Archives of Dermatological Research

, Volume 286, Issue 7, pp 408–413 | Cite as

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

  • K. Nomura
  • T. Sugawara
  • T. Sato
  • D. Sawamura
  • I. Hashimoto
  • Y. Sugita
  • J. Uitto
Original Contributions


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 α1(IV) and α2(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, α1(IV), α2(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.

Key words

Keratinocyte gene expression Fibroblast gene expression Basement membrane zone macromolecules Laminin Collagen type IV Bullons pemphigoid antigen 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Briggaman RA, Wheeler Jr. CE (1975) The epidermal-dermal junction. J Invest Dermatol 65: 71–84Google Scholar
  2. 2.
    Kleinman HK, Ebihara I, Killen PD, Sasaki M, Cannon FB, Yamada Y, Martin GR (1987) Genes for basement membrane proteins are coordinately expressed in differentiating F9 cells but not in normal adult murine tissues. Dev Biol 122: 373–378Google Scholar
  3. 3.
    Timpl R, Dziadek M (1986) Structure, development, and molecular pathology of basement membranes. Int Rev Exp Pathol 29: 1–111Google Scholar
  4. 4.
    Olsen D, Nagayoshi T, Fazio M, Peltonen J, Jaakkola S, Sanborn D, Sasaki T, Kuivaniemi H, Chu M-L, Deutzmann R, Timple R, Uitto J (1989) Human laminin: cloning and sequence analysis of cDNAs encoding A, B1 and B2 chains, and expression of the corresponding genes in human skin and cultured cells. Lab Invest 60: 772–782Google Scholar
  5. 5.
    Pikkarainen T, Eddy R, Fukushima Y, Byers M, Shows T, Pihlajaniemi T, Saraste M, Tryggvason K (1987) Human laminin chain: a multidomain protein with gene (LAMB1) locus in the q22 region of chromosome 7. J Biol Chem 262: 10454–10462Google Scholar
  6. 6.
    Pikkarainen T, Kallunki T, Tryggvason K (1988) Human laminin B2 chain: comparison of the complete amino acid sequence with the B1 chain reveals variability in sequence homology between different structural domains. J Biol Chem 263: 6751–6758Google Scholar
  7. 7.
    Kalluri R, Gunwar S, Reeders ST, Morrison KC, Mariyama M, Ebner KE, Noelken ME, Hudson BG (1991) Goodpasture syndrome. Localization of the epitope for the autoantibodies to the carboxyl-terminal region of the 3(IV) chain of basement membrane collagen. J Biol Chem 266: 24018–24024Google Scholar
  8. 8.
    Kleppel MM, Fan WW, Cheong HI, Michael AF (1992) Evidence for separate networks of classical and novel basement membrane collagen. Characterization of 3 (IV)-alport antigen heterodimer. J Biol Chem 267: 4137–4142Google Scholar
  9. 9.
    Stanley JR (1989) Pemphigus and pemphigoid as paradigms of organ-specific, autoantibody-mediated diseases. J Clin Invest 83: 1443–1448Google Scholar
  10. 10.
    Diaz LA, Ratrie III H, Saunders WS, Futamura S, Squiquera HL, Anhalt GJ, Guidice GJ (1990) Isolation of a human epidermal cDNA corresponding to the 180-kD autoantigen recognized by bullous pemphigoid and herpes gestationis sera. Immunolocalization of this protein to the hemidesmosome. J Clin Invest 86: 1088–1094Google Scholar
  11. 11.
    Stanley JR, Tanaka T, Mueller S, Klaus-Kovtun V, Roop D (1988) Isolation of complementary DNA for bullous pemphigoid antigen by use of patients' autoantibodies. J Clin Invest 82: 1864–1870Google Scholar
  12. 12.
    Sawamura D, Li K-H, Nomura K, Sugita Y, Christiano AM Uitto J (1991) Bullous pemphigoid antigen: cDNA cloning, cellular expression, and evidence for polymorphism of the human gene. J Invest Dermatol 96: 908–915Google Scholar
  13. 13.
    Sawamura D, Li K, Chu M-L, Uitto J (1991) Human bullous pemphigoid antigen (BPAG1). Amino acid sequences deduced from cloned cDNAs predict biologically important peptide segments and protein domains. J Biol Chem 266: 17784–17790Google Scholar
  14. 14.
    Prunieras M, Regnier M, Fougere S, Woodley D (1983) Keratinocytes synthesize basal-lamina proteins in culture. J Invest Dermatol 81: 74s-81sGoogle Scholar
  15. 15.
    Stanley JR, Hawley-Nelson P, Yaar M, Martin GR, Katz SI (1982) Laminin and bullous pemphigoid antigen are distinct basement membrane proteins synthesized by epidermal cells. J Invest Dermatol 78: 456–459Google Scholar
  16. 16.
    Woodley DT, Stanley JR, Reese MJ, O'Keefe EJ (1988) Human dermal fibroblasts synthesize laminin. J Invest Dermatol 90: 679–683Google Scholar
  17. 17.
    Stanley JR, Rubinstein N, Klaus-Kovtun V (1985) Epidermolysis bullosa acquisita antigen is synthesized by both human keratinocytes and human dermal fibroblasts. J Invest Dermatol 85: 542–545Google Scholar
  18. 18.
    Woodley DT, Briggaman RA, Gammon WR, Falk RJ, Reese MJ, Tomsick RS, O'Keefe EJ (1986) Epidermolysis bullosa acquisita antigen, a major cutaneous basement membrane component, is synthesized by human dermal fibroblasts and other cutaneous tissues. J Invest Dermatol 87: 227–231Google Scholar
  19. 19.
    Olsen DR, Uitto J (1989) Differential expression of type IV procollagen and laminin genes by fetal vs adult skin fibroblasts in culture: determination of subunit mRNA steady-state levels. J Invest Dermatol 93: 127–131Google Scholar
  20. 20.
    Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156–159Google Scholar
  21. 21.
    Davis LG, Dibner MD, Battey JF (1986) Basic methods in molecular biology. Preparation and analysis of RNA from eu-karyotic cells. Elsevier, New York, pp 129–156Google Scholar
  22. 22.
    Marchuk D, McCrohon S, Fuchs E (1984) Remarkable conservation of structure among intermediate filament genes. Cell 39: 491–498Google Scholar
  23. 23.
    Uitto J, Chung-Honet LC, Christiano A (1992) Molecular biology and pathology of type VII collagen. Exp Dermatol 1: 2–11Google Scholar
  24. 24.
    Parente MG, Chung LC, RyynÄnen J, Woodley DT, Wynn KC, Bauer EA, Mattei M-G, Chu ML, Uitto J (1991) Human type VII collagen: cDNA cloning and chromosomal mapping of the gene. Proc Natl Acad Sci USA 88: 6931–6935Google Scholar
  25. 25.
    Lankat-Buttgereit B, Kulozik M, Hunzelmann N, Krieg T (1991) Cytokines alter mRNA steady state levels for basement membrane proteins in human skin fibroblasts. J Dermatol Sci 2: 300–307Google Scholar
  26. 26.
    Dziadek M, Timpl R (1985) Expression of nidogen and laminin in basement membranes during mouse embryogenesis and in teratocarcinoma cells. Dev Biol 111: 372–382Google Scholar
  27. 27.
    Cornbrooks CJ, Carey DJ, McDonald JA, Timple R, Bunge RP (1983) In vivo and in vitro observations on laminin production by Schwann cells. Proc Natl Acad Sci USA 80: 3850–3854Google Scholar
  28. 28.
    Nissinen M, Vuolteenaho R, Boot-Handford R, Kallunki P, Tryggvason K (1991) Primary structure of the human laminin A chain. Limited expression in human tissues. Biochem J 276: 369–379Google Scholar
  29. 29.
    Marinkovich MP, Lunstrum GP, Keene DR, Burgeson RE (1992) The dermal-epidermal junction of human skin contains a novel laminin variant. J Cell Biol 119: 695–703Google Scholar
  30. 30.
    Stanley JR, Nelson PH, Yuspa SH, Shevach EM, Katz SI (1981) Characterization of bullous pemphigoid antigen: a unique basement membrane protein synthesized by epidermal cells. J Invest Dermatol 78: 456–459Google Scholar
  31. 31.
    Diaz LA, Marcelo CL (1978) Pemphigoid and pemphigus antigens in cultured epidermal cells. Br J Dermatol 98: 631–637Google Scholar
  32. 32.
    Barry I (1990) The polymerase chain reaction: a new method of using molecular genetics for medical diagnosis. N Engl J Med 322: 178–183Google Scholar

Copyright information

© Springer-Verlag 1994

Authors and Affiliations

  • K. Nomura
    • 1
  • T. Sugawara
    • 1
  • T. Sato
    • 1
  • D. Sawamura
    • 1
  • I. Hashimoto
    • 1
  • Y. Sugita
    • 2
  • J. Uitto
    • 3
  1. 1.Department of DermatologyHirosaki University School of MedicineHirosakiJapan
  2. 2.Department of DermatologyYokohama City University School of MedicineYokohamaJapan
  3. 3.Department of DermatologyJefferson Medical CollegePhiladelphiaUSA

Personalised recommendations