-2 ratio. This change coincides with the activation and suppression of the differentiation-related K3 gene and the basal cell-related K14 keratin gene, respectively. In addition, we show that polyamines, which are present in a high concentration in proliferating basal keratinocytes, can inhibit the binding of Sp1 to its cognate binding motif but not that of AP-2. These results suggest that the relatively low Sp1/AP-2 ratio as well as the polyamine-mediated inhibition of Sp1 binding to the E motif may account, in part, for the suppression of the K3 gene in corneal epithelial basal cells, while the elevated Sp1/AP-2 ratio may be involved in activating the K3 gene in differentiated corneal epithelial cells. Coupled with the previous demonstration that AP-2 activates the K14 gene in basal cells, the switch of the Sp1/AP-2 ratio during corneal epithelial differentiation may play a role in the reciprocal expression of the K3 and K14 genes in the basal and suprabasal cell layers.     

Suppression of the metastatic phenotype of a mouse skin carcinoma cell line independent of E-cadherin expression and correlated with reduced Ha-ras oncogene products

The HaCa4 cell line, derived from a mouse skin carcinoma induced by Harvey murine sarcoma virus, is highly tumorigenic when injected into nude mice and produces multiple metastases in the lungs. HaCa4 cells express high levels of viral Ha-ras oncogene products, anomalously synthesize the embryonic/simple epithelial keratin K8, and have lost the expression of the cell-cell adhesion receptor E-cadherin (E-CD). E-CD(+) cell clones (E62 and E24), obtained by transfection of an exogenous E-CD cDNA into HaCa4 cells, had a decreased ability to migrate through type IV collagen matrices. However, the E-CD (+) E62 clone remained as metastatic as the parental cell line, whereas the E24 clone, which does not take up the exogenous cDNA but spontaneously switches on the endogenous E-CD gene, suppressed the metastatic phenotype although it maintained its tumorigenicity. E24 cells had fivefold to sixfold lower levels of viral Ha-ras mRNA and p21 protein than the other cell lines. In addition, they did not synthesize K8 but rather switched on keratin K19. The comparison of E-CD proteins synthesized by E62 and E24 cell lines revealed no structural or functional differences because both localized at cell-cell contacts and associated with alpha-catenin, beta-catenin, and plakoglobin. Furthermore, E-CD was still expressed in metastatic lung nodules produced by E62 cells. These results suggest that suppression of the metastatic phenotype in E24 cells occurs independently of E-CD expression and correlates with decreased levels of the oncogenic ras p21 protein.     

Worldwide trends in suicide mortality, 1955-1989

Expression of keratin K5 (and K14) in multilayered epithelia occurs predominantly in the basal layer of proliferating keratinocytes. When a keratinocyte becomes committed to terminal differentiation, it moves out of the basal layer towards the epithelial surface. As part of this program of terminal differentiation, the expression of K5 (and K14) is downregulated in suprabasal cells, and new pairs of differentiation-specific keratins are expressed. To define the cis-acting DNA sequences required for K5 cell-type- and differentiation-specific expression, chimeric gene fusions between portions of the bovine keratin K5 locus and the Escherichia coli lacZ gene were used to generate transgenic mice. In the genomic fragment consisting of 5.3 kb of 5' flanking sequences, 6.1 kb corresponding to the body of the gene and 4.5 kb of 3' flanking sequences, the subfragment extending from -5300 bp to +138 bp was the smaller region that directed lacZ expression to stratified epithelia in a manner analogous to the endogenous keratin K5. Proximal sequences from -1300 bp to +138 bp were inactive. We also determined the expression pattern of keratin K5 during mouse development using an antiserum specific for mouse keratin K5. Expression was first detected in ectodermal cells of 11.5 days postcoitum embryos, and from day 13.5 postcoitum onwards K5 was detected in the precursors of most epithelia and organs which express K5 at adult stages. This pattern was reproduced, with few differences, by the construct with sequences from -5300 bp to +138 bp fused to the lacZ gene. These findings identify sequences between -5.3 kb and -1.3 kb of the bovine K5 gene as being important for cell-type- and differentiation-specific gene expression both during mouse development and in the adult.     

Mutation of a major keratin phosphorylation site predisposes to hepatotoxic injury in transgenic mice

Simple epithelia express keratins 8 (K8) and 18 (K18) as their major intermediate filament (IF) proteins. One important physiologic function of K8/18 is to protect hepatocytes from drug-induced liver injury. Although the mechanism of this protection is unknown, marked K8/18 hyperphosphorylation occurs in association with a variety of cell stresses and during mitosis. This increase in keratin phosphorylation involves multiple sites including human K18 serine-(ser)52, which is a major K18 phosphorylation site. We studied the significance of keratin hyperphosphorylation and focused on K18 ser52 by generating transgenic mice that overexpress a human genomic K18 ser52--> ala mutant (S52A) and compared them with mice that overexpress, at similar levels, wild-type (WT) human K18. Abrogation of K18 ser52 phosphorylation did not affect filament organization after partial hepatectomy nor the ability of mouse livers to regenerate. However, exposure of S52A-expressing mice to the hepatotoxins, griseofulvin or microcystin, which are associated with K18 ser52 and other keratin phosphorylation changes, resulted in more dramatic hepatotoxicity as compared with WT K18-expressing mice. Our results demonstrate that K18 ser52 phosphorylation plays a physiologic role in protecting hepatocytes from stress-induced liver injury. Since hepatotoxins are associated with increased keratin phosphorylation at multiple sites, it is likely that unique sites aside from K18 ser52, and phosphorylation sites on other IF proteins, also participate in protection from cell stress.     

Targeted expression of activated erbB-2 to the epidermis of transgenic mice elicits striking developmental abnormalities in the epidermis and hair follicles

The erbB-2 proto-oncogene belongs to a receptor tyrosine kinase family that includes the epidermal growth factor receptor, erbB-2, erbB-3, and erbB-4. erbB-2 is expressed in basal cells of the squamous epithelia and the outer root sheath of the hair follicles, but its function in epidermal development has not been well studied. To investigate its role in the skin, we created transgenic mice harboring an activated erbB-2 oncogene under the control of the human keratin 14 promoter. The keratin 14 promoter directed its expression to cells in which erbB-2 is normally expressed, whereas the activated receptor gene ensured increased signaling. All transgenic founder mice exhibited extensive and striking skin phenotype, including epidermal hyperplasia, preneoplasia, papilloma, hyperkeratosis, and dyskeratosis. The majority of the hair follicles were replaced by bizarre hyperproliferative intradermal squamous invaginations, whereas the rest of the follicles exhibited severe hyperplasia and disorganization. All but one of the transgenic mice died before or shortly after birth, probably as a consequence of defects in the skin and esophagus. These observations demonstrate that the skin is sensitive to erbB-2 signaling, suggesting an important role for this receptor tyrosine kinase in epidermal growth, differentiation, and hair follicle morphogenesis.     

Assembly of hair keratins in transfected epithelial cells

To define the interactions required for the filament assembly of differentiation-specific keratins, active copies of mouse hair keratin mHa1 and mHb4 genes were introduced into a rat kangaroo kidney epithelial cell line (PtK2) and a rat stratified squamous epithelial cell line (rat epidermal keratinocyte). In PtK2 transient transfectants, when introduced individually or in combination, mHa1 and mHb4 formed aggregates of ring-like structures of various sizes at the perinuclear region with no evidence of organization into a keratin network. These aggregates altered the distribution of the endogenous keratins and vimentin. In most of the cells carrying the ring-like structures of mHa1 and mHb4 around the nucleus, the endogenous keratin network collapsed and localized around the nucleus. Furthermore, the densely accumulated endogenous keratin surrounded the ring-like aggregates with partial co-localization. However, when transfected into the rat epidermal keratinocytes, mHa1 and mHb4 were able to co-localize with the well-developed cytoskeleton of endogenous keratins. These results showed that, in contrast to keratin pairs K5/K14 and K8/K18, the mHa1/mHb4 pair is unable to develop an extensive keratin network on its own and that there are possible differential abilities among these hair keratins and other keratins to form well-developed cytoplasmic networks.     

Altered epidermal cell growth control in vivo by inducible expression of transforming growth factor beta 1 in the skin of transgenic mice

An inducible bovine KIV* keratin gene promoter was used to target expression of latent or activated transforming growth factor beta 1 (TGF beta 1) to keratinocytes in transgenic mice. This short (2.2-kb) keratin 6 (K6) promoter element was generally silent in untreated animals but was induced in keratinocytes when placed in culture or, in vivo, in response to hyperplasia that follows topical application of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate. All of the K6-TGF beta 1 transgenic lines studied showed attenuation of the basal keratinocyte proliferative response to 12-O-tetradecanoylphorbol-13-acetate as a consequence of inducible TGF beta 1 gene expression. One of the six lines studied showed constitutive transgene expression at low levels in the skin, and this line had a 2- to 3-fold increase in epidermal DNA labeling index over control mice. Although in vitro TGF beta 1 is known to be a potent negative regulator of epithelial cell proliferation, in vivo TGF beta 1 has complex biological activities and can act as either a positive or negative regulator of keratinocyte proliferation.     

Susceptibility to hepatotoxicity in transgenic mice that express a dominant-negative human keratin 18 mutant

Keratins 8 and 18 (K8/18) are intermediate filament phosphoglycoproteins that are expressed preferentially in simple-type epithelia. We recently described transgenic mice that express point-mutant human K18 (Ku, N.-O., S. Michie, R.G. Oshima, and M.B. Omary. 1995. J. Cell Biol. 131:1303-1314) and develop chronic hepatitis and hepatocyte fragility in association with hepatocyte keratin filament disruption. Here we show that mutant K18 expressing transgenic mice are highly susceptible to hepatotoxicity after acute administration of acetaminophen (400 mg/Kg) or chronic ingestion of griseofulvin (1.25% wt/wt of diet). The predisposition to hepatotoxicity results directly from the keratin mutation since nontransgenic or transgenic mice that express normal human K18 are more resistant. Hepatotoxicity was manifested by a significant difference in lethality, liver histopathology, and biochemical serum testing. Keratin glycosylation decreased in all griseofulvin-fed mice, whereas keratin phosphorylation increased dramatically preferentially in mice expressing normal K18. The phosphorylation increase in normal K18 after griseofulvin feeding appears to involve sites that are different to those that increase after partial hepatectomy. Our results indicate that hepatocyte intermediate filament disruption renders mice highly susceptible to hepatotoxicity, and raises the possibility that K18 mutations may predispose to drug hepatotoxicity. The dramatic phosphorylation increase in nonmutant keratins could provide survival advantage to hepatocytes.     

Directed expression of keratin 16 to the progenitor basal cells of transgenic mouse skin delays skin maturation

We previously hypothesized that the type I keratin 16 (K16) plays a role in the process of keratinocyte activation that occurs in response to skin injury (Paladini, R.D., K. Takahashi, N.S. Bravo, and P.A. Coulombe. 1996. J. Cell Biol. 132:381-397). To further examine its properties in vivo, the human K16 cDNA was constitutively expressed in the progenitor basal layer of transgenic mouse skin using the K14 gene promoter. Mice that express approximately as much K16 protein as endogenous K14 display a dramatic postnatal phenotype that consists of skin that is hyperkeratotic, scaly, and essentially devoid of fur. Histologically, the epidermis is thickened because of hyperproliferation of transgenic basal cells, whereas the hair follicles are decreased in number, poorly developed, and hypoproliferative. Microscopically, the transgenic keratinocytes are hypertrophic and feature an altered keratin filament network and decreased cell-cell adhesion. The phenotype normalizes at approximately 5 wk after birth. In contrast, control mice expressing a K16-K14 chimeric protein to comparable levels are normal. The character and temporal evolution of the phenotype in the K16 transgenic mice are reminiscent of the activated EGF receptor- mediated signaling pathway in skin. In fact, tyrosine phosphorylation of the EGF receptor is increased in the newborn skin of K16 transgenic mice. We conclude that expression of K16 can significantly alter the response of skin keratinocytes to signaling cues, a distinctive property likely resulting from its unique COOH-terminal tail domain.     

Both conserved region 1 (CR1) and CR2 of the human papillomavirus type 16 E7 oncogene are required for induction of epidermal hyperplasia and tumor formation in transgenic mice

High-risk human papillomavirus type 16 (HPV-16) and HPV-18 are associated with the majority of human cervical carcinomas, and two viral genes, HPV E6 and E7, are commonly found to be expressed in these cancers. The presence of HPV-16 E7 is sufficient to induce epidermal hyperplasia and epithelial tumors in transgenic mice. In this study, we have performed experiments in transgenic mice to determine which domains of E7 contribute to these in vivo properties. The human keratin 14 promoter was used to direct expression of mutant E7 genes to stratified squamous epithelia in mice. The E7 mutants chosen had either an in-frame deletion in the conserved region 2 (CR2) domain, which is required for binding of the retinoblastoma tumor suppressor protein (pRb) and pRb-like proteins, or an in-frame deletion in the E7 CR1 domain. The CR1 domain contributes to cellular transformation at a level other than pRb binding. Four lines of animals transgenic for an HPV-16 E7 harboring a CR1 deletion and five lines harboring a CR2 deletion were generated and were observed for overt and histological phenotypes. A detailed time course analysis was performed to monitor acute effects of wild-type versus mutant E7 on the epidermis, a site of high-level expression. In the transgenic mice with the wild-type E7 gene, age-dependent expression of HPV-16 E7 correlated with the severity of epidermal hyperplasia. Similar age-dependent patterns of expression of the mutant E7 genes failed to result in any phenotypes. In addition, the transgenic mice with a mutant E7 gene did not develop tumors. These experiments indicate that binding and inactivation of pRb and pRb-like proteins through the CR2 domain of E7 are necessary for induction of epidermal hyperplasia and carcinogenesis in mouse skin and also suggest a role for the CR1 domain in the induction of these phenotypes through as-yet-uncharacterized mechanisms.     

Epidermolytic hyperkeratosis (bullous congenital ichthyosiform erythroderma). Genetic linkage to chromosome 12q in the region of the type II keratin gene cluster

Epidermolytic hyperkeratosis (EHK) is an autosomal dominant genodermatosis characterized by hyperkeratosis and blistering of the skin. Histopathology demonstrates suprabasilar blister formation with aggregation of tonofilaments. In this study, we tested the hypothesis that the EHK phenotype is linked to one of the suprabasilar keratins (KRT10 or KRT1) present in the types I and II keratin gene clusters in chromosomes 17q and 12q, respectively. For this purpose, Southern hybridizations were performed with DNA from a large kindred with EHK, consisting of 11 affected individuals in three generations. Segregation analysis with markers flanking the keratin gene clusters demonstrated linkage (Z = 3.61 at theta = 0) to a locus on 12q, while markers on 17q were excluded. These data implicate KRT1, the type II keratin expressed in suprabasilar keratinocytes, as a candidate gene in this family with EHK.     

Co-operation between follicular ornithine decarboxylase and v-Ha-ras induces spontaneous papillomas and malignant conversion in transgenic skin

Ornithine decarboxylase (ODC) is aberrantly regulated in tumor cells and results in high basal levels of ODC and polyamines in many epithelial tumors. To determine if elevated ODC/polyamine levels can co-operate with a mutant Ha-ras gene in mouse skin tumorigenesis, double transgenic mice were generated by breeding K6/ODC transgenic mice with TG.AC v-Ha-ras transgenic mice. A K6 keratin promoter drives the ODC transgene in K6/ ODC transgenic mice, which results in elevated ODC/ polyamine levels directed to the outer root sheath cells of hair follicles. TG.AC transgenic mice carry a v-Ha-ras transgene while still retaining two normal c-Ha-ras alleles. Transgenic mice that possess only the K6/ODC or the v-Ha-ras transgene did not develop tumors unless treated with either a carcinogen or a tumor promoter, respectively. However, a high percentage of double transgenic mice possessing both the K6/ODC and v-Ha-ras transgenes developed spontaneous tumors. All tumors were well-differentiated keratoacanthomas, some of which progressed to carcinomas within 2 months. The development and the maintenance of these ODC/ras tumors was ODC-dependent since alpha-difluoromethylornithine (DFMO), a specific ODC inhibitor, prevented the formation and caused the regression of these tumors. These findings indicate that ODC overexpression and an activated Ha-ras are sufficient to produce a high rate of malignant transformation in an animal model. The ODC/ras double transgenic mouse provides a simple in vivo model without the use of chemical carcinogens or tumor promoters in which to test downstream effectors that play a key role in mediating the development of epithelial tumors resulting from the cooperation between ODC and v-Ha-ras.     

Man-in-the-barrel syndrome caused by cervical spinal cord infarction

Ichthyosis bullosa of Siemens is a rare autosomal dominant skin disorder whose clinical findings are quite similar to those of epidermolytic hyperkeratosis. The differences between those two diseases include absence of erythroderma and different distributions in the skin in ichthyosis bullosa of Siemens. Recent studies have confirmed that ichthyosis bullosa of Siemens is caused by the mutation in the keratin 2e (K2e) gene, which is expressed in the upper spinous and granular layers. We have identified a sporadic case of ichthyosis bullosa of Siemens; based on diagnosis by histopathological findings, the K2e gene of the patient was analysed. Direct sequencing of PCR products revealed a single base change in sequences encoding the highly conserved end of the 2B rod domain segment of the K2e gene. This mutation results in substitution of the codon for glutamic acid by a codon for lysine in position 493 in K2e (E493K). Mutations of the K2e gene involving five different residue positions (Q187P, T485P, L490P, E493D, E493K and E494K) are known to cause ichthyosis bullosa of Siemens. Of these sites, E493, which is conserved in type I and type II keratin genes, is the most frequently altered amino acid in the K2e gene. These data together suggest that this codon constitutes a hot spot for mutations in the K2e gene.     

A novel H1 mutation in the keratin 1 chain in epidermolytic hyperkeratosis

We report a novel mutation in a case of epidermolytic hyperkeratosis that results in a proline for arginine substitution in the penultimate residue position of the H1 subdomain of the keratin 1 chain, which is near the beginning of the rod domain. This causes a severe clinical disease classified as PS-2. Therefore, the H1 subdomain is probably equally important for the maintenance of keratin intermediate filament integrity as the rod domain. Since earlier concepts had implied that mutations in the H1 subdomain produce milder disease, this case suggests that attempts to correlate mutations with disease presentation remain problematic.     

Phenotypic and functional characterization in vitro of a multipotent epithelial cell present in the normal adult human breast

The developmental relationships between the different mammary epithelial cell lineages in the human mammary gland are not well defined. To characterize human breast epithelial cells (HBEC) with progenitor activity, we used flow cytometry and single cell sorting to analyze the distribution of cellular phenotypes in primary cultures of reduction mammoplasties and their associated ability to generate colonies in 2-dimensional (D) and 3-D (collagen gel) culture systems. This approach allowed two distinct types of HBEC progenitor populations to be distinguished on the basis of their differential expression of the MUC-1 glycoprotein, CALLA/CD10 and epithelial-specific antigen (ESA). The first type of progenitor, which is enriched in the MUC-1+/CAL-LA-/ESA+ subpopulation, generated colonies of tightly arranged cells in 2-D cultures and small alveolar-like colonies with a central lumen when cultured in a collagen matrix. The cells produced in the colonies and derived from these MUC-1+/CALLA-/ESA+ progenitors were found to express typical luminal epitopes (keratin 8/18, keratin 19, MUC-1, ESA) and showed low levels of expression of myoepithelial epitopes (keratin 14 and CD44v6). The second type of progenitor, which is present in the MUC-1-to +/-/CALLA +/- to +/ESA+ subpopulation, generated mixed colonies of both luminal and myoepithelial cells when seeded in 2-D and 3-D cultures. In 2-D cultures, the centrally located cells exhibited a luminal morphology and expressed ESA, but were heterogeneous in their expression of MUC-1. Radiating from the periphery of these ESA+ HBEC were highly refractile ESA- teardrop-shaped myoepithelial-like cells. When cultured in a collagen matrix, these bipotent progenitors generated large branched colonies composed of a heterogeneous population of cells, with some of the progeny cells expressing luminal epitopes (keratin 8/18, keratin 19 and MUC-1) and others expressing myoepithelial epitopes (keratin 14 and CD44v6). A third type of progenitor, which became apparent is passaged HBEC cultures and was enriched in the MUC-1-/CALLA+/ESA- subpopulation, was found to generate colonies of cells with an exclusively myoepithelial phenotype. These results provide definitive evidence for the existence of multilineage HBEC progenitors in normal adult human mammary tissue. The phenotypic profile of these cells suggest that these multilineage progenitors are a relatively undifferentiated cell since they express low levels of MUC-1 and that they have a luminal location within the mammary epithelium since they are ESA+. Furthermore, we suggest that the MUC-1+/CALLA-/ESA+ and the MUC-1- to +/-/CALLA +/- to +/ESA+ progenitors we have identified and characterized are candidate in vivo alveolar and ductal progenitors, respectively.     

Lung-specific expression of adenovirus E3-14.7K in transgenic mice attenuates adenoviral vector-mediated lung inflammation and enhances transgene expression

Herein, we report that the adenovirus E3-14.7K protein inhibits the inflammatory response to adenovirus in transgenic mice in which the E3-14.7K gene was selectively expressed in the respiratory epithelium, using the human surfactant protein C (SP-C) promoter. E3-14.7K mRNA and protein were detected specifically in the lungs of SPC/E3-14.7K transgenic mice. Responses of the transgenic mice to Av1Luc1, an E1-E3-deleted Ad vector encoding the luciferase reporter gene, were examined, including vector transgene expression and lung inflammation. In wild-type mice, luciferase activity declined rapidly and was lost 14 days following Av1Luc1 administration. The loss of luciferase activity was associated with pulmonary infiltration by macrophages and lymphocytes. In heterozygous SPC/E3-14.7K mice, luciferase activity was increased by 7 days compared with control littermates, and pulmonary infiltration by macrophages was decreased. In homozygous (+/+) SPC/E3-14.7K mice, luciferase activity was increased 7, 14, and 21 days following administration compared with wild-type mice, and lung inflammation was markedly reduced. After Av1Luc1 administration, PCNA staining of bronchiolar and alveolar respiratory epithelial cells was decreased in SPC/E3-14.7K transgenic mice, indicating decreased epithelial cell proliferation, a finding consistent with the observed reduction in inflammation. CD4 and CD8 lymphocyte populations were only mildly altered, while humoral responses to adenoviral vectors were unchanged in the SPC/E3-14.7K mice. The E3-14.7K protein expressed selectively in respiratory epithelial cells suppresses Ad-induced pulmonary epithelial cell cytotoxicity and lung inflammation in vivo and prolongs reporter gene expression.     

Novel K5 and K14 mutations in German patients with the Weber-Cockayne variant of epidermolysis bullosa simplex

We report novel keratin 5 and 14 gene mutations in four unrelated German families with the localized subtype of the dominantly inherited blistering disease epidermolysis bullosa simplex Weber-Cockayne (MIM# 131800). The mutations are located in the keratin 14 L12 linker region (D273G), the keratin 5 L12 linker (M327K and D328H), and the H1 domain of keratin 5 (P156L). These mutations add to those previously reported and provide further evidence of phenotype-genotype correlations in epidermolysis bullosa simplex subtypes. The above mutations in mildly affected patients underline the relevance of the keratin linker regions for the epidermolysis bullosa simplex Weber-Cockayne phenotype and keratin filament integrity. In addition, they confirm that the gene segments encoding the linker regions represent hotspots for mutations.