Thyroid hormone-dependent expression of bullfrog tadpole collagenase gene

Collagenase has been suggested to play a pivotal role in tissue remodeling during anuran metamorphosis. RNA blot analysis and nuclear run-on assay showed that collagenase genes of caudal cells of tadpoles of the bullfrog, Rana catesbeiana, are up-regulated by thyroid hormone after only 24 h. The gene for collagenase was cloned from genome DNA of the bullfrog and its structure, including the 5'-upstream region with-1 kbp, was determined. The gene contained 4 exons with sequences of the enzyme active centre in exon 2. Cis-acting regulatory sequences were recognized in the 5'-upstream region as AP-1 and Sp1, which had also been found in the mammalian collagenase gene. A special sequence was noticed near -870 bp, 5'-AG-GTAAGAACAGGATA-3', that satisfies a general criterion for the identification of the thyroid hormone responsive element (TRE) proposed by Umesono et al. (1991) and is homologous to the element reported in genes of thyroid-hormone responsive proteins such as thyroid hormone receptor (TR) β, growth hormone, malic enzyme and myosin heavy chain. Results of the gel mobility shift assay supported the idea that the sequence functions as a TRE. The supershift procedure demonstrated that the complex formed in the gel mobility shift assay contains TR. The present study revealed the uniqueness of bullfrog collagenase as a metamorphosis-associated enzyme, because, to our knowledge, known mammalian collagenase genes do not contain THE in their transcription regulatory regions.