Cloning of human keratolinin cDNA: Keratolinin is identical with a cysteine proteinase inhibitor, cystatin A, and is regulated by Ca2+, TPA, and cAMP

[摘要] Keratolinin has been described as one of the precursor proteins of cornified cell envelope of keratinocytes, Using rabbit polyclonal anti-human keratolinin antibody, we isolated a cDNA clone of human keratolinin gene from a human lambda gt11 cDNA expression library that was constructed by random priming from poly(A)(+)RNA extracted from cultured normal human keratinocytes, Screening by rabbit anti-human keratolinin antibody detected one positive clone (HKL-1), The recombinant 12.5-kDa protein constructed from the clone reacted specifically with the anti-human keratolinin antibody, DNA sequence analysis revealed that HKL-1. clone was 448 bp long, and its putative amino acid sequence was identical with that of a human cysteine proteinase inhibitor, cystatin A, Western blot analysis showed that the commercially available recombinant cystatin A also reacted specifically with the anti-human keratolinin antibody, Northern blot analysis indicated that HKL-1 clone hybridizes with mRNA of about 0.5 kb, consistent with the size of the HKL-1 clone, The keratolinin mRNA was highly expressed in cultured human keratinocytes in high Ca2+ (1 mM); in low Ca2+ (0.05 mM), the keratolinin mRNA expression was significantly lower, Using SV40-transformed human keratinocytes (SVHK cells), we further analyzed the regulation of keratolinin mRNA. In low Ca2+ (0.05 mM), keratolinin mRNA in SVHK cells was marginally detectable, Upon shift to 1 mM calcium, keratolinin mRNA was markedly increased, The up-regulation of keratolinin mRNA was also observed by the treatment of SVHK cells with 10 ng TPA per ml or 100 mu M forskolin under low calcium conditions (0.05 mM). Our results indicate that keratolinin is identical with cystatin A, a cysteine proteinase inhibitor, and its expression is positively regulated by Ca2+, TPA, and forskolin.

[发布日期] 1997-06-01

[关键词] keratinocyte;cornified cell envelope;transglutaminase;psoriasis