The Endoplasmic Reticulum Stress Transducer BBF2H7 Suppresses Apoptosis by Activating the ATF5-MCL1 Pathway in Growth Plate Cartilage*

Background: The endoplasmic reticulum stress transducer BBF2H7 is expressed in proliferating chondrocytes. Results: Apoptosis is promoted in the cartilage of Bbf2h7−/− mice. BBF2H7 activates Atf5 transcription, followed by apoptosis suppression. Conclusion: BBF2H7 plays crucial roles in suppressing ER stress-induced apoptosis. Significance: BBF2H7 acts as a bifunctional regulator to promote cartilage extracellular matrix protein secretion and inhibit ER stress-induced apoptosis. 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.


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.
The endoplasmic reticulum (ER) 3 is a cellular organelle that regulates the synthesis, folding, and posttranslational modifications of secreted proteins. Various cellular stress conditions lead to accumulation of unfolded or misfolded proteins in the ER. These conditions are collectively termed ER stress (1)(2)(3). The ER responds to these perturbations by activating the unfolded protein response, which transduces signals to enhance the capacity for protein folding (4 -7), attenuate the synthesis of proteins (8 -10), and facilitate the degradation of unfolded proteins though ER stress transducers (11)(12)(13). Excessive and/or prolonged ER stress causes cells to undergo ER stress-induced apoptosis (14 -16). Recent advanced studies have revealed that ER stress also provides important signals for regulating cell differentiation and maturation or maintenance of cellular homeostasis (17)(18)(19)(20)(21)(22). However, it remains unclear how cells avoid the apoptosis induced by ER stress, which is necessary for the maintenance of biological functions.
Previously, we identified BBF2H7 (box B-binding factor 2 human homolog on chromosome 7) as a novel ER stress transducer (23). BBF2H7 is a basic leucine zipper (bZIP) transcription factor that belongs to the cyclic AMP-responsive element (CRE)-binding protein (CREB)/activating transcription factor (ATF) family (23). Although BBF2H7 is localized to the ER membrane under normal conditions, it is cleaved at the trans-membrane region in response to ER stress, and the processed N-terminal fragment of BBF2H7 containing the bZIP domain is translocated into the nucleus to promote the transcription of its target genes (23). BBF2H7 is preferentially expressed in proliferating chondrocytes in cartilage during the development of long bones (17). Bbf2h7-deficient (Bbf2h7 Ϫ/Ϫ ) mice exhibit severe chondrodysplasia and die from suffocation shortly after birth because of an immature chest cavity (17). Bbf2h7 Ϫ/Ϫ proliferating chondrocytes show an abnormally expanded rough ER containing aggregated extracellular matrix (ECM) proteins, such as type II collagen and cartilage oligomeric matrix protein, caused by decreased levels of Sec23a, which encodes a coat protein complex II component responsible for protein transport from the ER to the Golgi and is a BBF2H7 target gene in chondrocytes (17). The cartilage of Bbf2h7 Ϫ/Ϫ mice also contains a decreased number of chondrocytes (17), indicating that Bbf2h7 Ϫ/Ϫ chondrocytes are promoted toward apoptosis or attenuated proliferation during chondrogenesis. Although these findings suggest that BBF2H7 is involved in the apoptosis and/or proliferation of chondrocytes, the detailed relationships between BBF2H7 and these phenomena are still unknown.
Atf5 (activating transcription factor 5) is another member of the CREB/ATF family (24 -27). The ability of ATF5 to regulate pathways that are integrated with the transduction cascades controlling apoptosis has been investigated in cell types from the lymphocytic lineage (28). When ATF5 is stably expressed in an IL-3-dependent cell line, cell apoptosis is suppressed through cytokine deprivation (29). Inhibition of endogenous ATF5 activity by the introduction of a dominant-negative form of ATF5 leads to apoptosis in asynchronously growing cells, even in the presence of growth factors (28 -30). Interference with the ATF5 function results in glioma cell death in primary tumors (31,32). The downstream effector of the ATF5-mediated survival pathway is Mcl1 (myeloid cell leukemia sequence 1), which belongs the antiapoptotic B-cell leukemia/lymphoma 2 family and is a target gene of ATF5 in malignant glioma cells (33). These previous data indicate that ATF5 is an antiapoptotic factor that plays important roles in promoting survival and inhibiting apoptosis. Here, we show that BBF2H7 activated by ER stress directly up-regulates the ATF5-MCL1 antiapoptotic pathway to avoid ER stress-induced apoptosis caused by the production of abundant ECM proteins leading to an enhanced burden on the ER.

EXPERIMENTAL PROCEDURES
Generation of Bbf2h7 Ϫ/Ϫ Mice-Bbf2h7 Ϫ/Ϫ mice were previously established in our laboratory (17). In all studies comparing WT and Bbf2h7 Ϫ/Ϫ mice, sex-matched littermates derived from the mating of Bbf2h7 ϩ/Ϫ mice were used. The experimental procedures and housing conditions for the animals were approved by the Committee of Animal Experimentation at Hiroshima University.
Histological Analysis-The limbs of embryonic day 18.5 (E18.5) mice were fixed overnight in 10% formalin neutral buffer solution (Wako). The samples were then decalcified in Morse solution, embedded in paraffin, and sectioned at 5-m thickness. Hematoxylin-eosin (H&E) staining was performed according to standard protocols.
Skeletal Preparations-E18.5 mice were skinned, and muscles and organs were removed as possible. Mice were fixed with 99% ethanol (Wako) at room temperature. After staining of 0.15% Alcian blue (Sigma), they were cleared in 1% KOH (Wako) for 1 day. Then they were stained with 0.15% alizarin red S (Sigma).
TUNEL Staining-TUNEL assays were performed using the DeadEnd TM fluorometric TUNEL system (Promega), according to the manufacturer's protocol. For cells, primary cultured chondrocytes prepared from E18.5 WT and Bbf2h7 Ϫ/Ϫ mice were fixed in 4% PFA and then permeabilized in 0.1% sodium citrate and 0.1% Triton X-100. Cells were visualized under a fluorescence microscope. The number of positive cells was measured in five fields per well.
Immunohistochemistry-Limbs were fixed in 4% formalin and then decalcified with Morse's solution. Subsequently, the samples were dehydrated with ethanol, embedded in paraffin, and sectioned (5 m). The sections were stained with antibody and visualized under a fluorescence microscope (BX51, Olympus) or a confocal microscope (FV1000D, Olympus). The following antibody and dilution were used: anti-cleaved caspase 3 (1:200; Promega).
In Situ Hybridization-In situ hybridization was performed using digoxigenin-labeled Col2 (type II collagen) and Col10 (type X collagen) antisense RNA (cRNA) probes (17). The Col2 and Col10 cRNA probes were obtained by in vitro transcription in the presence of digoxigenin-labeled dUTP using various cDNAs subcloned into the pGEM-Teasy vector (Promega) as templates. Limbs were fixed in 4% formalin and then decalcified with Morse's solution. Frozen sections (5 m) were treated with 0.1% proteinase K for 5 min. After washing with PBS, the sections were refixed with 4% formalin for 20 min and treated with 0.1 M triethanolamine, 2.5% anhydrous acetic acid for 10 min. The sections were prehybridized for 1 h at 37°C in hybridization buffer (0.01% dextran sulfate, 0.01 M Tris-HCl, pH 8.0, 0.05 M NaCl, 50% formamide, 0.2% sarcosyl, 1ϫ Denhardt's solution, 0.5 mg/ml yeast tRNA, 0.2 mg/ml salmon testis DNA) and hybridized with the probes overnight at 55°C. After washing with 4ϫ saline sodium citrate buffer for 20 min at 60°C followed by 2ϫ saline sodium citrate buffer, 50% formamide for 30 min at 60°C, the sections were treated with RNase A in RNase buffer (10 mM Tris-HCl, pH 7.4, 1 mM 0.5 M EDTA, pH 8.0, 0.5 M NaCl) for 30 min at 37°C to remove non-hybridized probes. After RNase treatment, the sections were washed with 2ϫ saline sodium citrate buffer, 50% formamide for 30 min at 60°C and blocked with 1.5% blocking reagent in 100 mM Tris-HCl, pH 7.5, containing 150 mM NaCl for 60 min at room temperature. For detection of digoxigenin-labeled cRNA probes, an anti-digoxigenin antibody conjugated to alkaline phosphatase (Roche Applied Science) was used at a dilution of 1:500, and color was developed by incubation with 4-nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate solution.
Cell Cultures and Treatment-Primary rib chondrocytes were cultured as described previously (17). Briefly, chondrocytes were isolated from E18.5 mouse rib cartilage using 0.2% collagenase D (Roche Applied Science) after adherent connective tissue had been removed by 0.2% trypsin (Sigma) pretreat-ment. The isolated chondrocytes were maintained in ␣-MEM (Invitrogen) supplemented with 10% FCS. The medium was changed every 3 days and on the day of the assays to create identical conditions in each dish. Micromass cultures were performed as described previously (17). Briefly, mesenchymal cells were prepared from the limbs of E11.5 mice and digested with 0.1% trypsin and 0.1% collagenase D. Cells at 1 ϫ 10 7 cells/ml were plated and maintained in ␣-MEM supplemented with 100 ng/ml bone morphogenic protein-2 (Sigma), 50 g/ml ascorbic acid, and 5 nM ␤-glycerophosphate. The cells were treated with thapsigargin (Tg) (Wako), tunicamycin (Tm) (Sigma), staurosporine (STS) (Sigma), and etoposide (Etop) (Wako) for the indicated times. Adenovirus vectors expressing the mouse ATF5 were constructed using the AdenoX expression system (Clontech), according to the manufacturer's protocol. A recombinant adenovirus carrying the mouse BBF2H7 N terminus and Sec23a was generated previously (17).
Cell Proliferation and Cell Counting Assay-For the cell proliferating analysis using WST-8 solution (Dojindo), a total of 1 ϫ 10 4 cells/ml of primary cultured chondrocytes were plated in a 96-well microplate. 10 l of WST-8 was added to each well at the indicated times, and the plate was incubated for 2 h at 37°C. Absorbance was measured at 450 nm using a microplate reader (Bio-Rad). For the cell counting assay, cells were visualized under microscope and manually counted.
Luciferase Assay-A reporter gene carrying a 4.0-kb promoter region of Atf5 and the reporter construct mut CRE were kind gifts from Michael R. Green (University of Massachusetts Medical School). Primary chondrocytes were cultured for 4 days and then transfected with 0.2 g of pGL3 basic reporter plasmid carrying the firefly luciferase gene (Promega) and 0.02 g of reference plasmid pRL-SV40 carrying the Renilla luciferase gene under the control of the SV40 enhancer and promoter (Promega), together with 0.2 g of an effector protein expression plasmid, using Lipofectamine 2000 reagent (Invitrogen). After 24 h, the luciferase activities were measured using the Dual-Luciferase reporter assay system (Promega) and a GLO-MAX Multiϩ detection system (Promega), according to the manufacturer's protocol. The relative activity was defined as the ratio of firefly luciferase activity to Renilla luciferase activity.
Statistical Analysis-Statistical comparisons were made using the unpaired Student's t test. The difference between two samples was considered to be statistically significant for a p value of less than 0.05.

The Number of Chondrocytes Is Decreased in Bbf2h7
Ϫ/Ϫ Mice-BBF2H7 contains a transmembrane domain, a transcription activation domain, and a bZIP domain at its N terminus (supplemental Fig. 1A). We previously reported that Bbf2h7 Ϫ/Ϫ mice exhibit severe chondrodysplasia associated with short limbs, a protruding tongue, and a distended belly (supplemental Fig. 1B) (17). Skeletal preparations at E18.5 showed extreme hypoplasia of the craniofacial bones and a reduction in cartilage ECM proteins in the limbs (supplemental Fig. 1C) caused by a reduction in Sec23a, which is one of the target genes for BBF2H7 in chondrocytes and recruits other components of coat protein complex II vesicles, such as Sec13/ 31, followed by completion of the complex before transporting secretory proteins, including cartilage ECM proteins (17). H&E staining of Bbf2h7 Ϫ/Ϫ cartilage sections showed a lack of the typical columnar structure in the proliferating zone with a decreased number of chondrocytes (supplemental Fig. 2). These data indicate that chondrogenesis and cartilage formation are disrupted by the reduced secretion of cartilage ECM proteins and the significantly decreased number of chondrocytes in Bbf2h7 Ϫ/Ϫ mice. BBF2H7 may be involved in not only secretion of cartilage ECM proteins but also maintenance of the number of chondrocytes during chondrogenesis.
Apoptosis Is Promoted and Proliferation Is Inhibited in Bbf2h7 Ϫ/Ϫ Cells-It is well known that prolonged ER stress causes cells to undergo ER stress-induced apoptosis (14 -16). We reported that ER stress induced by the synthesis of abundant ECM proteins occurs during the differentiation of chon-drocytes (17). Furthermore, we showed that Bbf2h7 knockdown cells are vulnerable to ER stress-induced apoptosis (23). Therefore, we hypothesized that the decreased number of chondrocytes in Bbf2h7 Ϫ/Ϫ mice compared with WT mice is caused by the ER stress-induced apoptosis. To examine this possibility, we performed TUNEL staining of epiphyseal cartilage sections from WT and Bbf2h7 Ϫ/Ϫ mice (Fig. 1A). In the proliferating zone of cartilage, the number of TUNEL-positive cells was higher in Bbf2h7 Ϫ/Ϫ mice than in WT mice (Fig. 1, A  and B). The number of cells positive for cleaved caspase 3, a critical executioner of apoptosis, was higher in Bbf2h7 Ϫ/Ϫ mice than in WT mice (Fig. 1, C and D). Furthermore, the level of cleaved caspase 3 was increased in primary cultured Bbf2h7 Ϫ/Ϫ chondrocytes treated with Tg (an ER stressor) compared with WT chondrocytes (Fig. 2, A and B), coinciding with the in vivo data. These findings indicate that Bbf2h7 Ϫ/Ϫ chondrocytes were vulnerable to ER stress and that apoptosis was promoted compared with WT chondrocytes. We further investigated whether proliferation was inhibited in primary cultured Bbf2h7 Ϫ/Ϫ chondrocytes. The data from cell counting (Fig. 2C) and WST-8 (Fig. 2D) assays showed that the number of Bbf2h7 Ϫ/Ϫ chondrocytes was lower than that of WT chondrocytes. These findings suggest that inhibition of proliferation as well as progression of apoptosis was observed in Bbf2h7 Ϫ/Ϫ chondrocytes.
The ATF5-MCL1 Pathway Is Inhibited in Bbf2h7 Ϫ/Ϫ Chondrocytes-Previously, it was reported that BBF2H7 can activate the transcription of Atf5 in malignant glioma (33). ATF5 is an antiapoptotic factor that plays an important role in promoting the survival of various types of cells (28,29,34). Atf5 may be one of the direct target genes of BBF2H7 in chondrocytes, and it is possible that BBF2H7 is involved in the inhibition of apoptosis through up-regulation of Atf5. Therefore, we examined the expression levels of Atf5 during chondrocyte differentiation using micromass cultures of mesenchymal cells. Atf5 was transiently up-regulated during the differentiation of mesenchymal cells into chondrocytes, synchronizing with the up-regulation of Bbf2h7 (Fig. 3A). However, the expression of Atf5 was markedly inhibited in Bbf2h7 Ϫ/Ϫ cells (Fig. 3A). Because BBF2H7 is activated in response to ER stress, Atf5  OCTOBER 19, 2012 • VOLUME 287 • NUMBER 43

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could be induced in chondrocytes exposed to ER stress if Atf5 is a direct target of BBF2H7 in chondrocytes. Indeed, we found that Atf5 was induced in WT chondrocytes during ER stress (Fig. 3C). In contrast, the induction of Atf5 was significantly attenuated in Bbf2h7 Ϫ/Ϫ chondrocytes (Fig. 3C). Infection of Bbf2h7 Ϫ/Ϫ chondrocytes with an adenovirus expressing the BBF2H7 N terminus resulted in significant up-regulation of Atf5 (Fig. 3C). These findings support the idea that Atf5 may be a direct target of BBF2H7 in chondrocytes. It has been reported that ATF5 promotes cell survival by activating the transcription of Mcl1, an antiapoptotic Bcl2 family member (33). We investigated the expression levels of Mcl1 during the differentiation of mesenchymal cells into chondrocytes. There were no differences in the expression patterns between WT and Bbf2h7 Ϫ/Ϫ cells (Fig. 3A). However, when micromass-cultured cells were treated with Tg, the up-regulation of Mcl1 was inhibited in Bbf2h7 Ϫ/Ϫ cells (Fig. 3B). The expression of Mcl1 was lower in primary cultured Bbf2h7 Ϫ/Ϫ chondrocytes than in WT chondrocytes during ER stress (Fig. 3D). In Bbf2h7 Ϫ/Ϫ chondrocytes, Mcl1 expression was up-regulated by overexpression of the BBF2H7 N terminus (Fig. 3D). Furthermore, we generated adenoviruses expressing ATF5 to examine whether MCL1 was up-regulated by ATF5 in chondrocytes (Fig. 3E). We found that Mcl1 expression was significantly up-regulated by overexpression of ATF5 (Fig. 3F). Thus, we concluded that ATF5 activated by BBF2H7 up-regulates MCL1 expression in chondrocytes. However, it is possible that Mcl1 is mainly regulated by ATF5 and that its expression may be affected by other regulators.
We previously identified Sec23a as one of the target genes of BBF2H7 in cartilage (17). Therefore, we examined whether Sec23a expression affects Atf5 expression by infecting adenoviruses expressing Sec23a or ATF5 into Bbf2h7 Ϫ/Ϫ chondrocytes. As shown in Fig. 3, G and H, Sec23a overexpression did not affect ATF5 expression, and ATF5 overexpression did not affect Sec23a expression. We concluded that the ATF5-mediated pathway is independent of the Sec23a-mediated pathway.
Atf5 Is a Direct Target of BBF2H7 in Chondrocytes-To determine whether BBF2H7 acts on the Atf5 promoter region and activates the transcription of Atf5 in chondrocytes, we performed reporter assays with a reporter gene carrying the 4.0-kb promoter region of Atf5 (4.0kb-Luc) (Fig. 4A). In Bbf2h7 Ϫ/Ϫ chondrocytes transfected with the 4.0kb-Luc construct, the reporter activities were dramatically reduced (Fig. 4B). Furthermore, the reporter activities were significantly induced by expression of the BBF2H7 N terminus into Bbf2h7 Ϫ/Ϫ chondrocytes (Fig. 4B). The Atf5 promoter includes a CRE sequence (Ϫ632 to Ϫ639 bp), to which BBF2H7 can bind (32). Next, we performed promoter assays using the reporter construct mut CRE, which has a mutated CRE sequence (Fig. 4A). In chondrocytes transfected with the construct, the reporter activities were markedly reduced (Fig. 4B). Furthermore, we performed ChIP assays and detected a high level of BBF2H7 binding to the endogenous Atf5 promoter in chondrocytes expressing the BBF2H7 N terminus (Fig. 4, C-E). These findings indicate that  OCTOBER 19, 2012 • VOLUME 287 • NUMBER 43 BBF2H7 directly acts on the CRE sequence within the Atf5 promoter and facilitates its transcription in chondrocytes.

BBF2H7 Suppresses ER Stress-induced Apoptosis in Cartilage
The BBF2H7-ATF5-MCL1 Pathway Inhibits ER Stress-induced Apoptosis-It has been reported that ATF5 contributes to the inhibition of apoptosis (28,29). Apoptosis is induced via two main routes involving either mitochondria (internal pathway) (34) or activation of death receptors (external pathway) (35,36). Both pathways converge to activate caspases as the final executioners of cell death (37,38). In addition, it has been revealed that apoptosis is induced by the ER stress response (14 -16). Because BBF2H7 is activated in response to ER stress, the BBF2H7-ATF5-MCL1 pathway could specifically inhibit ER stress-induced apoptosis. To examine this possibility, we performed TUNEL staining using primary cultured WT and Bbf2h7 Ϫ/Ϫ chondrocytes treated with various agents (Fig. 5,  A-D). The numbers of cells for TUNEL-positive staining did not differ between WT and Bbf2h7 Ϫ/Ϫ chondrocytes treated with STS (Fig. 5A) or Etop (Fig. 5B), which do not induce ER stress. In Bbf2h7 Ϫ/Ϫ chondrocytes, the numbers of cells for TUNEL-positive staining were significantly higher than those of WT chondrocytes after treatment with Tm (Fig. 5C) or Tg (Fig. 5D), which are ER stressors. Furthermore, cleaved caspase 3 was increased in primary cultured Bbf2h7 Ϫ/Ϫ chondrocytes treated with the ER stressors, but not STS or Etop, compared with WT chondrocytes (Fig. 5, E and F). These data indicate that Bbf2h7 Ϫ/Ϫ chondrocytes are vulnerable to ER stress-induced apoptosis. Finally, we examined whether the ATF5-MCL1 pathway inhibits the ER stress-induced apoptosis in Bbf2h7 Ϫ/Ϫ chondrocytes. When adenoviruses expressing ATF5 were infected into Bbf2h7 Ϫ/Ϫ chondrocytes, the number of TUNEL-positive cells and the cleaved caspase 3 levels were decreased compared with non-treated Bbf2h7 Ϫ/Ϫ chondrocytes (Fig. 6, A-C). These findings indicate that BBF2H7-ATF5-MCL1 signaling specifically inhibits ER stress-induced apoptosis. Taken together, our data suggest that BBF2H7 plays an essential role in the inhibition of apoptosis induced by ER stress during chondrocyte differentiation (Fig. 6D).

DISCUSSION
We previously demonstrated that BBF2H7 is activated in response to ER stress and promotes chondrocyte differentiation and cartilage formation through regulation of cartilage ECM protein secretion by activating the transcription of Sec23a, a coat protein complex II component (17). Although ER stress activated by the synthesis of abundant ECM proteins is essential for chondrogenesis, the mechanisms for how cells prevent ER stress-induced apoptosis have remained unclear. In this study, we found that ATF5, another direct target of BBF2H7, inhibits ER stress-induced apoptosis and maintains the number of chondrocytes against ER stress. Therefore, BBF2H7 controls two essential mechanisms, namely 1) acceleration of ECM protein secretion followed by promotion of cartilage formation and 2) avoidance of ER stress-induced apoptosis caused by abundant cartilage ECM protein synthesis by activating the transcription of Atf5. In this context, our study strongly suggests that BBF2H7 possesses a critical ability to act as a bifunctional regulator activated by ER stress during chondrogenesis. Additionally, Bbf2h7 Ϫ/Ϫ chondrocyte proliferation was inhibited compared with WT chondrocytes. To maintain the number of chondrocytes, BBF2H7 may activate the transcription of proliferation-related genes as well as antiapoptotic factors.
We found that Atf5 is a target gene of BBF2H7 in chondrocytes. This conclusion is supported by the following facts: 1) Atf5 was significantly down-regulated in primary cultured chondrocytes from Bbf2h7 Ϫ/Ϫ mice; 2) exogenous expression of the BBF2H7 N terminus, the active form of BBF2H7, up-regulated Atf5 expression; 3) the promoter activities of the Atf5 gene were facilitated by BBF2H7; 4) a CRE sequence, to which BBF2H7 can bind, exists in the promoter region of Atf5; 5) the Atf5 promoter activities were drastically reduced by a mutation in the CRE sequence; and 6) BBF2H7 directly bound to the Atf5 promoter region. These findings and those in a previous report indicate that ATF5 activates the transcription of Mcl1 to suppress apoptosis (32). However, in micromass cultures of mesenchymal cells, the expression patterns of Mcl1 showed no significant differences between WT and Bbf2h7 Ϫ/Ϫ cells. We revealed that Mcl1 is mainly regulated by the BBF2H7-ATF5 pathway in chondrocytes. However, Mcl1 could be regulated by other factors as well as ATF5, or ATF5 could suppress the apoptosis to induce the expression of not only Mcl1 but also other target genes. The expression levels of various antiapoptotic factors should be precisely analyzed to elucidate the detailed mechanisms underlying the suppression of ER stress-induced apoptosis regulated by BBF2H7, ATF5, and MCL1.
The process of apoptosis is controlled by diverse cell signaling pathways from mitochondria; death receptors, such as FAS and TNF receptor; and the ER (39 -42). We previously demonstrated that chondrocytes have an increased ER burden caused by the synthesis and secretion of large amounts of ECM proteins during differentiation (17). It is inevitable that ER stress is augmented in various secretory cells, including chondrocytes, osteoblasts, goblet cells, and astrocytes (17)(18)(19)(20)(21)(22). These cells may activate specific pathways to avoid apoptosis induced by ER stress. However, the mechanism for how such ER stress activates only unfolded protein response signaling, which is essential for cell differentiation, and does not cause ER stressinduced apoptosis remains unclear. Our study has revealed that BBF2H7 plays a crucial role in suppressing ER stress-induced apoptosis caused by the synthesis of abundant ECM proteins by directly activating the ATF5-MCL1 antiapoptotic pathway. It is possible that the BBF2H7-ATF5-MCL1 pathway may also be activated during the differentiation of various other secretory cells.
We previously reported that Bbf2h7 Ϫ/Ϫ mice exhibit severe chondrodysplasia caused by decreased secretion of ECM proteins (17). It has also been reported that one of the etiologies of chondrodysplasia, such as thanatophoric dysplasia, is an increase in the number of apoptotic chondrocytes caused by the accumulation of mutated FGF receptor 3 in the ER lumen followed by extension of ER stress (43,44). It is possible that the chondrodysplasia observed in Bbf2h7 Ϫ/Ϫ mice causes not only the decrease in ECM proteins in the extracellular region but also the decrease in the number of chondrocytes through ER stress-induced apoptosis, similar to thanatophoric dysplasia. Further advanced studies, such as precise morphological analyses, are necessary to clarify the association between BBF2H7 and the pathogenesis of chondrodysplasia.