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H Shibata, M Kanzaki, T Takeuchi, J-i Miyazaki, and I Kojima


Activin A stimulates insulin secretion in pancreatic β-cells by a calcium-dependent mechanism. The present study was conducted to further characterize the effects of activin A in two glucose-responsive insulinoma cell lines, MIN6 and HIT-T15 cells. In HIT-T15 cells, activin A evoked an increase in cytoplasmic free calcium concentration, stimulated insulin secretion, maintained glucose responsiveness of the cells and inhibited DNA synthesis. However, activin A did not have any effect in MIN6 cells. Measurement of 125I-labeled activin A binding in MIN6 cells revealed that the number of binding sites was markedly reduced, suggesting that the refractoriness was due, at least partly, to the reduced numbers of the activin receptor. Stable transfectants of MIN6 cells that overexpressed the type II activin receptor were then developed. The transfected cells (MIN6-ActR cells) expressed ten times more 125I-labeled activin A-binding sites than parental cells and the apparent K d was 1·15 nm, which was nearly identical to that in parental cells. Affinity cross-linking in MIN6-ActR cells showed that a 90 kDa type II receptor as well as a 52 kDa protein, presumably follistatin, was markedly labeled with 125I-labeled activin A. Although MIN6-ActR cells expressed significant numbers of activin receptors, activin A did not induce immediate calcium-dependent responses in these cells. In contrast, activin A was capable of inducing long-term effects in MIN6-ActR cells; thus, reduction of the glucose concentration in culture medium from 25 to 5·5 mm for 4 days resulted in a remarkable loss of insulin response to glucose stimulation but this decline in response to glucose was prevented by the addition of activin A during culture. In addition, activin A inhibited DNA synthesis in MIN6-ActR cells. Hence, although activin A did not induce calcium-dependent responses, it evoked some calcium-independent effects in MIN6-ActR cells. Taken together, activin A elicits various effects in β-cells by both calcium-dependent and -independent mechanisms.

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S Kobayashi, H Shibata, I Kurihara, K Yokota, N Suda, I Saito, and T Saruta

Chicken ovalbumin upstream promoter-transcription factors (COUP-TFs) are orphan receptors involved in regulation of neurogenesis and organogenesis. COUP-TF family members are generally considered to be transcriptional repressors and several mechanisms have been proposed to underlie this activity. To explore novel transcriptional coregulators for COUP-TFs, we used the COUP-TFI as bait in a yeast two-hybrid screen of an adrenocortical adenoma cDNA library. We have identified Ubc9, a class E2 conjugating enzyme of small ubiquitin-related modifier (SUMO)-1 as a COUP-TFI corepressor. Ubc9 interacts with COUP-TFI in yeast and in glutathione S-transferase pulldown and coimmunoprecipitation assays. Fluorescence imaging studies show that both Ubc9 and COUP-TFI are colocalized in the nuclei of transfected COS-1 cells. The C-terminal region of Ubc9 encoding amino acids 59-158 interacts with the C-terminus of COUP-TFI encoding amino acids 383-403, in which transcriptional repression domains are located. Mammalian one-hybrid assays utilizing a variety of Ubc9 fragments fused to Gal4 DNA-binding domain show that a Ubc9 fragment encoding amino acids 1-89 contains autonomous transferrable repression domain. Transfection of Ubc9 into COS-1 cells markedly enhances transcriptional repression by Gal4 DNA-binding domain-fused to COUP-TFI(155-423), but not by Gal4-COUP-TFI(155-388) which lacks a repressor domain. Coexpression of a C-terminal deletion mutant of Ubc9(1-58), which fails to interact with COUP-TFI, but retains a transcriptional repression domain, has no effect on Gal4-COUP-TFI-mediated repression activity. These findings indicate that interaction of Ubc9 with COUP-TFI is crucial for the corepressor function of Ubc9. Overexpression of Ubc9 similarly enhances COUP-TFI-dependent repression of the promoter activity of the bovine CYP17 gene encoding steroid 17alpha-hydroxylase. In addition, the C93S mutant of Ubc9, which abrogates SUMO-1 conjugation activity, continues to function as a COUP-TFI corepressor. Our studies indicate that Ubc9 functions as a novel COUP-TFI corepressor, the function of which is distinct from its SUMO-1 conjugating enzyme activity.