The exposure of a cell to external stimuli triggers complex intracellular signaling cascades that result in finely tuned alterations in gene expression, enabling the cell to react in an appropriate manner to the stimulus. IEGs are operationally defined as genes whose rapid and usually transient activation for RNA transcription upon an external stimulus does not require de novo protein synthesis i.e. is directly linked to transduction mechanisms activated by the stimulus (Thomson et al. 1999). IEGs encoding inducible transcription factors regulate the secondary transcriptional responses appropriate for the particular stimulus to which a cell is exposed (Herdegen and Leah 1998). Following exposure to hypertonicity the rapid tonicity-induced expression of some IEGs encoding transcription factors was thus expected as part of the cellular response to the osmotic stress. Three IEGs were thus identified, namely Atf3, Verge, and Klf4 that showed a rapid tonicity-induced expression. To our knowledge none of these has previously been reported to be tonicity-induced. Increased Atf3 and Verge expression in whole tissue was markedly high. This may indicate that it occurs in cells that are widely distributed in brain parenchyma. In comparison, there was a relatively weak increase in Klf4 expression, suggesting that it may only occur in some cell subsets.
Activation of IEGs encoding transcription factors in response to environmental stress triggers the coordinated expression of functionally related effector genes that lead either to cell recovery, or alternatively, cell death. In order to evaluate the beneficial or detrimental roles of the rapid tonicity-induced activation of the above IEGs we looked at the transcriptional profiles for possible indices of apoptosis and focused our attention on the expression of DNA damage and cell death related genes, given the data previously reported in vitro. Cells exposed acutely to a strongly hypertonic medium (osmolality > 500 mOsm/kg) show DNA breaks, as well as up-regulation of proteins known to respond to genotoxic stress, such as p53 and GADDs. In the mIMCD3 renal cell line, DNA breaks appear within 15 min after acute elevation of osmolality from 300 to 500–600 mOsmol/kg by adding NaCl (Kultz and Chakravarty 2001). Acute elevation of NaCl also results in rapid increases in p53 abundance, p53 phosphorylation, and p53 transcriptional activity (Dmitrieva et al. 2000). High NaCl also increases the abundance of the proteins GADD34, 45, and, 153 (Kultz et al. 1998; Chakravarty et al. 2002). p53 protein is a transcription factor that is activated by phosphorylation/acetylation upon DNA damage caused by various genotoxic stresses that lead to the downstream transactivation of a wide variety of genes controlling the cell cycle, DNA repair, and apoptosis (Vousden 2000). p53 up-regulates the expression of Gadd45, a gene involved in the control of the cell cycle and DNA repair, as well as many proapoptotic genes such as Aip1, Apaf1, Bax, Bak, Perp, and Puma (Vousden and Lu 2002). In brain tissue of rats subjected to systemic hypertonicity, no increased expression of p53 was recorded. Furthermore, there was no increased expression of the p53-regulated genes, Gadd45, Aip1, Apaf1, Bax, Bak, Perp, and Puma. Indeed none of the apoptosis-related genes that gave a detectable hybridization signal on the microarrays showed an altered expression following systemic hypertonicity for up to 6 h. In particular, the proapoptotic genes, Bad, Bid, Bid3, Bik, Bnip3, and Bnip3L, which encode BH3-only proteins that are essential initiators of programmed cell death (Bouillet and Strasser 2002) did not show increased expression. Taken together, these data indicate that DNA damage inducing cell cycle arrest, DNA repair, and ultimately apoptosis is unlikely to occur to a significant extend in brain cells under our conditions of systemic hypertonicity. The sharp contrast with the data reported for renal cells in vitro is probably because of the fact that brain cells in vivo were subjected to a weaker hypertonic stress, which was applied more progressively. Altogether these observations suggest that the IEGs, whose activation we recorded, are not involved as primary signals for triggering apoptosis.
It must be noted that there exists selectivity of the tonicity-induced expression of IEGs encoding transcription factors. The expression of most of them was not affected by hypertonicity. Furthermore, among the different members of the same family, the expression of only one member appears to be tonicity-induced, e.g. Atf3 among the Atf family and Klf4 among the Klf family. For some families such as Fos/Jun, Egr or Ier, hypertonicity did not lead to an early increased expression of any of their members. Tissue expression of some genes that is increased by systemic hypertonicity is also increased by other stresses, albeit at different levels. Increased Atf3 expression following systemic hypertonicity is thus much larger than that reported following hypoxia (Tang et al. 2006). More significant is the marked difference in the overall profiles of stress-induced genes. Fos/jun as well as Egr gene families show a large increase in expression following ischemia (Soriano et al. 2000; Lu et al. 2004; Nagata et al. 2004) but no altered expression following systemic hypertonicity. In other words, although each of these cellular stresses may induce overlapping signaling pathways and target genes, the IEG expression profile is stress-specific. This specificity suggests that the activation of these IEGs represents the primary response of the cells for secondarily triggering the expression of the genes allowing adaptation to the stress. According to this view the tonicity-induced expression of ATF3, VERGE, and to a lesser extend KLF4, may activate genes encoding proteins involved in cellular osmoadaptation.