The nucleoside AICAriboside (5-amino-4-imidazolecarboxamide riboside) has been shown to inhibit glycolysis in isolated rat hepatocytes [Vincent, Bontemps and Van den Berghe (1992) Biochem. J. 281, 267-272]. The effect is mediated by AICA-ribotide (ZMP), the product of the phosphorylation of AICA-riboside by adenosine kinase. To assess the cell-type specificity of the effect, studies were conducted in rabbit cardiomyocytes, human erythrocytes and rat hepatoma FTO-2B cells. AICA-riboside had no effect on glycolysis in cardiomyocytes, and a slight stimulatory effect in erythrocytes, but inhibited glycolysis by 65% at 250 microM concentration in FTO-2B cells, although only when tissue-culture medium was replaced by Krebs-Ringer bicarbonate buffer. At 500 microM AICAriboside, ZMP remained undetectable in cardiomyocytes, but reached 0.65 mM in erythrocytes and 5 mM in FTO-2B cells. In the latter, AICAriboside provoked up to 2-fold elevations of glucose 6-phosphate and fructose 6-phosphate, accompanied by a decrease in fructose 1,6-bisphosphate. This indicated inhibition of 6-phosphofructo-1-kinase (PFK-1). Accordingly, in FTO-2B cell-free extracts, the activity of PFK-1, measured under physiological conditions, was inhibited by approx. 70% by 5 mM ZMP. ZMP had a less pronounced effect on the activity of PFK-1 in normal rat liver; it did not influence the activity of PFK-1 in rat muscle, rabbit heart and human erythrocytes. It is concluded that the inhibitory effect of AICAriboside on glycolysis is dependent on both (1) the capacity of the cells to accumulate ZMP and (2) the presence of target enzymes which are sensitive to ZMP.
Wehav~?stt&d the bindingofpurifkd~-1 on thebotine thyroglobulin gencpromotcr. DNasc I foolprintingcxperiments revealed three binding siw which auresponded in location to the A. B and C sites found in the rat thyroglobulin promoter. hlutants in the A and C regions showing r&u& binding of IXF-1, also exhibited largely decrtzzd promoter activity in transient exprcssiop experiments in primary-cultured dog thyrocyts TWY mutants in the B site that exhibited a reduced capaaty to hind ITF-I also displayed a drastically affcctcd transcriptional activity in tmnsient assays As in the rat sites A and C only are critical for promoter activity. these results suggest :hat fuli occupa~lcy of the T. site is required for thyroglobulin ptcrmntn activity in the cow only.
INTR0DUCTiONThe expression of t floglobulin (Tg) gene is restricted to the follicular cells of the thyroid gland. The expression of the gene is under the positive control of thyrotropin which acts mainly via the intracellular messenger cAMP [I]. This control occurs. at least partly, at the transcriptional level [2,3]. It has been shown that the specific expression of a reporter-gene, the chloramphenicol acetyltran&rase (CAT) gcnc. rcquircd only the first 250 bp of the bovine Tg promoter when transfccted in thyroid cells in primary culture [4]. Likewise. 170 bp of the rat Tg promoter wcrc suficicnt lo direct the cell-type-specific expression of the same reporter gent when transfmed in FRTL-5 cells [SJ The contra! of gene expression depends largely on the binding of transcription factors on promoter sequences. A DNA sequence repeated three times in the rat T'g promoter has been sho&m to interact in footprinting experiments with a thyroid-specific nuclear factor called TTF-1 (s]. The lTF-1 protein hzs bccu purified and Cbdracterized by cDNA cloning i7]_In this study. we compare the binding of TTF-1 on both rat and bovine promoters and its functional role in ?hc control of the cxpmsion of ihe corresponding gcna. Although ITF-l WCO@ZCS ilrra s~qu~na in bolh promotcrz in DIQ.sc I footprinting assays, o&y two caqmccs appear functionally rclcvaot in the rat promoter while occupancy of the three sites seems n_ scntial to sustain normal activity of the bovine promoter.
MATERIALS AND METWDS
We have investigated the interaction of a nuclear factor(s) with the promoter region of the thyroglobulin (Tg) gene, which is only expressed in differentiated thyroid cells under the positive control of the pituitary hormone thyrotropin (TSH) via a cAMP-dependent pathway. Using the mobility shift assay, we first demonstrated that a thyroid nuclear factor interacts with a short segment of 60 bp (-136 - -77) which is conserved among species in the regulatory region of the Tg gene. A specific binding site was then localized in a subfragment of 20 bp located between -126 bp and -107 bp relative to the transcription initiation site. The corresponding nuclear factor is absent in a tissue which does not express the Tg gene. This factor differs from previously identified factors shown to mediate a direct cAMP response since the observed binding is neither competed out by the cAMP responsive element (CRE) nor by the activator protein 2 (AP2) binding site. This trans-acting factor represents a new candidate intermediate in the regulation of transcription by a cAMP dependent mechanism.
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