RT–PCR analysis of ATF3ΔZip2 mRNA in A23187-treated HUVECs. (A) Primers used in this study and their locations in the human ATF3 gene are shown. Exons are indicated by boxes, and labeled as A, B, C, D and E as in Liang et al. (33). Exons D′ and E′ are novel ones described in the text. Initiation codon ATG in exon B is shown under primer 5′. Stop codons for the full-length ATF3 (*), ATF3ΔZip (*1) and ATF3ΔZip2 (*2) are shown. (B) HUVECs (3 × 10⁵ cells) were treated with 10 µM A23187 for 4 h, and total RNA (1 µg) was assayed for RT–PCR using primer 5′ and primer 3′ (lanes 1–3), primer 5′ and primer Zip3 (lanes 4–6), or primer Zip4 and primer 3′ (lanes 7–9). Lanes 1, 4 and 7 are derived from untreated cells; all the other lanes are from A23187-treated cells with (lanes 3, 6 and 9) or without (lanes 2, 5 and 8) RT. Bands of 546, 858 and 767 bp represented products of full-length ATF3, ATF3ΔZip2a and ATF3ΔZip2b, respectively (lane 3). 554 bp was the product derived from ATF3ΔZip2a and b (lane 6); note that the primer set used in this experiment (primer 5′ and primer Zip3) does not distinguish ATF3ΔZip2a from ATF3ΔZip2b. 510 and 419 bp represented ATF3ΔZip2a and ATF3ΔZip2b, respectively (lane 9). M, the 123 bp DNA size marker.

Splicing events for generating ATF3ΔZip2a and b and comparison of their nucleotide and amino acid sequences. (A) Splicing events for generating ATF3ΔZip2a and b and their nucleotide and amino acid sequences are compared with those for full-length ATF3 and ATF3ΔZip as described in Liang et al. (33). Exon D′ is comprised of a 76 base sequence of intron C (shaded squares) that precedes exon D. Exon E′ is an original intron D of 91 bp (dotted square) and transcribed into ATF3ΔZip2a mRNA. Leucine residues in the zipper region were marked. (B) Schematic representations of the full-length ATF3, ATF3ΔZip and ATF3ΔZip2 are shown. Basic, Zip, A and R represent the basic, leucine zipper, activation, repression region, respectively (21).

Semi-quantitative measurement of ATF3ΔZip2 and full ATF3 mRNA. Total RNA (1 µg) from the A23187-treated HUVECs was subjected to RT–PCR using primer 5′ and primer 3′ and the expression of full ATF3 and ATF3ΔZip2 was measured at various cycles of PCR. A typical experiment is shown in (A). In (B), the logarithm of density of each band was plotted against the number of PCR cycles, and the ratio of expression of ATF3ΔZip2 to full ATF3 was determined from data at the linear amplification range of PCR. Results were means ± SE bars of three independent experiments. The amount of ATF3ΔZip2 represents a total of ATF3ΔZip2a and b.

Western blot analysis of ATF3ΔZip2 protein in A23187-treated HUVECs. HUVECs (3 × 10⁵ cells) were treated with 10 µM A23187 for 8 h. Whole-cell extracts (20 µg protein) were prepared and separated on a 15% SDS–PAGE and subjected to western blot analysis using anti-ATF3 antibody (21) as described in Materials and Methods. Lane 1, bacterially expressed full-length His-tagged ATF3. The asterisk denotes a proteolytic artifact. Lane 2, control untreated HUVECs; lane 3, A23187-treated HUVECs; lane 4, Flag-tagged ATF3ΔZip2 expressed in Cos-7 cells. NS, the non-specific 35 kDa protein in HUVECs.

EMSA of recombinant ATF3ΔZip2. Full-length ATF3 and ATF3ΔZip2 were expressed as GST-fusion proteins as described in the Materials and Methods. (A) Purified GST–ATF3 fusion protein was analyzed by western blot using monoclonal anti-GST antibody. Lane 1, GST-full ATF3; lane 2, GST–ATF3ΔZip2; lane 3, GST. (B) Recombinant ATF3 proteins were assayed for their DNA binding activity by EMSA as detailed in the Materials and Methods. Lane 1, probe only; lane 2, 2.5 µg GST–full-length ATF3; lanes 3–5, 5 µg GST–full-length ATF3 in the absence (lane 3) or presence of wild-type ATF/CRE sequence (lane 4) and mutated sequence (lane 5), respectively; lanes 6 and 7, 2.5 and 5 µg GST–ATF3ΔZip2; lanes 8 and 9, 2.5 and 5 µg GST.

Nuclear localization of ATF3ΔZip2. ATF3ΔZip2 protein N-terminally fused to GFP (pEGFPATF3ΔZip2) or HA-tag (pHAATF3ΔZip2) was transiently expressed in Cos-7 cells as described in the Materials and Methods. At 24 h post-transfection, GFP was detected in cells transfected with empty vector [pEGFP (a and b] or pEGFPATF3ΔZip2 (c and d). HA-tagged ATF3 protein was detected by monoclonal anti-HA antibody in cells transfected with empty vector [pHA (e and f)] or pHAATF3ΔZip2 (g and h). Nuclei were also stained by DAPI in (b, d, f and h).

Effects of ATF3ΔZip2 on transcriptional repression by ATF3. (A) Cos-7 cells were transfected with pME (lane 1), pME-Flag-ATF3 (lane 2) or pME-Flag-ATF3ΔZip2 (lane 3) and their whole-cell extract were analyzed by western blot using monoclonal anti-Flag antibody. (B) Cos-7 cells were co-transfected with pLuc-4xATF and increasing amounts of pME-Flag-ATF3 or pME-Flag-ATF3ΔZip2. At 24 h post-transfection, cell extracts were assayed for luciferase activity as detailed in the Materials and Methods. Results represent mean ± SE from three independent experiments. Statistically significant inhibition or activation are indicated (*P < 0.05; **P < 0.005).

Induced expression of ATF3ΔZip2 in response to various stimuli. Cells were stimulated with various agents and their total RNA (1 µg) was isolated and subjected to RT–PCR. The full-length ATF3 mRNA was assayed using primer 5′ plus primer 3′, and ATF3ΔZip2 mRNA using primer 5′ plus primer Zip3. The cycle numbers of PCR for full-length ATF3 and ATF3ΔZip2 were 24 and 28 cycles, respectively. (A) HUVECs were untreated (lane 1) or stimulated with 10 µM A23187 (lane 2), 3 mM homocysteine (lane 3), 2 µM thapsigargin (lane 4), 10 µg/ml tunicamycin (lane 5), 0.05 mM hydrogen peroxide (lane 6), 10 ng/ml TNF-α (lane 7) for 2 h. (B) Saos2 cells were untreated (lane 2), or treated with A23187 (lane 3), homocysteine (lane 4), thapsigargin (lane 5), tunicamycin (lane 6) and TNF-α (lane 7) as in (A), and with 0.1 µg/ml MMS for 4 h (lane 9), 10 µM MG132 for 14 h (lane 10), and 2.5 µM colchicine for 1.5 h (lane 11). Lanes 1 and 8, A23187-treated HUVECs as the positive control. (C) Esophageal cancer cell lines over-expressing ATF3 (29) were analyzed. Lane 1, A23187-treated HUVECs as the positive control; lanes 2–6, KYSE70, KYSE150, KYSE170, KYSE190 and KYSE200 cell lines, respectively.