title: Sp1/Sp3 and DNA-methylation contribute to basal transcriptional activation of human podoplanin in MG63 versus Saos-2 osteoblastic cells.
abstract: BACKGROUND
  Podoplanin is a membrane mucin that, among a series of tissues, is expressed on late osteoblasts and osteocytes. Since recent findings have focussed on podoplanin's potential role as a tumour progression factor, we aimed at identifying regulatory elements conferring PDPN promoter activity. Here, we characterized the molecular mechanism controlling basal PDPN transcription in human osteoblast-like MG63 versus Saos-2 cells.
  
  
  RESULTS
  We cloned and sequenced 2056 nucleotides from the 5'-flanking region of the PDPN gene and a computational search revealed that the TATA and CAAT box-lacking promoter possesses features of a growth-related gene, such as a GC-rich 5' region and the presence of multiple putative Sp1, AP-4 and NF-1 sites. Reporter gene assays demonstrated a functional promoter in MG63 cells exhibiting 30-fold more activity than in Saos-2 cells. In vitro DNase I footprinting revealed eight protected regions flanked by DNaseI hypersensitive sites within the region bp -728 to -39 present in MG63, but not in Saos-2 cells. Among these regions, mutation and supershift electrophoretic mobility shift assays (EMSA) identified four Sp1/Sp3 binding sites and two binding sites for yet unknown transcription factors. Deletion studies demonstrated the functional importance of two Sp1/Sp3 sites for PDPN promoter activity. Overexpression of Sp1 and Sp3 independently increased the stimulatory effect of the promoter and podoplanin mRNA levels in MG63 and Saos-2 cells. In SL2 cells, Sp3 functioned as a repressor, while Sp1 and Sp3 acted positively synergistic. Weak PDPN promoter activity of Saos-2 cells correlated with low Sp1/Sp3 nuclear levels, which was confirmed by Sp1/Sp3 chromatin immunoprecipitations in vivo. Moreover, methylation-sensitive Southern blot analyses and bisulfite sequencing detected strong methylation of CpG sites upstream of bp -464 in MG63 cells, but hypomethylation of these sites in Saos-2 cells. Concomitantly, treatment with the DNA methyltransferase inhibitor 5-azaCdR in combination with trichostatin A (TSA) downregulated podoplanin mRNA levels in MG63 cells, and region-specific in vitro methylation of the distal promoter suggested that DNA methylation rather enhanced than hindered PDPN transcription in both cell types.
  
  
  CONCLUSIONS
  These data establish that in human osteoblast-like MG63 cells, Sp1 and Sp3 stimulate basal PDPN transcription in a concerted, yet independent manner, whereas Saos-2 cells lack sufficient nuclear Sp protein amounts for transcriptional activation. Moreover, a highly methylated chromatin conformation of the distal promoter region confers cell-type specific podoplanin upregulation versus Saos-2 cells.
authors.affiliation: Institute of Clinical Pathology, Medical University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria. brigitte.hantusch@meduniwien.ac.at
authors.forename: Brigitte
authors.initials: B
authors.lastname: Hantusch
authors.forename: Romana
authors.initials: R
authors.lastname: Kalt
authors.forename: Sigurd
authors.initials: S
authors.lastname: Krieger
authors.forename: Christina
authors.initials: C
authors.lastname: Puri
authors.forename: Dontscho
authors.initials: D
authors.lastname: Kerjaschki
mesh_terms: D045744:Cell Line, Tumor; D019175:DNA Methylation; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000502:physiology*; D010006:Osteoblasts / Q000378:metabolism*; D012516:Osteosarcoma; D011401:Promoter Regions, Genetic / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000502:physiology*; D051705:Sp3 Transcription Factor / Q000502:physiology*; D015533:Transcriptional Activation; D000098853:Podoplanin
chemical_list: D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D016329:Sp1 Transcription Factor; D051705:Sp3 Transcription Factor; C117468:PDPN protein, human; D000098853:Podoplanin; C497121:SP3 protein, human