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Effects of nuclear factor I phosphorylation on calpastatin (CAST) gene variant expression and subcellular
The Minh Vo1, Rebecca Burchett1, Miranda Brun1
1Department of Oncology, University of Alberta, Cross Cancer Institute, Edmonton, Alberta T6G 1Z2, Canada.
Abstract:
Malignant glioma (MG) is the most lethal primary brain tumor. In addition to having inherent resistance to radiation treatment and chemotherapy, MG cells are highly infiltrative, rendering focal therapies ineffective. Genes involved in MG cell migration and glial cell differentiation are up-regulated by hypophosphorylated nuclear factor I (NFI), which is dephosphorylated by the phosphatase calcineurin in MG cells. Calcineurin is cleaved and thereby activated by calpain proteases, which are, in turn, inhibited by calpastatin (CAST). Here, we show that the CAST gene is a target of NFI and has NFI-binding sites in its intron 3 region. We also found that NFI-mediated regulation of CAST depends on NFI's phosphorylation state. We noted that occupation of CAST intron 3 by hypophosphorylated NFI results in increased activation of an alternative promoter. This activation resulted in higher levels of CAST transcript variants, leading to increased levels of CAST protein that lacks the N-terminal XL domain. CAST was primarily present in the cytoplasm of NFI-hypophosphorylated MG cells, with a predominantly perinuclear immunostaining pattern. NFI knockdown in NFI-hypophosphorylated MG cells increased CAST levels at the plasma membrane. These results suggest that NFI plays an integral role in the regulation of CAST variants and CAST subcellular distribution. Along with the previous findings indicating that NFI activity is regulated by calcineurin, these results provide a foundation for further investigations into the possibility of regulatory cross-talk between NFI and the CAST/calpain/calcineurin signaling pathway in MG cells.
Insights
Nuclear factor I (NFI) regulates the CAST gene in malignant glioma (MG) cells. This regulation affects CAST protein levels and its location, suggesting a role in MG cell behavior.
Area of Science:
- Neuro-oncology
- Molecular Biology
- Cell Biology
Background:
- Malignant glioma (MG) is a deadly brain tumor with poor treatment outcomes.
- MG cells exhibit resistance to therapy and invasive growth, driven by factors like cell migration and differentiation.
- Nuclear factor I (NFI) influences these processes, and its activity is modulated by calcineurin and calpain/calpastatin (CAST) signaling.
Purpose of the Study:
- To investigate the relationship between NFI and the CAST gene in malignant glioma cells.
- To determine how NFI phosphorylation state affects CAST gene regulation and protein expression.
- To elucidate the role of NFI in controlling CAST subcellular localization.
Main Methods:
- Analysis of NFI binding sites within the CAST gene.
- Assessment of NFI-mediated CAST gene activation based on NFI phosphorylation.
- Evaluation of CAST transcript and protein variants.
- Immunostaining to determine CAST subcellular localization in MG cells.
- NFI knockdown experiments to observe effects on CAST localization.
Main Results:
- The CAST gene is a direct transcriptional target of NFI, with binding sites in intron 3.
- Hypophosphorylated NFI activates an alternative CAST promoter, increasing specific CAST variants.
- These variants lead to increased CAST protein levels, localized primarily in the cytoplasm.
- NFI knockdown alters CAST localization to the plasma membrane.
Conclusions:
- NFI plays a critical role in regulating CAST gene expression and protein localization in malignant glioma.
- NFI's phosphorylation state dictates its control over CAST variants and subcellular distribution.
- These findings suggest potential cross-talk between NFI and the CAST/calpain/calcineurin pathway in MG pathogenesis.
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