Regulation of NFI by calcineurin in malignant glioma Calcineurin regulates Nuclear Factor I dephosphorylation and activity in malignant glioma cell lines

Malignant gliomas (MG), comprising grades III and IV astrocytomas, are the most common adult brain tumors. These tumors are highly aggressive with a median survival of less than two years. Nuclear Factor I (NFI) is a family of transcription factors that regulates the expression of glial genes in the developing brain. We have previously shown that regulation of the brain fatty acid-binding protein (B-FABP) and glial fibrillary acidic protein (GFAP) genes in MG cells is dependent on the phosphorylation state of NFI, with hypophosphorylation of NFI correlating with GFAP and B-FABP expression. Importantly, NFI phosphorylation is dependent on phosphatase activity that is enriched in GFAP/B-FABP+ve cells. Using chromatin immunoprecipitation, we show that NFI occupies the GFAP and B-FABP promoters in NFI-hypophosphorylated GFAP/B-FABP+ve MG cells. NFI occupancy, NFI-dependent transcription activity and NFI phosphorylation are all modulated by the serine/threonine phosphatase calcineurin. Importantly, a cleaved form of calcineurin, associated with increased phosphatase activity, is specifically expressed in NFI-hypophosphorylated GFAP/B-FABP+ve MG cells. Calcineurin in GFAP/B-FABP+ve MG cells localizes to the nucleus. In contrast, calcineurin is primarily found in the cytoplasm of GFAP/B-FABP-ve cells, suggesting a dual mechanism for calcineurin activation in MG. Finally, our results demonstrate that calcineurin expression is upregulated in areas of high infiltration/migration in grade IV astrocytoma tumor tissue. Our data suggest a critical role for calcineurin in NFI transcriptional regulation and in the determination of MG infiltrative properties. Malignant gliomas (MG), comprising grades III and IV astrocytomas, are the most common adult brain tumors. These tumors have a dismal prognosis with a median survival of less than two years (1). MGs are highly infiltrative, resulting in recurrence despite aggressive treatment including surgical resection, radiotherapy, and chemotherapy (2). MGs have traditionally been hypothesized to arise from astrocytes as tumor cells express glial fibrillary acidic protein (GFAP), an intermediate filament protein expressed in differentiated astrocytes (3). More recent findings suggest that these tumors 1 http://www.jbc.org/cgi/doi/10.1074/jbc.M113.455832 The latest version is at JBC Papers in Press. Published on July 9, 2013 as Manuscript M113.455832 Copyright 2013 by The American Society for Biochemistry and Molecular Biology, Inc. by guest on July 19, 2013 http://www.jbc.org/ Downloaded from Regulation of NFI by calcineurin in malignant glioma may arise from less differentiated glial cell types (4,5). MG tumors express brain fatty acid-binding protein (B-FABP) (6), a marker of radial glial cells. Radial glial cells have been shown to have neural precursor cell properties as defined by the ability to self renew and differentiate into glial and neuronal cells (7-11). B-FABP expression correlates with decreased survival in grade IV astrocytomas (12-14) and B-FABP expression increases MG cell migration and is associated with sites of infiltration in MG tumors (5,15). Expression of B-FABP and GFAP in MG cells is regulated by nuclear factor I (NFI) (16,17). The NFI family of transcription factors consists of four genes: NFIA, NFIB, NFIC, NFIX, all of which can bind to the consensus binding site 5’TTGGCN5GCCAA 3’ as a homodimer or heterodimer, to regulate target gene expression (18,19). While the N-terminal DNA-binding domain is highly conserved in all four NFIs, the Cterminal domain shows divergence among family members (20). Our lab has demonstrated that specific NFIs have distinct effects on NFIdependent promoter activity (16). Furthermore, NFIs can either activate or repress transcription from NFI-dependent promoters, and regulation by NFI is both tissueand promoter contextdependent (16,20). In addition to B-FABP and GFAP, NFI consensus binding sites have been identified in many brain specific promoters (21), and NFIs have been shown to be regulators of glial cell differentiation (22-24). Nfia-/and Nfib-/mice exhibit agenesis of the corpus callosum, enlargement of lateral ventricles, and reduction of specific glial cell populations (25-27). In addition, Nfib-/mice have defects in lung maturation (26,28). Nfix-/mice show enlargement of lateral ventricles and a host of skeletal defects (29). Unlike Nfia, Nfib, and Nfix knock-out mice, Nfic-/mice have defects in tooth root development, but no apparent brain defects (30,31). In the developing spinal cord, NFIA and NFIB control glial fate specification (22). At early stages of development, both NFIA and NFIB are necessary for the maintenance of neural progenitor cells including radial glial cells. At later stages of development, NFIA regulates the migration and differentiation of these precursor cells into astrocytes (22). NFIA has also been shown to be critical for astrocyte differentiation of neural precursor cells in the developing brain (23). The B-FABP and GFAP promoters each contain three NFI consensus binding sites (16,17,32). Based on chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assays, NFI binds to all three NFI consensus sites in both genes. In addition, we have shown that modulation of NFI expression alters BFABP and GFAP promoter activity, as well as endogenous expression of B-FABP and GFAP in MG cell lines (16). Our data indicate that NFI is differentially phosphorylated in different MG cell lines and that NFI phosphorylation state correlates with expression of B-FABP and GFAP; i.e., NFI is hyperphosphorylated in MG cell lines that do not express B-FABP or GFAP and is hypophosphorylated in MG cell lines that express B-FABP and GFAP (17). Intriguingly, this differential phosphorylation appears to be due to a phosphatase activity that is specifically present in MG cell lines with hypophosphorylated NFI (17). Thus, regulation of NFI dephosphorylation may be vital to the control of neural/glial gene expression in MG. Calcineurin is a calcium-dependent serine/threonine phosphatase (33) composed of two subunits: calcineurin A (CNA; PP2B), the catalytic subunit (33), and calcineurin B (CNB), a regulatory calcium binding subunit (34). Calcineurin plays a wide variety of biological functions, linking calcium signalling to multiple outputs ranging from immediate cellular responses to long term alterations in gene expression (35,36). In the brain, calcineurin is highly expressed, and plays important roles in synaptic plasticity (3739). In developing cerebellar granule neurons, calcineurin signalling activates NFAT binding to NFI target genes, blocking NFI occupancy. As these neurons mature, binding of NFAT is temporally downregulated resulting in an increase in NFI binding to target genes (40). A more direct link between calcineurin and NFI comes from the observation that calcineurin is able to activate the transactivation domain of NFIC in fibroblasts (41). Here, we investigate the regulation of NFI dephosphorylation and activity in MG cell lines. We show that calcineurin regulates NFI dephosphorylation and activity in MG cell lines. In addition, we identify a cleaved form of CNA that is specific to MG cell lines with