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Identification of lympho-epithelial Kazal-type inhibitor 2 in human skin as a kallikrein-related peptidase 5-specific protease inhibitor.

Meyer-Hoffert U, Wu Z, Schröder JM - PLoS ONE (2009)

Bottom Line: Recombinant LEKTI-2 inhibited KLK5 but not KLK7, 14 or other serine proteases tested including trypsin, plasmin and thrombin.LEKTI-2 immune-expression was focally localized at the stratum granulosum and stratum corneum at palmar and plantar sites in close localization to KLK5.At sites of plantar hyperkeratosis, LEKTI-2 expression was increased.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany.

ABSTRACT
Kallikreins-related peptidases (KLKs) are serine proteases and have been implicated in the desquamation process of the skin. Their activity is tightly controlled by epidermal protease inhibitors like the lympho-epithelial Kazal-type inhibitor (LEKTI). Defects of the LEKTI-encoding gene serine protease inhibitor Kazal type (Spink)5 lead to the absence of LEKTI and result in the genodermatose Netherton syndrome, which mimics the common skin disease atopic dermatitis. Since many KLKs are expressed in human skin with KLK5 being considered as one of the most important KLKs in skin desquamation, we proposed that more inhibitors are present in human skin. Herein, we purified from human stratum corneum by HPLC techniques a new KLK5-inhibiting peptide encoded by a member of the Spink family, designated as Spink9 located on chromosome 5p33.1. This peptide is highly homologous to LEKTI and was termed LEKTI-2. Recombinant LEKTI-2 inhibited KLK5 but not KLK7, 14 or other serine proteases tested including trypsin, plasmin and thrombin. Spink9 mRNA expression was detected in human skin samples and in cultured keratinocytes. LEKTI-2 immune-expression was focally localized at the stratum granulosum and stratum corneum at palmar and plantar sites in close localization to KLK5. At sites of plantar hyperkeratosis, LEKTI-2 expression was increased. We suggest that LEKTI-2 contributes to the regulation of the desquamation process in human skin by specifically inhibiting KLK5.

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Spink9 mRNA expression in human skin and keratinocytes.(A) Expression profile of Spink9 mRNA. Fragments were obtained after RT-PCR amplification on human multiple tissue cDNAs with primers specific to the human GAPDH and Spink9. Lanes are labelled according to the template tissue. The Spink9 fragments are of 175 bp in size. H2O (no cDNA) and RT-control (no RNA template) were used as negative controls. (B) Spink9 mRNA expression in cultured primary keratinocytes. Quantitative realtime PCR was conducted on RT-PCR products of total RNA samples collected from keratinocytes treated with 1.0 mM CaCl2 for the indicated time. Bar graphs represent the relative mRNA expression of Spink9 against GAPDH. Data are obtained from three independent experiments with different sources of keratinocytes and are indicated as the mean+/−SD. * indicates significant (p<0.05); ** indicates significant (p<0.01).
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pone-0004372-g003: Spink9 mRNA expression in human skin and keratinocytes.(A) Expression profile of Spink9 mRNA. Fragments were obtained after RT-PCR amplification on human multiple tissue cDNAs with primers specific to the human GAPDH and Spink9. Lanes are labelled according to the template tissue. The Spink9 fragments are of 175 bp in size. H2O (no cDNA) and RT-control (no RNA template) were used as negative controls. (B) Spink9 mRNA expression in cultured primary keratinocytes. Quantitative realtime PCR was conducted on RT-PCR products of total RNA samples collected from keratinocytes treated with 1.0 mM CaCl2 for the indicated time. Bar graphs represent the relative mRNA expression of Spink9 against GAPDH. Data are obtained from three independent experiments with different sources of keratinocytes and are indicated as the mean+/−SD. * indicates significant (p<0.05); ** indicates significant (p<0.01).

Mentions: To investigate the cellular source of LEKTI-2, both RT-PCR and real-time RT-PCR were used to determine its mRNA expression. Expression of Spink9 mRNA was detected in skin samples from foreskin and cultured primary keratinocytes (Fig. 3A). In addition, its expression was also detected in thymus, tonsils, testis, placenta and brain but not in other tissue samples tested (Fig. 3A). In cultured primary keratinocytes, the expression level of Spink9 mRNA was increased up to 10-fold over the time course during calcium-induced differentiation, suggesting that Spink9 is produced by epithelial terminally differentiating keratinocytes.


Identification of lympho-epithelial Kazal-type inhibitor 2 in human skin as a kallikrein-related peptidase 5-specific protease inhibitor.

Meyer-Hoffert U, Wu Z, Schröder JM - PLoS ONE (2009)

Spink9 mRNA expression in human skin and keratinocytes.(A) Expression profile of Spink9 mRNA. Fragments were obtained after RT-PCR amplification on human multiple tissue cDNAs with primers specific to the human GAPDH and Spink9. Lanes are labelled according to the template tissue. The Spink9 fragments are of 175 bp in size. H2O (no cDNA) and RT-control (no RNA template) were used as negative controls. (B) Spink9 mRNA expression in cultured primary keratinocytes. Quantitative realtime PCR was conducted on RT-PCR products of total RNA samples collected from keratinocytes treated with 1.0 mM CaCl2 for the indicated time. Bar graphs represent the relative mRNA expression of Spink9 against GAPDH. Data are obtained from three independent experiments with different sources of keratinocytes and are indicated as the mean+/−SD. * indicates significant (p<0.05); ** indicates significant (p<0.01).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2631147&req=5

pone-0004372-g003: Spink9 mRNA expression in human skin and keratinocytes.(A) Expression profile of Spink9 mRNA. Fragments were obtained after RT-PCR amplification on human multiple tissue cDNAs with primers specific to the human GAPDH and Spink9. Lanes are labelled according to the template tissue. The Spink9 fragments are of 175 bp in size. H2O (no cDNA) and RT-control (no RNA template) were used as negative controls. (B) Spink9 mRNA expression in cultured primary keratinocytes. Quantitative realtime PCR was conducted on RT-PCR products of total RNA samples collected from keratinocytes treated with 1.0 mM CaCl2 for the indicated time. Bar graphs represent the relative mRNA expression of Spink9 against GAPDH. Data are obtained from three independent experiments with different sources of keratinocytes and are indicated as the mean+/−SD. * indicates significant (p<0.05); ** indicates significant (p<0.01).
Mentions: To investigate the cellular source of LEKTI-2, both RT-PCR and real-time RT-PCR were used to determine its mRNA expression. Expression of Spink9 mRNA was detected in skin samples from foreskin and cultured primary keratinocytes (Fig. 3A). In addition, its expression was also detected in thymus, tonsils, testis, placenta and brain but not in other tissue samples tested (Fig. 3A). In cultured primary keratinocytes, the expression level of Spink9 mRNA was increased up to 10-fold over the time course during calcium-induced differentiation, suggesting that Spink9 is produced by epithelial terminally differentiating keratinocytes.

Bottom Line: Recombinant LEKTI-2 inhibited KLK5 but not KLK7, 14 or other serine proteases tested including trypsin, plasmin and thrombin.LEKTI-2 immune-expression was focally localized at the stratum granulosum and stratum corneum at palmar and plantar sites in close localization to KLK5.At sites of plantar hyperkeratosis, LEKTI-2 expression was increased.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany.

ABSTRACT
Kallikreins-related peptidases (KLKs) are serine proteases and have been implicated in the desquamation process of the skin. Their activity is tightly controlled by epidermal protease inhibitors like the lympho-epithelial Kazal-type inhibitor (LEKTI). Defects of the LEKTI-encoding gene serine protease inhibitor Kazal type (Spink)5 lead to the absence of LEKTI and result in the genodermatose Netherton syndrome, which mimics the common skin disease atopic dermatitis. Since many KLKs are expressed in human skin with KLK5 being considered as one of the most important KLKs in skin desquamation, we proposed that more inhibitors are present in human skin. Herein, we purified from human stratum corneum by HPLC techniques a new KLK5-inhibiting peptide encoded by a member of the Spink family, designated as Spink9 located on chromosome 5p33.1. This peptide is highly homologous to LEKTI and was termed LEKTI-2. Recombinant LEKTI-2 inhibited KLK5 but not KLK7, 14 or other serine proteases tested including trypsin, plasmin and thrombin. Spink9 mRNA expression was detected in human skin samples and in cultured keratinocytes. LEKTI-2 immune-expression was focally localized at the stratum granulosum and stratum corneum at palmar and plantar sites in close localization to KLK5. At sites of plantar hyperkeratosis, LEKTI-2 expression was increased. We suggest that LEKTI-2 contributes to the regulation of the desquamation process in human skin by specifically inhibiting KLK5.

Show MeSH
Related in: MedlinePlus