KRT6A Research Articles

Single-Nucleotide-Specific siRNA Targeting in a Dominant-Negative Skin Model

RNA interference offers a novel approach for developing therapeutics for dominant-negative genetic disorders. The ability to inhibit expression of the mutant allele without affecting wild-type gene expression could be a powerful new treatment option. Targeting the single-nucleotide keratin 6a (K6a) N171K mutation responsible for the rare monogenic skin disorder pachyonychia congenita (PC), we demonstrate that small interfering RNAs (siRNAs) can potently and selectively block expression of mutant K6a. To test whether lead siRNAs could discriminate mutant mRNA in the presence of both wild-type and mutant forms, a dominant-negative PC cell culture model was developed. As predicted for a dominant-negative disease, simultaneous expression of both wild-type and mutant K6a resulted in defective keratin filament formation. Addition of mutant-specific siRNAs allowed normal filament formation, suggesting selective inhibition of mutant K6a. The effectiveness of our siRNA in skin was tested by co-delivering a firefly luciferase/mutant K6a bicistronic reporter construct and mutant-specific siRNAs to mouse footpads. Potent inhibition of the fluorescent reporter was demonstrated using the Xenogen IVIS200 in vivo imaging system. Additionally, wild type-specific siRNAs knocked down the expression of pre-existing endogenous K6a in human keratinocytes. These results suggest that efficient delivery of these "designer siRNAs" may allow effective treatment of numerous genetic disorders including PC.

Therapeutic Interference: A Step Closer for Pachyonychia Congenita?

The identification of mutations in keratin genes as the cause of several inherited skin disorders raised the possibility that molecular-based therapies might be developed to treat these conditions. In this issue, Smith et al. (2007) have identified small interfering RNAs that specifically and potently silence keratin 6a expression. These molecules have great promise as therapeutic agents for the treatment of pachyonychia congenita.

Transformation of human urothelial cells (UROtsa) by as and cd induces the expression of keratin 6a.

BackgroundCadmium and arsenite can directly and malignantly transform the UROtsa cell line. The tumor heterotransplants produced from these transformed cells have histologic features consistent with human bladder cancer. Previous microarray analysis of total RNA from the parental and transformed cells suggested that keratin 6a was overexpressed as a result of cell transformation.ObjectivesOur goals were to verify overexpression of keratin 6a in Cd2+- and As3+-transformed UROtsa cells, the corresponding tumor heterotransplants, and human bladder cancer biopsy specimens and to assess what factors may be involved in keratin 6a overexpression.MethodsExpression was assessed with real-time polymerase chain reaction, Western blot analysis, and immunohistochemistry. We used the effect of addition and deletion of potential growth factors in the cell culture growth medium to assess possible pathways used in keratin 6a overexpression.ResultsCd2+- and As3+-transformed cells grown in serum-containing growth medium, as well as the derived tumor heterotransplants, overexpressed keratin 6a mRNA and protein compared with UROtsa cells grown in serum-containing growth medium. Immunostaining of keratin 6a in tumor heterotransplants showed focal staining of the tumor cells that was localized to the cytoplasm. Focal immunostaining of keratin 6a was also found in some but not all archival patient specimens of high-grade bladder cancer, confirming translation of the results to human bladder cancer. Studies on growth factor deletion and addition indicated that the level of keratin 6a expression was regulated by the presence of both insulin and epidermal growth factor (EGF). In contrast, growth factors had no effect on the elevated levels of keratin 6a expression found in transformed UROtsa cells.ConclusionsOur present studies suggest that keratin 6a expression may be a biomarker for malignant urothelial cells that possess an activated EGF and or insulin growth factor pathway.

ΔNp63 isoforms regulate CD44 and keratins 4, 6, 14 and 19 in squamous cell carcinoma of head and neck

The human p63 gene codes for multiple protein isoforms and is commonly over-expressed in squamous cell carcinoma of head and neck (SCCHN). This expression is predominantly of the DeltaN- and beta-isoforms, the former lacking the p53-related transactivation domain. p63 can activate or repress transcription of p53 and p73 target genes, but also has unique transcriptional targets and, unlike other p53 family members, is required for normal development and differentiation of squamous epithelia. We have identified novel targets of p63, using microarray analysis of SCCHN cells that stably over-express individual DeltaNp63 isoforms. All three isoforms induced expression of the cancer stem cell marker, CD44, with the DeltaNp63beta isoform showing strongest induction. Using chromatin immunoprecipitation, we were unable to show direct binding of p63 to the CD44 promoter, but found that p63 specifically increased expression of CD44 lacking variant exon 2. Each of the DeltaNp63 isoforms up-regulated expression of keratins 6A and 14 and down-regulated expression of keratins 4 and 19, in keeping with their expression patterns in SCCHN. The data strengthen the idea that p63 has key roles in regulating normal and abnormal differentiation processes through both induction and repression of genes with opposite functions. The identification of up-regulation and differential splicing of CD44 following p63 over-expression indicates roles in the regulation of adhesion, metastasis and the cancer stem cell phenotype.

Keratin Expression Provides Novel Insight into the Morphogenesis and Function of the Companion Layer in Hair Follicles

Hair follicles cycle between stages of growth (anagen) and metabolic quiescence (telogen) throughout life. In mature follicles, transition from telogen back into anagen involves the activation, proliferation, and differentiation of epithelial stem cells located in the bulge, a specialization of the outer root sheath. Recent studies identified keratin 6a (K6a) transcripts as enriched in bulge epithelial stem cells in mouse skin. We used messenger RNA probes, antibodies, a LacZ reporter mouse model, and whole-mount staining assays to investigate the regulation of mK6a during mouse postnatal hair cycling, and compare it to mK75, a companion layer (Cl) marker. We find that mK75 regulation parallels that of inner root sheath (IRS) markers, with expression onset at anagen IIIa above the new hair bulb and subsequent spreading towards the bulge. Although also occurring in the Cl, mK6a expression begins at anagen IIIb in differentiating cells located proximal to the bulge, and subsequently spreads towards the hair bulb. mK6a and mK75 thus exhibit temporally distinct, and spatially opposed, expression patterns in the Cl during postnatal anagen. These findings provide novel insight into the morphogenesis and properties of the Cl, and raise the distinct possibility that it is an integral part of the IRS compartment.

Identification of a Keratin 4 Mutation in a Chemically Induced Mouse Mutant that Models White Sponge Nevus

With the goal of increasing the number of genetic entry points for studying physiologic processes and human disease, large-scale, systematic, chemical mutagenesis projects in mice have been initiated in several different centers. We have been studying mouse mutants that exhibit dominantly inherited defects in either skin and/or hair color. Here, we describe a bright coat color mutant, Bright coat color 1 (Bcc1), which develops light-colored hair at 4 weeks of age, and when homozygous exhibits oral leukoplakia and blistering, and growth retardation. We identified a missense mutation in mutant animals that predicts an N154S amino-acid substitution in the 1A domain of Keratin 4 (encoded by the Krt2-4 gene), a region known to be mutated in human patients with white sponge nevus (WSN). Bcc1 recapitulates the gross pathologic, histologic, and genetic aspects of the human disorder, WSN.

A novel missense mutation L468Q of keratin 6a in pachyonychia congenita type 1

Pachyonychia congenita is an autosomal dominant disorder that usually develops in early infancy. The major features of the syndrome are hypertrophic nail dystrophy, palmoplantar keratoderma and oral leucokeratosis, accompanied by other ectodermal defects, according to subtype. To analyse the K6a gene mutation in a sporadic Chinese patient with pachyonychia congenita type 1 (PC-1) and to explore the relationship between the genotype and phenotype of PC-1. Genomic DNA was extracted from peripheral blood of the patient with PC-1 and 100 unrelated controls. The whole coding region of K6a gene was amplified using long-range polymerase chain reaction (PCR); nested PCR was then used to amplify the mutation 'hot-spot' of the K6a gene. The PCR products were directly sequenced to detect the mutation. A novel missense mutation L468Q in the helix 2B domain of the K6a polypeptide was identified in the patient but not in the healthy individuals from the family and 100 unrelated control individuals. We describe this mutation for the first time, and provide further evidence that the helix boundary motif sequences of K6a are a mutation 'hot-spot'.

SiRNA‐Mediated Selective Inhibition of Mutant Keratin mRNAs Responsible for the Skin Disorder Pachyonychia Congenita

RNA interference offers a novel approach for treating genetic disorders including the rare monogenic skin disorder pachyonychia congenita (PC). PC is caused by mutations in keratin 6a (K6a), K6b, K16, and K17 genes, including small deletions and single nucleotide changes. Transfection experiments of a fusion gene consisting of K6a and a yellow fluorescent reporter (YFP) resulted in normal keratin filament formation in transfected cells as assayed by fluorescence microscopy. Similar constructs containing a single nucleotide change (N171K) or a three-nucleotide deletion (N171del) showed keratin aggregate formation. Mutant-specific small inhibitory RNAs (siRNAs) effectively targeted these sites. These studies suggest that siRNAs can discriminate single nucleotide mutations and further suggest that "designer siRNAs" may allow effective treatment of a host of genetic disorders including PC.

A severe case of pachyonychia congenita type I due to a novel proline mutation in keratin 6a

A severe case of pachyonychia congenita type I due to a novel proline mutation in keratin 6a

A role for thyroid hormone in wound healing through keratin gene expression.

The importance of thyroid hormone (TH) in wound healing is not well understood. To gain insight, we evaluated the impact of TH deficiency on wound-healing genes in cultured keratinocytes. By RT-PCR, keratin 6a (K6a) and 16 (K16) gene expression in TH replete cells was 3.8- (P < 0.005) and 1.9-fold (P < 0.05) greater, respectively, than expression in TH-deficient cells. By real-time PCR, TH replete cell expression of K6a, K16, and K17 was greater than in deficient cells: 18- (P < 0.001), 10- (P < 0.001), and 4-fold (P < 0.005), respectively. To examine TH requirement for optimal wound healing, we contrasted TH-deficient vs. ip T(3)-treated mice. Four days after wounding, ip T(3)-treated mice had twice the degree of wound closure as hypothyroid mice (P < 0.001). By RT-PCR, K6a and K17 gene expression from control mouse skin was greater than from hypothyroid mouse skin: 5- (P < 0.001) and 1.7-fold (P < 0.05), respectively. T(3) is necessary for the keratinocyte proliferation required for optimal wound healing. T(3) exerts influence by stimulating expression of the wound-healing keratin genes. Thus, for hypothyroid patients undergoing surgery that cannot be delayed until euthyroidism is achieved, our data support T(3) treatment for the perioperative period.

In this Issue

In this Issue

A reporter transgene based on a human keratin 6 gene promoter is specifically expressed in the periderm of mouse embryos.

We report the developmental regulation of a lacZ reporter transgene fused to the promoter region of the human keratin 6a gene. In mouse embryos, the transgene is expressed in the periderm (the outermost layer of embryonic epidermis), as are the endogenous keratin 6 alpha and beta genes. A subset of periderm cells, localized to temporary epithelial fusions, is known to contain keratin 6 protein, and we find that these cells also harbor LacZ enzymatic activity.

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.

Delayed wound healing in keratin 6a knockout mice.

Keratin 6 (K6) expression in the epidermis has two components: constitutive expression in the innermost layer of the outer root sheath (ORS) of hair follicles and inducible expression in the interfollicular epidermis in response to stressful stimuli such as wounding. Mice express two K6 isoforms, MK6a and MK6b. To gain insight into the functional significance of these isoforms, we generated MK6a-deficient mice through mouse embryonic stem cell technology. Upon wounding, MK6a was induced in the outer ORS and the interfollicular epidermis including the basal cell layer of MK6a(+/+) mice, whereas MK6b induction in MK6a(-/-) mice was restricted to the suprabasal layers of the epidermis. After superficial wounding of the epidermis by tape stripping, MK6a(-/-) mice showed a delay in reepithelialization from the hair follicle. However, the healing of full-thickness skin wounds was not impaired in MK6a(-/-) animals. Migration and proliferation of MK6a(-/-) keratinocytes were not impaired in vitro. Furthermore, the migrating and the proliferating keratinocytes of full-thickness wounds in MK6a(-/-) animals expressed neither MK6a nor MK6b. These data indicate that MK6a does not play a major role in keratinocyte proliferation or migration but point to a role in the activation of follicular keratinocytes after wounding. This study represents the first report of a keratin null mutation that results in a wound healing defect.

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 (Δ2B-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 Δ2B-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 Δ2B-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.

Stimulating pediatric cases for lunch

Pachyonychia congenita (PC) comprises a group of rare autosomal dominant genetic disorders that involve ectodermal dysplasia. It is characterized by hypertrophic nail dystrophy, focal palmoplantar keratoderma, follicular keratoses, and oral leukokeratosis. Historically, PC has been subdivided into two subtypes, PC-1 or PC-2, on the basis of clinical presentation. However, differential diagnosis based on clinical grounds, especially in young and/or not fully penetrant patients, can be difficult. In addition, clinical analysis of the large case series has shown that there is considerable phenotypic overlap between these two subtypes recently. Based on the advent of molecular genetics and the identification of the genes causing PC, more specific nomenclature has been adopted. Therefore, diagnosis at the molecular level is useful and important to confirm the clinical impression. In this report, we describe two typical cases of PC with mutation analysis revealed a small deletion (514_516delACC, Asn172del) and a point mutation (487 G > A, GAG → AAG, Glu163Lys) in the KRT6A gene.

Antiphospholipids for dermatologists

Pachyonychia congenita (PC) comprises a group of rare autosomal dominant genetic disorders that involve ectodermal dysplasia. It is characterized by hypertrophic nail dystrophy, focal palmoplantar keratoderma, follicular keratoses, and oral leukokeratosis. Historically, PC has been subdivided into two subtypes, PC-1 or PC-2, on the basis of clinical presentation. However, differential diagnosis based on clinical grounds, especially in young and/or not fully penetrant patients, can be difficult. In addition, clinical analysis of the large case series has shown that there is considerable phenotypic overlap between these two subtypes recently. Based on the advent of molecular genetics and the identification of the genes causing PC, more specific nomenclature has been adopted. Therefore, diagnosis at the molecular level is useful and important to confirm the clinical impression. In this report, we describe two typical cases of PC with mutation analysis revealed a small deletion (514_516delACC, Asn172del) and a point mutation (487 G > A, GAG → AAG, Glu163Lys) in the KRT6A gene.

Defining a Region of the Human Keratin 6a Gene That Confers Inducible Expression in Stratified Epithelia of Transgenic Mice

Injury to the epidermis and other stratified epithelia triggers a repair response involving the rapid induction of several genes, including keratin 6 (K6). The signaling pathways and mechanisms presiding over this induction in keratinocytes at the wound edge remain to be defined. We reported previously that of the multiple genes encoding K6 isoforms in human, K6a is dominant in skin epithelia (Takahashi, K., Paladini, R., Coulombe, P. A. (1995) J. Biol. Chem. 270, 18581-18592). Using bacterial LacZ as a reporter gene in transgenic mice, we show that the proximal 5.2 kilobases of 5'-upstream sequence from the K6a gene fails to direct sustained expression in any adult tissue, including those where K6 is constitutively expressed (e.g. hair follicle, nail, oral mucosa, tongue, esophagus, forestomach). In contrast, the proximal 960 base pairs of 5'-upstream sequence suffice to mediate an induction of beta-galactosidase expression in a near-correct spatial and temporal fashion after injury to epidermis and other stratified epithelia. Transgene expression also occurs following topical application of phorbol esters, all-trans-retinoic acid, or 2-4-dinitro-1-fluorobenzene, all known to induce K6 expression in skin. Our data show that critical regulatory sequences for this inducibility are located between -960 and -550 bp in the 5'-upstream sequence of K6a and that their activity is influenced by enhancer element(s) located between -2500 and -5200 base pairs. These findings have important implications for the control of gene expression after injury to stratified epithelia.