Myofibroblast phenotype and apoptosis in keloid and palmar fibroblasts in vitro.

Keloid formation is a wound healing response, which fails to resolve and leads to formation of a raised collagen mass extending beyond the original wound margins. Keloids are typically excluded from palms and soles. Therefore we compared keloid and palmar fibroblasts in vitro using fibroblasts from nonaffected individuals as controls. Collagen I, alpha-smooth muscle actin and thrombospondin-1 were found at higher levels in keloid than in palmar fibroblasts. These differences were ameliorated by addition of TGFbeta1. The potential for resolution of the wound healing response was estimated analyzing apoptosis during serum starvation. Annexin V and TUNEL assays showed that palmar fibroblasts underwent faster apoptosis, than did the keloid fibroblasts, and started detaching. Addition of TGFbeta1 counteracted this effect. The weak expression of the myofibroblast phenotype and the advanced apoptosis of palmar fibroblasts suggest mechanisms for the exclusion of keloids from palmar sites.

Characterization of an immortalized cell line from a patient with epidermolytic hyperkeratosis.

The most frequent mutation that causes the autosomal dominant skin disease epidermolytic hyperkeratosis (EHK) is an arginine to histidine substitution at position 10 in the 1A segment of the rod domain of keratin 10. As an initial step toward developing a strategy for treating EHK, a cell line, EH18-1, was established after keratinocytes derived from an EHK patient with this mutation were immortalized by a recombinant retrovirus encoding the E6 and E7 genes of human papillomavirus type 18. EH18-1 cells synthesize considerable amounts of keratin 10 mRNA and protein when maintained in either submerged cultures or in organotypic cultures. When grown in organotypic culture, EH18-1 cells form multiple layers and express keratin 10 and filaggrin predominantly in the upper layers. Thus, the EH18-1 cell line exhibits several morphological and biochemical markers of terminal epidermal differentiation. A semiquantitative reverse transcriptase polymerase chain reaction assay for keratin 10 mRNA was developed to distinguish between expression of the normal and the mutant alleles. The EH18-1 keratinocyte cell line will be useful in developing protocols for gene therapy of EHK that may be monitored by reverse transcriptase polymerase chain reaction of either allele.

Preferential sites in keratin 10 that are mutated in epidermolytic hyperkeratosis.

Epidermolytic hyperkeratosis (EH) is a rare autosomal dominant skin disease. Recent studies in our laboratory established genetic linkage to the type II keratin gene locus on chromosome 12q in one family with EH and identified a single amino acid mutation in keratin 1 that is responsible for the disease. Other point mutations in the keratin 1 or keratin 10 genes have now been reported in other patients with EH. We have examined a series of probands with EH in order to develop a catalog of mutations in keratin 10. Using direct sequencing of PCR-amplified genomic DNA, we have identified mutations in six families, in which five mutations occur in the beginning of the 1A rod domain of keratin 10-namely, two ARg10 to His, one Arg10 to Cys, and Asn8 to His, and a Tyr14 to Asp. This region contains highly conserved residues among all keratins. An additional mutation (Leu103 to Gln) was found in the conserved region late in the 2B rod domain in keratin 10. We developed several allele-specific assays to assess the frequency of these mutations in the general population. No evidence was found for the presence of such changes in unaffected individuals. In vitro functional assays performed with peptides corresponding to the 1A mutations in these families show severely diminished capacity to disaggregate preformed keratin intermediate filaments, in comparison with a wild-type control peptide. Results from this work support the hypothesis that the beginning of the 1A rod domain segment in keratin 10 contains preferential sites for disease-causing mutation in EH. This should be of considerable use when developing prenatal diagnostic tests and biologically based therapies for this disease.

Mutations in the H1 and 1A domains in the keratin 1 gene in epidermolytic hyperkeratosis.

In the autosomal dominant disorder epidermolytic hyperkeratosis, the structural integrity of the keratin intermediate filaments is altered in the suprabasal layers of the epidermis. We and others have used genetic linkage studies and mutation analysis to establish that single amino acid substitutions in either the keratin 1 or keratin 10 chains can cause epidermolytic hyperkeratosis. However, a larger database of mutations is required to better understand the relationship between specific mutations in these keratin chains and their effect on keratin filament structure. A larger database will also provide a catalog that may be useful for genetic counseling purposes. In this paper, we report the identification of three new mutations of the keratin 1 chain of epidermolytic hyperkeratosis probands in highly conserved residues in the H1 or beginning of the 1A rod domain segments. These correspond to regions involved in molecular overlaps between neighboring molecules in keratin filaments. Using an in vitro assay, synthetic peptides bearing these substitutions show diminished capacity to disassemble preformed filaments in vitro in comparison to the wild type peptides. Moreover, analyses of all mutations in epidermolytic hyperkeratosis known to date demonstrate remarkable clustering in the molecular overlap region. We conclude that non-conservative substitutions in the overlap region are likely to interfere with normal keratin filament structure and function, leading to pathology.

Profilaggrin is a major epidermal calcium-binding protein.

Profilaggrin is a major highly phosphorylated protein component of the keratohyalin granules of mammalian epidermis. It contains 10 to 12 tandemly repeated filaggrin units and is processed into the intermediate filament-associated protein filaggrin by specific dephosphorylation and proteolysis during terminal differentiation of the epidermal cells. Later, filaggrin itself is degraded to free amino acids that participate in maintenance of epidermal flexibility. The present paper describes the structural organization of the 5' region of the human profilaggrin gene as well as the amino terminus of the profilaggrin protein. The primary profilaggrin transcript consists of three exons and two introns. The first exon (exon I) is only 54 bp and is untranslated. The coding sequences are distributed between exon II (159 bp) and exon III, which contains the information for 10 to 12 filaggrin repeats (972 bp each) and the 3' noncoding sequences. A very large intron separates exons I and II. The combination of a very short exon I with an unusually long intron 1 makes the structure of the profilaggrin gene unique among the epidermally expressed genes investigated so far. Comparison of the expression patterns revealed by primer extension and RNase protection analysis of foreskin epidermal and cultured keratinocyte RNAs suggests that alternately spliced messages, which are different from profilaggrin mRNA, are transcribed from the profilaggrin gene system at earlier stages of epidermal differentiation. The amino terminus of profilaggrin exhibits a significant homology to the small calcium-binding S100-like proteins. It contains two alpha-helical regions, termed EF-hands, that bind calcium in vitro. This is the first example of functional calcium-binding domains fused to a structural protein. We suggest that in addition to its role in filament aggregation and the maintenance of epidermal flexibility, profilaggrin may play an important role in the differentiation of the epidermis by autoregulating its own processing in a calcium-dependent manner or by participating in the transduction of calcium signal in epidermal cells.

A leucine----proline mutation in the H1 subdomain of keratin 1 causes epidermolytic hyperkeratosis.

Epidermolytic hyperkeratosis is an autosomal dominant disorder affecting the structural integrity of the suprabasal layers of human epidermis. We have recently documented in one family linkage of the disease phenotype to the cluster of type II keratins. We have now identified a leucine----proline amino acid substitution in the conserved H1 subdomain of keratin 1 that is present only in affected family members. Using a quantitative assay and electron microscopy with synthetic peptides, we show that, whereas the wild-type H1 peptide rapidly disassembles preformed keratin filaments in vitro, the mutant peptide does this far less efficiently. Therefore the mutation in keratin 1 is likely to cause defective keratin filaments and hence a defective cytoskeleton in the epidermal cells in vivo.

Chromosomal organization of Xenopus laevis oocyte and somatic 5S rRNA genes in vivo.

We describe the chromosomal organization of the major oocyte and somatic 5S RNA genes of Xenopus laevis in chromatin isolated from erythrocyte nuclei. Both major oocyte and somatic 5S DNA repeats are associated with nucleosomes; however, differences exist in the organization of chromatin over the oocyte and somatic 5S RNA genes. The repressed oocyte 5S RNA gene is protected from nuclease digestion by incorporation into a nucleosome, and the entire oocyte 5S DNA repeat is assembled into a loosely positioned array of nucleosomes. In contrast, the potentially active somatic 5S RNA gene is accessible to nuclease digestion, and the majority of somatic 5S RNA genes appear not to be incorporated into positioned nucleosomes. Evidence is presented supporting the stable association of transcription factors with the somatic 5S RNA genes. Histone H1 is shown to have a role both in determining the organization of nucleosomes over the oocyte 5S DNA repeat and in repressing transcription of the oocyte 5S RNA genes.

Differential DNase I sensitivity of the two complementary nucleosomal DNA strands in cycloheximide-treated Ehrlich ascites tumor cells.

The accessibility of the two complementary DNA strands in newly replicated chromatin of Ehrlich ascites tumor (EAT) cells grown under conditions of cycloheximide-inhibited protein synthesis was studied by analysis of the DNase I digestion of isolated nuclei. Bulk DNA was labeled with 14C-thymidine and the newly synthesized strands - with bromodeoxyuridine and 3H-thymidine. The DNase I digests were fractionated in two successive CsCl density gradient centrifugations to obtain a dense fraction containing 15-20% newly replicated DNA. Analysis of the distribution of 14C-labeled parental DNA fragments complementary to the 3H-nascent strand has shown that the 14C-labeled fragments prevail in the region of 30-50 nucleotides. Simulation experiments using the rate constants for DNase I attack show that this result may be explained by an enhanced accessibility at the nucleosomal 5'-end region of the parental strands, where the H2a-H2b dimer interacts with DNA. This asymmetry seems to be induced by interactions in the chromatin.