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PDGF Up-regulates CSF-1 Gene Transcription in Ameloblast-like Cells

¹ Departments of Pathology and
² Medicine, and
³ Dental School, University of Texas Health Science Center, 7703 Floyd Curl Drive, and South Texas Veterans Health Care System, Audie Murphy Division, San Antonio, TX 78229, USA; and
⁴ School of Dentistry, University of Alabama at Birmingham, Birmingham, AL 35294, USA

View larger version (29K): [in this window] [in a new window]   Figure 1. Analysis of PDGF in MEOE-3M cells. (A) Expression profile of MEOE-3M cells. Total RNA was isolated from cells in complete medium, and the mRNA expression profile of indicated genes was determined by RT-PCR. PCR products were analyzed in 1% agarose gels stained with ethidium bromide. Normal mouse incisor mRNA was used as a positive control. (B) Dose-response graph for the effect of PDGF-BB or PDGF-AA on MEOE-3M DNA synthesis. Cells in serum-free DMEM were incubated with increasing concentrations of PDGF isoforms and pulsed with [3H]-TdR for 4 hrs prior to being harvested at 24 hrs. Control wells were incubated with SF medium, 20 ng/mL albumin, or 10% FCS alone. Data represent the mean ± SE of 2 separate experiments, each performed in triplicate wells. ***p < 0.001; *p < 0.05 vs. SF medium.

View larger version (47K): [in this window] [in a new window]   Figure 2. Effects of PDGF isoforms on CSF-1 mRNA (A and B, upper panel) in MEOE-3M. Cells were placed in serum-free medium overnight and treated with 20 ng/mL of PDGF-BB, PDGF-AA, or albumin. At indicated time-points, cells were harvested for total RNA. Northern blots (20 µg/lane) were hybridized with 32P-labeled CSF-1 probe; blots were stripped and re-hybridized with a 36B4 probe. Densitometry analysis of Northern blots (A and B, lower panel). Data are expressed as fold change of the ratio of the CSF-1 band to the corresponding 36B4 band in treated cells, compared with control untreated cells, at time 0 or 6 hrs. (C) CSF-1 protein quantification by ELISA. Supernatants were collected for analysis of CSF-1 protein levels from experiments in panel A and at extended time-points. Data represent the mean ± SE of 2 separate experiments. ***p < 0.001; **p < 0.01; *p < 0.05 vs. control.

View larger version (34K): [in this window] [in a new window]   Figure 3. Identification of PDGF-BB response region in CSF-1 promoter by deletion analysis. (A) Schematic showing cutback segments of CSF-1 promoter used in panel B. (B) MEOE-3M cells, stably transfected with the indicated deletion constructs, were seeded into 24-well dishes at a density of 1 x 105/well. After 2 days, cells were placed in serum-free DMEM overnight, then incubated with or without PDGF-BB (20 ng/mL) for 12 hrs; cell lysates were assayed for luciferase activity. The bar graph shows the fold increase in promoter activity stimulated by PDGF relative to promoter activity without PDGF. PDGF-BB did not have a significant effect in cells transfected with the empty vector. Data represent the mean ± SE of 3 separate experiments, each performed in triplicate wells. ***p < 0.001 vs. –0.455 kb; (a) p > 0.05 vs. –0.455 kb.

View larger version (93K): [in this window] [in a new window]   Figure 4. Characterization of nuclear protein-DNA complex by EMSA. A double-stranded oligonucleotide representing –982 bp to –1021 bp and containing the AGGAAA sequence was end-labeled with [gamma32P]ATP and incubated without nuclear extract (lane 1) or with nuclear extracts isolated from untreated (lane 2) or PDGF-BB-treated (lanes 3 to 8) cells. For competition analysis, nuclear extracts were incubated with cold specific (SP, lane 4) or non-specific (NSP, lane 5) oligonucleotide (100-fold in excess of the labeled probe) prior to incubation with the radiolabeled probe. Nuclear extracts were incubated with specific antibodies against Pea3 (lane 6), PU.1 (lane 7), or Elf-1 (lane 8) before being incubated with radiolabeled probe. (A, B) Arrows indicate the 2 DNA-protein complexes.

Journal of Dental Research, Vol. 87, No. 1, 33-38 (2008)
DOI: 10.1177/154405910808700105


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