GATA2 is critical for the maintenance of cellular identity in differentiated mast cells derived from mouse bone marrow.

GATA2 plays a crucial role for the mast cell fate decision. We herein demonstrate that GATA2 is also required for the maintenance of the cellular identity in committed mast cells derived from mouse bone marrow (BMMCs). The deletion of the GATA2 DNA binding domain (GATA2ΔCF) in BMMCs resulted in a loss of the mast cell phenotype and an increase in the number of CD11b- and/or Ly6G/C-positive cells. These cells showed the ability to differentiate into macrophage- and neutrophil-like cells but not into eosinophils. Although the mRNA levels of basophil-specific genes were elevated, CD49b, a representative basophil marker, never appeared on these cells. GATA2 ablation led to a significant upregulation of C/EBPα, and forced expression of C/EBPα in wild-type BMMCs phenocopied the GATA2ΔCF cells. Interestingly, simultaneous deletion of the Gata2 and Cebpa genes in BMMCs restored the aberrant increases of CD11b and Ly6G/C while retaining the reduced c-Kit expression. Chromatin immunoprecipitation assays indicated that GATA2 directly binds to the +37-kb region of the Cebpa gene and thereby inhibits the RUNX1 and PU.1 binding to the neighboring region. Upregulation of C/EBPα following the loss of GATA2 was not observed in cultured mast cells derived from peritoneal fluid, whereas the repression of c-Kit and other mast cell-specific genes were observed in these cells. Collectively, these results indicate that GATA2 maintains cellular identity by preventing Cebpa gene activation in a subpopulation of mast cells, whereas it plays a fundamental role as a positive regulator of mast cell-specific genes throughout development of this cell lineage.

Effects of dimethyl sulphoxide and dexamethasone on mRNA expression of myogenesis- and muscle proteolytic system-related genes in mouse myoblastic C2C12 cells.

We examined the time course of mRNA expression of myogenic cell differentiation- and muscle proteolytic system-related genes in cultures of C2C12 cells during differentiation from myoblasts to myotubes. Furthermore, we treated C2C12 myotubes with dimethyl sulphoxide (DMSO) and dexamethasone (Dex), and examined changes in these mRNA levels. Myogenin (Myog), Atrogin1, forkhead box O1 (Foxo1) and Capn1 mRNA levels increased in C2C12 cells differentiating from myoblasts to myotubes, whereas Myf5 mRNA levels decreased. Although genes such as MRF4, Foxo3a, UbB, Capn1 and MuRF1 mRNAs in the myotubes were affected by DMSO exposure, mRNA levels of other genes were not markedly affected by exposure to 0.02% or 0.5% DMSO. Myf5, MRF4, Atrogin1, Foxo3 and MuRF1 mRNA levels were elevated by Dex at all time points, Cbl and Capn1 mRNA levels were significantly elevated by Dex at 8 h, and Myog mRNA levels were significantly elevated by Dex at 24 h. However, CtsH mRNA levels decreased significantly with Dex at 24 h. This study provides a useful database of gene profiles that are differentially expressed throughout myogenic cell differentiation and the muscle proteolytic system.

Effects of dimethyl sulfoxide and dexamethasone on mRNA expression of housekeeping genes in cultures of C2C12 myotubes.

We used quantitative real-time RT-PCR to investigate the effects of dimethyl sulfoxide (DMSO) and dexamethasone (Dex) on the mRNA expression levels of the housekeeping genes beta-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-glucuronidase (GUSB), hypoxanthine phosphoribosyltransferase 1 (HPRT1), phosphoglycerate kinase 1 (PGK1), peptidylprolyl isomerase A (PPIA), and transferrin receptor (TFRC) in cultures of C2C12 myotubes. The ratios of ACTB mRNA levels to the HPRT1 mRNA level in C2C12 cells that were differentiating from myoblast cells to myotubes decreased from 0 to 120h of culture, whereas the ratios of TFRC mRNA levels to the HPRT1 mRNA level increased from 0 to 120h of culture. The ratios of GAPDH, GUSB, PGK1, and PPIA mRNA levels to the HPRT1 mRNA level remained constant from 0 to 120h of culture. All housekeeping gene mRNA levels were unaffected by exposure to DMSO concentrations of 0.1% or less. The GAPDH mRNA level was increased by Dex, while the ACTB and PGK1 mRNA levels were significantly decreased by Dex. The GUSB, PPIA, and TFRC mRNA levels were unaffected by exposure to Dex. GUSB, HPRT1, and PPIA are thus suitable internal controls for evaluating mRNA expression levels in cultures of C2C12 cells.