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.
Highlights
Malignant glioma (MG) is the most lethal primary brain tumor
Analysis of ChIP-on-chip data obtained with this pan-specific nuclear factor I (NFI) antibody and FABP7/glial fibrillary acidic protein (GFAP)-expressing U251 MG cells resulted in the identification of CAST as a putative target of NFI [36]
To verify protein binding to these three putative NFI recognition sites, we performed gel shift assays with nuclear extracts prepared from U87 and U251 MG cells
Summary
All four NFIs are expressed in cells of glial origin as well as glioblastoma cells, with a number of NFI targets identified in these glial-like cell types [14, 15, 19]. Protein binding to the C2 and C3 oligonucleotides was examined using nuclear extracts prepared from both NFIhyperphosphorylated U87 and NFI-hypophosphorylated U251 MG cells [14, 15] These cell lines express all four NFIs, with similar amounts of NFIA RNA and higher levels of NFIB, NFIC, and NFIX RNA in U251 compared with U87 MG cells (Fig. S1A) [14]. Supershifted bands were observed when antiNFIC antibody was incubated with either the C2 or C3 probe in the presence of either U87 or U251 nuclear extracts, indicating that NFIC is present in both C2– and C3–protein complexes (Fig. 3A). Supershift experiments with anti-AP2 antibody had no effect on the migration of either the C2–protein or C3–protein complexes These results suggest that all four NFIs may bind to C2 and C3 oligonucleotides or interact with protein–C2 or –C3 complexes, in both. The migration patterns of the shifted complexes were similar in both U87 and U251 MG cells, suggesting that exogenous NFIs are not subjected to the same phosphorylation/dephosphorylation process as endogenous NFIs
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
