Array analysis of gene expression in connexin-43 null astrocytes

Abstract

Connexin-43 (Cx43) is the most abundant gap junction protein in brain, where it is found primarily between astrocytes. Although the morphology of astrocytes from Cx43-null (knockout, KO) mice is similar to that of wild-type (WT) astrocytes, KO astrocytes exhibit reduced growth rate in culture. To evaluate the impact of deletion of Cx43 on other genes, including those encoding cell cycle proteins, we used DNA arrays to determine expression patterns in cultured astrocytes from sibling Cx43-null and WT mice. RNA samples extracted from astrocytes cultured from WT and Cx43-null neonatal mice were dye labeled and individually cohybridized with a reference of labeled cDNAs pooled from a variety of tissues on 8 gene arrays containing 8,975 mouse DNA sequences. Normal variability in expression of each gene was evaluated and incorporated into "expression scores" to statistically compare expression levels between WT and KO samples. In Cx43-null astrocytes, 4.1% of the 4,998 adequately quantifiable spots were found to have significantly (P < 0.05) decreased hybridization compared with controls, and 9.4% of the spots showed significantly higher hybridization. The significantly different spots corresponded to RNAs encoding 252 known proteins, many not previously linked to gap junctions, including transcription factors, channels and transporters, cell growth and death signals, enzymes and cell adhesion molecules. These data indicate a surprisingly high degree of impact of deletion of Cx43 on other astrocyte genes, implying that gap junction gene expression alters numerous processes in addition to intercellular communication.

Fig. 1

Variability in spot hybridization among all four arrays for wild-type (WT) and connexin-43 (Cx43) null astrocytes. A and B: histogram of relative estimated variability (REV) values for WT and Cx43-null astrocytes. For WT astrocytes, the mean REV value was lower and the distribution more sharply peaked (as reflected in higher kurtosis value). C: comparison of the REV values for the individual spots that were quantifiable on all four arrays from both genotypes. Note that spots with REV values >100 in one genotype generally corresponded to much lower REV values in the other genotype. KO, knockout.

Fig. 2

Categorization of significantly (P < 0.05) altered expression of identified genes in Cx43-null compared with WT astrocytes. A: of the 252 significantly altered identified genes, 35% (88 genes) were downregulated and 65% (164) were upregulated. B: distribution of altered genes in functional categories. JAE, junction-adhesion-extracellular matrix; CY, cytoskeletal; T1, transport into the cell; T2, transport within the cell; CS, cell signaling; CSD, cell cycle-shape-differentiation; TR, transcription; EnMet, energy and metabolism; OG, organelle genetics; UNKf, unknown function. See text for expanded description of categories. C and D: relative percentages of up- and downregulated genes of each functional category in astrocytes of Cx43-null compared with WT mice.

Fig. 3

Significant (P < 0.05) coordination among the expression of cell cycle-shape-differentiation (CSD) genes in WT cortical astrocytes (A) that were significantly regulated in the Cx43-null astrocytes (B). Gene designations are within boxes; lines between boxes indicate significant coordinations of the expression of paired genes in the WT cortical astrocytes (numbers atop lines provide correlation coefficients; antagonistic coordinations are highlighted in yellow). Colors of boxes in B indicate whether a gene was significantly upregulated (red) or downregulated (green) in Cx43-null astrocytes. Red lines in the cluster at the bottom indicate that regulation in Cx43 KO samples was opposite to that predicted by the coordination analysis in the WT astrocytes.

Fig. 4

Significant coordinations among the expression of genes related to programmed cell death in WT cortical astrocytes. Gene designations are within boxes; lines between boxes indicate significant coordinations of the expressions of paired genes in the WT cortical astrocytes (values on lines provide correlation coefficients). Colors of boxes indicate whether a gene was significantly upregulated (red) or downregulated (green) in Cx43-null astrocytes, while colors of lines indicate whether the coordination was synergistic (blue) or antagonistic (red).