Quantitative analysis of the synthesis and secretion of type VII collagen in cultured human dermal fibroblasts with a sensitive sandwich enzyme-linked immunoassay.
Type VII collagen is the major component of anchoring fibrils in the epidermal basement membrane. Its expression has been analyzed by immunostaining or Northern blotting, but rarely at the protein level. In this study, we have quantitatively examined the effects of ascorbic acid and various cytokines/growth factors on the protein synthesis and secretion of type VII collagen by human dermal fibroblasts in culture, using a developed, highly sensitive sandwich enzyme-linked immunoassay with two kinds of specific monoclonal antibodies against the non-collagenous domain-1. Ascorbic acid and its derivative induced a twofold increase in type VII collagen synthesis, and markedly increased the secretion of type VII collagen into the medium when compared with the control culture. This effect was not influenced by the presence of transforming growth factor-beta1 (TGF-beta1). The synthesis of type VII collagen was elevated by TGF-beta1, platelet-derived growth factor, tumor necrosis factor-alpha, and interleukin-1beta, but not by TGF-alpha. Thus, our data indicate that the synthesis and secretion of type VII collagen in human dermal fibroblasts are regulated by ascorbate and the enhancement of type VII collagen gene expression by cytokines/growth factors is accompanied with elevated production of type VII collagen at the protein level.
Amano
Satoshi
S
Shiseido Life Science Research Center, Yokohama, Japan. satoshi.amano@to.shiseido.co.jp
Ogura
Yuki
Y
Akutsu
Nobuko
N
Nishiyama
Toshio
T
D000293:Adolescent; D000328:Adult; D000367:Age Factors; D000368:Aged; D000369:Aged, 80 and over; D001205:Ascorbic Acid / Q000502:physiology; D002478:Cells, Cultured; D002648:Child; D002675:Child, Preschool; D024143:Collagen Type VII / Q000032:analysis / Q000096:biosynthesis* / Q000378:metabolism; D016207:Cytokines / Q000502:physiology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005347:Fibroblasts / Q000378:metabolism*; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008875:Middle Aged; D012867:Skin / Q000378:metabolism*; D053773:Transforming Growth Factor beta1 / Q000502:physiology; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology
D024143:Collagen Type VII; D016207:Cytokines; D053773:Transforming Growth Factor beta1; D014409:Tumor Necrosis Factor-alpha; D001205:Ascorbic Acid