Messadi DV^1,2 , Le A¹, Berg S¹, Huang G¹, Zhuang W¹ and Bertolami CN³

¹ School of Dentistry, ² Dental Research Institute, University of California, Los Angeles, California, ³ School of Dentistry,University of California, San Francisco, California, USA

Received 1/8/98 Accepted 2/3/98

4. RESULTS

4.1. Effect of TGF- beta 1 on PDGF receptors subunits: RadioImmunoBinding assay

By RadioImmunoBinding assay all cell lines expressed both PDGF-alpha and beta receptors before TGF- beta 1 treatment but only at low levels. Scar cells showed higher PDGF-beta receptor levels than NSk. TGF- beta 1 treatment induced no significant increase in PDGF-beta receptors expression for any of the cell lines i.e: NSk, NSc and Keloid "figure 1". In case of PDGF-alpha receptor , TGF- beta 1 treatment upregulated the expression of the alpha receptor subunit on keloid fibroblasts only (p<0.01) with no effect on NSk (p> 0.05) and NSc fibroblasts (p>0.05), "figure 2". All testing of differences between groups of data utilized the Student t test; significance was established at a p value of < 0.05.

Figure 1. Effect of TGF- beta 1 on PDGF-beta receptor cell surface expression. Using a RadioImmunoBinding assay, normal skin (NSk), normal scar (NSc) and Keloid (kel) fibroblasts were examined. Half of the subconfluent cultures were preincubated with TGF- beta 1 (10ng/mL) for 24 hours. Monoclonal mouse anti-human PDGF-beta receptor antibody was added at a concentration range of (0-20 nM/mL). Data are expressed as average disintegrations per minute (dpm) of [¹²⁵I]-conjugated mouse secondary antibody per 10⁴ cells per well. Each data point represents the average of values for triplicate wells for each cell line.

Figure 2: Comparison of PDGF-alpha receptor cell surface expression after TGF- beta 1 treatment. In normal skin (NSk), normal scar (NSc) and Keloid (kel) fibroblasts. Half of the subconfluent cultures were preincubated with TGF- beta 1 (10ng/mL) for 24 hours. Monoclonal mouse anti-human PDGF-alpha receptor antibody was added at a concentration range of (0-20 nM/mL). Data are expressed as average disintegrations per minute (dpm) of [¹²⁵I]-conjugated mouse secondary antibody per 10⁴ cells per well. Each data point represents the average of values for triplicate wells for each cell line.

4.2 TGF- beta 1 Modulation of PDGF-alpha and beta receptor Protein Synthesis

Protein synthesis as determined by Western analysis is shown in "figure 3, and relative optical densities shown in table 1". The amount of PDGF-alpha receptor subunit in keloid fibroblasts was shown to be increased after TGF- beta 1 stimulation for 24 hours, whereas no changes was observed with NSk or NSc fibroblasts "figure 3A, table 1". In the case of PDGF-beta, there was no significant increase in PDGF-beta receptor expression for any of the cell line tested whether with or without TGF- beta 1 stimulation "figure 3B, table 1".

Figure 3: Effects of TGF- beta 1 on PDGF alpha and beta receptor protein levels. Immunoprecipitates prepared from equivalent numbers of cells were analyzed by Western blot. Half of the cells were pre-incubated with (+) or without (-) TGF- beta 1 (10 ng/mL) for 24 hours. Lanes 1 keloid I (+), lanes 2 keloid I (-), lanes 3 NSc (+), lanes 4 NSc(-), lanes 5 keloid II (+), lane 6 keloid II (-), lanes 7 NSk (+) and lanes 8 NSk (-). (A:top) Analyzed with anti-PDGF-alpha receptor antibody. (B:bottom) Analyzed with anti-PDGF-beta receptor antibody.

4.3 TGF- beta 1 Effect on PDGF-alpha and beta receptor mRNA Expression

Northern analysis showed that 24-hr treatment with 10 ng/mL of TGF- beta 1 upregulated the mRNA expression of the 6.5- kb PDGF-alpha receptor transcript for keloid scar tissues. There was also a slight upregulation for NSc, but there was no effect on NSk "figure 4A and relative optical densities shown in table 2". In contrast to the results of the alpha receptor; the PDGF-beta-receptor transcript (5.6 kb) mRNA levels were not altered after TGF- beta 1 treatment for any of the cell lines tested "figure 4B, table 2". Levels of PDGF- alpha and PDGF-beta receptor mRNA are presented relative to beta-Actin mRNA levels.

Figure 4: Northern Blot analysis. Total cellular RNAs were isolated from the different cell lines after stimulation with (+) or without (-) TGF- beta 1 (10 ng/mL) for 24 hours. Lanes 1 keloid I (-), lanes 2 keloid I (+), lanes 3 NSc (-), lanes 4 NSc (+), lanes 5 NSk (-), lanes 6 NSk (+), lanes 7 keloid II (-), lanes 8 keloid II (+). Equal amounts of RNAs were loaded in each lane, electrophoresed, transferred to nitrocellulose membrane and hybridized with (A:top) 32P-labeled PDGF-alpha receptor cDNA probe. (B; bottom) 32P-labeled PDGF-beta receptor cDNA probe. beta -Actin was used as a control probe.