Regulation of gene expression in hepatic cells by the mammalian Target of Rapamycin (mTOR)

Abstract

Background: We 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 findings: The 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/significance: There 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.

Figure 1. Microarray analysis of the effect of rapamycin on gene expression in hepatic cells.

WB-F344 and WB311 cells were exposed to DMSO vehicle or rapamycin (50 nM) for 24 hr. Total RNA was prepared and processed for microarray analysis. Panel A shows a clustering analysis and heat map for the expression of all gene features that showed a significant change in response to rapamycin across the four experimental conditions (two cell lines, each plus and minus rapamycin). The accompanying Venn diagram shows the gene features affected in one cell line, the other or in both. Panel B shows a magnification of the gene expression pattern of the 106 gene features in the middle box of Panel A, representing those that were affected by rapamycin in both cell lines. The blue boxes at the top and in the middle of the heat map represent gene features that were regulated in opposite directions in the WB-F344 and WB311 cells after treatment with rapamycin. The gene features that are not included in the blue boxes represent genes that were co-regulated in the two cell lines.

Figure 2. The effect of rapamycin on the expression of components of the Myc signaling network.

WB-F344 cells were treated with rapamycin for 6 hr (Panel A) or 24 hr (Panel B). At the end of the incubation period, total RNA was prepared from triplicate wells for each condition. RNA (21 µg) was analyzed by multiplex RNase protection assay. Results are expressed as the mean + 1SD. The asterisks indicate a significant difference from the control group as determined by unpaired t-test. The densitometry units are arbitrary and should not be used to compare the relative expression of one gene versus another, only the effect of rapamycin. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 3. Enrichment of c-Myc targets among rapamycin targets in hepatic cell lines.

Microarray results identifying genes affected by rapamycin in WB-F344 cells (Upper Panel) and WB311 cells (Lower Panel) were compared to c-Myc targets. The latter were identified as genes whose expression was altered in response to c-myc induction by OHT in HOMycER12 fibroblasts. The expected (unfilled), observed (black), upregulated (red) and downregulated (green) c-Myc targets regulated in response to rapamycin are shown for the temporal response clusters 1, 2 and 3 in the HOMycER12 fibroblasts and for the total population of genes affected in these cells. *, P<0.05 versus corresponding control were determined by chi square analysis.

Figure 4. Distribution and number of canonical E-box elements (CACGTG) among rapamycin sensitive genes.

Identification of the E-boxes and their distribution was performed as described in the methods section. The promoter sequences spanning −3 kb upstream to +500 bases downstream of the transcriptional start site (TSS) were used in this analysis. Distribution (frequency as a function of position relative to +1, the TSS) of each E-box sequence is shown for WB-F344 cells (Panel A) and WB311 cells (Panel B). The distribution of the number of E-boxes along the promoters of genes affected by rapamycin is shown for the WB-F344 cells (Panel C) and the WB311 cells (Panel D). Density (Y-axis) is a probability function representing the normalized frequency of E-boxes. Black lines denote the total number of E-boxes detected in the database, red lines represent E-boxes in the promoters of genes upregulated by rapamycin and blue lines represent the genes downregulated by rapamycin.

Figure 5. Enrichment of c-Myc targets among rapamycin targets in rat fibroblasts.

TGR-1 cells (c-myc^+/+; Upper Panel) and c-HO15.19 cells (c-myc ^−/−; Lower Panel) were treated with rapamycin (50 nM) for 24 hr. At the end of that time, cells were processed for the preparation of RNA, which was used for microarray analysis. The genes affected by rapamycin were compared to c-Myc targets that were identified as genes whose expression was altered in response to c-Myc induction by OHT in HOMycER12 fibroblasts. The expected (unfilled), observed (black), upregulated (red) and downregulated (green) c-Myc targets regulated in response to rapamycin are shown for the temporal response clusters 1, 2 and 3 in the HOMycER12 fibroblasts and for the total population of genes affected in these cells. *, P<0.05 versus corresponding control were determined by chi square analysis.

Figure 6. Bipartite network of pathways and genes identified as rapamycin responsive in rat fibroblasts.

The microarray analyses described for Figure 5 were analyzed for networks of pathways that were significantly affected by rapamycin. Pathways are represented by circles and genes by squares. Gray circles indicate non significant pathways. Red and blue circles indicate significant pathways that are overrepresented and underrepresented, respectively. The green tone on the squares indicates the degree of pathways membership (from light green representing genes connected to few pathways to dark green for hubs). Panel A shows the pathways and related critical genes affected by rapamycin in TGR-1 cells. Panel B shows the pathways affected by rapamycin in HO15.19 cells. The Mapk1 gene appeared as critical but was not connected to any significant pathways. No significant gene was associated with the overrepresented pathways.