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Role of collagen XVII in migrating keratinocytes
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JSID Abstracts / Journal of Dermatological Science 69 (2013) e1–e46
bullous dermolysis of the newborn (BDN), granular C7 within basal cells was detected by standard anti-C7 mAb. Interestingly, 3 C7like mAbs reacted linearly with BMZ but not with intracellular C7 in BDN skin. These findings suggest that the 3 C7-like mAbs react with maturation-related unique epitope(s) on human C7, which are absent within cultured DJM-1 cells or basal cells of BDN skin. http://dx.doi.org/10.1016/j.jdermsci.2012.11.383 P03-07
2011 JSID’s Fellowship Shiseido Award Role of collagen XVII in migrating keratinocytes Wataru Nishie 1,∗ , Machiko Nishimura 1 , Daisuke Sawamura 2 , Hiroshi Shimizu 1 1
Department of Dermatology, Hokkaido University Graduate School of Medicine, Sapporo, Japan 2 Department of Dermatology, Hirosaki University Graduate School of Medicine, Hirosaki, Japan Collagen XVII (COL17) is one of the main molecules in the hemidesmosomes of basal keratinocytes. The most important role of COL17 is to maintain stable adhesion between the epidermis and the underlying dermis, and congenital and acquired dysfunctions of the molecule leads to the respective blistering skin diseases of non–Herlitz junctional epidermolysis bullosa (nHJEB) and bullous pemphigoid. In contrast, keratinocytes must detach from the underlying dermis when they migrate. Therefore, the adhesive function and expression of COL17 in migrating keratinocytes should be tightly regulated in a context-dependent manner. However, the regulation mechanisms and detailed expression patterns of COL17 in migrating keratinocytes remain unknown. To address this, we produced keratinocytes that stably express N-terminal GFP-tagged COL17 (GFP–COL17) by introducing a corresponding cDNA into the COL17–null keratinocytes obtained from a patient with nHJEB. GFP–COL17 was confirmed to be accurately incorporated into the cell membrane on which the extracellular domain of the molecule could be constitutively shed from the cell surface as observed in normal human keratinocytes. As expected, confocal microscopy demonstrated that GFP–COL17 was expressed in the hemidesmosomes. Interestingly, GFP–COL17 was also detectable entirely on the cell membrane, suggesting that COL17 can be expressed not only in the hemidesmosomes but also in the cell membrane. Notably, GFP–COL17 was also observed in some parts of the lamellipodia and the filopodia, indicating that COL17 may play a distinct role in regulating keratinocyte migration. In summary, the present study suggests a novel role of COL17 in keratinocyte migration that may be distinct from the molecule’s roles as a hemidesmosomal protein. http://dx.doi.org/10.1016/j.jdermsci.2012.11.384 P03-08 The transcriptional role of BBF2H7, ER stress transducer, during collagen biosynthesis in dermal fibroblasts Takeshi Katayoshi ∗ , Seiko Ishikura, Hiroshi Saeki, Kentaro Naito DHC Corporation Laboratories, Division 2, Chiba, Japan BBF2 human homolog on chromosome 7 (BBF2H7), a member of the CREB/ATF family, is an endoplasmic reticulum (ER) stress transducer. BBF2H7 functions as a transcription factor to regulate expression of Sec23A, which encodes a coat protein complex
II component responsible for protein transport from the ER to the Golgi. We have previously shown that BBF2H7 in normal human dermal fibroblasts (NHDFs) is involved in collagen synthesis in response to insulin growth factor-I (IGF-I), and that the BBF2H7Sec23A pathway is required for ER-to-Golgi collagen trafficking. However, it is uncertain whether this pathway is also engaged in other cytokine stimulus-responsive collagen synthesis in dermal fibroblasts. Herein, we demonstrate a new pathway activating BBF2H7 in response to transforming growth factor-beta (TGF-), which maintains dermal collagen homeostasis as well as IGF-I. TGF increased the expression levels of mRNA and protein of type I collagen (COL1) in NHDFs. The expression of BBF2H7 markedly elevated at earlier time points, whereas that of Sec23A was very similar to that of COL1. Furthermore, BBF2H7 knockdown caused a significant reduction in the expression of COL1 and Sec23A induced by TGF- stimulation. These results suggest that BBF2H7 in NHDFs regulates the expression of COL1 and Sec23A in response to TGF. Pretreatment of SB431542, a specific TGF- receptor type I kinase inhibitor, completely suppressed the expression of BBF2H7 and Sec23A induced by TGF- stimulation, indicating that TGF-inducible activation of BBF2H7-Sec23A pathway is triggered by a ligand binding event. Our results raise the possibility that BBF2H7 plays a key role in dermal collagen synthesis constitutively regulated via several routes, contributing to provide novel anti-ageing strategies. http://dx.doi.org/10.1016/j.jdermsci.2012.11.385 P03-09 An approach to fabricate the polymer hollow microneedle that targets dermal lymphatic vessels in the skin Satoshi Hirakawa 1,∗ , Noriyuki Tamaru 2 , Yoshiki Tokura 1
Ogai 2 , Yuko
Wada 2 , Takuya
1
Department of Dermatology, Hamamatsu University School of Medicine, Hamamatsu, Japan 2 Advanced Micromachining Devices Promoting Office, ASTI CORPORATION, Hamamatsu, Japan Cutaneous lymphatic vessels absorb and transport interstitial fluid and macromolecules from the skin to blood stream. Of particular importance, lymphatic vessels play an essential role in absorbing several antigens which are required for T-cell-based immune response. Previous studies have shown that dermal injection of tracers such as colloidal carbon or fluorescence-labeled dextran sulfate represents an established technique that visualizes the absorption and transport of the macromolecules by cutaneous lymphatic vessels. However, lack of feasible needles tightly limited the advance of research that clarifies the biological function of lymphatic vessels. In order to quantitate and visualize the absorption and transport of cutaneous lymphatic vessels, we fabricated novel polymer microneedles in combination with tilted puncture system. The microneedles compose an array that facilitates efficient injection of tracers into the dermis. A single microneedle is composed of polyglycolic acid and fabricated by 250 m in width and 1.5 mm in length with vertical hollow so that the microneedles can puncture the area where initial lymphatic vessels are localized. The surface of the microneedle is treated with O2 plasma prior to injection. This modification enabled to detect the depth of insertion. The resistance force measurement assay further revealed that the array assists microneedles to puncture the mouse skin. The assay contributes to optimize the insertion. These results indicate that our microneedle array can be utilized for the quantitative injection of macromolecules
JSID Abstracts / Journal of Dermatological Science 69 (2013) e1–e46
bullous dermolysis of the newborn (BDN), granular C7 within basal cells was detected by standard anti-C7 mAb. Interestingly, 3 C7like mAbs reacted linearly with BMZ but not with intracellular C7 in BDN skin. These findings suggest that the 3 C7-like mAbs react with maturation-related unique epitope(s) on human C7, which are absent within cultured DJM-1 cells or basal cells of BDN skin. http://dx.doi.org/10.1016/j.jdermsci.2012.11.383 P03-07
2011 JSID’s Fellowship Shiseido Award Role of collagen XVII in migrating keratinocytes Wataru Nishie 1,∗ , Machiko Nishimura 1 , Daisuke Sawamura 2 , Hiroshi Shimizu 1 1
Department of Dermatology, Hokkaido University Graduate School of Medicine, Sapporo, Japan 2 Department of Dermatology, Hirosaki University Graduate School of Medicine, Hirosaki, Japan Collagen XVII (COL17) is one of the main molecules in the hemidesmosomes of basal keratinocytes. The most important role of COL17 is to maintain stable adhesion between the epidermis and the underlying dermis, and congenital and acquired dysfunctions of the molecule leads to the respective blistering skin diseases of non–Herlitz junctional epidermolysis bullosa (nHJEB) and bullous pemphigoid. In contrast, keratinocytes must detach from the underlying dermis when they migrate. Therefore, the adhesive function and expression of COL17 in migrating keratinocytes should be tightly regulated in a context-dependent manner. However, the regulation mechanisms and detailed expression patterns of COL17 in migrating keratinocytes remain unknown. To address this, we produced keratinocytes that stably express N-terminal GFP-tagged COL17 (GFP–COL17) by introducing a corresponding cDNA into the COL17–null keratinocytes obtained from a patient with nHJEB. GFP–COL17 was confirmed to be accurately incorporated into the cell membrane on which the extracellular domain of the molecule could be constitutively shed from the cell surface as observed in normal human keratinocytes. As expected, confocal microscopy demonstrated that GFP–COL17 was expressed in the hemidesmosomes. Interestingly, GFP–COL17 was also detectable entirely on the cell membrane, suggesting that COL17 can be expressed not only in the hemidesmosomes but also in the cell membrane. Notably, GFP–COL17 was also observed in some parts of the lamellipodia and the filopodia, indicating that COL17 may play a distinct role in regulating keratinocyte migration. In summary, the present study suggests a novel role of COL17 in keratinocyte migration that may be distinct from the molecule’s roles as a hemidesmosomal protein. http://dx.doi.org/10.1016/j.jdermsci.2012.11.384 P03-08 The transcriptional role of BBF2H7, ER stress transducer, during collagen biosynthesis in dermal fibroblasts Takeshi Katayoshi ∗ , Seiko Ishikura, Hiroshi Saeki, Kentaro Naito DHC Corporation Laboratories, Division 2, Chiba, Japan BBF2 human homolog on chromosome 7 (BBF2H7), a member of the CREB/ATF family, is an endoplasmic reticulum (ER) stress transducer. BBF2H7 functions as a transcription factor to regulate expression of Sec23A, which encodes a coat protein complex
II component responsible for protein transport from the ER to the Golgi. We have previously shown that BBF2H7 in normal human dermal fibroblasts (NHDFs) is involved in collagen synthesis in response to insulin growth factor-I (IGF-I), and that the BBF2H7Sec23A pathway is required for ER-to-Golgi collagen trafficking. However, it is uncertain whether this pathway is also engaged in other cytokine stimulus-responsive collagen synthesis in dermal fibroblasts. Herein, we demonstrate a new pathway activating BBF2H7 in response to transforming growth factor-beta (TGF-), which maintains dermal collagen homeostasis as well as IGF-I. TGF increased the expression levels of mRNA and protein of type I collagen (COL1) in NHDFs. The expression of BBF2H7 markedly elevated at earlier time points, whereas that of Sec23A was very similar to that of COL1. Furthermore, BBF2H7 knockdown caused a significant reduction in the expression of COL1 and Sec23A induced by TGF- stimulation. These results suggest that BBF2H7 in NHDFs regulates the expression of COL1 and Sec23A in response to TGF. Pretreatment of SB431542, a specific TGF- receptor type I kinase inhibitor, completely suppressed the expression of BBF2H7 and Sec23A induced by TGF- stimulation, indicating that TGF-inducible activation of BBF2H7-Sec23A pathway is triggered by a ligand binding event. Our results raise the possibility that BBF2H7 plays a key role in dermal collagen synthesis constitutively regulated via several routes, contributing to provide novel anti-ageing strategies. http://dx.doi.org/10.1016/j.jdermsci.2012.11.385 P03-09 An approach to fabricate the polymer hollow microneedle that targets dermal lymphatic vessels in the skin Satoshi Hirakawa 1,∗ , Noriyuki Tamaru 2 , Yoshiki Tokura 1
Ogai 2 , Yuko
Wada 2 , Takuya
1
Department of Dermatology, Hamamatsu University School of Medicine, Hamamatsu, Japan 2 Advanced Micromachining Devices Promoting Office, ASTI CORPORATION, Hamamatsu, Japan Cutaneous lymphatic vessels absorb and transport interstitial fluid and macromolecules from the skin to blood stream. Of particular importance, lymphatic vessels play an essential role in absorbing several antigens which are required for T-cell-based immune response. Previous studies have shown that dermal injection of tracers such as colloidal carbon or fluorescence-labeled dextran sulfate represents an established technique that visualizes the absorption and transport of the macromolecules by cutaneous lymphatic vessels. However, lack of feasible needles tightly limited the advance of research that clarifies the biological function of lymphatic vessels. In order to quantitate and visualize the absorption and transport of cutaneous lymphatic vessels, we fabricated novel polymer microneedles in combination with tilted puncture system. The microneedles compose an array that facilitates efficient injection of tracers into the dermis. A single microneedle is composed of polyglycolic acid and fabricated by 250 m in width and 1.5 mm in length with vertical hollow so that the microneedles can puncture the area where initial lymphatic vessels are localized. The surface of the microneedle is treated with O2 plasma prior to injection. This modification enabled to detect the depth of insertion. The resistance force measurement assay further revealed that the array assists microneedles to puncture the mouse skin. The assay contributes to optimize the insertion. These results indicate that our microneedle array can be utilized for the quantitative injection of macromolecules