The activity of ornithine decarboxylase (OD-Case; L-ornithine carboxy-lyase, EC 4.1.1.17) in rabbit costal chondrocytes in culture increased markedly after addition of parathyroid hormone (PTH), ...reaching a maximum 4 to 5 hr after PTH addition. The increase in ODCase activity was followed by increase in the intracellular concentrations of polyamines, especially putrescine, which increased in 6 hr to about 3-fold that of untreated cultures. The induction of ODCase by PTH was not observed in L, 3T3, HeLa, buffalo rat liver, or BHK cells. Retinyl acetate and retinoic acid both inhibited expression of the differentiated phenotype of chondrocytes by rabbit costal chondrocytes in culture within 3 days after their addition, as judged by morphological change and decrease in sulfate incorporation into glycosaminoglycans but did not inhibit cell proliferation. PTH could not induce an increase in ODCase in de-differentiated cells that had been pretreated with retinyl acetate or retinoic acid for 3 days; but 4 days after removal of the retinoids, these de-differentiated cells regained the ability to synthesize ODCase in response to PTH. These facts suggest that the induction of ODCase and the formation of putrescine by PTH are good markers of the differentiated phenotype of cultured chondrocytes.
Neuroactive steroids are known to modulate excitability in neurons. Neuronal activity during early development is critical to normal development of the brain. A neuroactive steroid, pregnenolone, was ...administered (10 microg/g) to rats from postnatal day 3 (PD 3) through PD 7. Dopamine (DA), serotonin (5-HT) and their metabolites were measured in the striatum. The results showed that neonatal treatment with pregnenolone increases DA and 5-HT turnover in the striatum at 3 weeks of age. The increased 5-HT turnover in the pregnenolone-treated animals was normalized at 14 weeks of age whereas the DA turnover in the pregnenolone-treated group was lower than in the control group. The present study indicated that pregnenolone treatment during the neonatal period induced abnormal development of the striatal dopaminergic function in adulthood.
To analyze the regulatory mechanism of connective tissue growth factor expression, the 3′-untranslated region (3′-UTR) of CTGF cDNA was amplified from HeLa cell RNA. Direct nucleotide sequencing ...revealed a single major population in the amplicon, which was nearly identical to other sequences. Subsequently, the effect of the 3′-UTR on gene expression was evaluated. When it was fused downstream of a firefly luciferase gene, the 3′-UTR strongly repressed luciferase gene expression. Interestingly, the repressive effect of the antisense 3′-UTR appeared to be more prominent than that of the sense one. Together with the fact that several consensus sequences for regulatory elements are found in it, these results suggest the involvement of multiple sets of regulatory elements in the CTGF 3′-UTR.
To clarify the chondrocyte-specific regulatory mechanism of connective tissue growth factor (
ctgf) gene expression, we analyzed the functionality and DNA–protein interaction of the CTGF promoter. ...Comparative luciferase assay of the CTGF promoter deletion mutants among HCS-2/8 chondrocytic cells and fibroblastic cells revealed that a 110-bp region in the promoter was crucial for the HCS-2/8-specific transcriptional enhancement. Subsequent competitive gel shift assay revealed that transcription factors in HCS-2/8 nuclei bound to a 60-bp portion in the corresponding region. Relative luciferase activity from a CTGF promoter with mutant TGF-β response element (TbRE) was 16.9% lower than that from an intact promoter. On the other hand, relative luciferase activity from a CTGF promoter with 4
bp point mutations at 30
bp upstream of the TbRE was 47.7% lower than that from the intact one. The binding activity of HCS-2/8 nuclear factor(s) to the sequence over the 4-bp was remarkably higher than that of any nuclear extract from other types of cells. Therefore, we entitled the sequence `TRENDIC', a transcription enhancer dominant in chondrocytes, which stands for a novel enhancer for chondrocyte-specific CTGF gene expression.
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