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.

Connexins, cell motility, and the cytoskeleton

“…In some systems both effects, regulation of the cytoskeleton and migration, have been observed [Iacobas et al, 2003;Xu et al, 2006;Elias et al, 2007]. This strengthens the notion of a functional relationship of Cxs and the cytoskeleton and we will peruse this further in the following chapter.…”

“…A cDNA microarray analysis of cultured Cx43 KO astrocytes revealed significant changes in several genes with diverse functional properties [Iacobas et al, 2003]. Among the regulated genes, seven corresponded to cytoskeletal proteins, which included microtubule and microfilament associated proteins.…”

“…GJ influence on cell motility [Iacobas et al, 2003;Xu et al, 2006;Elias et al, 2007] may therefore be conveyed by a cytoskeletal motor involving myosin II. Protein 4.1 is a multifunctional membrane protein that was originally identified as an abundant protein in erythrocytes, where it stabilizes spectrin-actin association.…”

Mathematical model of morphogen electrophoresis through gap junctions

“…An important aspect is the identification of gene transcription modulated by the intracellular arrival of GJC-dependent morphogens into cells. Although these studies are just beginning in our laboratory in the context of the asymmetric gene cascade, others have begun to characterize, using transcriptome analysis, the genetic targets of gap junction-mediated signaling (Iacobas et al, 2003(Iacobas et al, , 2004(Iacobas et al, , 2005Kim et al, 2005). It is now abundantly clear that a large number of important gene networks are potentially controlled by GJC signaling and these networks represent excellent targets for future molecular dissection.…”

Connexin subtype expression during oral carcinogenesis: A pilot study in patients with oral squamous cell carcinoma

“…King et al (4) described a correlation between endogenous Cx43 mRNA and protein expression and increased growth control, with decreased growth capacity in HeLa cervical cancer cells. Cx43 knockout in mice leads to astrocytes exhibiting altered expression of genes associated with apoptosis, cell growth, transcription factors (10,11), and increased susceptibility of mice to pulmonary neoplasia (6). Cx26 and Cx43 expression in MDA-MB-231 breast cancer cells (5) provided similar results.…”