Complete sequence of a hair-like intermediate filament type II keratin gene

The Intermediate Filament (IF) superfamily comprises several multigene families, of which the two keratin families are the largest. The keratin IF genes are expressed in epithelial tissues in differentiation-specific patterns and recently we reported the sequence and expression of a hair IF type II keratin gene (KRT2.9). Two related genes were present in the cosmid containing KRT2.9 and we have now sequenced one of them and found that it encodes a hair-like IF type II protein (KRT2.13). However, KRT2.13 is not expressed in the hair follicle. Interestingly there is significant sequence homology between introns 1, 5 and 6 of KRT2.13 and KRT2.9 to suggest gene conversion of these regions or possibly conservation of functional sequences.     

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.     

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.     

Evidence for the presence of sodium- and potassium-dependent adenosine triphosphatase alpha1 and beta1 subunit isoforms and their probable role in blastocyst expansion in the preattachment horse conceptus

Two Caribbean hair sheep breeds, the St. Croix (SC) and Barbados Blackbelly (BB), are found in the United States, and the SC has led to the development of the Katahdin (K), a synthetic breed of hair sheep. These breeds have mature ewe BW ranging from 32 to 54 kg (for BB and SC) and from 55 to 73 kg (K). Hair sheep and hair sheep crosses have lower rectal temperatures and respiration rates than wool breeds and a lower DMI and water intake. There are indications of increased resistance to internal parasites in hair sheep. Although hair sheep are seasonal breeders under U.S. photoperiodic conditions, they tend to perform better under accelerated lambing systems than traditional wool breeds. Fertility, prolificacy, and lamb survival is high in BB and SC, but hair x wool crossbred ewes tend to have a higher level of fertility than hair and wool parent breeds. Ewe productivity is also higher in hair x wool crosses than in wool crosses, particularly when adjusted for ewe BW or under accelerated lambing systems. Hair sheep have a lower ADG and intake of high-energy diets, as well as a lower gain/feed ratio, than wool breeds. Growth rates tend to be higher in SC than in BB. Differences in carcass characteristics are inconsistent between hair and wool breeds. Production characteristics of hair sheep, particularly hair x wool crosses, make them suitable for low-input, sustainable production systems that do not require high growth rates and large carcasses. There is a need to preserve the existing U.S. hair sheep germplasm base in support of such systems.     

Genetic analysis, using P22 challenge phage, of the nitrogen activator protein DNA-binding site in the Klebsiella aerogenes put operon

Protection of dietary lipids in a protein matrix prevents biohydrogenation in ruminants and increases the availability of polyunsaturated fatty acids. This alters the composition of the tissue lipids, including the membrane phospholipids, which are important substrates for signal transduction. This study investigates the effects of a diet containing protected fatty acids on the activities of key intracellular kinases in the skin. Two groups of six sheep were offered either a control diet or one containing protected cottonseed, a source of linoleic acid (C18:2), for 3 months. Skin was taken from August to October, and analysed for protein kinase C (PKC), protein kinase A (PKA), mitogen-activated protein kinase (MAPK), phosphotyrosine activity and epidermal growth factor (EGF) receptor content. Skin and wool samples were also taken to measure changes in the fibre characters and follicle function. At the end of the experiment, the mean linoleic acid content of skin phospholipids from sheep fed the protected diet was twice that of the controls. In both groups, PKC activity was significantly elevated in skin taken during September and October compared with August values. However, activities measured in the experimental sheep were higher than in controls. This coincided with a decline in wool production. PKA activity decreased significantly in both groups between August and October. MAPK activities did not alter during the experiment. Western analyses did not reveal differences in phosphotyrosine-positive or EGF receptor bands between the groups.     

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.     

The genetic basis of epidermolysis bullosa simplex with mottled pigmentation

Epidermolysis bullosa simplex (EBS) is a group of autosomal dominant skin diseases characterized by blistering, due to mechanical stress-induced degeneration of basal epidermal cells. It is now well-established that the three major subtypes of EBS are genetic disorders of the basal epidermal keratins, keratin 5 (K5) and keratin 14 (K14). Here we show that a rare subtype, referred to as EBS with mottled pigmentation (MP), is also a disorder of these keratins. Affected members of two seemingly unrelated families with EBS-MP had a C to T point mutation in the second base position of codon 24 of one of two K5 alleles, leading to a Pro: Leu mutation. This mutation was not present in unaffected members nor in 100 alleles from normal individuals. Linkage analyses mapped the defect to this type II keratin gene (peak logarithm of odds score at phi = 0 of 3.9), which is located on chromosome 12q11-q13. This provides strong evidence that this mutation is responsible for the EBS-MP phenotype. Only conserved between K5 and K6, and not among any of the other type II keratins, Pro-24 is in the nonhelical head domain of K5, and only mildly perturbs the length of 10-nm keratin filaments assembled in vitro. However, this part of the K5 head domain is likely to protrude on the filament surface, perhaps leading to additional aberrations in intermediate filament architecture and/or in melanosome distribution that are seen ultrastructurally in patients with the mutation.     

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.     

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.     

Location of a high-sulphur protein in developing wool follicles using peroxidase-labelled antibody

Antiserum to a high-sulphur protein (SCMK-B2) from wool was purified to obtain the immunoglobin G fraction, and this was labelled with horse-radish peroxidase. The labelled antibody was then applied to sections of sheep skin embedded in glycol methacrylate in order to locate the distribution of high-sulphur protein within the lower hair shaft. Regions containing the highest concentration of high-sulphur protein were the cuticle of the hair shaft and the keratogenous zone of the cortex.     

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 role of Hsp60 in autoimmune diabetes: analysis in a transgenic model

A pathogenic role for self-reactive cells against the stress protein Hsp60 has been proposed as one of the events leading to autoimmune destruction of pancreatic beta cells in the diabetes of nonobese diabetic (NOD) mice. To examine this hypothesis, we generated transgenic NOD mice carrying a murine Hsp60 transgene driven by the H-2E alpha class II promoter. This would be expected to direct expression of the transgene to antigen-presenting cells including those in the thymus and so induce immunological tolerance by deletion. Detailed analysis of Hsp60 expression revealed that the endogenous gene is itself expressed strongly in thymic medullary epithelium (and weakly in cortex) yet fails to induce tolerance. Transgenic mice with retargeted Hsp60 showed overexpression of the gene in thymic cortical epithelium and in bone marrow-derived cells. Analysis of spontaneous T-cell responses to a panel of self and heterologous Hsp60 antigens showed that tolerance to the protein had not been induced, although responses to an immunodominant 437-460 epitope implicated in disease were suppressed, probably indicating an epitope shift. This correlated with changes in disease susceptibility: insulitis in transgenic mice was substantially reduced so that pathology rarely progressed beyond periislet infiltration. This was reflected in a substantial reduction in hyperglycemia and disease. These data indicate that T cells specific for some epitopes of murine Hsp60 are likely to be involved in the islet-cell destruction that occurs in NOD mice.     

Making a connection: direct binding between keratin intermediate filaments and desmosomal proteins

In epidermal cells, keratin intermediate filaments connect with desmosomes to form extensive cadherin-mediated cytoskeletal architectures. Desmoplakin (DPI), a desmosomal component lacking a transmembrane domain, has been implicated in this interaction, although most studies have been conducted with cells that contain few or no desmosomes, and efforts to demonstrate direct interactions between desmoplakin and intermediate filaments have not been successful. In this report, we explore the biochemical nature of the connections between keratin filaments and desmosomes in epidermal keratinocytes. We show that the carboxy terminal "tail" of DPI associates directly with the amino terminal "head" of type II epidermal keratins, including K1, K2, K5, and K6. We have engineered and purified recombinant K5 head and DPI tail, and we demonstrate direct interaction in vitro by solution-binding assays and by ligand blot assays. This marked association is not seen with simple epithelial type II keratins, vimentin, or with type I keratins, providing a possible explanation for the greater stability of the epidermal keratin filament architecture over that of other cell types. We have identified an 18-amino acid residue stretch in the K5 head that is conserved only among type II epidermal keratins and that appears to play some role in DPI tail binding. This finding might have important implications for understanding a recent point mutation found within this binding site in a family with a blistering skin disorder.     

Calbindin-D28k fails to protect hippocampal neurons against ischemia in spite of its cytoplasmic calcium buffering properties: evidence from calbindin-D28k knockout mice

We report the sequences of seven new cytoplasmic intermediate filament (IF) proteins of the cephalochordate Branchiostoma. The eight sequences currently known describe four subfamilies (A, B, C and D). All eight IF proteins show the short-length version of the coil 1b subdomain found in vertebrates and lack the additional 42 residues present in all nuclear lamins and the protostomic IF proteins. Although the lancelet is considered to be the closest relative of the vertebrates, it is difficult to relate its IF subfamilies unambiguously to a particular type I-IV subfamily of vertebrates. C1 and C2 have tail domains with two 64 residue repeats of coiled coil-forming ability, a structural feature unknown for IF proteins from vertebrates or protostomia. The epidermal protein D1 shows only a slightly better identity score with vertebrate type II keratins than with type III proteins, but the D1 gene organization is that of type III proteins. The same holds for A1, A2, B1, B2 and C2 genes, although the latter has an additional and uniquely positioned intron. Antibodies (Ab) raised against recombinant C2 and D1 proteins reveal these proteins in epidermis, some internal epithelia and parts of the spinal cord. The results on exonic sequences, gene organization and expression suggest that Branchiostoma IF proteins may retain a largely archetypal condition, whereas the vertebrates have established the well-known type I-IV IF system.