Regulation of Brain Fatty Acid-binding Protein Expression by Differential Phosphorylation of Nuclear Factor I in Malignant Glioma Cell Lines

Abstract

Brain fatty acid-binding protein (B-FABPMalignant gliomas are believed to be derived from the astrocytic cell lineage because they contain bundles of cytoplasmic glial fibrillary acidic protein (GFAP), 1 an intermediate filament protein specifically expressed in differentiated astrocytes.There is an inverse relationship between the number of GFAPpositive cells and aggressive behavior in glioma tumors. Glioblastoma multiforme, the most common and aggressive glioma, often have low GFAP expression, while low grade astrocytomas usually have high levels of GFAP (1-4). In vitro studies directly correlate GFAP expression with a less aggressive behavior (5). Transfection of a GFAP expression vector into GFAP(Ϫ) malignant glioma cells results in decreased cell proliferation and decreased growth in soft agar (6, 7). Conversely, transfection of a GFAP antisense vector into a GFAP(ϩ) line results in undetectable GFAP expression and increased proliferation rate, anchorage-independent growth, and invasiveness (8).We have previously shown that GFAP(ϩ) malignant glioma lines express a second glial cell marker, brain fatty acid-binding protein (B-FABP) (9). Of 15 malignant glioma lines tested, 5 co-expressed B-FABP and GFAP, 8 expressed neither gene, while 2 had low levels of B-FABP and undetectable levels of GFAP. B-FABP is a 15-kDa protein normally found in the radial glial cells of the developing central nervous system as well as in select glial cell populations of the adult brain including glia limitans cells and Bergmann glial cells (10, 11). B-FABP expression has been implicated in the establishment of the radial glial fiber system which serves to guide immature migrating neurons to their correct location in the central nervous system (10, 12). Addition of anti-B-FABP antibody to primary cultures of cerebellar cells prevents both the extension of radial glial processes and the migration of neuronal cells along these processes, suggesting a role for B-FABP in relaying inductive signals required for glial cell differentiation.It is generally believed that radial glial cells are converted into astrocytes once neuronal migration in the developing brain is complete (13). Co-expression of GFAP and B-FABP in the same malignant glioma cells (9) therefore suggests that these tumors are derived from cells that have the potential of expressing proteins that are normally produced at different stages in the glial differentiation pathway. We are studying the regulation of the B-FABP gene in order to identify transcription factors involved in the regulation of glial genes in malignant glioma and understand the basis for the variation in B-FABP expression in different malignant glioma lines. By sequencing and DNase I footprinting, we have identified two NFI-binding sites in the promoter region of the B-FABP gene. We present evidence that a phosphatase specifically expressed in B-FABP(ϩ) cells is responsible for differential expression of the B-FABP gene in malignant glioma lines.

Nuclear Factor-1-X Regulates Astrocyte-specific Expression of the α1-Antichymotrypsin and Glial Fibrillary Acidic Protein Genes

“…and neurons, including myelin basic protein, brain fatty acid-binding protein, S100B, mouse neurofilament L, ␥-aminobutyric acid type A receptor, promoter of JC virus, and proteolipid protein (42)(43)(44).…”

Calcineurin Regulates Nuclear Factor I Dephosphorylation and Activity in Malignant Glioma Cell Lines

“…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.…”

“…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).…”

“…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 the four genes NFIA, NFIB, NFIC, and NFIX, all of which can bind to the consensus binding site 5Ј-TTGGCN 5 GCCAA-3Ј as a homodimer or heterodimer to regulate target gene expression (18,19).…”

“…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.…”