Partial limitation of cellular functions and compensatory modulation of unfolded protein response pathways caused by double-knockout of ATF6α and ATF6β

Abstract

Mammalian cells have three types of endoplasmic reticulum (ER) stress-sensing molecules: ATF6, IRE1, and PERK. Among these, ATF6 is unique in that it is processed in an ER-stress-specific manner and functions as a transcription factor for the activation of anti-ER stress genes (such as BiP). ATF6 is known to have two homologues, ATF6α and ATF6β, and a greater understanding of their functions has been achieved through analyses using cultured cells. Physiological functions are also gradually being investigated in mice lacking ATF6α or ATF6β. However, little is known about the effects on mouse organisms of the deletion of both the ATF6α and ATF6β genes, since such double-knockout (DKO) mice suffer embryonic lethality at an early developmental stage. In this study, we generated and analyzed ATF6 DKO mice in which embryonic lethality was evaded by using Cre/loxP technology. Pancreatic β cell-specific ATF6 DKO mice were born normally and lived without dysregulation of blood-glucose levels but had a reduced tolerance to glucose. Islets isolated from ATF6 DKO mice also showed low production and secretion of insulin and mild enhancement of IRE1 and PERK activity. We further examined the developmental abnormalities of systemic ATF6 DKO mice. The phenotypes of ATF6α^-/-; ATF6β^-/- mice were similar to those previously reported, but ATF6α^+/-; ATF6β^-/- and ATF6α^-/-; ATF6β^+/- mice showed embryonic lethality at middle developmental stages, unlike those reported. Analysis of embryonic fibroblasts derived from these mice revealed that ATF6α and ATF6β have a gene-dose-dependent functional redundancy and display distinct differences in their ability to induce BiP expression. (250 words).

Keywords: BiP; ER stress; Insulin; Pancreatic β cell.

Fig. 1

Design and establishment of ATF6α and ATF6β conditional knockout mice. a, b Schematic diagrams showing the targeting vector and the indicated genotype in the mouse ATF6α and ATF6β loci, respectively. “Neo” and “TK” indicate expression units of the neomycin-resistance gene and thymidine kinase gene for positive and negative selection, respectively. Closed and open arrowheads indicate FRT and loxP elements, respectively. WT: wild-type allele, Vec: targeting vector, Rec: homologous recombination allele, flox: flox allele, ∆bZIP: ∆bZIP allele, H: HindIII site, and B: BamHI. c, d Southern blot analysis of mouse genome DNA with the indicated genotype. For genotyping of ATF6α and ATF6β alleles, mouse genome DNA was digested with HindIII and BamHI, respectively. The upper panels show autoradiographs with the probes indicated in a and b, respectively. The lower panels show EtBr staining of genome DNA as a loading control. e, f PCR analysis of mouse genome DNA with the indicated genotype. The position of primers is schematically indicated in a and b, respectively.

Fig. 2

Body weight (upper) and blood glucose (lower) of mice with pancreatic β cell-specific deletion of ATF6α and ATF6β (DKO). Mice with ATF6α^flox/flox and ATF6β^flox/flox (flox) were used as controls. The plots show the mean, and the error bars denote the S.E.M. (n = 5).

Fig. 3

Glucose tolerance of mice and isolated islets with pancreatic β cell-specific deletion of ATF6α and ATF6β (DKO). a, Time course of the blood-glucose level (upper) and the blood-insulin level (lower) in mice after oral administration of glucose. The plots show the mean and error bars denote the S.E.M. (n = 5). b, Relative level of insulin protein secreted from the islets after low- or high-glucose stimulation. All data are normalized to the no-glucose treatment. The columns show the mean, and the error bars denote the S.E.M. (n = 3). Mice and the isolated islets with ATF6α^flox/flox and ATF6β^flox/flox (flox) were used as controls. The asterisks indicate statistically significant results.

Fig. 4

Expression levels of insulin protein and mRNA in islets isolated from wild-type or ATF6-mutant mice. a, Each panel shows the Western blot signals as detected by anti-insulin antibody or anti-GAPDH antibody. Signals of GAPDH and images from SDS-PAGE were used as a loading control. b, Expression levels of insulin protein and mRNA as measured by ELISA and quantitative PCR analysis, respectively. The columns show the mean, and the error bars denote the S.E.M. (n = 3). The asterisks indicate statistically significant results. + = wild-type allele; ∆ = ∆bZIP allele.

Fig. 5

Expression levels or phosphorylation levels of ER-stress-associated molecules in isolated islets with pancreatic β cell-specific deletion of ATF6α and ATF6β (DKO). IRE1α, PERK, and eIF2α were evaluated from the phosphorylation level of the protein. ATF4 was evaluated from the expression level of the protein. The other values were evaluated from the expression level of mRNA. Islets with ATF6α^flox/flox and ATF6β^flox/flox (flox) were used as a control. The columns show the mean, and the error bars denote the S.E.M. (n = 3). The asterisks indicate statistically significant results.

Fig. 6

Numbers of living offspring obtained from matings between ATF6-mutant mice. a, Number of living pups obtained from matings between ATF6α^+/flox; ATF6β^+/flox mice. b, Number of living pups obtained from matings between ATF6α^+/∆bZIP; ATF6β^+/∆bZIP mice. c, Number of living embryos obtained from matings between ATF6α^+/∆bZIP; ATF6β^+/∆bZIP mice. The viability of the embryos was judged from the beating of their hearts.

Fig. 7

Characterization of embryos and MEFs with ATF6α^+/∆bZIP; ATF6β^{∆bZIP/∆bZIP} or ATF6α^{∆bZIP/∆bZIP}; ATF6β^+/∆bZIP. a, Typical images of embryos and placentas with the indicated ATF6 genotype at E11.5 stage (scale bar: 5 mm). b, Typical images of MEFs with the indicated ATF6 genotype (scale bar: 50 µm). c, Typical growth curves for MEFs with the indicated ATF6 genotype (DT: doubling time calculated from the growth curve). The MEFs with ATF6α^+/+; ATF6β^+/+ (wild-type) were used as a control. + = wild-type allele, and ∆ = ∆bZIP allele.

Fig. 8

Comparison of ATF6 levels and BiP levels between various ATF6 mutant MEFs. a, Expression levels of ATF6α mRNA, ATF6β mRNA, and BiP mRNA in ATF6 mutant MEFs cultured under normal conditions. MEFs with ATF6α^+/+ and ATF6β^+/+ (wild-type) were used as controls. The asterisks indicate statistically significant results. b, Expression levels of BiP mRNA in ATF6 mutant MEFs cultured under ER stress conditions (Tun, Thg, and DTT). Normal conditions were used as a control. + = wild-type allele, ∆ = ∆bZIP allele, Tun: tunicamycin, Thg: Thapsigargin, DTT: dithiothreitol. The columns indicate the mean, and the error bars denote the S.E.M. (n = 3).