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S Vianello, L Brazzoduro, L Dalla Valle, P Belvedere, and L Colombo

Myostatin, a member of the transforming growth factor-beta superfamily, is a negative regulator of skeletal muscle mass in mammals. We have studied myostatin expression during embryonic and post-hatching development in zebrafish by semiquantitative RT-PCR. The transcript is present in just-fertilized eggs and declines at 8 h post-fertilization (hpf), suggesting a maternal origin. A secondary rise occurs at 16 hpf, indicating the onset of embryonic transcription at the time of muscle cell differentiation. The level of myostatin mRNA decreases slightly at 24 hpf, when somitogenesis is almost concluded, and rises again at and after hatching, during the period of limited muscle hyperplastic growth that is typical of slow-growing, small fish. In the adult muscle, we found the highest expression of myostatin mRNA and protein, which were detectable by Northern and Western blot analyses respectively. Although only the precursor protein form was revealed in the adult lateral muscle, we demonstrated that zebrafish myostatin is proteolytically processed and secreted in cultured cells, as is its mammalian counterpart. These results suggest that myostatin may play an important regulatory role during myogenesis and muscle growth in fish, as it does in mammals. In chronically stressed fish, grown from 16 days post-fertilization to adulthood in an overcrowded environment, we observed both depression of body growth and a diminished level of myostatin mRNA in the adult muscle, as compared with controls. We propose that chronic stunting in fish brings about a general depression of muscle protein synthesis which does not spare myostatin.

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L Dalla Valle, V Toffolo, A Nardi, C Fiore, P Bernante, R Di Liddo, PP Parnigotto, and L Colombo

Expression analysis by reverse transcriptase (RT)-PCR indicates that human adipose tissue is not likely to perform de novo synthesis of steroid hormones from cholesterol because the mRNAs of cytochromes P450scc and P450c17, and of the steroidogenic-related proteins, steroidogenic acute regulatory protein and steroidogenic factor 1, were not detected. Instead, our data support an intracrine role of adipose tissue, in which adrenal dehydroepiandrosterone sulfate (DHEA-S), the most abundant circulating androgen in man, is selectively uptaken, desulfated, and converted into bioactive androgens and estrogens. Three organic anion-transporting polypeptides-B, -D, and -E, presumably involved in DHEA-S transmembrane transport, were demonstrated at the mRNA level. While sulfotransferase expression was not found, the occurrence of steroid sulfatase (STS), converting DHEA-S to DHEA, was established at the mRNA, protein and catalytic activity levels. The 5′-rapid amplification of cDNA ends analysis showed that STS transcription in adipose tissue is regulated by the use of two promoters which differ from the prevalent placental one. The adipose transcripts contain a distinct untranslated first exon, 0a or 0b, followed by a common partially translated exon 1b, and nine other exons that are also shared by the main placental transcript. The presence of an upstream open reading frame in the new transcript variants could lead to an N-terminal divergence restricted to the cleavable signal peptide and thus not interfering with the catalytic activity of the mature STS protein. The adipose transcripts are also present in the placenta as minor isoforms. Western blotting revealed the characteristic ~64 kDa band of STS in both the placenta and adipose tissue. The specific enzymatic activity of STS in adipocytes was 118 pmol/106 cells per hour, about 50–100 times lower than in the placenta. A similar rate of [3H] DHEA-S uptake plus desulfation was measured in preadipo-cytes and adipocytes, equivalent to 40–45 pmol/106 cells per hour. Thus, an excessive accumulation of fat may out-compete other peripheral organs that are also dependent on intracrine DHEA-S utilization, especially when the adrenal production is low or declining with aging.

Free access

H A Sterle, E Valli, F Cayrol, M A Paulazo, D J Martinel Lamas, M C Diaz Flaqué, A J Klecha, L Colombo, V A Medina, G A Cremaschi, and M L Barreiro Arcos

We have shown in vitro that thyroid hormones (THs) regulate the balance between proliferation and apoptosis of T lymphoma cells. The effects of THs on tumor development have been studied, but the results are still controversial. Herein, we show the modulatory action of thyroid status on the in vivo growth of T lymphoma cells. For this purpose, euthyroid, hypothyroid, and hyperthyroid mice received inoculations of EL4 cells to allow the development of solid tumors. Tumors in the hyperthyroid animals exhibited a higher growth rate, as evidenced by the early appearance of palpable solid tumors and the increased tumor volume. These results are consistent with the rate of cell division determined by staining tumor cells with carboxyfluorescein succinimidyl ester. Additionally, hyperthyroid mice exhibited reduced survival. Hypothyroid mice did not differ significantly from the euthyroid controls with respect to these parameters. Additionally, only tumors from hyperthyroid animals had increased expression levels of proliferating cell nuclear antigen and active caspase 3. Differential expression of cell cycle regulatory proteins was also observed. The levels of cyclins D1 and D3 were augmented in the tumors of the hyperthyroid animals, whereas the cell cycle inhibitors p16/INK4A (CDKN2A) and p27/Kip1 (CDKN1B) and the tumor suppressor p53 (TRP53) were increased in hypothyroid mice. Intratumoral and peritumoral vasculogenesis was increased only in hyperthyroid mice. Therefore, we propose that the thyroid status modulates the in vivo growth of EL4 T lymphoma through the regulation of cyclin, cyclin-dependent kinase inhibitor, and tumor suppressor gene expression, as well as the stimulation of angiogenesis.