The early response gene IEX-1 attenuates NF-κB activation in 293 cells, a possible counter-regulatory process leading to enhanced cell death

Abstract

The early response gene IEX-1 is involved in the regulation of cellular growth and survival, and its expression is related to stress-, growth-and deathinducing signals. Addressing the role of IEX-1 in the promotion of apoptosis, we investigated the effect of IEX-1 on nuclear factor-jB (NF-jB) activation. Stably transfected HEK-293 cells conditionally overexpressing IEX-1 exhibit decreased levels of NF-jB activity, either basal or TNFa induced, as shown by gel-shift and luciferase reporter gene assay. Furthermore, activated p65 accumulated in the nuclei of 293 cells to a lower degree, if IEX-1 expression was increased. This inhibited NF-jB activation was preceded by an altered turnover of IjBa and phospho-IjBa. In addition, IEX-1 expression also inhibited the activity of the 26S-proteasome, as shown by a fluorometric proteasome assay. Conversely, disruption of IEX-1 expression in 293 cells by stable transfection with specific anti-IEX-1 hammerhead ribozymes increased NF-jB activity, and accelerated the degradation of IjBa. Along with these opposite effects of IEX-1 expression and IEX-1 disruption on NF-jB activation, the sensitivity of 293 cells towards various apoptotic stimuli also changed. In contrast to ribozymetransduced 293 cells that were significantly less sensitive to apoptosis, this sensitivity was enhanced if IEX-1 expression was increased. Our data suggest that IEX-1 -itself an NF-jB target gene -inhibits the activation of this transcription factor, and hereby may counteract the antiapoptotic potential of NF-jB.

Immediate early gene X-1 interacts with proteins that modulate apoptosis

“…IEX-1 enhances apoptosis in some cells, whereas in other cell systems, IEX-1 protects against apoptosis [7,8,[11][12][13]19,22,23]. Our findings show that IEX-1 may function in the control of apoptosis by directly binding to a variety of proteins associated with either the enhancement or inhibition of apoptosis and that differential binding and activation may provide an explanation for the varied effects of this protein in different cells.…”

“…The human immediate early gene X-1 (IEX-1, also known as Dif2, and murine orthologs gly96 and PRG1) plays a role in the control of apoptosis and cellular growth [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20]. The gene (gly96) was initially identified in mouse fibroblasts as a serum-regulated, glycosylated, immediate early gene [1].…”

“…Increased expression of IEX-1 is associated with an increase in the growth rate of keratinocytes and HeLa cells, and disruption of IEX-1 gene expression in HeLa cells and 293 cells is associated with a decrease in cellular proliferation [3,7,8,21]. IEX-1 has been shown to be induced in cardiomyocytes and vascular smooth muscle cells by mechanical stretch, and overexpression of IEX-1 in vascular smooth muscle cells protects against stretch-induced hypertrophy [9,15] IEX-1 is regulated by a variety of factors.…”

“…The messenger RNA of a similar gene (p22/PRG1) is induced in rat pancreatic cells by pituitary adenylate cyclase activating peptide (PACAP) [20]. The human ortholog of gly96 and PRG1, IEX-1, was identified as an X-radiation induced in fibroblasts by Kondratyev et al [2], and as a UV-radiation and 1α, 25-dihydroxyvitamin D 3 -regulated gene in human keratinocytes [3,4].Increased expression of IEX-1 is associated with an increase in the growth rate of keratinocytes and HeLa cells, and disruption of IEX-1 gene expression in HeLa cells and 293 cells is associated with a decrease in cellular proliferation [3,7,8,21]. IEX-1 has been shown to be induced in cardiomyocytes and vascular smooth muscle cells by mechanical stretch, and overexpression of IEX-1 in vascular smooth muscle cells protects against stretch-induced hypertrophy [9,15] IEX-1 is regulated by a variety of factors.…”

“…For example, in keratinocytes [22], HeLa cells [7], hepatocyte (Hc) [19], and 293 cells [8], IEX-1 increases the rate of apoptosis. With respect to the induction of apoptosis, the effect of IEX-1 can be explained partially by its inhibitory effect on survival signals.…”