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Effect of TGF-beta1 on cell cycle regulatory proteins in LPS-stimulated normal mouse B lymphocytes
C Bouchard1, W H Fridman, C Sautès
1Laboratoire d'Immunologie Cellulaire et Clinique, INSERM U255, Institut Curie, Paris, France.
Abstract:
The cell cycle events accompanying TGF-beta1-induced growth arrest of normal mouse resting B lymphocytes stimulated by LPS were investigated. We showed that TGF-beta1 prevents the retinoblastoma protein (pRb) phosphorylation and induces growth arrest in mid- to late G1. To explore the molecular basis of the effect of TGF-beta1, we analyzed the in vitro kinase activities of cyclin/cyclin-dependent kinase (cdk) complexes involved in the progression through G1 phase and in the G1/S transition, by using the glutathione S-transferase-pRb fusion protein as a substrate. Cdk2-associated kinase activity was strongly induced in mitogen-treated B cells. It was dramatically inhibited by TGF-beta1 as were the cyclin E- and cyclin A-dependent kinase activities. TGF-beta1 treatment had no significant effect on the expression of two G1/S phase proteins, cyclin E and cdk2. In contrast, the appearance of cyclin A, occuring in late G1 phase, was almost totally inhibited by TGF-beta1. We also showed that expression of the cdk inhibitor protein p27Kip1 decreased as cells progressed through the G1 phase. An accumulation of p27 was found in TGF-beta1-treated cells, showing that TGF-beta1 prevented LPS-induced decline of p27. Finally we found that the lack of kinase activity associated with cyclin E/cdk2 complexes was correlated with increased amounts of cdk2- and cyclin E-bound p27. Overall, these results suggest that both cyclin A and cdk2 may be active participants in the TGF-beta1-induced cell cycle arrest in normal mouse B cells and indicate the involvement of p27 in this mechanism.
Insights
Transforming growth factor-beta1 (TGF-beta1) halts mouse B lymphocyte cell cycle progression in G1 phase. This occurs via inhibition of cyclin-dependent kinases (cdks) and accumulation of the p27Kip1 inhibitor, preventing retinoblastoma protein phosphorylation.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- B lymphocytes play a crucial role in adaptive immunity.
- Cell cycle regulation is essential for lymphocyte proliferation and differentiation.
- Transforming growth factor-beta1 (TGF-beta1) is a cytokine with diverse biological effects, including roles in immune regulation.
Purpose of the Study:
- To investigate the cell cycle events induced by TGF-beta1 in normal mouse B lymphocytes.
- To elucidate the molecular mechanisms underlying TGF-beta1-mediated growth arrest in G1 phase.
- To identify the specific cyclin-dependent kinase (cdk) pathways affected by TGF-beta1.
Main Methods:
- Primary mouse B lymphocytes were stimulated with lipopolysaccharide (LPS) and treated with TGF-beta1.
- Cell cycle progression was monitored by analyzing retinoblastoma protein (pRb) phosphorylation.
- In vitro kinase assays were performed using glutathione S-transferase-pRb fusion protein as a substrate to measure cyclin/cdk complex activities.
- Expression levels of cyclins (E, A), cdks (cdk2), and cdk inhibitors (p27Kip1) were assessed.
Main Results:
- TGF-beta1 induced G1 growth arrest by preventing pRb phosphorylation.
- TGF-beta1 significantly inhibited cyclin E/cdk2 and cyclin A/cdk2 kinase activities.
- TGF-beta1 treatment did not alter cyclin E or cdk2 expression but strongly inhibited cyclin A appearance.
- TGF-beta1 caused an accumulation of p27Kip1, which was found bound to cdk2 and cyclin E, correlating with reduced kinase activity.
Conclusions:
- TGF-beta1-induced G1 arrest in mouse B cells involves the inhibition of cyclin A and cdk2 kinase activities.
- The accumulation of p27Kip1 is a key mechanism mediating TGF-beta1's effect on cell cycle progression.
- These findings highlight the role of TGF-beta1 in regulating B lymphocyte proliferation through specific cell cycle control points.