Expression profiling after activation of amino acid deprivation response in HepG2 human hepatoma cells

Abstract

Dietary protein malnutrition is manifested as amino acid deprivation of individual cells, which activates an amino acid response (AAR) that alters cellular functions, in part, by regulating transcriptional and posttranscriptional mechanisms. The AAR was activated in HepG2 human hepatoma cells, and the changes in mRNA content were analyzed by microarray expression profiling. The results documented that 1,507 genes were differentially regulated by P < 0.001 and by more than twofold in response to the AAR, 250 downregulated and 1,257 upregulated. The spectrum of altered genes reveals that amino acid deprivation has far-reaching implications for gene expression and cellular function. Among those cellular functions with the largest numbers of altered genes were cell growth and proliferation, cell cycle, gene expression, cell death, and development. Potential biological relationships between the differentially expressed genes were analyzed by computer software that generates gene networks. Proteins that were central to the most significant of these networks included c-myc, polycomb group proteins, transforming growth factor β1, nuclear factor (erythroid-derived 2)-like 2-related factor 2, FOS/JUN family members, and many members of the basic leucine zipper superfamily of transcription factors. Although most of these networks contained some genes that were known to be amino acid responsive, many new relationships were identified that underscored the broad impact that amino acid stress has on cellular function.

Keywords: liver gene expression; microarray; nutrient sensing; starvation.

Fig. 1.

Validation of differentially regulated genes in response to the amino acid response (AAR). Total RNA was isolated from HepG2 hepatoma cells incubated for 4 h in either control medium [Dulbecco's modified Eagle's medium (DMEM)] or in DMEM containing 5 mM histidinol (HisOH). After isolation of total RNA, real-time quantitative PCR (RT-qPCR) was used to measure the mRNA expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a negative control and 8 genes regulated by HisOH treatment. Data are expressed as a ratio to the GAPDH mRNA for each gene and plotted relative to the value for the DMEM control condition. Results are presented as averages ± SD of assays in triplicate. CBX4, chromobox protein homolog 4; UTX, ubiquitously transcribed X chromosome tetratricopeptide; HOXA3, homeobox A3; RhoB, Ras homolog gene family-member B; MLL2, myeloid/lymphoid or mixed-lineage leukemia 2; KLF5, Kruppel-like factor 5; HSPA1A, heat shock 70-kDa protein 1A; FAM13A1, family with sequence similarity 13 member A1.

Fig. 2.

Comparison of microarray data from 3 independent studies. Genes that were identified as amino acid responsive based on 3 independent investigations were evaluated for overlap, and data are presented as a Venn diagram. In addition to the results from the present study (HepG2+HisOH), the data from Lee et al. (38) (HepG2-Cys) and Deval et al. (15) (MEF-Leu) were included in the analysis. As indicated in parentheses, the number of total genes analyzed was 1,173 (present study), 1,009 (Lee et al.), and 728 (Deval et al). The 38 genes common to all 3 investigations are listed in Supplemental Table S3.

Fig. 3.

Networks that contain significant numbers of genes associated with the AAR. The subset of 1,507 differentially expressed AAR-regulated genes (P < 0.001 and a 2-fold change or greater; Supplemental Table S2) was analyzed to identify potential interactions between gene products with the information compiled in the Ingenuity Knowledge Base. Each possible network was assigned a score that is the negative log of Fisher's exact test P value, and only networks with a score of at least 20 (P value of 10⁻²⁰) were analyzed further (Supplemental Table S3). Networks 1 (A), 2 (B), and 3 (C) contained the greatest number of “focus genes,” while network 23 (D) is presented as an additional example of a network containing both known and newly identified genes associated with the AAR. Keys explain the symbols used and gene interactions shown. Intensity of pink-to-red symbols indicates degree of upregulation of gene expression and intensity of green symbols indicates downregulation in response to the AAR.

Fig. 4.

Heat maps of up- or downregulated genes involved in network 3 and network 23. Each column represents an individual sample incubated for 4 h in either control medium DMEM (M) or DMEM containing 5 mM HisOH (H). A probe set for each gene is depicted in each row, labeled with the gene name for that probe set. Gene expression level is depicted as intensity of red (upregulated) and blue (downregulated) boxes.