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PU.1 acts as tumor suppressor for myeloma cells through direct transcriptional repression of IRF4
N Ueno1, N Nishimura1, S Ueno1
1Departments of Hematology, Rheumatology and Infectious Diseases, Kumamoto University Graduate School of Medicine, Chuo-ku, Kumamoto, Japan.
Abstract:
We previously reported that PU.1 is downregulated in the majority of myeloma cell lines and primary myeloma cells of certain myeloma patients, and conditional expression of PU.1 in such myeloma cell lines induced cell cycle arrest and apoptosis. We found downregulation of IRF4 protein in the U266 myeloma cell line following induction of PU.1. Previous studies reported that knockdown of IRF4 in myeloma cell lines induces apoptosis, prompting us to further investigate the role of IRF4 downregulation in PU.1-induced cell cycle arrest and apoptosis in myeloma cells. PU.1 induced downregulation of IRF4 at the protein level, cell cycle arrest and apoptosis in six myeloma cell lines. Chromatin immunoprecipitation (ChIP) revealed that PU.1 directly binds to the IRF4 promoter, whereas a reporter assay showed that PU.1 may suppress IRF4 promoter activity. Stable expression of IRF4 in myeloma cells expressing PU.1 partially rescued the cells from apoptosis induced by PU.1. As it was reported that IRF4 directly binds to the IRF7 promoter and downregulates its expression in activated B cell-like subtype of diffuse large B cell lymphoma cells, we performed ChIP assays and found that IRF4 directly binds the IRF7 promoter in myeloma cells. It is known that IRF7 positively upregulates interferon-β (IFNβ) and induces apoptosis in many cell types. Binding of IRF4 to the IRF7 promoter decreased following PU.1 induction, accompanied by downregulation of IRF4 protein expression. Knockdown of IRF7 protected PU.1-expressing myeloma cells from apoptosis. Furthermore, IFNβ, which is a downstream target of IRF7, was upregulated in myeloma cells along with IRF7 after PU.1 induction. Finally, we evaluated the mRNA expression levels of PU.1, IRF4 and IRF7 in primary myeloma cells from patients and found that PU.1 and IRF7 were strongly downregulated in contrast to the high expression levels of IRF4. These data strongly suggest that PU.1-induced apoptosis in myeloma cells is associated with IRF4 downregulation and subsequent IRF7 upregulation.
Insights
PU.1 expression induces myeloma cell death by downregulating IRF4 and upregulating IRF7. This pathway involves PU.1 binding to the IRF4 promoter, leading to apoptosis and interferon-beta production in myeloma cells.
Area of Science:
- * Molecular Biology
- * Cancer Research
- * Immunology
Background:
- * PU.1 is downregulated in most myeloma cell lines and patient samples.
- * PU.1 re-expression induces cell cycle arrest and apoptosis in myeloma cells.
- * IRF4 knockdown is known to induce apoptosis in myeloma cells.
Purpose of the Study:
- * To investigate the role of IRF4 downregulation in PU.1-mediated apoptosis.
- * To elucidate the mechanism by which PU.1 induces apoptosis in myeloma cells.
- * To explore the relationship between PU.1, IRF4, IRF7, and interferon-beta in myeloma.
Main Methods:
- * Chromatin immunoprecipitation (ChIP) assays to determine transcription factor binding.
- * Reporter assays to assess promoter activity.
- * Stable cell line expression and knockdown experiments.
- * Analysis of mRNA and protein expression levels.
Main Results:
- * PU.1 directly binds the IRF4 promoter and downregulates IRF4 protein expression.
- * PU.1 induces cell cycle arrest and apoptosis in six myeloma cell lines.
- * IRF4 re-expression partially rescues myeloma cells from PU.1-induced apoptosis.
- * PU.1 induction leads to decreased IRF4 binding to the IRF7 promoter.
- * IRF7 knockdown protects PU.1-expressing myeloma cells from apoptosis.
- * Interferon-beta is upregulated in myeloma cells following PU.1 induction.
- * Primary myeloma cells show downregulated PU.1 and IRF7, with high IRF4 expression.
Conclusions:
- * PU.1-induced apoptosis in myeloma is mediated by IRF4 downregulation.
- * The pathway involves IRF4 binding to the IRF7 promoter, leading to IRF7 upregulation.
- * Upregulated IRF7 subsequently increases interferon-beta production, contributing to apoptosis.
- * Findings suggest a novel therapeutic strategy targeting the PU.1/IRF4/IRF7 axis in myeloma.
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