Related Experiment Videos
Down-regulation of platelet-derived growth factor receptor expression during terminal differentiation of 3T3-L1
1Cancer Research Center, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
Abstract:
The transcription and expression of platelet-derived growth factor (PDGF) receptors (PDGFRs) is down-regulated as a consequence of entry into the replicative cell cycle (Vaziri, C., and Faller, D. V. (1995) Mol. Cell. Biol. 15, 1244-1253). In this study, we have investigated the expression of PDGFRs during terminal differentiation, a process in which cells exit from the cell cycle. When treated with appropriate hormonal stimuli, 3T3-L1 fibroblasts initiate a differentiation program resulting in conversion to lipid-accumulating, adipocyte-like cells. Pre-adipocytes express amounts of PDGFalphaR and PDGFbetaR mRNA and protein that are similar to levels expressed in other murine 3T3 fibroblasts. In contrast, the expression of both alpha and beta receptor transcripts is greatly reduced in differentiated 3T3-L1 cells. The loss of PDGFR mRNA following induction of differentiation precedes morphological conversion as well as the induction of many adipocyte-specific genes. The amounts of cell surface PDGFR protein diminish in parallel with the mRNA levels during differentiation, as shown by Western blotting and PDGF-binding assays. The reduced expression of PDGFRs does not reflect a general down-regulation of growth factor receptors, as expression of the type 1 FGFR is unaffected by terminal differentiation. The PDGFbetaR promoter drives strong expression of a luciferase reporter gene in pre-adipocytes, but not in differentiated cells, indicating that the decrease in PDGFR expression following induction of differentiation is a transcriptionally regulated event. Decreased PDGFR expression in differentiated cells is associated with impaired biological responsiveness to PDGF, as shown by reduced activation of mitogen-activated protein-kinase following PDGF stimulation, and decreased chemotactic responsiveness to PDGF. Our data suggest that PDGFR down-regulation is an important mechanism for reducing PDGF-responsiveness in terminally differentiated 3T3-L1 cells.
Insights
Terminal differentiation of 3T3-L1 fibroblasts down-regulates platelet-derived growth factor receptors (PDGFRs) transcriptionally. This reduces cell responsiveness to PDGF signaling, impacting cell cycle exit and adipogenesis.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Platelet-derived growth factor receptors (PDGFRs) expression is known to decrease upon entry into the replicative cell cycle.
- Terminal differentiation involves cells exiting the cell cycle, a process distinct from replicative entry.
Purpose of the Study:
- To investigate PDGFR expression during terminal differentiation of 3T3-L1 fibroblasts.
- To determine the regulatory mechanisms and functional consequences of PDGFR down-regulation in differentiating cells.
Main Methods:
- Quantitative analysis of PDGFalphaR and PDGFbetaR mRNA and protein levels in pre-adipocytes and differentiated 3T3-L1 cells.
- Western blotting and PDGF-binding assays to assess receptor protein levels.
- Luciferase reporter assays to evaluate PDGFR promoter activity.
- Mitogen-activated protein-kinase activation assays and chemotaxis assays to assess PDGF signaling responsiveness.
Main Results:
- PDGFR alpha and beta mRNA and protein levels significantly decrease in differentiated 3T3-L1 cells compared to pre-adipocytes.
- PDGFR down-regulation occurs transcriptionally, as evidenced by reduced promoter activity in differentiated cells.
- Fibroblast growth factor receptor 1 (FGFR1) expression remains unaffected, indicating specificity of PDGFR down-regulation.
- Differentiated cells exhibit impaired PDGF-induced mitogen-activated protein-kinase activation and reduced chemotaxis.
Conclusions:
- Terminal differentiation of 3T3-L1 fibroblasts leads to a significant, transcriptionally regulated decrease in PDGFR expression.
- Reduced PDGFR levels diminish cellular responsiveness to PDGF, playing a role in the differentiation process.
- PDGFR down-regulation is a specific event not associated with general growth factor receptor down-regulation during differentiation.