calpastatin and Glioma

Topics: Binding Sites; Calcium-Binding Proteins; Cell Movement; Gene Expression Regulation, Neoplastic; Glioma; Humans; Mutation; Neurofibromin 1; Phosphorylation; Promoter Regions, Genetic; Subcellular Fractions; Tumor Cells, Cultured

Malignant gliomas (MG), including grades III and IV astrocytomas, are the most common adult brain tumors. These tumors are highly aggressive with a median survival of less than 2 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; FABP7) 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 transcriptional 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 up-regulated 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.

Topics: Adult; Astrocytoma; Calcineurin; Calcium-Binding Proteins; Cell Line, Tumor; Chromatin Immunoprecipitation; Cyclosporine; Glioma; Humans; Immunohistochemistry; Immunoprecipitation; Ionomycin; NFI Transcription Factors; Phosphorylation; Promoter Regions, Genetic; Protein Binding; Protein Transport

According to differences in mobility on SDS-polyacrylamide gel electrophoresis, calpastatins (inhibitor proteins of the calcium-dependent proteinase calpain) are classified into the tissue type (100-120 kDa) and the erythrocyte type (70 kDa), which lacks the amino-terminal domains (domains L and 1). We investigated the molecular diversity of calpastatin in human hematopoietic cells by Western-blot analysis and by the reverse-transcription-polymerase-chain reaction method. While the mononuclear and polymorphonuclear cells in peripheral blood showed the tissue type (110 and 114 kDa), a cell line of erythroid cells (JK-1) showed both the tissue type (110 kDa) and the erythrocyte type (70 kDa) at approximately equal ratios. When the lysate of JK-1 cells was incubated in the presence of ATP, the 110-kDa form was degraded much faster than the 70-kDa form. In human erythrocytes, the 110-kDa form was identified as the tissue type by an antibody recognizing domain L, and this form was also present in addition to the predominant 70-kDA form. JK-1 cells, as well as nucleated cells in peripheral blood, contained calpastatin mRNA with exon-3-deleted. Glioblastoma and fibroblast cell lines expressed the nondeleted calpastatin mRNA in addition to the deletion type, and they showed bands corresponding to 117 kDa as well as 110 and 114 kDa. The 117-kDa band was detectable by an anti-exon 3 peptide antibody. These results suggest that diversity among the tissue type calpastatins is caused by both alternative splicing and post-translational processing whereas the apparent conversion from the tissue type to the erythrocyte type is caused by proteolytic processing.

Topics: Adenosine Triphosphate; Blotting, Western; Calcium-Binding Proteins; Cell Line; Electrophoresis, Polyacrylamide Gel; Exons; Fibroblasts; Gene Deletion; Glioma; Hematopoietic Stem Cells; Humans; Magnesium Chloride; Molecular Weight; Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured