Endoplasmic Reticulum Stress Induction of Insulin-like Growth Factor-binding Protein-1 Involves ATF4

Alexandre Marchand,Robert Barouki,Laurent Magne,Michèle Garlatti,Céline Tomkiewicz

doi:10.1074/jbc.m602157200

Abstract

Endoplasmic reticulum (ER) stress is sensed by cells in different physiopathological conditions in which there is an accumulation of unfolded proteins in the ER. A coordinated adaptive program called the unfolded protein response is triggered and includes translation inhibition, transcriptional activation of a set of genes encoding mostly intracellular proteins, and ultimately apoptosis. Here we show that insulin-like growth factor (IGF)-binding protein-1 (IGFBP-1), a secreted protein that modulates IGF bioavailability and has other IGF-independent effects, is potently induced during ER stress in human hepatocytes. Various ER stress-inducing agents were able to increase IGFBP-1 mRNA levels, as well as cellular and secreted IGFBP-1 protein up to 20-fold. A distal regulatory region of the human IGFBP-1 gene (-6682/-6384) containing an activating transcription factor 4 (ATF4) composite site was required for promoter activation upon ER stress. Mutation of the ATF4 composite site led to the loss of IGFBP-1 regulation. Electrophoretic mobility shift assay revealed an ER stress-inducible complex that was displaced by an ATF4 antibody. Knockdown of ATF4 expression using two specific small interfering RNAs impaired up-regulation of IGFBP-1 mRNA, which highlights the relevance of ATF4 in endogenous IGFBP-1 gene induction. In addition to intracellular proteins involved in secretory and metabolic pathways, we conclude that ER stress induces the synthesis of secreted proteins. Increased secretion of IGFBP-1 during hepatic ER stress may thus constitute a signal to modulate cell growth and metabolism and induce a systemic adaptive response.

Highlights

The endoplasmic reticulum (ER)3 is the site of synthesis, folding, and modification of secretory and cell surface proteins as well as the resident proteins of the secretory pathway
ER Stress Induces Insulin-like growth factor-binding proteins (IGFBPs)-1 mRNA in Primary Cultures of Human Hepatocytes—The mRNAs of primary cultures of human hepatocytes treated or not for 24 h with 2 ␮g/ml tunicamycin or Me2SO were analyzed by Northern blot using probes for IGFBP-1, for markers of ER stress: the chaperones BiP, GRP94, and Herp as well as for 18 S ribosomal RNA
We asked whether other components of the insulin-like growth factor (IGF)/IGFBP system were regulated by tunicamycin

Summary

Introduction

The endoplasmic reticulum (ER) is the site of synthesis, folding, and modification of secretory and cell surface proteins as well as the resident proteins of the secretory pathway. In addition to the recovery from ER stress, the UPR initiates proapoptotic pathways that could lead to programmed cell death if the stress is sustained [9] The diversity of these responses is mediated by three ER transmembrane transducer proteins that sense the accumulation of unfolded protein in the ER lumen and activate signaling pathways. The second transmembrane ER kinase, IRE1, is an endoribonuclease that splices XBP1 (X-box binding protein-1) mRNA in response to ER stress; this generates the potent bZIP transcription factor, XBP1, which regulates the transcription of a set of UPR target genes [11]. Ire1/XBP1 and ATF6 pathways are specific to ER stress, the PERK pathway shares phosphorylation of eIF2␣ and the induced “integrated stress response” with unrelated stress: amino acid deprivation, viral infection, and heme deficiency that activate specific kinases [8, 21]

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Conclusion