Thyroid hormone (TH) receptor (TR) expression begins early in development in all vertebrates when circulating TH levels are absent or minimal, yet few developmental roles for unliganded TRs have been established. Unliganded TRs are expected to repress TH-response genes, increase tissue responsivity to TH, and regulate the timing of developmental events. Here we examined the role of unliganded TRα in gene repression and development in Xenopus tropicalis. We used transcription activator-like effector nuclease gene disruption technology to generate founder animals with mutations in the TRα gene and bred them to produce F1 offspring with a normal phenotype and a mutant phenotype, characterized by precocious hind limb development. Offspring with a normal phenotype had zero or one disrupted TRα alleles, and tadpoles with the mutant hind limb phenotype had two truncated TRα alleles with frame shift mutations between the two zinc fingers followed by 40–50 mutant amino acids and then an out-of-frame stop codon. We examined TH-response gene expression and early larval development with and without exogenous TH in F1 offspring. As hypothesized, mutant phenotype tadpoles had increased expression of TH-response genes in the absence of TH and impaired induction of these same genes after exogenous TH treatment, compared with normal phenotype animals. Also, mutant hind limb phenotype animals had reduced hind limb and gill responsivity to exogenous TH. Similar results in methimazole-treated tadpoles showed that increased TH-response gene expression and precocious development were not due to early production of TH. These results indicate that unliganded TRα delays developmental progression by repressing TH-response genes.
During development in all vertebrates, thyroid hormone receptors (TRs) are expressed before as well as during and after the peak in plasma thyroid hormone (TH) levels. Previously, we established a role for unliganded TRα in gene repression and developmental timing using tadpoles of TRα knockout (TRαKO) frogs. Here, we examined the role of liganded TRα on growth, development, and intestinal remodeling during natural and TH-induced metamorphosis. Disrupted TRα had little effect on growth during the larval period, but after metamorphosis, TRαKO juveniles grew more slowly than wild-type (WT) juveniles. TRαKO tadpoles developed faster throughout premetamorphosis when TH was low or absent, and despite their decreased responsivity to exogenous TH, TRαKO tadpoles not only were able to complete TH-dependent metamorphosis but also did so earlier than WT tadpoles. In contrast to external morphology, larval epithelial cell apoptosis and adult cell proliferation of intestinal remodeling were delayed in TRαKO tadpoles. Also, TRαKO intestines did not shrink in length to the full extent, and fewer intestinal folds into the lumen were present in TRαKO compared with WT juveniles. Such delayed remodeling occurred despite higher premetamorphic expression levels of TH target genes important for metamorphic progression-namely, TRβ, Klf9, and ST3. Furthermore, the decreased TH-dependent intestinal shrinkage was consistent with reduced TH response gene expression during natural and TH-induced metamorphosis. As in the TRα null mouse model, TRαKO frogs had statistically significant but surprisingly mild growth and development phenotypes with normal survival and fertility.
A wealth of data implicates that ErbB receptors have essential roles in tumor development. Probiotic bacteria are known to exert an anti-cancer activity in animal studies. Bacillus polyfermenticus (B.P.), a probiotic bacterium, has been clinically used for a variety of gastrointestinal disorders in East Asia. Here we investigated the effect of B.P. on the growth of tumors and its putative mechanism of actions. Conditioned medium of B.P. cultures (B.P. CM) inhibited the growth of human colon cancer cells including HT-29, DLD-1 and Caco-2 cells. Moreover, B.P. CM suppressed colony formation of HT-29 cells cultured on soft agar and reduced carcinogen-induced colony formation of normal colonocytes. Furthermore, data from the mouse xenograft model of human colon cancer cells showed reduced tumor size in B.P. CM-injected mice when compared to E.coli conditioned medium-injected mice. Exposure of B.P. CM to HT-29 cells for 24 h, 48 h and 2 weeks reduced ErbB2 and ErbB3 protein expression as well as mRNA levels. Moreover, cyclin D1 expression which is required for ErbB-dependent cell transformation was decreased by B.P. CM. Furthermore, transcription factor E2F-1 which regulates cyclin D1 expression was also decreased by B.P. CM. These results show that B.P. inhibits tumor growth and its anti-cancer activity occurs, at least in part, through suppressing ErbB2 and ErbB3. Taken together, our study suggests that this probiotic may be clinically used as a prophylactic treatment to prevent colon cancer development.
Spadefoot toad species display extreme variation in larval period duration, due in part to evolution of thyroid hormone (TH) physiology. Specifically, desert species with short larval periods have higher tail tissue content of TH and exhibit increased responsiveness to TH. To address the molecular basis of larval period differences, we examined TH receptor (TR) expression across species. Based on the dual function model for the role of TR in development, we hypothesized that desert spadefoot species with short larval periods would have 1) late onset of TR expression prior to the production of endogenous TH and 2) higher TR levels when endogenous TH becomes available. To test these hypotheses, we cloned fragments of TRα and TRβ genes from the desert spadefoot toads Scaphiopus couchii and Spea multiplicata and their non-desert relative Pelobates cultripes and measured their mRNA levels in tails using quantitative PCR in the absence (premetamorphosis) or presence (natural metamorphosis) of TH. All species express TRα and TRβ from the earliest stages measured (from just after hatching), but S. couchii, which has the shortest larval period, had more TRα throughout development compared to P. cultripes, which has the longest larval period. TRβ mRNA levels were similar across species. Exogenous T3 treatment induced faster TH-response gene expression kinetics in S. couchii compared to the other species, consistent with its increased TRα mRNA expression and indicative of a functional consequence of more TRα activity at the molecular level. To directly test whether higher TRα expression may contribute to shorter larval periods, we overexpressed TRα via plasmid injection into tail muscle cells of the model frog Xenopus laevis and found an increased rate of muscle cell death in response to TH. These results suggest that increased TRα expression evolved in S. couchii and contribute to its higher metamorphic rates.
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