[논문]Effects of human collagen α-1 type I-derived proteins on collagen synthesis and elastin production in human dermal fibroblasts

Hwang, Su Jin; Kim, Su Hwan; Seo, Woo-Young; Jeong, Yelin; Shin, Min Cheol; Ryu, Dongryeol; Lee, Sang Bae; Choi, Young Jin; Kim, KyeongJin

doi:10.5483/bmbrep.2021.54.6.038

Effects of human collagen α-1 type I-derived proteins on collagen synthesis and elastin production in human dermal fibroblasts 원문보기

BMB reports, v.54 no.6, 2021년, pp.329 - 334

Hwang, Su Jin (ABIOTECH Co., Ltd.) , Kim, Su Hwan (Department of Agricultural Biotechnology, Seoul National University) , Seo, Woo-Young (ABIOTECH Co., Ltd.) , Jeong, Yelin (Department of Biomedical Sciences, College of Medicine, Inha University) , Shin, Min Cheol (Department of Biomedical Sciences, College of Medicine, Inha University) , Ryu, Dongryeol (Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine) , Lee, Sang Bae (Division of Life Sciences, Jeonbuk National University) , Choi, Young Jin (Department of Agricultural Biotechnology, Seoul National University) , Kim, KyeongJin (Department of Biomedical Sciences, College of Medicine, Inha University)

Abstract ▼ AI-Helper

Collagen type I is the most abundant form of collagen in human tissues, and is composed of two identical α-1 type I chains and an α-2 type I chain organized in a triple helical structure. A previous study has shown that human collagen α-2 type I (hCOL1A2) promotes collagen synthesis, wound healing, and elastin production in normal human dermal fibroblasts (HDFs). However, the biological effects of human collagen α-1 type I (hCOL1A1) on various skin properties have not been investigated. Here, we isolate and identify the hCOL1A1-collagen effective domain (CED) which promotes collagen type I synthesis. Recombinant hCOL1A1-CED effectively induces cell proliferation and collagen biosynthesis in HDFs, as well as increased cell migration and elastin production. Based on these results, hCOL1A1-CED may be explored further for its potential use as a preventative agent against skin aging.

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표/그림 (4)

그림 Fig. 1. Identification of hCOL1A1 domain for the synthesis of collagen type I. (A) Schematic representing full-length hCOL1A1 and its various deletion mutants. (B and C) Human dermal fibroblasts (HDFs) were transfected with plasmids encoding hemagglutinin (HA), as the negative control (NC), HA-tagged full length hCOL1A2 (FL), and each of the deletion mutants (N-terminal chain [N], middle chain [M], or C-terminal chain [C]). Forty-eight hours after transfection, cells were collected and cell lysates were analyzed by immunoblotting with anti-HA and anti-tubulin antibodies (B). The amount of collagen type I in transfected HDF culture media was measured with an enzyme-linked immunosorbent assay (ELISA) kit (C). (D and E) HDF cells were transfected with plasmids encoding HA (NC) and each of the indicated mutants (small middle A [SMA], small middle B [SMB], small middle C [SMC], small middle D [SMD], or small middle E [SME]). Forty-eight hours after transfection, cells were collected and cell lysates were analyzed by immunoblotting with anti-HA and anti-tubulin antibodies (D). The amount of collagen type I was measured with an ELISA kit in a transfected HDF-cultured media (E). Results are presented as mean ± SD of three independent experiments. Student's t-test was used for statistical analyses (*P < 0.05, **P < 0.005).
그림 Fig. 2. Identification of purified hCOL1A1-CED. (A) Coomassie Blue staining of uninduced (without IPTG), induced TCP (total cell protein), or isolated hCOL1A1-CED showed a prominent band; arrows indicate that hCOL1A1-CED was identified using (B) anti-His and (C) antihCOL1A1 antibodies by western blot analysis. (D) Identification of hCOL1A1-CED protein in the gel band of isolated hCOL1A1-CED by liquid chromatography-mass spectrometry. The panels show the representative MS/MS spectrum for the identified peptides of GESGPSGPAGPTGAR (1297.61724 m/z, 2+).
그림 Fig. 3. hCOL1A1-CED stimulates cell proliferation and collagen synthesis in HDFs. (A) HDF cells were treated with EGF (0.1 and 1 μg/ml) and hCOL1A1-CED (1 and 10 μg/ml) for 48 h. Cell proliferation, induced by EGF and hCOL1A1-CED, was determined by 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. (B and C) HDF cells were treated with EGF (1 μg/ml; 166.7 nM), hCOL1A1-CED (10 μg/ml; 840.9 nM), or TGF-β1 (10 ng/ml; 781.3 pM) for 48 h. The amount of collagen type I in the HDF culture media and mRNA levels of Col1a1 in HDF cells were measured by enzyme-linked immunosorbent assay (B) or qRT-PCR (C), respectively. Cell proliferation was determined by MTT assay (D). (E) HDF cells were treated with EGF (1 μg/ml), TGF-β1 (10 ng/ml), or hCOL1A1-CED (1 and 10 μg/ml), for 48 h. Cells were collected 48 h post-treatment and cell lysates were analyzed by immunoblotting with anti-His (upper panel) or anti-α-tubulin antibodies, respectively. Results are presented as the mean ± standard deviation of three independent experiments. Student’s t-test was used for statistical analyses (*P < 0.05, **P < 0.005).
그림 Fig. 4. hCOL1A1-derived protein increases the migration efficiency and elastin production, but not fibronectin production. (A) HaCaT cells were treated with TGF-β1 (10 ng/ml), EGF (1 μg/ml), or hCOL1A1- CED (10 μg/ml) and then subjected to transwell migration assays. Migration was monitored for up to 20 h. The same fields were photographed immediately after treatment and after 20 h, and images were superimposed using Adobe Photoshop. Areas were measured using Image J. A representative result is shown. (B) In vitro wound healing assay in HaCaT (top) or HDF (bottom) cells with TGFβ-1 (10 ng/ml) or hCOL1A1-CED at the indicated concentration. The cells were photographed immediately after wounding and 16 hours later, and the pictures were superimposed. A representative result is shown. (C and D) HDF cells were treated with TGF-β1 (10 ng/ml), EGF (1 μg/ml), and hCOL1A1-CED (10 μg/ml) for 48 h. The amount of elastin (C) and fibronectin (D) in the HDF culture media was measured using enzyme-linked immunosorbent assay. (E and F) Melanin contents in B16F10 melanoma cells (E) or expression of indicated genes in HDF cells (F) after treatment with TGFβ-1 or hCOL1A1. Results are presented as the mean ± standard deviation of three independent experiments. Student's t-test was used for statistical analyses (*P < 0.05, **P < 0.005).

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