Cloning of Human Keratolinin cDNA: Keratolinin Is Identical with a Cysteine Proteinase Inhibitor, Cysteine A, and Is Regulated by Ca2+, TPA, and cAMP

Abstract

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 Agt11 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 upregulation of keratolinin mRNA was also observed by the treatment of SVHK cells with 10 ng TPA per ml or 100 microM 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.