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. 2012 Oct 19;287(43):36190-200.
doi: 10.1074/jbc.M112.373746. Epub 2012 Aug 30.

The endoplasmic reticulum stress transducer BBF2H7 suppresses apoptosis by activating the ATF5-MCL1 pathway in growth plate cartilage

Soutarou Izumi  1 , Atsushi Saito, Soshi Kanemoto, Noritaka Kawasaki, Rie Asada, Hideo Iwamoto, Mami Oki, Hidetaka Miyagi, Mitsuo Ochi, Kazunori Imaizumi
Affiliations

The endoplasmic reticulum stress transducer BBF2H7 suppresses apoptosis by activating the ATF5-MCL1 pathway in growth plate cartilage

Soutarou Izumi et al. J Biol Chem. .

Abstract

BBF2H7 (box B-binding factor 2 human homolog on chromosome 7) is a basic leucine zipper transmembrane transcription factor that belongs to the cyclic AMP-responsive element-binding protein (CREB)/activating transcription factor (ATF) family. This novel endoplasmic reticulum (ER) stress transducer is localized in the ER and is cleaved in its transmembrane region in response to ER stress. BBF2H7 has been shown to be expressed in proliferating chondrocytes in cartilage during the development of long bones. The target of BBF2H7 is Sec23a, one of the coat protein complex II components. Bbf2h7-deficient (Bbf2h7(-/-)) mice exhibit severe chondrodysplasia, with expansion of the rough ER in proliferating chondrocytes caused by impaired secretion of extracellular matrix (ECM) proteins. We observed a decrease in the number of proliferating chondrocytes in the cartilage of Bbf2h7(-/-) mice. TUNEL staining of the cartilage showed that apoptosis was promoted in Bbf2h7(-/-) chondrocytes. Atf5 (activating transcription factor 5), another member of the CREB/ATF family and an antiapoptotic factor, was also found to be a target of BBF2H7 in chondrocytes. ATF5 activated the transcription of Mcl1 (myeloid cell leukemia sequence 1), which belongs to the antiapoptotic B-cell leukemia/lymphoma 2 family, to suppress apoptosis. Finally, we found that the BBF2H7-ATF5-MCL1 pathway specifically suppressed ER stress-induced apoptosis in chondrocytes. Taken together, our findings indicate that BBF2H7 is activated in response to ER stress caused by synthesis of abundant ECM proteins and plays crucial roles as a bifunctional regulator to accelerate ECM protein secretion and suppress ER stress-induced apoptosis by activating the ATF5-MCL1 pathway during chondrogenesis.

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Figures

FIGURE 1.
FIGURE 1.
Apoptosis is promoted in the cartilage of Bbf2h7−/− mice. A, H&E staining (top panels) and TUNEL staining (bottom panels) of cartilage sections from the tibias of E18.5 WT and Bbf2h7−/− mice. Bars, 50 μm. B, numbers of TUNEL-positive proliferating chondrocytes in A. The data shown are means ± S.D. (error bars) (n = 6). *, p < 0.05, unpaired Student's t test. C, immunohistochemical analysis of cleaved caspase 3 (c-CASP3) in the tibias of E18.5 WT and Bbf2h7−/− mice. Bars, 50 μm. D, numbers of c-CASP3-positive proliferating chondrocytes in C. The data shown are means ± S.D. (n = 4). *, p < 0.05, unpaired Student's t test.
FIGURE 2.
FIGURE 2.
Apoptosis is promoted and proliferation is inhibited in Bbf2h7−/− cells. A, Western blotting of c-CASP3 in primary cultured chondrocytes. Cells were treated with Tg (1 μm) for the indicated times. B, quantitative analysis of c-CASP3 expression in A. The data are means ± S.D. (error bars) (n = 3). *, p < 0.05, unpaired Student's t test. C and D, cell counting (C) and WST-8 (D) assays using primary cultured chondrocytes. Bbf2h7−/− chondrocytes show inhibited proliferation compared with WT chondrocytes. The data are means ± S.D. (n = 4). *, p < 0.05; **, p < 0.01, unpaired Student's t test.
FIGURE 3.
FIGURE 3.
BBF2H7 is involved in the up-regulation of Atf5 and Mcl1. A, real-time PCR analyses of Bbf2h7, Atf5, and Mcl1 in mesenchymal cells prepared from WT and Bbf2h7−/− mice and maintained as micromass cultures for 1, 4, 7, 10, and 13 days. The data are means ± S.D. (error bars) (n = 3). The WT expression level/β-actin level of each gene at 1 day was set as 1.0 on the y axis. B, real-time PCR analyses of Atf5 (left) and Mcl1 (right) in mesenchymal cells prepared from WT and Bbf2h7−/− mice and maintained as micromass cultures for 4 days. The cells were treated with Tg (1 μm) for 12 h. The data are means ± S.D. (n = 3). *, p < 0.05, unpaired Student's t test. C, real-time PCR analysis of Atf5 using primary cultured chondrocytes. The cells were treated with Tg (1 μm) for the indicated times. The data are means ± S.D. (n = 3). *, p < 0.05, unpaired Student's t test. D, real-time PCR analysis of Mcl1 using primary cultured chondrocytes. The cells were treated with Tg (1 μm) for the indicated times. The data are means ± S.D. (n = 3). *, p < 0.05, unpaired Student's t test. E, Western blotting analysis of ATF5 (top) and quantitative analysis of ATF5 expression in the top panel (bottom) using primary cultured chondrocytes. Although ATF5 expression was down-regulated in Bbf2h7−/− cells, that was induced by the infection of adenovirus expressing ATF5. mock, empty vector. F, real-time PCR analyses of Mcl1 in primary cultured chondrocytes. The data are means ± S.D. (n = 3). *, p < 0.05, unpaired Student's t test. G, real-time PCR analyses of Atf5 (left) and Mcl1 (right) using primary cultured chondrocytes. Atf5 and Mcl1 expression are not affected by overexpression of Sec23a. The data are means ± S.D. (n = 3). *, p < 0.05, unpaired Student's t test. H, real-time PCR analysis of Sec23a using primary cultured chondrocytes. Sec23a expression is not affected by overexpression of ATF5. The data are means ± S.D. (n = 3). *, p < 0.05, unpaired Student's t test.
FIGURE 4.
FIGURE 4.
Atf5 is a direct target of BBF2H7. A, scheme of the Atf5 promoter region and reporter constructs. The 4.0-kb Luc construct consists of the 4-kb Atf5 promoter region. The mut CRE reporter construct has a mutation in the CRE sequence (shown as lowercase letters). Luc, luciferase gene. B, reporter assays using primary cultured chondrocytes. A vector expressing the BBF2H7 N terminus was cotransfected with each reporter construct. The data are means ± S.D. (error bars) (n = 3). **, p < 0.01, unpaired Student's t test. C, The top panel shows a schematic representation of the Atf5 promoter and the annealing sites of the primer set used in the ChIP assays. The bottom panel shows the results of PCR amplification of the Atf5 promoter region containing the CRE sequence (−632 to −639). Chondrocytes were infected with an adenovirus for overexpression of the BBF2H7 N terminus. mock, empty vector. D, quantitative analysis of immunoprecipitation with an anti-BBF2H7 antibody in C. The data are means ± S.D. (n = 3). **, p < 0.01, unpaired Student's t test. The immunoprecipitation/BBF2H7 (IP:BBF2H7) level/input level of mock was set as 1.0 on the y axis. E, real-time PCR amplification of the Atf5 promoter region containing the CRE sequence (−632 to −639). Chondrocytes were infected with an adenovirus for overexpression of the BBF2H7 N terminus, followed by immunoprecipitation with an anti-BBF2H7 antibody.
FIGURE 5.
FIGURE 5.
Bbf2h7−/− chondrocytes are vulnerable to ER stress-induced apoptosis. A–D, TUNEL staining in primary cultured WT and Bbf2h7−/− chondrocytes treated with STS (100 nm) (A), Etop (100 nm) (B), Tm (3 μg/ml) (C), or Tg (1 μm) (D) for the indicated times. The data are means ± S.D. (error bars) (n = 3). *, p < 0.05, unpaired Student's t test. E, Western blotting of c-CASP3 in primary cultured WT and Bbf2h7−/− chondrocytes. The cells were treated with STS, Etop, Tm, or Tg at the above concentrations for the indicated times. F, quantitative analysis of c-CASP3 expression in E. The data are means ± S.D. (n = 3). *, p < 0.05, unpaired Student's t test.
FIGURE 6.
FIGURE 6.
The BBF2H7-ATF5-MCL1 pathway inhibits ER stress-induced apoptosis. A, Western blotting of c-CASP3 in primary cultured chondrocytes. The cells were treated with Tg (1 μm) for the indicated times. mock, empty vector. B, quantitative analysis of c-CASP3 expression in A. The data are means ± S.D. (error bars) (n = 3). *, p < 0.05, unpaired Student's t test. C, TUNEL staining in primary cultured WT and Bbf2h7−/− chondrocytes treated with Tg (1 μm) for the indicated times. The introduction of ATF5 in Bbf2h7−/− chondrocytes inhibits ER stress-induced apoptosis. The data are means ± S.D. (n = 3). *, p < 0.05, unpaired Student's t test. D, proposed model for the BBF2H7-mediated signaling pathway in the secretion of cartilage ECM proteins and inhibition of ER stress-induced apoptosis during chondrocyte differentiation.
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