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Decreased TJ Components in Atopic Dermatitis: A Possible Mechanism of Impaired Outside-In Barrier in Atopic Dermatitis
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JSID Abstracts / Journal of Dermatological Science 69 (2013) e1–e46
P05-03[C09-02] Keratinocyte-specific PRSS3 participates in the desquamation process via degradation of LEKTI Masashi Miyai 1,∗ , Haruyo Yamanishi 1 , Yuuko Matsumoto 1 , Mami Yamamoto 1,2 , Toshihiko Hibino 1 1 2
Shiseido Research Center, Yokohama, Japan Department of Dermatology, Tokyo Medical University, Tokyo, Japan
Kallikrein-related peptidases (KLKs) and the lymphoepitherial Kazal-type inhibitor (LEKTI) play important roles in corneocyte desquamation. It is suggested that KLKs such as KLK5 and 7 are released from LEKTI because of acidic pH at the skin surface and degrades corneodesmosomes, resulting in the removal of surface corneocytes. It is still obscure whether the KLK-LEKTI interaction is easily broke in such a weak acidic pH(5.5-6.5). Recently we reported cloning of a new PRSS3 gene product, keratinocytespecific mesotrypsin from the cDNA library. The aim of this study is to elucidate involvement of PRSS3 in the desquamation process. We constructed various recombinant LEKTI proteins, D2, D2-5, D2-6, D2-7, D5, D6, D6-9, D7, D7-9 and D10-15. All of these recombinants inhibited KLK5 with different extent. However, they did not show any inhibitory effect on PRSS3. Interestingly we found that single domain LEKTI, D5 and D6 were degraded by PRSS3. We then investigated pH susceptibility between KLK5 and each LEKTI domain. KLK5 was incubated with D5, or D6 in different pH buffers ranging from pH 3.5 to pH 9.0 and residual activities were measured using Gln-Ala-Arg-MCA. KLK5 was active between pH 5.5 to pH 9 and that D5 as well as D6 completely suppressed KLK5 activity in the same pH range. Other LEKTI domain constructs gave similar results. In order to elucidate whether PRSS3 could participate in desquamation process, localization in human skin was examined with immunoelectron microscopy. PRSS3 was localized in the cytoplasm of granular cells and intercellular spaces of the cornified layer. Collectively our results suggest that PRSS3 is actively involved in the desquamation process via degradation of the intrinsic inhibitor, LEKTI. http://dx.doi.org/10.1016/j.jdermsci.2012.11.423 P05-04[C09-03] Co-localization of kallikrein5 and profilaggrin in keratohyalin granules and reduction of filaggrin monomers by kallikrein5 downmodulation Jun-ichi Tokura 1
Sakabe 1,∗ , Isao
Ohta 2 , Satoshi
Hirakawa 1 , Yoshiki
1 Department of Dermatology, Hamamatsu University School of Medicine, Hamamatsu, Japan 2 Research Equipment Center, Hamamatsu University School of Medicine, Hamamatsu, Japan
Kallikrein5 (KLK5) is known as an enzyme involved in epidermal desquamation. We have recently identified KLK5 as one of profilaggrin processing enzymes by LC/MS/MS analysis. However, the localization of KLK5 remains unfully identified in association with profilaggrin, and the functional significance of KLK5 in live keratinocytes needs to be addressed. Here, we sought to demonstrate co-localization of KLK5 and profilaggrin by immunoelectron microscopy and to provide evidence for the functional activity of KLK5 by shRNA analysis. Normal human skin specimens were stained for profilaggrin and KLK5 with rabbit anti-human KLK5 antibody and mouse anti-human profilaggrin/filaggrin antibody, respectively. They were subsequently incubated with 20 nm gold particle-conjugated rabbit IgG Fc-specific and 40 nm gold
particle-conjugated mouse IgG Fc-specific antibodies We found that KLK5 and profilaggrin were co-localized as represented by the two types of gold particles in the keratohyalin granule accentuated with uranyl acetate. As a functional assay, the expression of KLK5 was downmodulated by shRNA, and inhibition of profilaggrin processing and resultant reduction of filaggrin monomers were assessed by Western blotting and transepidermal electrical resistance in normal human epidermal keratinocytes (NHEKs). NHEKs were incubated with KLK5 shRNA lentiviral particles and subsequently cultured in a 1.2 mM calcium condition. We found that filaggrin monomers were reduced by the KLK5 blockade. KLK5 is a serine protease and well known as corneodesmosome protease and PAR-2 ligand. In addition to these functions, the present study further confirmed that KLK5 strongly participates in profilaggrin processing. http://dx.doi.org/10.1016/j.jdermsci.2012.11.424 P05-05[C09-04] Decreased TJ Components in Atopic Dermatitis: A Possible Mechanism of Impaired Outside-In Barrier in Atopic Dermatitis Yuki Takuo 1,∗ , Aya Komiya 1 , Shotaro Ito 2 , Junko Ishikawa 2 , Yoshito Takahashi 1 1 Innovative Beauty Science Laboratory, Kanebo Cosmetics Inc., Kanagawa, Japan 2 Biological Science Laboratories, Kao Corporation, Tochigi, Japan
In this study, we examined changes in the TJ barrier in atopic dermatitis associated with a defective outside-in barrier. We then investigated the relationship between the TJ barrier and the outside-in barrier using a human skin equivalent with weakened TJ barrier.An analysis of DNA microarrays in atopic dermatitis showed diminished amounts of claudin-1, -4, -7, -8 and -17 mRNAs. Immunohistochemical and western blot analyses revealed diminished expression of claudin-4 (including claudin-1 shown by De Benedetto et al. (2011)), but no occludin or ZO-1 was observed. Knockdown experiments of claudin-4 in human epidermal keratinocytes weakened the TJ barrier, suggesting that the TJ barrier was weakened in atopic dermatitis.We then investigated the relationship between the outside-in barrier and TJs in human skin equivalents treated with GST-C-CPE (a polypeptide with claudin-4 inhibitory property). Human skin equivalent models possess functional TJs that completely prevent the diffusion of a paracellular tracer. However, after four days of incubation with GST-C-CPE, the TJs failed to prevent the diffusion of the paracellular tracer. In this model with weakened TJ barrier, an increase in the penetration of Lucifer Yellow through the stratum corneum down to the basal layer was observed, accompanied by an impairment of pro-filaggrin and polar lipid processing.We, therefore, suggest that an impaired TJ function promoted the entry of external substances by affecting stratum corneum formation. Further, our study suggests that a weakened TJ barrier contributes to its defective Outside-In Barrier in atopic dermatitis. http://dx.doi.org/10.1016/j.jdermsci.2012.11.425
JSID Abstracts / Journal of Dermatological Science 69 (2013) e1–e46
P05-03[C09-02] Keratinocyte-specific PRSS3 participates in the desquamation process via degradation of LEKTI Masashi Miyai 1,∗ , Haruyo Yamanishi 1 , Yuuko Matsumoto 1 , Mami Yamamoto 1,2 , Toshihiko Hibino 1 1 2
Shiseido Research Center, Yokohama, Japan Department of Dermatology, Tokyo Medical University, Tokyo, Japan
Kallikrein-related peptidases (KLKs) and the lymphoepitherial Kazal-type inhibitor (LEKTI) play important roles in corneocyte desquamation. It is suggested that KLKs such as KLK5 and 7 are released from LEKTI because of acidic pH at the skin surface and degrades corneodesmosomes, resulting in the removal of surface corneocytes. It is still obscure whether the KLK-LEKTI interaction is easily broke in such a weak acidic pH(5.5-6.5). Recently we reported cloning of a new PRSS3 gene product, keratinocytespecific mesotrypsin from the cDNA library. The aim of this study is to elucidate involvement of PRSS3 in the desquamation process. We constructed various recombinant LEKTI proteins, D2, D2-5, D2-6, D2-7, D5, D6, D6-9, D7, D7-9 and D10-15. All of these recombinants inhibited KLK5 with different extent. However, they did not show any inhibitory effect on PRSS3. Interestingly we found that single domain LEKTI, D5 and D6 were degraded by PRSS3. We then investigated pH susceptibility between KLK5 and each LEKTI domain. KLK5 was incubated with D5, or D6 in different pH buffers ranging from pH 3.5 to pH 9.0 and residual activities were measured using Gln-Ala-Arg-MCA. KLK5 was active between pH 5.5 to pH 9 and that D5 as well as D6 completely suppressed KLK5 activity in the same pH range. Other LEKTI domain constructs gave similar results. In order to elucidate whether PRSS3 could participate in desquamation process, localization in human skin was examined with immunoelectron microscopy. PRSS3 was localized in the cytoplasm of granular cells and intercellular spaces of the cornified layer. Collectively our results suggest that PRSS3 is actively involved in the desquamation process via degradation of the intrinsic inhibitor, LEKTI. http://dx.doi.org/10.1016/j.jdermsci.2012.11.423 P05-04[C09-03] Co-localization of kallikrein5 and profilaggrin in keratohyalin granules and reduction of filaggrin monomers by kallikrein5 downmodulation Jun-ichi Tokura 1
Sakabe 1,∗ , Isao
Ohta 2 , Satoshi
Hirakawa 1 , Yoshiki
1 Department of Dermatology, Hamamatsu University School of Medicine, Hamamatsu, Japan 2 Research Equipment Center, Hamamatsu University School of Medicine, Hamamatsu, Japan
Kallikrein5 (KLK5) is known as an enzyme involved in epidermal desquamation. We have recently identified KLK5 as one of profilaggrin processing enzymes by LC/MS/MS analysis. However, the localization of KLK5 remains unfully identified in association with profilaggrin, and the functional significance of KLK5 in live keratinocytes needs to be addressed. Here, we sought to demonstrate co-localization of KLK5 and profilaggrin by immunoelectron microscopy and to provide evidence for the functional activity of KLK5 by shRNA analysis. Normal human skin specimens were stained for profilaggrin and KLK5 with rabbit anti-human KLK5 antibody and mouse anti-human profilaggrin/filaggrin antibody, respectively. They were subsequently incubated with 20 nm gold particle-conjugated rabbit IgG Fc-specific and 40 nm gold
particle-conjugated mouse IgG Fc-specific antibodies We found that KLK5 and profilaggrin were co-localized as represented by the two types of gold particles in the keratohyalin granule accentuated with uranyl acetate. As a functional assay, the expression of KLK5 was downmodulated by shRNA, and inhibition of profilaggrin processing and resultant reduction of filaggrin monomers were assessed by Western blotting and transepidermal electrical resistance in normal human epidermal keratinocytes (NHEKs). NHEKs were incubated with KLK5 shRNA lentiviral particles and subsequently cultured in a 1.2 mM calcium condition. We found that filaggrin monomers were reduced by the KLK5 blockade. KLK5 is a serine protease and well known as corneodesmosome protease and PAR-2 ligand. In addition to these functions, the present study further confirmed that KLK5 strongly participates in profilaggrin processing. http://dx.doi.org/10.1016/j.jdermsci.2012.11.424 P05-05[C09-04] Decreased TJ Components in Atopic Dermatitis: A Possible Mechanism of Impaired Outside-In Barrier in Atopic Dermatitis Yuki Takuo 1,∗ , Aya Komiya 1 , Shotaro Ito 2 , Junko Ishikawa 2 , Yoshito Takahashi 1 1 Innovative Beauty Science Laboratory, Kanebo Cosmetics Inc., Kanagawa, Japan 2 Biological Science Laboratories, Kao Corporation, Tochigi, Japan
In this study, we examined changes in the TJ barrier in atopic dermatitis associated with a defective outside-in barrier. We then investigated the relationship between the TJ barrier and the outside-in barrier using a human skin equivalent with weakened TJ barrier.An analysis of DNA microarrays in atopic dermatitis showed diminished amounts of claudin-1, -4, -7, -8 and -17 mRNAs. Immunohistochemical and western blot analyses revealed diminished expression of claudin-4 (including claudin-1 shown by De Benedetto et al. (2011)), but no occludin or ZO-1 was observed. Knockdown experiments of claudin-4 in human epidermal keratinocytes weakened the TJ barrier, suggesting that the TJ barrier was weakened in atopic dermatitis.We then investigated the relationship between the outside-in barrier and TJs in human skin equivalents treated with GST-C-CPE (a polypeptide with claudin-4 inhibitory property). Human skin equivalent models possess functional TJs that completely prevent the diffusion of a paracellular tracer. However, after four days of incubation with GST-C-CPE, the TJs failed to prevent the diffusion of the paracellular tracer. In this model with weakened TJ barrier, an increase in the penetration of Lucifer Yellow through the stratum corneum down to the basal layer was observed, accompanied by an impairment of pro-filaggrin and polar lipid processing.We, therefore, suggest that an impaired TJ function promoted the entry of external substances by affecting stratum corneum formation. Further, our study suggests that a weakened TJ barrier contributes to its defective Outside-In Barrier in atopic dermatitis. http://dx.doi.org/10.1016/j.jdermsci.2012.11.425