Nuclear factor Y controls the basal transcription activity of the mouse platelet-derived-growth-factor beta-receptor gene.

To determine the regulatory mechanism of the expression of the mouse platelet-derived growth factor (PDGF) beta-receptor gene, a 1.9-kb 5' flanking genomic fragment was cloned and analyzed. Site-directed mutagenesis of a CCAAT motif, located 60 bp upstream of the transcriptional-start site, completely abolished the promoter activity [Ballagi, A. E., Ishisaki, A., Nelin, J.-O. & Funa, K. (1995) Biochem. Biophys. Res. Commun. 210, 165-1751. The sequence around the intact CCAAT motif was protected by in vitro DNase-I-footprinting analysis. Electrophoresis-mobility-shift assays with anti-[nuclear factor Y(NF-Y)]Ig revealed binding of the NF-Y complex to the CCAAT box. Furthermore, the double-stranded oligonucleotides corresponding to the sequence around the CCAAT motif were conjugated with DNA-affinity magnetic beads. The binding proteins were affinity purified and identified as the NF-Y transcription factor by western blotting. Our results indicate that NF-Y controls the basal transcription activity of the mouse PDGF beta-receptor gene.

Isolation and characterization of the mouse PDGF beta-receptor promoter.

The PDGF beta-receptor expression is tightly regulated during embryonic development and in several physiological and pathological situations. To determine the regulatory mechanism of the receptor, a 1.9 kb 5' flanking genomic fragment of the mouse PDGF beta-receptor gene was cloned and analyzed by functional promoter assays. The fragment was shown to exert promoter activity in the luciferase expression vector system in mouse NIH 3T3 fibroblast and NB41 neuroblastoma cell lines as well as rat ST15A cerebellar cell lines. Functional studies on deletion mutants revealed several putative regulatory sequences. The deletion mutants acted similarly in NB41 cells and in ST15A cells, both of neuronal origin, but differently in the NIH 3T3 fibroblasts. No TATA box was found in the analyzed promoter region, however, site directed mutagenesis of a CCAAT motif, located 60 basepair upstream of the transcriptional start site, almost completely abolished the promoter activity in all cell types.

Platelet-derived growth factor receptor expression after neural grafting in a rat model of Parkinson's disease.

Platelet-derived growth factor (PDGF) has trophic effect on dopaminergic neurons in vitro. We have previously shown dynamic changes in the expression of PDGF in embryonic mesencephalic grafts and surrounding host striatal tissue following intracerebral transplantation in a rat model of Parkinson's disease. In this study the expression of the PDGF receptors was examined in the same model using immunohistochemistry. Most ventral mesencephalic (VM) cells from E13-E15 rat embryos possessed both PDGF alpha- and beta-receptors before implantation. Double immunofluorescence staining revealed that about 10% of the cells also expressed tyrosine hydroxylase (TH). The PDGF alpha-receptor was detectable in the graft up to 1 wk after transplantation but had disappeared at 3 wk. In the host tissue, scattered glial cells were positive for the alpha-receptor but the expression was unchanged following transplantation. The beta-receptor expression almost completely disappeared from the grafted tissue by 4 h following transplantation, and only a few cells of the host striatum showed immunoreactivity. However, after 3 wk beta-receptor positive cells were again detectable in the graft. These cells appeared to be endothelial cells as identified by an antibody against von Willebrand's factor. Our data suggest that PDGF might act locally on embryonic dopaminergic cells in an autocrine or juxtacrine manner before and shortly after transplantation, and on surrounding glial cells in a paracrine manner after transplantation. Furthermore, PDGF-BB might influence neovascularization in the graft.

Regulation of GAD expression in islets of Langerhans occurs both at the mRNA and protein level.

The expression of the autoantigen glutamate decarboxylase in islets of Langerhans was investigated under different culture conditions, which affect the functional activity of the beta-cell. Using immunoprecipitations and analyses of enzyme activity, an increase in glutamate decarboxylase was detected in rat islets cultured at a glucose concentration of 11 mmol/l compared with those cultured at 5.6 mmol/l glucose. To determine whether the change was induced at the level of mRNA expression, total RNA was extracted from rat islets cultured at 5.6 or 11 mmol/l glucose, reverse transcribed and amplified by the polymerase chain reaction. Comparative quantitation in a phosphor imager revealed a significantly higher (82%, P < 0.005) content of glutamate decarboxylase mRNA in islets cultured at 11 mmol/l glucose. In parallel, human recombinant interleukin-1 beta, and diazoxide were tested for their effects on the expression of glutamate decarboxylase. Islets cultured at 11 mmol/l glucose in the presence of 40 U/ml of interleukin-1 beta, showed a 63% decrease (P < 0.005) in enzyme activity compared with those cultured at 11 mmol/l glucose alone, and similar decreases were noted on analysis of glutamate decarboxylase biosynthesis and mRNA. Islets cultured at 11 mmol/l glucose in the presence of 22.5 mg/ml diazoxide exhibited a significant reduction in enzyme activity (59%; P < 0.001) compared with those cultured at 11 mmol/l glucose only. This reduction, however, was not accompanied by a decrease in the content of glutamate decarboxylase mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

PDGF-BB exerts trophic activity on cultured GABA interneurons from the newborn rat cerebellum.

Platelet-derived growth factor (PDGF) is a well known mitogen for mesenchyme-derived cells and glial cells. Its presence in neuronal cells of the central nervous system has only recently been described. We have shown earlier that neurons of newborn rat brains in culture express PDGF beta-receptors and that PDGF-BB, a homodimer of PDGF B-chain, increases survival and promotes neurite outgrowth of newborn cerebellar cells (Smits et al., Proc. Natl Acad. Sci. USA, 88, 8159-8163, 1991). In this study, the effects of PDGF on early postnatal rat cerebellar cells were further explored. By using chemically defined serum-free medium, we have established primary cell cultures of rat cerebella (postnatal day 4-5) containing 70-80% neuronal cells. During the first 10 days in vitro, no difference in total cell number was found between PDGF-BB-treated and untreated cultures. After this time period, however, increased survival of the PDGF-BB-treated cells was found. Within the first 10 days in vitro, the addition of PDGF-BB to the cultures resulted in a relative increase in survival of interneurons expressing glutamic acid decarboxylase (GAD), the GABA biosynthetic enzyme. Moreover, addition of PDGF-BB in the untreated cell culture resulted in a rapid increase of GAD mRNA. These results show that PDGF-BB acts as a trophic factor on GABAergic interneurons of the cerebellum by up-regulating GAD synthesis and prolonging the survival of these cells. Furthermore, in situ hybridization revealed that there are scattered cells present in the early postnatal cerebellum that express PDGF beta-receptor mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)