Kenta Fujimoto

Expression of matrix metalloproteinase genes in regressing or remodeling organs during amphibian metamorphosis

Several matrix metalloproteinases (MMP) are induced by thyroid hormone (TH) during the climax of amphibian metamorphosis and play a pivotal role in the remodeling of the intestine and the regressing tail and gills by degrading the extracellular matrix (ECM). We compared MMP gene expression levels precisely by quantitative real‐time reverse transcription‐polymerase chain reaction. The expression of MMP genes increases prominently at Nieuwkoop and Faber (NF) stages 60, 60–61 and 62 in the intestine, gills and tail, respectively, when the drastic morphological changes start in each organ. Gene expression analysis in the TH‐treated tadpoles and cell line revealed that MMP mRNAs are upregulated in response to TH quickly within several hours to low levels and then increase in a day to high levels. All TH‐induced MMP genes have TH response elements (TREs). The presence of high affinity TREs in MMP genes correlates with early TH‐induction. Based on these results, we propose that TH stimulates the transcription of MMP genes through TREs within several hours to low levels and then brings about the main increase of mRNAs by TH‐induced transcriptional factors, including TH receptor β, in a cell type‐specific transcriptional environment.

One of the duplicated matrix metalloproteinase-9 genes is expressed in regressing tail during anuran metamorphosis

The drastic morphological changes of the tadpole are induced during the climax of anuran metamorphosis, when the concentration of endogenous thyroid hormone is maximal. The tadpole tail, which is twice as long as the body, shortens rapidly and disappears completely in several days. We isolated a cDNA clone, designated as Xl MMP‐9TH, similar to the previously reported Xenopus laevis MMP‐9 gene, and showed that their Xenopus tropicalis counterparts are located tandemly about 9 kb apart from each other in the genome. The Xenopus MMP‐9TH gene was expressed in the regressing tail and gills and the remodeling intestine and central nervous system, and induced in thyroid hormone‐treated tail‐derived myoblastic cultured cells, while MMP‐9 mRNA was detected in embryos. Three thyroid hormone response elements in the distal promoter and the first intron were involved in the upregulation of the Xl MMP‐9TH gene by thyroid hormone in transient expression assays, and their relative positions are conserved between X. laevis and X. tropicalis promoters. These data strongly suggest that the MMP‐9 gene was duplicated, and differentiated into two genes, one of which was specialized in a common ancestor of X. laevis and X. tropicalis to be expressed in degenerating and remodeling organs as a response to thyroid hormone during metamorphosis.

Regulation of thyroid hormone sensitivity by differential expression of the thyroid hormone receptor during Xenopus metamorphosis.

During amphibian metamorphosis, a series of dynamic changes occur in a predetermined order. Hind limb morphogenesis begins in response to low levels of thyroid hormone (TH) in early prometamorphosis, but tail muscle cell death is delayed until climax, when TH levels are high. It takes about 20 days for tadpoles to grow from early prometamorphosis to climax. To study the molecular basis of the timing of tissue‐specific transformations, we introduced thyroid hormone receptor (TR) expression constructs into tail muscle cells of Xenopus tadpoles. The TR‐transfected tail muscle cells died upon exposure to a low level of thyroxine (T4). This cell death was suggested to be mediated by type 2 iodothyronine deiodinase (D2) that converts T4 to T3—the more active form of TH. D2 mRNA was induced in the TR‐overexpressing cells by low levels of TH. D2 promoter contains a TH‐response element (TRE) with a lower affinity for TR. These results show that the TR transfection confers the ability to respond to physiological concentrations of TH at early prometamorphosis to tail muscle cells through D2 activity and promotes TH signaling. We propose the positive feedback loop model to amplify the cells ability to respond to low levels of T4.

Expression profiles of the duplicated matrix metalloproteinase-9 genes suggest their different roles in apoptosis of larval intestinal epithelial cells during Xenopus laevis metamorphosis.

Matrix metalloproteinases (MMPs) play a pivotal role in development and/or pathogenesis through degrading extracellular matrix (ECM) components. We have previously shown that Xenopus MMP‐9 gene is duplicated. To assess possible roles of MMP‐9 and MMP‐9TH in X. laevis intestinal remodeling, we here analyzed their expression profiles by in situ hybridization and show that their expression is transiently up‐regulated during thyroid hormone‐dependent metamorphosis. Of interest, MMP‐9TH mRNA is strictly localized in the connective tissue and most highly expressed just beneath the larval epithelium that begins to undergo apoptosis. On the other hand, cells expressing MMP‐9 mRNA become first detectable in the connective tissue and then, after the start of epithelial apoptosis, also in the larval epithelium. These results strongly suggest that MMP‐9TH is responsible in the larval epithelial apoptosis through degrading ECM components in the basal lamina, whereas MMP‐9 is involved in the removal of dying epithelial cells during amphibian intestinal remodeling. Developmental Dynamics 236:2338–2345, 2007.

Promotion of cathepsin L activity in newt spermatogonial apoptosis induced by prolactin

We previously showed that prolactin (PRL) induces apoptosis in newt secondary spermatogonia and indicated that caspase activity is involved in the apoptosis. Since it was recently reported that Z‐VAD‐fmk, a pan‐caspase inhibitor, blocks activity of cysteine cathepsins as well, we examined whether cathepsin is involved in the newt spermatogonial apoptosis. We found cathepsin L activity in the testis that was elevated by PRL in organ culture of testis, while E‐64d, a lysosomal cysteine protease inhibitor, and Z‐VAD‐fmk suppressed it and chromosomal condensation. These results suggest that cathepsin L activity play a pivotal role in PRL‐induced spermatogonial apoptosis.

Low temperature promotes annexin V expression in newt testis

Abstract We examined the effect of low temperatures on annexin V expression in newt testis. When newts were transferred to a low temperature (12°C), up-regulation of annexin V protein was observed in secondary spermatogonia. In primary spermatocytes, high levels of annexin V expression were observed at both 12°C and 22°C, but at 12°C the protein was localized in part of the cytoplasm of primary spermatocytes. These results indicate that in newt testis annexin V is a cold-sensitive protein, suggesting the possibility that annexin V might have a cold stress-related function in newt germ cells.