The Mallory body as an aggresome: in vitro studies.

Prior in vivo studies supported the concept that Mallory bodies (MBs) are aggresomes of cytokeratins 8 and 18. However, to test this hypothesis an in vitro model is needed to study the dynamics of MB formation. Such a study is difficult because MBs have never been induced in tissue culture. Therefore, MBs were first induced in vivo in drug-primed mice and then primary cultures of hepatocytes from these mice were studied. Two approaches were utilized: 1. Primary cultures were transfected with plasmids containing the sequence for cytokeratin 18 (CK 18) tagged with green fluorescent protein (GFP). 2. Immunofluorescent staining was used to localize the ubiquitin-proteasome pathway components involved in MB-aggresome complex formation in primary hepatocyte cultures. The cells were double stained with a ubiquitin antibody and one of the following antibodies: CK 8, CK 18, tubulin, mutant ubiquitin (UBB+1), transglutaminase, phosphothreonine, and the 20S and 26S proteasome subunits P25 and Tbp7, respectively. In the first approach, fluorescence was observed in keratin filaments and MBs 48 h after the cells were transfected with the CK 18 GFP plasmid. Nascent cytokeratin 18 was preferentially concentrated in MBs. Less fluorescence was observed in the normal keratin filaments. This indicated that MBs continued to form in vitro. The immunofluorescent staining of the hepatocytes showed that CK 8 and 18, ubiquitin, mutant ubiquitin (UBB+1), P25, Tbp7, phosphothreonine, tubulin, and transglutaminase were all located at the border or the interior of the MB. These results support the concept that MBs are aggresomes of CK 8 and CK18 and are a result of inhibition of the ubiquitin-proteasome pathway of protein degradation possibly caused by UBB+1.

Characterization of mouse Frizzled-3 expression in hair follicle development and identification of the human homolog in keratinocytes.

Frizzled genes encode a family of Wnt ligand receptors, which have a conserved cysteine-rich Wnt binding domain and include both transmembrane and secreted forms. Work by others has shown that experimental perturbation of Wnt signaling results in aberrant hair formation, hair growth, and hair structure. To date, however, there is no information on the contribution of individual Frizzled proteins to hair development. We now report that Frizzled-3 expression in skin is restricted to the epidermis and to the developing hair follicle. Northern analysis on total mouse skin mRNA revealed a single Frizzled-3 transcript of 3.7 kb. Reverse transcription-polymerase chain reaction and in situ hybridization analysis revealed Frizzled-3 expression in epidermal and hair follicle keratinocytes. Frizzled-3 transcripts are first detected in discrete foci in the developing epidermis of 13 d embryos and later in the hair follicle placodes of 15 d embryos, suggesting a role for this Frizzled isoform in follicle development. In 17 d embryos and 1 d old newborn mice Frizzled-3 expression is limited to suprabasal keratinocytes and is not seen in pelage follicles until 3 d postpartum. In 7 d old neonatal skin, Frizzled-3 is expressed throughout the epidermis and in the outer cell layers of hair follicles. We have also identified the mRNA encoding human Frizzled-3 in epidermal keratinocytes and in the HaCaT keratinocyte cell line. Human Frizzled-3 mRNA encodes a 666 amino acid protein with 97.8% identity to the mouse protein. The human Frizzled-3 gene was mapped using a radiation-hybrid cell line panel to the short arm of chromosome 8 between the markers WI-1172 and WI-8496 near the loci for the Hypotrichosis of Marie Unna and Hairless genes.

Post-transcriptional regulation of the gli1 oncogene by the expression of alternative 5' untranslated regions.

The oncogene GLI1 is involved in the formation of basal cell carcinoma and other tumor types as a result of the aberrant signaling of the Sonic hedgehog-Patched pathway. In this study, we have identified alternative GLI1 transcripts that differ in their 5' untranslated regions (UTRs) and are generated by exon skipping. These are denoted alpha-UTR, beta-UTR, and gamma-UTR according to the number of noncoding exons possessed (three, two, and one, respectively). The alpha- and beta-UTR forms represent the major Gli1 transcripts expressed in mouse tissues, whereas the gamma-UTR is present at relatively low levels but is markedly induced in mouse skin treated with 12-O-tetradecanoylphorbol 13-acetate. Transcripts corresponding to the murine beta and gamma forms were identified in human tissues, but significantly, only the gamma-UTR form was present in basal cell carcinomas and in proliferating cultures of a keratinocyte cell line. Flow cytometry analysis determined that the gamma-UTR variant expresses a heterologous reporter gene 14-23-fold higher than the alpha-UTR and 5-13-fold higher than the beta-UTR in a variety of cell types. Because expression of the gamma-UTR variant correlates with proliferation, consistent with a role for GLI1 in growth promotion, up-regulation of GLI1 expression through skipping of 5' noncoding exons may be an important tumorigenic mechanism.

Analysis of mouse keratin 6a regulatory sequences in transgenic mice reveals constitutive, tissue-specific expression by a keratin 6a minigene.

The analysis of keratin 6 expression is complicated by the presence of multiple isoforms that are expressed constitutively in a number of internal stratified epithelia, in palmoplantar epidermis, and in the companion cell layer of the hair follicle. In addition, keratin 6 expression is inducible in interfollicular epidermis and the outer root sheath of the follicle, in response to wounding stimuli, phorbol esters, or retinoic acid. In order to establish the critical regions involved in the regulation of keratin 6a (the dominant isoform in mice), we generated transgenic mice with two different-sized mouse keratin 6a constructs containing either 1.3 kb or 0.12 kb of 5' flanking sequence linked to the lacZ reporter gene. Both constructs also contained the first intron and the 3' flanking sequence of mouse keratin 6a. Ectopic expression of either transgene was not observed. Double-label immunofluorescence analyses demonstrated expression of the reporter gene in keratin 6 expressing tissues, including the hair follicle, tongue, footpad, and nail bed, showing that both transgenes retained keratinocyte-specific expression. Quantitative analysis of beta-galactosidase activity verified that both the 1.3 and 0.12 kb keratin 6a promoter constructs produced similar levels of the reporter. Notably, both constructs were constitutively expressed in the outer root sheath and interfollicular epidermis in the absence of any activating stimulus, suggesting that they lack the regulatory elements that normally silence transcription in these cells. This study has revealed that a keratin 6a minigene contains critical cis elements that mediate tissue-specific expression and that the elements regulating keratin 6 induction lie distal to the 1.3 kb promoter region.

Identification of a novel mutation in keratin 1 in a family with epidermolytic hyperkeratosis.

Epidermolytic hyperkeratosis (EHK) is a hereditary skin disorder typified by blistering due to cytolysis. One in 100,000 individuals is affected by this autosomal-dominant disease. The onset of the disease phenotype is typically at birth. Histological and ultrastructural examination of the epidermis shows a thickened stratum corneum and tonofilament clumping around the nucleus of suprabasal keratinocytes. Linkage studies localized the disease genes on chromosomes 12q and 17q which contain the type II and type I keratin gene clusters. Recently, several point mutations in the genes encoding the suprabasal keratins, K1 and K10, have been reported in EHK patients. We have investigated a large kindred affected by EHK and identified a new point mutation in the 2B region of keratin 1 (I107T), resulting from a T to C transition in codon 478.

Expression of MK6a dominant-negative and C-terminal mutant transgenes in mice has distinct phenotypic consequences in the epidermis and hair follicle.

Mouse keratin 6a (MK6a) is constitutively expressed in a single cell layer of the outer root sheath (ORS) of hair follicles, but its synthesis can be induced in interfollicular epidermis including the basal cell layer in response to perturbing stimuli. A basally inducible human K6 (HK6) isoform has not been described, and it is not clear which of the known HK6 isoforms is expressed in the ORS. In this study we show that expression of a dominant-negative MK6a construct (Delta2B-P) in the interfollicular epidermis caused severe blistering and neonatal lethality, suggesting that mutations in a yet to be identified basally expressed HK6 isoform might result in a severe blistering phenotype. Surviving Delta2B-P animals showed transgene expression only in isolated epidermal cells and not in all cells of the ORS, but nevertheless developed severe alopecia. Expression of two different C-terminal mutant transgenes also caused alopecia while a third C-terminal mutant had no phenotypic conse- quences. Electron microscopy revealed that Delta2B-P expression resulted in the collapse of keratin filaments, while destruction of hair follicles in the two phenotypic C-terminal mutant lines occurred in the absence of filament abnormalities. The latter finding indicates that the innermost ORS cells are uniquely sensitive to expression of even slightly altered K6 proteins, suggesting that mutations affecting an HK6 isoform expressed in this cell layer could result in alopecia in humans as well.

The mouse keratin 6 isoforms are differentially expressed in the hair follicle, footpad, tongue and activated epidermis.

Keratin 6 (K6) is expressed constitutively in a variety of internal stratified epithelia as well as in palmoplantar epidermis and in specialized cells of the hair follicle. K6 expression can also be induced by hyperproliferative conditions as in wound healing or by conditions that perturb normal keratinocyte function. The functional significance of the expression of K6 on keratinocyte biology under these disparate conditions is not known. Here we report on the characterization of two isoforms of mouse K6 that are encoded by separate genes. The two genes (denoted K6a and K6b) are linked, have the same orientation and are actively transcribed. Sequence analysis revealed, that although they encode almost identical products, they have distinctly different regulatory regions, suggesting that the two K6 genes would be differentially expressed. In an attempt to define the expression characteristics of the K6 isoforms, we produced transgenic mice with each gene after modifying the C-terminal sequences to enable detection of the transgenic proteins with specific antibodies. The constitutive expression of the K6a transgene paralleled that of the endogenous genes in all K6 expressing tissues, except in the tongue. The K6b transgene was also expressed in these tissues but, in contrast to K6a, was only expressed in suprabasal cells. Both K6 transgenes were also induced in the interfollicular epidermis in response to phorbol esters, with K6a induced in all layers of the treated epidermis, while K6b was expressed only in suprabasal cells. These studies suggest that the K6 isoforms have overlapping yet distinct expression profiles.

Characterization of loricrin regulation in vitro and in transgenic mice.

We have previously shown that the promoter of a 6.5 kb mouse loricrin clone contains a functional AP-1 element and directs tissue-specific, but not differentiation-specific, expression. We now report the isolation of a 14-kb genomic clone containing an additional 7 kb of genomic sequence. The additional sequences limit expression of a reporter construct to differentiated keratinocytes in culture. The expression of the 6.5-kb and 14-kb loricrin constructs were also analyzed in transgenic mice. Significantly, loricrin was found in all layers of the epidermis of the 6.5-kb transgenics, including basal and spinous cells. The expression of the 14-kb clone was indistinguishable from that of the endogenous gene, confirming that the additional sequences contain negative regulatory elements that restrict loricrin expression to the granular layer in vivo. In addition, we show the AP-1 element localized in the loricrin proximal promoter is necessary but not sufficient for expression of the loricrin gene in vivo in transgenic mice. Finally, to gain further insight into how AP-1 family members regulate expression of the loricrin gene, we co-transfected the loricrin reporter constructs with expression plasmids for various fos and jun family members and demonstrated that c-Fos/Jun-B heterodimers could mimic the differentiation-specific induction of loricrin.

An asparagine to threonine substitution in the 1A domain of keratin 1: a novel mutation that causes epidermolytic hyperkeratosis.

Epidermolytic hyperkeratosis (EHK) is a congenital, autosomal dominant disorder of cornification characterized by hyperkeratosis and blister formation. The clinical manifestations are heterogeneous, with respect to the extent of body surface involvement, palmar and plantar hyperkeratosis and the presence of erythroderma. Point mutations in the genes encoding the suprabasal-specific keratins, keratins 1 and 10 have been identified in EHK patients. The inappropriate amino acid substitutions cause a collapse of the keratin filament network, resulting in cytolysis of the involved keratinocytes. We report a severe case of EHK with a single base pair mutation that causes a threonine for asparagine substitution in residue 8 (N8T) of the 1A region of the keratin 1 protein. This is the region involved in molecular overlaps between neighboring keratin heterodimers. These findings suggest that even conservative amino acid substitutions in overlap regions can cause tonofilament clumping.

A novel substitution in keratin 10 in epidermolytic hyperkeratosis.

Epidermolytic hyperkeratosis is characterized by tonofilament clumping, cytolysis, and blister formation in suprabasal keratinocytes. It has been shown that the tonofilament aggregates in these areas are composed of keratin 1 (K1) and keratin 10 (K10), and several K1 and K10 point mutations have been identified as the molecular basis of epidermolytic hyperkeratosis. In this report we identify a novel, single base pair substitution resulting in an amino acid exchange from tyrosine to serine at residue 14 within the conserved 1A region of K10 (Y14S). This A to C transversion in codon 160 was only present in the affected individual and was associated with a very severe disease phenotype. Our observations are in agreement with previous reports documenting that this tyrosine residue, located at the beginning of the rod domain of type I keratins, is particularly sensitive to amino acid substitutions, and that alterations in this residue can have deleterious effects on filament assembly and stability.

A novel mutation in the 1A domain of keratin 2e in ichthyosis bullosa of Siemens.

Ichthyosis bullosa of Siemens (IBS) is a rare autosomal dominant skin disorder with clinical features similar to epidermolytic hyperkeratosis (EHK). Both diseases have been linked to the type II keratin cluster on chromosome 12q. Hyperkeratosis and blister formation are relatively mild in IBS compared with EHK, and the lysis of keratinocytes is restricted to the upper spinous and granular layers of the epidermis of IBS patients, whereas in EHK lysis occurs in the lower spinous layer. Recently, mutations in the helix initiation and termination motifs of keratin 2e (K2e) have been described in IBS patients. The majority of the mutations reported to date lie in the 2B region. In this report, we have examined a large kindred in which the disease was originally diagnosed as EHK and mapped to the type II keratin cluster on chromosome 12q. Molecular analysis revealed a novel amino acid substitution at the beginning of the conserved 1A region of the rod domain (I4N) of K2e, resulting from a T to A transversion in codon 188.

Prenatal diagnosis for keratin mutations to exclude transmission of epidermolytic hyperkeratosis.

Epidermolytic hyperkeratosis (bullous congenital ichthyosiform erythroderma) is an autosomal dominant skin disorder caused by mutations in keratins 1 and 10. We have used direct gene sequencing to ascertain the status of a 15 week fetus of parents whose first child was affected with this disorder. The parents show no clinical signs of epidermolytic hyperkeratosis but were concerned about the possibility of transmitting the disorder due to germline mosaicism. Molecular analysis of the affected son revealed a G to A mutation in codon 156 of keratin 10, resulting in an arginine to histidine substitution within the highly conserved 1A region. Codon 156 has been previously identified as a mutational hot spot and substitutions of this arginine residue are very common in epidermolytic hyperkeratosis patients. Analysis of genomic DNA isolated from amniotic cells showed that the fetus did not harbour this mutation and a healthy infant was eventually born that was unaffected by this disorder.

A novel mutation in the L12 domain of keratin 5 in the Köbner variant of epidermolysis bullosa simplex.

We have identified a novel mutation within the linker L12 region of keratin 5 (K5) in a family with the Köbner variant of epidermolysis bullosa simplex. The pattern of inheritance of the disorder in this family is consistent with an autosomal dominant mode of transmission. Affected individuals develop extensive and generalized blistering at birth or early infancy but in later years clinical manifestations are largely confined to palmoplantar surfaces. Direct sequencing of polymerase chain reaction products revealed a T to C transition within codon 323 of K5 in affected individuals, resulting in a valine to alanine substitution of the seventh residue within the L12 linker domain. This mutation was not observed in unaffected family members or in 100 K5 alleles of unrelated individuals with normal skin. The other critical regions of K5 and K14 were unremarkable in this family except for common polymorphisms that have been previously described. The valine at position 7 of the L12 domain is absolutely conserved in all type II keratins, and in other intermediate filament subunits as well, which suggests that this residue makes an important contribution to filament integrity. Secondary structure analysis revealed that alanine at this position markedly reduces both the hydrophobicity and the beta-sheet nature of the L12 domain. This is the first report of a mutation at this position in an intermediate filament subunit and reinforces the importance of this region to filament biology.

A novel dinucleotide mutation in keratin 10 in the annular epidermolytic ichthyosis variant of bullous congenital ichthyosiform erythroderma.

Annular epidermolytic ichthyosis has recently been delineated as a distinct clinical phenotype within the spectrum of epidermolytic keratinization disorders. The pattern of inheritance of the disorder is consistent with an autosomal dominant mode of transmission. Here we report a second incidence of this disorder in a family with two affected generations. The proband suffered from bullous ichthyosis and had bouts of disease activity associated with the development of numerous annular and polycyclic erythematous, hyperkeratotic plaques on the trunk and the proximal extremities. Histologic examination showed the typical pathology of epidermolytic hyperkeratosis, and ultrastructural analysis revealed abnormal keratin filament networks and tonofilament clumping with a perinuclear distribution. Molecular analysis revealed a novel tandem CG to GA 2-bp mutation in the same allele of keratin 10 in affected individuals, resulting in an arginine to glutamate substitution at residue 83 (R83E) of the 2B helical segment. We conclude that annular epidermolytic ichthyosis should be considered a variant of bullous congenital ichthyosiform erythroderma.

A transgenic mouse model that recapitulates the clinical features of both neonatal and adult forms of the skin disease epidermolytic hyperkeratosis.

Keratins are the major structural proteins of keratinocytes, which are the most abundant cell type in the mammalian epidermis. Mutations in epidermal keratin genes have been shown to cause severe blistering skin abnormalities. One such disease, epidermolytic hyperkeratosis (EHK), also known as bullous congenital ichthyosiform erythroderma, occurs as a result of mutations in highly conserved regions of keratins K1 and K10. Patients with EHK first exhibit erythroderma with severe blistering, which later is replaced by thick patches of scaly skin. To assess the effect of a mutated K1 gene on skin biology and to produce an animal model for EHK, we removed 60 residues from the 2B segment of HK1 and observed the effects of its expression in the epidermis of transgenic mice. Phenotypes of the resultant mice closely resembled those observed in the human disease, first with epidermal blisters, then later with hyperkeratotic lesions. In neonatal mice homozygous for the transgene, the skin was thicker, with an increased labeling index, and the spinous cells showed a collapse of the keratin filament network around the nuclei, suggesting that a critical concentration of the mutant HK1, over the endogenous MK1, was required to disrupt the structural integrity of the spinous cells. Additionally, footpad epithelium, which is devoid of hair follicles, showed blistering in the spinous layer, suggesting that hair follicles can stabilize or protect the epidermis from trauma. Blisters were not evident in adult mice, but instead they showed a thick, scaly hyperkeratotic skin with increased mitosis, resulting in an increased number of corneocytes and granular cells. Irregularly shaped keratohyalin granules were also observed. To date, this is the only transgenic model to show the typical morphology found in the adult form of EHK.

Mutation of a type II keratin gene (K6a) in pachyonychia congenita.

Pachyonychia congenita (PC) is a rare autosomal dominant condition characterized by multiple ectodermal abnormalities. Patients with Jadassohn-Lewandowsky Syndrome (MIM #167200; PC-1) have nail defects (onchyogryposis), palmoplantar hyperkeratosis, follicular hyperkeratosis and oral leukokeratosis. Those with the rarer Jackson-Lawler Syndrome (MIM #167210; PC-2) lack oral involvement but have natal teeth and cutaneous cysts. Ultra-structural studies have identified abnormal keratin tonofilaments and linkage to the keratin gene cluster on chromosome 17 has been found in PC families. Keratins are the major structural proteins of the epidermis and associated appendages and the nail, hair follicle, palm, sole and tongue are the main sites of constitutive K6, K16 and K17 expression. Furthermore, mutations in K16 and K17 have recently been identified in some PC patients. Although we did not detect K16 or K17 mutations in PC families from Slovenia, we have found a heterozygous deletion in a K6 isoform (K6a) in the affected members of one family. This 3 bp deletion (AAC) in exon 1 of K6a removes a highly conserved asparagine residue (delta N170) from position 8 of the 1A helical domain (delta N8). This is the first K6a mutation to be described and this heterozygous K6a deletion is sufficient to explain the pathology observed in this PC-1 family.

The proximal promoter of the mouse loricrin gene contains a functional AP-1 element and directs keratinocyte-specific but not differentiation-specific expression.

Loricrin gene expression is limited to terminally differentiating keratinocytes of stratified squamous epithelia. To define the regulatory elements that mediate the expression of the loricrin gene, we replaced the loricrin coding sequences from a 6.5-kilobase genomic fragment with the chloramphenicol acetyltransferase gene and transfected this construct into cultured mouse keratinocytes. High expression levels were observed in both undifferentiated as well as differentiating cells. Transgenic mice bearing a similar construct, but with beta-galactosidase as the reporter gene, corroborated these in vitro findings and showed tissue- and cell type-specific, but not differentiation-specific expression. Deletion analysis of the promoter region determined that sequences up to -60 base pairs from the start of transcription could be removed without significant loss of promoter activity. Within these proximal 60 base pairs is an evolutionarily conserved AP-1 element that is recognized by both purified c-Jun and AP-1 factors from keratinocytes in vitro. Mutation of this AP-1 site abolished the activity of the loricrin promoter. These studies show that elements directing expression of the loricrin gene to the stratified squamous epithelia are contained within a 6.5-kilobase genomic fragment, and those elements required to restrict expression to differentiated keratinocytes lie outside this region.