Expression of laminin, type IV procollagen and 230 kDa bullous pemphigoid antigen genes by keratinocytes and fibroblasts in culture: application of the polymerase chain reaction for detection of small amounts of messenger RNA.
In order to clarify whether keratinocytes and/or fibroblasts express genes encoding basement membrane zone macromolecules, we examined laminin, type IV collagen and 230 kDa bullous pemphigoid antigen (BPAG1) gene expression in keratinocytes and fibroblasts in culture. Northern transfer analysis revealed the presence of specific mRNA transcripts for alpha 1(IV) and alpha 2(IV) chains of type IV collagen as well as B1 and B2 chains of laminin in both fibroblast and keratinocyte RNA. Laminin A mRNA, however, was detected in fibroblasts but not in keratinocytes. In contrast, BPAG1 mRNA was detected in keratinocytes but not in fibroblasts using the same RNA preparations. A polymerase chain reaction (PCR) using laminin A and BPAG1-specific primers produced amplified DNAs with the predicted sizes in reverse-transcripted cDNA derived from keratinocyte and fibroblast RNA, respectively. These results provide evidence that normal human skin keratinocytes and fibroblasts express genes encoding laminin A, B1, B2, alpha 1(IV), and BPAG1 at a steady-state level. Moreover, the PCR for detecting small amounts of mRNA suggested that both keratinocytes and fibroblasts can be utilized for the analysis of DNA mutations in inherited skin diseases affecting the basement membrane zone, such as epidermolysis bullosa.
Nomura
K
K
Department of Dermatology, Hirosaki University School of Medicine, Japan.
Sugawara
T
T
Sato
T
T
Sawamura
D
D
Hashimoto
I
I
Sugita
Y
Y
Uitto
J
J
D001324:Autoantigens / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D015152:Blotting, Northern; D002352:Carrier Proteins; D002478:Cells, Cultured; D003094:Collagen; D003598:Cytoskeletal Proteins; D004247:DNA / Q000032:analysis / Q000235:genetics; D000071821:Dystonin; D005347:Fibroblasts / Q000737:chemistry* / Q000166:cytology* / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D015603:Keratinocytes / Q000737:chemistry* / Q000166:cytology* / Q000378:metabolism; D007797:Laminin / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins; D024041:Non-Fibrillar Collagens; D010391:Pemphigoid, Bullous / Q000276:immunology; D016133:Polymerase Chain Reaction; D011347:Procollagen / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D000097805:Collagen Type XVII
D001324:Autoantigens; D002352:Carrier Proteins; D003094:Collagen; D003598:Cytoskeletal Proteins; D004247:DNA; D000071821:Dystonin; D007797:Laminin; D009419:Nerve Tissue Proteins; D024041:Non-Fibrillar Collagens; D011347:Procollagen; D012333:RNA, Messenger; D000097805:Collagen Type XVII; C486268:DST protein, human