The v-ras oncogene inhibits the expression of differentiation markers and facilitates expression of cytokeratins 8 and 18 in mouse keratinocytes.

Cultured mouse keratinocytes can be initiated in vitro by the introduction of a v-rasHa gene by viral transduction. Previous studies indicated that v-rasHa-transduced keratinocytes have a high proliferation rate in medium with 0.05 mM Ca2+ and resist terminal differentiation in medium with greater than 0.

Expression of murine epidermal differentiation markers is tightly regulated by restricted extracellular calcium concentrations in vitro.

Epidermal differentiation is characterized by a series of coordinated morphological and biochemical changes which result in a highly specialized, highly organized, stratified squamous epithelium. Among the specific markers expressed in differentiating epidermis are (a) two early spinous cell proteins, keratins 1 and 10 (K1 and K10); and (b) two later granular cell proteins, filaggrin and a cornified envelope precursor (CE). In vitro, epidermal basal cells are selectively cultured in 0.

Density-dependent modulation of synthesis of keratins 1 and 10 in the human keratinocyte line HACAT and in ras-transfected tumorigenic clones.

The spontaneous human keratinocyte line HaCaT and c-Ha-ras oncogene-transfected cell clones are capable of expressing an unusually broad spectrum of keratins, not observed so far in epithelial cells. This expression is, however, strongly modulated by environmental conditions, including cell density. Both cells of the nontumorigenic HaCaT line and the tumorigenic HaCaT-ras clones, I-7 and II-3 (giving rise to benign and malignant tumors, respectively), constitutively expressed the keratins K5, K6, K14, K16 and K17, which are also common in cultures of normal keratinocytes.

Isolation of complementary DNA for bullous pemphigoid antigen by use of patients' autoantibodies.

Autoantibodies from bullous pemphigoid (BP) patients define a 230-kD protein found in the basement membrane of stratified squamous epithelia. The purpose of this study was to isolate and characterize a cDNA clone with coding sequences for BP antigen. Poly(A+) RNA derived from total RNA of cultured keratinocytes was used, with oligo-dT priming, to construct a cDNA library in the lambda gt11 expression vector, which was screened by the immunoperoxidase method with one BP serum.

In situ hybridization analysis of keratin gene expression in human ameloblastomas.

Complementary DNA (cDNA) clones corresponding to the 55 kDa (K 14) and 59 kDa (K 10) keratins were used as probes for in situ hybridization analysis for the expression of keratin genes in human ameloblastomas and in oral mucosa. Transcripts for either the K 14 keratin or the K 10 keratin were restricted in their spatial distribution within stratified epithelia consistent with the stage of differentiation of the keratinocyte: the K 14 keratin gene transcript was restricted to the basal cell layers of the mucosa, while the K 10 keratin transcript was expressed predominantly in suprabasal cells, within the granular and prickle layers. In contrast, only the K 14 keratin transcript could be identified within the epithelial cells of human ameloblastomas.

The complete sequence of the human intermediate filament chain keratin 10. Subdomainal divisions and model for folding of end domain sequences.

We present the complete amino acid sequence of the human keratin 10 (type I) intermediate filament chain expressed in terminally differentiated epidermal cells. Comparisons of this sequence with its mouse and bovine counterparts allow us to describe structural features of the functional end domains. First, sections of their respective end domains are highly conserved and permit a redefinition of earlier models for their subdomainal organization.

Sequential development of aneuploidy, keratin modifications, and gamma-glutamyltransferase expression in mouse skin papillomas.

To elucidate the role and timing of expression of different premalignant and malignant markers in tumor promotion, we correlated alterations in keratin patterns and gamma-glutamyltransferase (GGT) expression with the chromosomal status of individual mouse skin papillomas. Papillomas were induced by 7,12-dimethylbenz[a]anthracene initiation and 12-O-tetradecanoylphorbol-13-acetate promotion. Individual tumors were randomly sampled at 20 and 35 weeks of promotion.

Transcriptional control of high molecular weight keratin gene expression in multistage mouse skin carcinogenesis.

Monospecific antikeratin antisera and specific complementary DNA probes were used to analyze expression of keratin genes in newborn mouse skin and skin papillomas and carcinomas by indirect immunofluorescence, immunoblotting, and in situ hybridization. Tumors were induced by initiation with 7,12-dimethylbenz[a]anthracene and promotion with 12-O-tetradecanoylphorbol-13-acetate. Type I epidermal keratin K14 protein (Mr 55,000) is found in all living layers of the newborn skin but is most abundant in the lower strata.

The expression and modulation of IL-1 alpha in murine keratinocytes.

Murine and human keratinocytes produce an IL-1-like factor that appears to be similar if not identical to monocyte-derived IL-1. IL-1 may be an important mediator in cutaneous inflammatory responses, however, little is currently known concerning factors that may modulate IL-1 expression in keratinocytes. To address this issue we examined the effect of LPS, UV, and the cell differentiation state on murine keratinocyte IL-1 mRNA expression.

Aberrant expression during two-stage mouse skin carcinogenesis of a type I 47-kDa keratin, K13, normally associated with terminal differentiation of internal stratified epithelia.

Specific keratin cDNA probes and monospecific antikeratin antisera were used to analyze mouse epidermis and epidermal tumors for the expression of a type I 47-kDa keratin, K13, normally associated with terminal differentiation of internal stratified epithelia. We demonstrated that this keratin was virtually absent from the entire body epidermis at various stages of development. Also, it was not detected in various forms of acute and chronic epidermal hyperproliferation or in epidermal cells cultured under conditions that favored either cell proliferation or in vitro differentiation.

Molecular cloning of mouse epidermal cystatin A and detection of regulated expression in differentiation and tumorigenesis.

A gamma gt 11 cDNA expression library representing mouse epidermis mRNA was screened with polyclonal rabbit antiserum directed against 10-13 kDa epidermal antigens that had previously been shown to be regulated during epidermal differentiation. A cDNA clone was detected and isolated and its identity as the coding sequence for one of the antigens was confirmed by translation of hybrid-selected mRNA from mouse epidermis. The cDNA sequence predicted a peptide homologous to the reported sequence of rat epidermis cystatin A, a thiol proteinase inhibitor.

Signal transduction for proliferation and differentiation in keratinocytes.

In mouse and human epidermis, the Ca2+ environment of the basal cell layer is substantially below serum Ca2+, while that of the granular cell layer is unusually high. Reduction of extracellular Ca2+ concentration (Cao) in the medium of keratinocyte cultures maintains a basal cell phenotype while serum Ca2+ concentrations induce terminal differentiation. Measurements of intracellular Ca2+ (Cai) by the use of Fura 2 and digital imaging technology reveal that Cai increases 10-20-fold in response to an increase in Cao and remains elevated.

The gene for mouse epidermal filaggrin precursor. Its partial characterization, expression, and sequence of a repeating filaggrin unit.

Filaggrin is an important keratin intermediate filament-associated protein of terminally differentiated mammalian epidermis. Its aberrant expression has been implicated in a number of keratinizing disorders. We have isolated and sequenced a cDNA clone to mouse filaggrin, of 1.

Cultivation of murine hair follicles as organoids in a collagen matrix.

Techniques are described for the isolation and cultivation of functionally intact mouse hair follicles. Follicles were isolated by collagenase digestion of dermis from 5-day-old mice and purified by differential centrifugation and filtration. Purified follicles were cultured in a Type 1 collagen matrix using Medium 199 and 8% fetal calf serum as the basic nutrient.

Abnormal expression and processing of keratins in pupoid fetus (pf/pf) and repeated epilation (Er/Er) mutant mice.

The pupoid fetus (pf) and repeated epilation (Er) mutations of mice result in a failure of epidermal differentiation in homozygotes. Expression of the epidermal keratins has been followed in pf/pf and Er/Er mice by two-dimensional gel electrophoresis, and by immunohistochemistry and Western blotting using polyclonal antibodies that are monospecific for individual keratin polypeptides. Our results show that expression of the differentiation-specific keratins (K1 and K10) is delayed in both the pf/pf and Er/Er mutants and that, when these keratins do appear later in development, they are localized in the deeper layers of the thickened mutant epidermis.

Assignment of the Ly-6--Ril-1--Sis--H-30--Pol-5/Xmmv-72--Ins-3--Krt-1--Int-1 --Gdc-1 region to mouse chromosome 15.

Previous work has demonstrated linkage between Ly-6, H-30, and a locus, Ril-1, that affects susceptibility to radiation-induced leukemia. Results or preliminary linkage analyses suggested further that the cluster might be linked to Ly-11 on the proximal portion of mouse chromosome 2. Using molecular probes to examine somatic cell lines and recombinant inbred and congenic strains of mice, we have re-evaluated these linkage relationships.

Regulated expression of differentiation-associated keratins in cultured epidermal cells detected by monospecific antibodies to unique peptides of mouse epidermal keratins.

Monospecific antibodies to mouse epidermal keratins were generated in rabbits and guinea pigs by injecting synthetic peptides of unique keratin sequences. The sequences were deduced from nucleotide sequences of cDNA clones representing basal (K14) and suprabasal (K1 and K10) cell-specific and hyperproliferative (K6) keratins of both the type-I and type-II subclasses. By applying single-and double-label immunofluorescence analysis, the expression of keratin peptides was analyzed in cultured keratinocytes maintained in the basal or suprabasal cell phenotypes.

An activated Harvey ras oncogene produces benign tumours on mouse epidermal tissue.

Studies of the mutagenic action required for specific chemicals to produce benign or malignant tumours suggest that in mouse skin at least two genetic events occur before carcinoma formation. The isolation of an activated form of the c-rasH gene from skin papillomas has provided evidence that this gene may be a target for the first mutation, which could constitute the initiating mutation in skin carcinogenesis. In vitro studies indicate that the v-rasH gene of Harvey murine sarcoma virus (Ha-MSV), a replication-defective transforming retrovirus, could impart a conditional initiated phenotype on cultured keratinocytes by blocking their ability to differentiate terminally and arresting them in a late basal cell stage of maturation.

Regulated synthesis of low-molecular-weight antigens in keratinocyte cell cultures.

Proteins from mouse epidermis cytosol extracts react on immunoblots with a polyclonal rabbit antiserum raised against rat skin calcium-binding protein (SCaBP), a parvalbumin of the panniculus carnosus. Three mouse epidermal proteins with molecular weights between 10-12K, which are distinct from SCaBP, are recognized by the antiserum. The synthesis of these proteins in keratinocyte culture is modulated by Ca++, as is the differentiation of the keratinocytes.