A missense mutation within the helix initiation motif of the keratin K71 gene underlies autosomal dominant woolly hair/hypotrichosis.

Woolly hair (WH) is an abnormal variant of tightly curled hair, which is frequently associated with hypotrichosis. Non-syndromic forms of WH can show either autosomal-dominant WH (ADWH) or autosomal-recessive WH (ARWH) inheritance patterns. ARWH has recently been shown to be caused by mutations in either the lysophosphatidic acid receptor 6 (LPAR6) or lipase H (LIPH) gene. More recently, a mutation in the keratin K74 (KRT74) gene has been reported to underlie ADWH. Importantly, all of these genes are abundantly expressed in the inner root sheath (IRS) of human hair follicles. Besides these findings, the molecular mechanisms underlying hereditary WH have not been fully disclosed. In this study, we identified a Japanese family with ADWH and associated hypotrichosis. After exclusion of known causative genes, we discovered the heterozygous mutation c.422T>G (p.Phe141Cys) within the helix initiation motif of the IRS-specific keratin K71 (KRT71) gene in affected family members. We demonstrated that the mutant K71 protein led to disruption of keratin intermediate filament formation in cultured cells. To our knowledge, it is previously unreported that the KRT71 mutation is associated with a hereditary hair disorder in humans. Our findings further underscore the crucial role of the IRS-specific keratins in hair follicle development and hair growth in humans.

Kaempferol enhanced the intracellular thioredoxin system in normal cultured human keratinocytes.

The effects of kaempferol on redox-associated enzymes in normal human keratinocytes were studied. It enhanced the gene expression of thioredoxin reductase 1 and thioredoxin. Flavonols had higher ability to induce the expression of these genes than the other anti-oxidants and the other flavonoids tested. Furthermore, kaempferol and quercetin increased the activity of thioredoxin reductase in normal human keratinocytes.

Up-regulation of serpin SCCA1 is associated with epidermal barrier disruption.

BACKGROUND: Parakeratosis, the persistent presence of nuclei in the stratum corneum (SC) is associated with serious disruption of skin barrier function. Squamous cell carcinoma antigen 1 (SCCA1) is strongly up-regulated in inflamed and parakeratotic skin. OBJECTIVE: To find a biochemical marker for the SC barrier disruption, especially the disruption associated with parakeratosis. METHODS: An ELISA assay system was established to quantify SCCA1 in the extract of tape-stripped cornified cells. Transepidermal water loss (TEWL) and other skin parameters were measured and compared with the amount of SCCA1. Localization of SCCA1 was investigated immunohistochemically in various skin diseases with parakeratosis. Nuclei and SCCA1 on the skin surface were detected by staining of corniocytes collected on an adhesive-coated slide glass. RESULTS: SCCA1 showed strong up-regulation in lesional skin with psoriasis (466-fold), hayfever skin caused by Japanese ceder pollen (232-fold) and sun-exposed skin of healthy individuals (90-fold) compared to their normal sun-protected skin. The increased levels of SCCA1 were well correlated with increased values of TEWL and the number of parakeratotic cells in the SC. Furthermore, subjects with high levels of SCCA1 in the epidermis were more susceptible to barrier disruption by external stimuli, and this was accompanied with a further increase of SCCA1. We confirmed that localization of SCCA1 was limited to parakeratotic areas by using the skin surface staining technique. Immunohistochemical study also demonstrated that SCCA1 was always present at high levels in parakeratotic epidermis. CONCLUSION: All of our findings indicate that SCCA1 plays an important role in the induction of epidermal barrier disruption. SCCA1 may be a critical determinant of barrier function in the epidermis.

Purification and characterization of active caspase-14 from human epidermis and development of the cleavage site-directed antibody.

Restricted expression of caspase-14 in differentiating keratinocytes suggests the involvement of caspase-14 in terminal differentiation. We purified active caspase-14 from human cornified cells with sequential chromatographic procedures. Specific activity increased 764-fold with a yield of 9.1%. Purified caspase-14 revealed the highest activity on WEHD-methylcoumaryl-amide (MCA), although YVAD-MCA, another caspase-1 substrate, was poorly hydrolyzed. The purified protein was a heterodimer with 17 and 11 kDa subunits. N-terminal and C-terminal analyses demonstrated that the large subunit consisted of Ser(6)-Asp(146) and N-terminal of small subunit was identified as Lys(153). We successfully developed an antiserum (anti-h14D146) directed against the Asp(146) cleavage site, which reacted only with active caspase-14 but not with procaspase-14. Furthermore we confirmed that anti-h14D146 did not show any reactivity to the active forms of other caspases. Immunohistochemical analysis demonstrated that anti-h14D146 staining was mostly restricted to the cornified layer and co-localized with some of the TUNEL positive-granular cells in the normal human epidermis. UV radiation study demonstrated that caspase-3 was activated and co-localized with TUNEL-positive cells in the middle layer of human epidermis. In contrast, we could not detect caspase-14 activation in response to UV. Our study revealed tightly regulated action of caspase-14, in which only the terminal differentiation of keratinocytes controls its activation process.

Keratinocytes synthesize enteropeptidase and multiple forms of trypsinogen during terminal differentiation.

Members of the trypsin-like and chymotrypsin-like kallikrein family are important in the desquamation process. In this study, we isolated cDNA clones encoding trypsinogen 4 (brain trypsinogen) and a previously unreported isoform of trypsinogen from a human keratinocyte cDNA library. The nucleotide sequence of the new isoform only differs from those of trypsinogen 3 (mesotrypsinogen) and trypsinogen 4 in an exon encoding the N-terminal region, indicating that this isoform is an alternative splicing variant of the mesotrypsinogen gene PRSS3. Both isoforms contained the sequence DDDDK-I, a putative cleavage site for activation by enteropeptidase. Thus, after activation, mesotrypsin would be produced. Immunohistochemical and in situ hybridization studies revealed that trypsinogens were expressed and localized in the upper epidermis, especially in the granular layer. In cultured keratinocytes, enteropeptidase mRNA was expressed at the confluent stage, and its expression was strongly upregulated after air exposure. Interestingly, it was synthesized and localized only at the granular layer, suggesting that the generation of active mesotrypsin is restricted to this layer. The enteropeptidase-cleavage product was also found at the same layer. When a skin equivalent model was cultured in the medium without air exposure, the cornified layer was not formed, and many cells expressed trypsinogens and enteropeptidase. Those cells were found to be TUNEL positive. Because mesotrypsin is resistant to naturally occurring trypsin inhibitors, confined expression of the isoforms of mesotrypsinogens and enteropeptidase may indicate that mesotrypsin is involved in keratinocyte terminal differentiation.

Expressions of rod and cone photoreceptor-like proteins in human epidermis.

Previous reports have suggested the existence of photoreceptors for visible radiation at the surface of the human body. Rhodopsin is a well-known photosensitive protein found in the rod cells of the retina and detects light/dark contrast. Cone opsins are also photosensitive receptors in the cone cells of the retina and detect colour. Here, we describe immunochemical studies using anti-rhodopsin and anti-opsin antibodies on human skin. Both mouse retina and human epidermis showed clear immunoreactivity with each antibody. Interestingly, immunoreactivity against longer-wavelength opsin antibody was observed in the basal layer of the epidermis, while immunoreactivity against rhodopsin and shorter-wavelength opsin was observed in the upper layer. PCR analysis confirmed the expression of rhodopsin-like and opsin-like genes in human retina and the skin. These results suggest that a series of proteins, which play a crucial role in visual perception, are expressed in human epidermis.

The FoxO3a gene is a key negative target of canonical Notch signalling in the keratinocyte UVB response.

Notch signalling has an important role in skin homeostasis, promoting keratinocyte differentiation and suppressing tumorigenesis. Here we show that this pathway also has an essential anti-apoptotic function in the keratinocyte UVB response. Notch1 expression and activity are significantly induced, in a p53-dependent manner, by UVB exposure of primary keratinocytes as well as intact epidermis of both mouse and human origin. The apoptotic response to UVB is increased by deletion of the Notch1 gene or down-modulation of Notch signalling by pharmacological inhibition or genetic suppression of 'canonical' Notch/CSL/MAML1-dependent transcription. Conversely, Notch activation protects keratinocytes against apoptosis through a mechanism that is not linked to Notch-induced cell cycle withdrawal or NF-kappaB activation. Rather, transcription of FoxO3a, a key pro-apoptotic gene, is under direct negative control of Notch/HERP transcription in keratinocytes, and upregulation of this gene accounts for the increased susceptibility to UVB of cells with suppressed Notch signalling. Thus, the canonical Notch/HERP pathway functions as a protective anti-apoptotic mechanism in keratinocytes through negative control of FoxO3a expression.

Skin surface electric potential as an indicator of skin condition: a new, non-invasive method to evaluate epidermal condition.

We previously demonstrated that the skin surface electric potential, which has been long recognized as a parameter of emotional or physiological state, is generated by epidermal keratinocytes and is strongly associated with the ion concentration gradient in the epidermis. Thus, at temperatures below the threshold of sweating, the potential provides a measure of the epidermal ion concentration gradient, which in turn is related to epidermal homeostasis and pathology. In the present study, we established a new, non-invasive method to measure skin surface electric potential. In healthy skin, calcium ion was localized in the uppermost epidermis and the gradation disappeared by tape stripping. Skin surface potential was also disappeared by tape stripping. Moreover, environmental humidity affected the potential, whereas temporary hydration of the stratum corneum did not affect it. These results suggest that the skin surface electric potential may be an indicator of the pathophysiology of the living layer of epidermis, and thus may be useful as a new parameter to evaluate skin condition.

Serpin squamous cell carcinoma antigen inhibits UV-induced apoptosis via suppression of c-JUN NH2-terminal kinase.

Protection from ultraviolet (UV) irradiation is a fundamental issue for living organisms. Although melanin's critical role in the protection of basal keratinocytes is well understood, other factors remain essentially unknown. We demonstrate that up-regulation of squamous cell carcinoma antigen-1 (SCCA1) suppresses c-Jun NH2-terminal kinase-1 (JNK1) and thus blocks UV-induced keratinocyte apoptosis. We found that serpin SCCA1 is markedly elevated in the top layers of sun-exposed or UV-irradiated epidermis. UV-induced apoptosis was significantly decreased when SCCA was overexpressed in 3T3/J2 cells. It was significantly increased when SCCA was down-regulated with small interfering RNA in HaCaT keratinocytes. A search for SCCA-interacting molecules showed specific binding with phosphorylated JNK. Interestingly, SCCA1 specifically suppressed the kinase activity of JNK1. Upon exposure of keratinocytes to UV, SCCA1 was bound to JNK1 and transferred to the nucleus. Involucrin promoter-driven SCCA1 transgenic mice showed remarkable resistance against UV irradiation. These findings reveal an unexpected serpin function and define a novel UV protection mechanism in human skin.