Regulation of Gene Expression in Hepatic Cells by the Mammalian Target of Rapamycin (mTOR)

Abstract

BackgroundWe investigated mTOR regulation of gene expression by studying rapamycin effect in two hepatic cell lines, the non-tumorigenic WB-F344 cells and the tumorigenic WB311 cells. The latter are resistant to the growth inhibitory effects of rapamycin, thus providing us with an opportunity to study the gene expression effects of rapamycin without confounding effects on cell proliferation.Methodology/Principal FindingsThe hepatic cells were exposed to rapamycin for 24 hr. Microarray analysis on total RNA preparations identified genes that were affected by rapamycin in both cell lines and, therefore, modulated independent of growth arrest. Further studies showed that the promoter regions of these genes included E-box-containing transcription factor binding sites at higher than expected rates. Based on this, we tested the hypothesis that c-Myc is involved in regulation of gene expression by mTOR by comparing genes altered by rapamycin in the hepatic cells and by c-Myc induction in fibroblasts engineered to express c-myc in an inducible manner. Results showed enrichment for c-Myc targets among rapamycin sensitive genes in both hepatic cell lines. However, microarray analyses on wild type and c-myc null fibroblasts showed similar rapamycin effect, with the set of rapamycin-sensitive genes being enriched for c-Myc targets in both cases.Conclusions/SignificanceThere is considerable overlap in the regulation of gene expression by mTOR and c-Myc. However, regulation of gene expression through mTOR is c-Myc-independent and cannot be attributed to the involvement of specific transcription factors regulated by the rapamycin-sensitive mTOR Complex 1.

Profiling of the fetal and adult rat liver transcriptome and translatome reveals discordant regulation by the mechanistic target of rapamycin (mTOR)

“…These findings are in keeping with our previous studies on a panel of hepatic cell lines in which we showed that effects of rapamycin on gene expression disproportionately affected genes whose promoter regions contained E-boxes (19). Although this indicated the possibility of a transcriptional control mechanism involving c-Myc, we went on to show that the rapamycin-induced effects were independent of c-Myc.…”

“…The results of our studies indicated resistance to the growth inhibitory effect of rapamycin at the level of G1 cell-cycle progression (13) and translation of 5=TOP mRNAs (12,14). We have also had a long-standing interest in the regulation of hepatic gene expression by mTOR (13,19,21). In the present study, we have extended these observations by characterizing the effect of rapamycin on the hepatic transcriptome and translatome in the late gestation fetal rat and in the adult rat.…”

The physiology and pathophysiology of rapamycin resistance

“…53 Our initial studies focused on the WB-F344 and WB311 cell lines. One goal of these studies was to identify rapamycin-induced and rapamycin-suppressed ) and total ribosomal protein S6.…”

“…Roughly equivalent numbers of genes were upregulated and downregulated in response to rapamycin (60 downregulated, 66 upregulated), a finding that recapitulated our earlier results in hepatic cell lines. 33,53 In order to characterize the effects of rapamycin on gene expression in fetal liver, results were analyzed using two separate instruments, Gene Set Enrichment Analysis (GSEA), 57,58 and Ingenuity Pathways Analysis (IPA; Ingenuity ® Systems, www.ingenuity.com). Results using GSEA did not disclose a significant overrepresentation of any transcription factor binding domains.…”

Functionally conserved effects of rapamycin exposure on zebrafish

“…In addition, microarray analyses testing the extent of global alterations in the transcriptome in yeast and in mammals contribute to the understanding of the mechanisms and pathways associated with the effect of rapamycin (7)(8)(9)(10)(11)(12). In vivo studies indicate that rapamycin results in developmental delay in different organisms including Drosophila melanogaster, zebrafish and mice (6,13,14).…”