The growth factors PDGF-AA and PDGF-BB have previously been shown to be potent mitogens for human periodontal ligament (hPDL) cells in vitro. Additionally, the mitogenic response to PDGF-AA has been shown to be specifically inhibited by TGF-beta. The purpose of the present investigation was to examine the binding of PDGF-AA and PDGF-BB, and the modulation of PDGF binding by TGF-beta, in hPDL cells. Scatchard analysis identified an average of 32,000 PDGF-AA high-affinity binding sites per cell with a dissociation constant (Kd) of 0.66 nM and an average of 36,000 PDGF-BB binding sites per cell with a dissociation constant (kd) of 0.44 nM. After treatment with TGF-beta, the receptor number for PDGF-AA was found to specifically decrease by approximately 50%, with no change in binding affinity. This reduced number of binding sites was shown to correlate with both a decrease in levels of receptor tyrosine phosphorylation and a decreased number of alpha receptor subunits. Northern blot analysis identified the TGF-beta-mediated decrease in PDGF alpha receptor subunit mRNA levels. PDGF-BB showed little change in the number of binding sites or in the binding affinity with TGF-beta treatment, and the data were consistent with an increase in the number of beta receptor subunits. These results demonstrate nearly equivalent numbers of receptors for both PDGF-AA and PDGF-BB in hPDL cells. Also, modulation of PDGF binding, by TGF-beta, was shown to result in a reduced number of alpha receptor subunits with an increase in the number of beta receptor subunits.

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Platelet derived growth factor (PDGF) is thought to play a significant role in bone repair and regeneration. We previously demonstrated that PDGF-AA binding can be modulated by interleukin-1 (IL-1). We now report that TNF-alpha significantly reduces PDGF-AA binding by decreasing the number of PDGF-alpha receptor subunits on the surface of normal human osteoblastic cells. This inhibition is likely due to a decrease in synthesis of PDGF-alpha receptors since TNF-alpha causes a relatively rapid decrease in PDGF-alpha receptor mRNA levels as determined by Northern blot analysis. The physiologic importance of this inhibition is demonstrated by a TNF-alpha induced decrease in PDGF-AA stimulated tyrosine kinase activity. When saturating concentrations of TNF-alpha were used, the addition of IL-1 further inhibited PDGF-AA binding and further decreased surface expression of PDGF-alpha receptors. In contrast, other mediators such as IL-6, PTH, 1,25(OH)2 vit D3, hydrocortisone, PGE2, bFGF, and IGF-1 had no effect. These results suggest that binding to the PDGF-alpha receptor is decreased by the strong pro-inflammatory cytokines such as IL-1 beta and TNF-alpha rather than as a general response to mediators important in bone resorption or bone formation. TNF-alpha and IL-1 are often co-expressed during destructive inflammatory processes. Thus, TNF-alpha and IL-1 may work in concert to limit the response of osteoblastic cells to PDGF-AA during periods of osseous inflammation.

Monocyte chemoattractant protein-1 (MCP-1) is induced in chronic osseous inflammation, and is temporally and spatially correlated with monocyte recruitment. We investigated the mechanism of MCP-1 regulation in a human osteoblastic cell line in response to IFN-γ, a potent mediator of the immune inflammatory response. Nuclear run-on and stability studies demonstrated that IFN-γ stimulated MCP-1 transcription and did not enhance mRNA stabilization. Using MCP-1 promoter/reporter gene constructs, we determined that IFN-γ-enhanced MCP-1 transcription is regulated by a 29-bp element located at −227 relative to the ATG start codon. This element contains a 13-bp CT-rich sequence (GCTTCCCTTTCCT) adjacent to a IFN-γ activation site (GAS). Since deletion of the CT sequence enhanced both the magnitude and duration of IFN-γ-stimulated, GAS-mediated transcription, we have termed it the IFN response-inhibitory sequence (IRIS). The combined IRIS/GAS sequence is highly conserved in mouse, rat, and bovine MCP-1 genes. In gel-shift assays, nuclear extracts from IFN-γ-stimulated osteoblastic cells formed two specific inducible bands with labeled IRIS/GAS DNA. Both bands were supershifted by anti-STAT1 Abs, but not by Abs to STAT2, p48(ISGF-3γ), IFN-regulatory factor-1, or IFN-regulatory factor-2. Formation of one of the bands required the presence of the IRIS moiety. IRIS/GAS DNA also formed a number of specific complexes with constitutively expressed factors, none of which were affected by the above Abs. These studies establish a mechanism for IFN-γ-stimulated MCP-1 expression and identify a complex element that regulates MCP-1 gene transcription.