Transcriptional Regulation of the Platelet-Derived Growth Factor beta-receptor

Sammanfattning: The Platelet Derived-Growth Factor (PDGF) receptors and ligands are expressed in cells of predominantly mesenchymal origin. Binding of the ligands to the receptors stimulates cell proliferation and other cellular responses such as differentiation and cell movement. Aberrant activity of PDGF has been associated with cell proliferative diseases, for example, several types of cancer, atherosclerosis and rheumatoid arthritis. Transcription the PDGF beta-receptor (PDGFRB) gene is controlled by a promoter containing a CCAAT motif, binding the transcription factor NF-Y, which has been shown to be crucial for initiation of transcription. PDGFRB expression is cell cycle regulated and peaks in early G1. The proto-oncogene product c-Myc is induced through PDGFR and establishes a negative feedback loop via binding of NF-Y, and thereby down-regulating PDGFRB. The aim of this thesis is to further investigate the mechanisms behind the cell-cycle-dependent regulation of the PDGFRB promoter.Paper I: The ubiquitous transcription factor Sp1 up-regulated the PDGFRB promoter activity and two oligonucleotides containing sequences from an area just upstream of the CCAAT motif bound Sp1 in vitro. Sp1 increased the promoter activity independently of an intact CCAAT motif. On the other hand, removal of the Sp1 binding sites reduced NF-Y induced activation, indicating an interaction between the two transcription factors.Paper II and III: The p53 tumour suppressor family member p73alfa down-regulated PDGFRB expression whereas an oncogenic form of p73alfa, DeltaNp73alfa, which lacks the amino terminal transactivation domain, increased the promoter activity. P73alfa bound the same region of NF-Y as c-Myc, however, assays performed in c-Myc-/- fibroblasts showed that p73alfa repressed the PDGFRB promoter independently of c-Myc. The transcriptional co-activators p300 and PCAF, which harbours intrinsic histone acetyl transferase activity, up-regulated the PDGFRB promoter. Immuno-precipitation studies showed that p73alfa, but not DeltaNp73, binds histone de-acetylase HDAC1 that also affected the promoter activity negatively when over-expressed. CHIP assays further showed that DeltaNp73 binds the promoter when PDGFRB expression is high (early G1) and that binding of HDAC1 coincided with weak DeltaNp73 binding and low PDGFRB expression. Paper IV: The transforming viral protein SV40 Large T (LT) antigen bound the promoter and decreased PDGFRB transcription. Mutants of LT unable to bind the tumour suppressors p53 and retinoblastoma protein (pRb), respectively, did not repress the promoter. LT was also unable to repress the PDGFRB promoter in c-Myc-, p53- and pRb-deficient cells, respectively. In conclusion, LT mediated repression of PDGFRB requires p53, pRb and c-Myc. Furthermore, p53 was shown to interact with the PDGFRB promoter and stimulate transcription in a way that requires the Sp1 binding sites.