Abstract
Mammalian cells respond to protein or amino acid (AA) limitation by activating a number of signaling pathways, collectively referred to as the AA response (AAR), that modulate a range of cellular functions, including transcriptional induction of target genes. This study demonstrates that in hepatocellular carcinoma cells, expression of c-JUN, JUN-B, c-FOS, and FOS-B was induced by the AAR, whereas JUN-D, FRA-1, and FRA-2 were not. Of the four activated FOS/JUN members, c-JUN made the largest contribution to the induction of several known AAR target genes. For several human liver, prostate, and ovarian cell lines, the AAR-induced increase in c-JUN expression was greater in transformed cells compared with nontransformed counterparts, an effect independent of cell growth rate. Thus far, the best characterized AA-responsive genes are all transcriptionally activated by ATF4, but the AAR-dependent induction of c-JUN transcription was ATF4-independent. The increased expression of c-JUN was dependent on ATF2 and on activation of the MEK-ERK and JNK arms of the MAPK signaling pathways. Formation of c-JUN-ATF2-activated heterodimers was increased after AA limitation, and c-JUN or ATF2 knockdown suppressed the induction of c-JUN and other AAR target genes. AA deprivation triggers a feed-forward process that involves phosphorylation of existing c-JUN protein by JNK and subsequent auto-activation of the c-JUN gene by recruitment of c-JUN and ATF2 to two AP-1 sites within the proximal promoter. The results document the novel observation that AP-1 sequences within the c-JUN gene can function as transcriptional amino acid-response elements.
Highlights
To survey the amino acid (AA)-dependent regulation of all family members in a systematic manner, HepG2 cells were incubated in 2 mM HisOH for 0 –24 h, and at specific time points the mRNA content for each was measured by quantitative reverse transcriptase PCR (qPCR)
1) In human hepatoma cells transcription from c-JUN, c-FOS, JUN-B, and FOS-B genes is activated by the AA response (AAR), whereas that for JUN-D, FRA-1, and FRA-2 is not
5) The MEK-ERK and JNK arms of the MAPK pathways are required for the increase in the c-JUN mRNA. 6) The abundance of the transcriptionally active heterodimer containing p-cJUN/p-ATF2 is greater following activation of the AAR, and this dimer contributes to the AAR-induced transcription by binding to AP-1 sites within the c-JUN promoter. 7) c-JUN and ATF2 contribute to the induction of downstream AAR target genes by enhancing the abundance of the newly synthesized activating transcription factor 4 (ATF4) protein
Summary
Each of the four AAR-induced JUN/FOS members, along with an ASNS promoter-luciferase reporter construct, was transiently expressed in HepG2 cells to determine whether they influenced ASNS regulation (supplemental Fig. 1). In contrast to the inhibition of ASNS mRNA, the induction of c-JUN mRNA after activation of the AAR was not affected by blocking ATF4 expression; there was slight enhancement of the mRNA level in the ATF4 knockdown cells treated with HisOH (Fig. 2B).
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