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Stat3-dependent induction of p19INK4D by IL-10 contributes to inhibition of macrophage proliferation
A M O'Farrell1, D A Parry, F Zindy
1Department of Molecular Biology, DNAX Research Institute, Palo Alto, CA 94304, USA. anne_marie.ofarrell@pharma.novartis.com
Abstract:
We have previously reported that IL-10 inhibits proliferation of normal bone marrow-derived macrophages and of the monocyte/macrophage cell line J774. Activation of Stat3 was shown to be necessary and sufficient to mediate inhibition of proliferation. To investigate further the mechanism of growth arrest, we examined the effect of IL-10 on expression of cell cycle inhibitors. We found that IL-10 treatment increases expression of the cyclin-dependent kinase inhibitors p19INK4D and p21CIP1 in macrophages. IL-10 cannot induce p19INK4D expression or block proliferation when Stat3 signaling is blocked by a dominant negative Stat3 or a mutant IL-10Ralpha which does not recruit Stat3 in J774 cells, whereas p21CIP1 induction is not affected. An inducibly active Stat3 (coumermycin-dimerizable Stat3-Gyrase B), which suppresses J774 cell proliferation, also induced p19INK4D expression. Sequencing of the murine p19INK4D promoter revealed two candidate Stat3 binding sites, and IL-10 treatment activated a reporter gene controlled by this promoter. These data suggest that Stat3-dependent induction of p19INK4D mediates inhibition of proliferation. Enforced expression of murine p19INK4D cDNA J774 cells significantly reduced their proliferation. Use of antisense p19INK4D and analysis of p19INK4D-deficient macrophages confirmed that p19INK4D is required for optimal inhibition of proliferation by IL-10, and indicated that additional IL-10 signaling events contribute to this response. These data indicate that Stat3-dependent induction of p19INK4D and Stat3-independent induction of p21CIP1 are important components of the mechanism by which IL-10 blocks proliferation in macrophages.
Insights
Interleukin-10 (IL-10) inhibits macrophage proliferation by increasing cell cycle inhibitors. This process involves Stat3-dependent induction of p19INK4D and Stat3-independent induction of p21CIP1.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Interleukin-10 (IL-10) is known to inhibit the proliferation of macrophages.
- Signal transducer and activator of transcription 3 (Stat3) activation is crucial for IL-10-mediated proliferation inhibition.
Purpose of the Study:
- To elucidate the molecular mechanisms by which IL-10 induces growth arrest in macrophages.
- To investigate the role of cell cycle inhibitors in IL-10-induced macrophage proliferation inhibition.
Main Methods:
- Analysis of cell cycle inhibitor expression (p19INK4D, p21CIP1) in macrophages treated with IL-10.
- Utilizing dominant-negative Stat3 and mutant IL-10Ralpha to assess Stat3 dependency.
- Reporter gene assays to confirm Stat3 binding to the p19INK4D promoter.
- Gene silencing (antisense) and knockout approaches to evaluate p19INK4D function.
Main Results:
- IL-10 treatment increased the expression of p19INK4D and p21CIP1 in macrophages.
- Stat3 signaling was essential for IL-10-induced p19INK4D expression and proliferation inhibition, but not for p21CIP1 induction.
- Activated Stat3 directly induced p19INK4D expression, with evidence of Stat3 binding sites in the p19INK4D promoter.
- p19INK4D was required for optimal IL-10-mediated proliferation inhibition, though additional IL-10 signaling pathways also contribute.
Conclusions:
- Stat3-dependent induction of p19INK4D is a key mechanism for IL-10-induced macrophage growth arrest.
- Stat3-independent induction of p21CIP1 also contributes to IL-10's anti-proliferative effects.
- These findings provide a deeper understanding of IL-10's role in regulating macrophage proliferation.