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7/21/2019 Analysis of ATF3, A Transcription Factor Induced by Physiological Stresses and Modulated by Gadd153 Chop10

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MOLECULAR ANDCELLULAR BIOLOGY, Mar. 1996, p. 11571168 0270-7306/96/$04.000Copyright 1996, American Society for Microbiology

Analysis of ATF3, a Transcription Factor InPhysiological Stresses and Modulated by gaddBENJAMIN P. C. CHEN,1 CURT D. WOLFGANG,1 ANDTSONWI

Ohio State Biochemistry Program1 and Department of Medical Biochemistry and NeuOhio State University, Columbus, Ohio 43210

Received 19 September 1995/Returned for modification 15 November 1995/Accepted

We demonstrate that ATF3, a member of the ATF/CREB family of transcription

variety of stressed tissues: mechanically injured liver, toxin-injured liver, blood-depzure brain. We also demonstrate that an ATF3-interacting protein, gadd153/Chop1heterodimer with ATF3: the heterodimer, in contrast to the ATF3 homodimer, does n

AMP response element consensus site and does not repress transcription. InterestiChop10 are expressed in inverse but overlapping manners during the livers respons(CCl

4): the level of gadd153/Chop10 mRNA is high in the normal liver and grea

treatment; the level of ATF3 mRNA, on the other hand, is low in the normal liver anCCl

4 treatment. We hypothesize that in nonstressed liver, gadd153/Chop10 inhibi

ATF3 by forming an inactive heterodimer with it, whereas in CCl4

-injured liver, t


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order to reveal the subtissue distribution of ATF3 mRNA. Inthis report, we demonstrate that ATF3 is induced in a varietyof stressed tissues: mechanically injured liver, toxin-injured

liver, blood-deprived heart, and postseizure brain. We furtherdemonstrate that an ATF3-interacting protein, gadd153/Chop10 (gadd stands for growth arrest and DNA-damaging;Chop stands for C/EBP-homologous protein), functions as anegative regulator of ATF3 by forming an inactive het-erodimer with it. Significantly, ATF3 and gadd153/Chop10 areexpressed in inversely but overlapping manners during thelivers response to the hepatoxin carbon tetrachloride (CCl4).We discuss a hypothesis to explain these observations.

MATERIALS AND METHODS

Isolation of cDNAs encoding ATF3-interacting proteins. A COS-1 cell cDNAexpression library was constructed in the EXloxT7 expression vector (Novagen)by using a Riboclone cDNA synthesis kit (Promega). The primary library wasamplified in Escherichia coli ER1647, and the amplified library was introducedinto E. coli BL21 (DE3/LysS) to express the T7 gene 10 fusion protein andscreened by radiolabeled HisK-ATF3 fusion protein as described previously (31).HisK-ATF3 was expressed by using the pET-HisK vector (8), which providestandem phosphorylation sites for heart muscle kinase (HMK) and labeled byheart muscle kinase (Sigma) in vitro in the presence of [-32P]ATP (4). pEX-

(i) Hepatectomy.Seven-week-old(midventral lobes) from each rat whours after hepatectomy, the rats wecollected.

(ii) CCl4treatment.Eight-week-oinjected with 0.08 ml of 100% CCllater, the rats were sacrificed and thsaline instead of CCl4. For the timedescribed above and sacrificed at th

(iii) Alcohol treatment. Four-wetized, and intragastrically injected

weight; 50% lethal dose according twere sacrificed and the livers were cof alcohol.

(iv) Coronary artery ligation. Sevleft anterior descending (LAD) coroischemia. Two hours later, the rats w

ventricle wall were collected. Shamwithout the coronary artery ligation

(v) Coronary artery ligation and

thetized with pentobarbital, and theby tying a slipknot, using a silk sutureperfused by removing the suturesacrificed and the region of the left artery was collected. Sham-operatethe coronary artery ligation and rep

(vi) PTZ treatment. Seven-week-pentylenetetrazole (PTZ; Sigma p6

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in PBS to remove the background due to the endogenous biotin in the liver.gadd153/Chop10 antibody was affinity purified and used at a dilution of 1:500.

RESULTS

ATF3 is induced by a variety of physiological stresses. Todetermine the expression pattern of ATF3 in regeneratingliver, we examined rat livers at 2 h after partial hepatectomy byin situ hybridization. As shown in Fig. 1A and B, the antisenseRNA probe detected signals in regenerating liver but did notdetect any signals in the normal liver, indicating that the ATF3mRNA level increases during liver regeneration. The hybrid-ization specificity was demonstrated by the observation that thesense RNA probe did not hybridize to either normal or regen-

erating liver (data not shown). The signals detected by theantisense RNA probe were patchy and spread throughout theliver and did not correspond to any specific structure of liver.

Because the liver is the major organ that metabolizes toxins,we examined the expression pattern of ATF3 in liver at 3.5 h(see Discussion) after intragastric injection of the hepatoxinCCl4. As shown in Fig. 1C and D, ATF3 was expressed in cellsaround central veins; it was, however, not expressed in the restof the liver. Alcohol, another hepatoxin, also induced ATF3

level in stressed tissues is nencoding bZip proteins.

ATF3 heterodimerizes wit

observations prompted us tomediates responses to physian important question: W

ATF3? Since ATF3 is inducent injuries, we propose thdetermined by the physiolinteracting proteins in the this possibility, we screeneradiolabeled ATF3 to isolatals and Methods). We isolbZip protein gadd153/Chop

protein blot analysis, radiocontains the bZip region, bitself but not to ATF1; thChop10 was consistently grlanes 1 to 3). Furthermore, gadd153/Chop10 (Fig. 2B, lthe interaction between ATnonspecific. The leucine zipessary for the interaction be

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(Fig. 5B). The observation transcriptional repression byassay also supports the no

interact with each other in vATF3 and gadd153/Chopliver in inverse but overlastrated that gadd153/Chop1

we then asked whether thisvant. As a first step toward ined the expression of rat gamodels described above. Weing reasons. First, C/EBP p(also named LAP and NF-ILregulate liver cell-specific

references 38 and 44). It isalso involved in regulating gadd153/Chop10 is inducedand inflammation (43, 64), ulated by some of the treaUsing Northern blot analysgadd153/Chop10 after CCl4and B, gadd153/Chop10 mRtreated liver, low at 1.5 h af

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FIG. 5. gadd153/Chop10 relieved the repression of E-selectin promoter by ATF3. (A) E-selectinCAT reporter waspCGN-p50 and pCGN-p65 (NF-B), pCG-ATF3, and pCG-gadd153. The plasmid DNA carrying only the cytomegalovcalcium phosphate-DNA mixes to ensure that each transfection mix contained the same amount of promoter. (B) Mumutated ELAM-1 site (TCACGACGGT) was cotransfected with DNA expressing ATF3 or NF-B as indicated. The ave

h

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9/15FIG. 7. Both ATF3 and gadd153/Chop10 proteins were present in the liver at 17.5 h after CCl4treatment. (A) gadd1

i i di d b i hi h i (L f ) Th li i ll d 17 5 h f CCl

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