Chondrocyte Proliferation Regulated by Secreted Luminal Domain of ...

Molecular Cell

Article Chondrocyte Proliferation Regulated by Secreted Luminal Domain of ER Stress Transducer BBF2H7/CREB3L2 Atsushi Saito,1 Soshi Kanemoto,1 Yizhou Zhang,1 Rie Asada,1 Kenta Hino,1 and Kazunori Imaizumi1,* 1Department of Biochemistry, Institute of Biomedical & Health Sciences, University of Hiroshima, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2013.11.008

SUMMARY

The endoplasmic reticulum (ER) stress transducer BBF2H7/CREB3L2 is an ER-resident transmembrane transcription factor. In response to physiological ER stress, it is processed at the transmembrane region to generate a cytoplasmic N terminus, which contains a basic leucine zipper (bZIP) domain, and luminal C terminus. The BBF2H7 N terminus functions as a transcription factor to promote the expression of ER-Golgi trafficking-related genes and plays crucial roles in chondrocyte differentiation. Here, we found that the BBF2H7 C terminus is secreted into the extracellular space as a signaling molecule for cell-to-cell communication. The secreted BBF2H7 C terminus directly binds to both Indian hedgehog and its receptor Patched-1, followed by activation of Hedgehog signaling, resulting in promoting the proliferation of neighboring chondrocytes. The dual N- and C-terminal functions of BBF2H7 triggered by physiological ER stress may allow chondrocytes to simultaneously regulate distinct cellular events for differentiation and proliferation in developing cartilage.

INTRODUCTION Eukaryotic cells adapt to endoplasmic reticulum (ER) dysfunction due to accumulation of unfolded proteins by producing signals from the ER to the cytosol or nucleus. These signaling pathways are collectively known as unfolded protein responses (UPRs) (Ron, 2002; Kaufman, 2002). Recent studies have shown that UPR signaling is involved not only in dealing with unfolded proteins but also in biological regulation, including development and differentiation (Reimold et al., 2001; Zhang et al., 2006; Vecchi et al., 2009). The canonical branches of the UPR are mediated by three ER membrane-bound proteins: PERK (Harding et al., 1999), IRE1 (Tirasophon et al., 1998), and ATF6 (Yoshida et al., 2000). These ER stress transducers possess an ER stress-sensing function in their ER luminal domains (Bertolotti et al., 2000; Shen et al., 2002) and play important roles in cell

survival after ER stress. Additionally, novel types of ER stress transducers that share sequence similarity with ATF6, and are structurally included in the CREB/ATF family, have been identified: Luman/CREB3 (Lu et al., 1997), OASIS/CREB3L1 (Kondo et al., 2005; Murakami et al., 2009), BBF2H7/CREB3L2 (Kondo et al., 2007; Saito et al., 2009), CREBH/CREB3L3 (Omori et al., 2001; Zhang et al., 2006), and CREB4/AIbZIP/CREB3L4 (Qi et al., 2002; Cao et al., 2002; Adham et al., 2005). These proteins are transmembrane basic leucine zipper (bZIP) transcription factors that are cleaved at the transmembrane domain by regulated intramembrane proteolysis (RIP) (Ye et al., 2000). Cleaved cytoplasmic N termini containing transcription activation and bZIP domains translocate into the nucleus to promote gene expression. Although the N-terminal sequences are similar to each other, the ER luminal C-terminal domains share little homology and have no recognized functional domains in their amino acid (aa) sequences. Bbf2h7 was originally identified by its involvement in gene translocation of low-grade fibromyxoid sarcoma (LGFMS) as a result of a fusion event between the fused in sarcoma (FUS) gene on chromosome 16 and the Bbf2h7 gene on chromosome 7 (Storlazzi et al., 2003). We have previously demonstrated that BBF2H7 is cleaved at the transmembrane region in response to ER stress, and its N terminus translocates into the nucleus to promote transcription of target genes (Kondo et al., 2007). BBF2H7 is preferentially expressed in chondrocytes of developing cartilage (Saito et al., 2009). Bbf2h7-deficient (Bbf2h7/) mice exhibit severe chondrodysplasia. Proliferating chondrocytes of Bbf2h7/ mice show abnormal expansion of the ER that contains aggregated extracellular matrix (ECM) proteins. We identified Sec23a, which is responsible for protein transport from the ER to the Golgi apparatus, as a target of BBF2H7 (Saito et al., 2009). BBF2H7 was activated in response to physiological ER stress caused by production of abundant cartilage matrix proteins during chondrogenesis (Saito et al., 2009). These previous findings indicate that the BBF2H7-Sec23a pathway activated by physiological ER stress plays crucial roles in chondrogenesis by activation of ECM protein secretion. The Hedgehog (Hh) signaling pathway is essential for numerous processes, including cell proliferation, differentiation, and tissue patterning during embryonic development (Varjosalo and Taipale, 2008). Although the overall activity of the pathway is diminished after embryogenesis, recent reports show that uncontrolled activation of the Hh signaling pathway results in Molecular Cell 53, 127–139, January 9, 2014 ª2014 Elsevier Inc. 127

Molecular Cell BBF2H7 C Terminus Promotes Cell Proliferation

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Figure 1. Cell Proliferation Is Inhibited in Bbf2h7–/– Cells

(A and B) Hematoxylin and eosin-stained femur cartilage from (A) WT and (B) Bbf2h7/ mice at E18.5. Proliferating zones are indicated by arrows and the dotted lines. Bars indicate 100 mm. (C) The number of proliferating chondrocytes in (A) and (B) (n = 3). (D and E) Immunohistochemical analysis of proliferating cell nuclear antigen (PCNA) in the proliferating zone of the tibia at E18.5 of (D) WT and (E) Bbf2h7/ mice. Bottom panels show higher magnifications of the white boxes in top panels. Bars indicate 100 mm (upper panels) and 10 mm (lower panels). (legend continued on next page)

128 Molecular Cell 53, 127–139, January 9, 2014 ª2014 Elsevier Inc.


the initiation of cancer formation and the survival of tumors including LGFMS (Jiang and Hui, 2008; Mo¨ller et al., 2011). The Hh signaling pathway is initiated by three Hh ligands (Sonic hedgehog [Shh], Desert hedgehog [Dhh], and Indian hedgehog [Ihh]) in mammals (Ingham and McMahon, 2001). Hh ligands are secreted into the extracellular space and bind to the transmembrane Hh receptor, Patched-1 (Ptch1) (Marigo et al., 1996). Another transmembrane protein, Smoothened, is negatively regulated by Ptch1 in the absence of Hh ligands. Once Hh ligands bind to Ptch1, Smoothened is relieved of the negative regulation. The derepressed Smoothened promotes the activation of glioma-associated oncogene (Gli) transcription factors (Ruiz i Altaba, 1998; Denef et al., 2000). Gli proteins translocate into the nucleus to promote transcription of Hh target genes such as Ptch1, Gli1, Forkhead box l1 (Foxl1), Cyclin D1, and Cyclin E1 (Duman-Scheel et al., 2002; Katoh and Katoh, 2009). Diverse Hh signaling is also modulated by several membraneassociated proteins including cell-adhesion molecule-related/ downregulated by oncogenes (Cdon), biregional Cdon-binding protein (Boc), and growth arrest-specific 1 (Gas1) (Liu et al., 2001; Tenzen et al., 2006). Although these proteins cannot act as ligands of Ptch1, they specifically bind to Hh ligands and act as a component of ligand-receptor complexes that activate Hh-dependent signaling. Ihh is expressed in prehypertrophic chondrocytes of developing cartilage and diffuses to the periarticular region to induce parathyroid hormone-related protein (PTHrP) expression. PTHrP binds to its receptor to promote chondrocyte proliferation and inhibit hypertrophic differentiation (Vortkamp et al., 1996; Lanske et al., 1996; St-Jacques et al., 1999). Thus, the Ihh-PTHrP pathway affects both chondrocyte proliferation and differentiation and eventually regulates the length of developing cartilage. However, the detailed mechanisms enabling Ihh diffused from prehypertrophic chondrocytes to efficiently bind to its receptor in the periarticular region and induce the expression of PTHrP are still unknown. Here, we found that the BBF2H7 C terminus processed in response to physiological ER stress is secreted from chondrocytes into the extracellular space as a signaling molecule and interacts with Ihh and its receptor Ptch1 of neighboring chondrocytes followed by promoting their proliferation. Our findings provide an insight into the regulation of cellular functions via cell-to-cell communication triggered by the UPR. RESULTS Inhibited Proliferation of Bbf2h7–/– Cells Bbf2h7/ mice exhibit severe chondrodysplasia and show abnormal expansion of the ER that contains aggregated ECM proteins (Saito et al., 2009). This abnormality of the ER is caused by downregulation of Sec23a, which is responsible for protein

trafficking from the ER to the Golgi apparatus. This observation suggests that BBF2H7 and its transcriptional targets play crucial roles in chondrogenesis, at least by facilitating ECM protein secretion. In addition to the ER abnormality, we found a significant decrease in the number of chondrocytes in the proliferating zone (Figures 1A–1C). As shown in a previous report, the decreased number of chondrocytes is partially caused by apoptosis (Izumi et al., 2012). However, the number of apoptotic cells in Bbf2h7/ mice was less than 10% of the reduced cell number (Figure S1A available online), indicating that apoptosis is not a major cause of the significant loss of chondrocytes in Bbf2h7/ mice. Thus, we asked whether cell proliferation was inhibited in Bbf2h7/ chondrocytes. The number of cells immunoreactive for proliferating cell nuclear antigen (PCNA), a marker of proliferating cells, was significantly reduced in the proliferating zone of Bbf2h7/ mice (Figures 1D–1F). Bromodeoxyuridine (BrdU) incorporation was also dramatically reduced (Figures 1G and 1H), and the number of cells in G0/G1 phase was higher, and those in S and M phases were lower in primary cultured Bbf2h7/ chondrocytes (Figure 1I). Furthermore, the expressions of cell cycle-related genes were significantly reduced in Bbf2h7/ chondrocytes (Figures 1J and S1B). Taken together, we concluded that cell proliferation is inhibited in Bbf2h7/ chondrocytes, and the major cause of the decreased chondrocyte number in Bbf2h7/ mice is impaired cell proliferation. Cleaved BBF2H7 C Terminus Is Secreted into the Extracellular Space To analyze the molecular mechanisms for the inhibited cell growth in Bbf2h7/ cells, we examined cell growth rates of Bbf2h7/ cells transfected with BBF2H7 full-length or processed fragments (N and C terminus) that are generated from BBF2H7 full-length by RIP (Figures S2A and S2B). Transfection of full-length BBF2H7 into Bbf2h7/ cells (primary cultured chondrocytes, Figure 2A; mouse embryonic fibroblasts [MEFs], Figure S2C) completely rescued the cell proliferation defects. However, the BBF2H7 N terminus alone, which acts as a transcription factor, did not improve the impaired cell proliferation. Surprisingly, the defects were almost completely rescued by expression of the BBF2H7 C-terminal luminal domain. These results suggest that the C terminus of BBF2H7 has the potential to promote cell proliferation. To investigate the subcellular localization of cleaved BBF2H7 fragments, MEFs were transfected with FLAG (N terminus)- and HA (C terminus)-tagged BBF2H7 (FLAG-BBF2H7-HA) (Figure 2B) under the control of doxycycline (DOX) (tet-off system). Expression of FLAG-BBF2H7-HA was induced within 6 hr after removal of DOX (Figure 2C). Importantly, full-length FLAG-BBF2H7HA was cleaved into its N- and C-terminal fragments in this system, albeit without ER stress stimuli. To follow the subcellular

(F) Percentages of PCNA-positive cells in the proliferating zone of (D) and (E) (n = 3). (G) BrdU incorporation of primary chondrocytes. Lower panels show overlapping of BrdU (red) and DAPI (blue). Bars indicate 20 mm. (H) Percentages of BrdU-positive cells in (G) (n = 3). (I) Flow cytometric analysis of primary chondrocytes. Green indicates G0/G1 phase, yellow indicates S phase, and blue indicates M phase. Arrows indicate the peaks in M phase. (J) Real-time PCR analysis of cell cycle-related genes in primary chondrocytes. Data shown are the ratios of each gene to b-actin (n = 5). All error bars represent the mean ±SD; *p < 0.05; **p < 0.01. See also Figure S1 and Tables S1 and S2.

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localization of the fragments, continued FLAG-BBF2H7-HA synthesis was shut off by addition of DOX. FLAG-tagged N termini were initially observed in the ER and gradually accumulated in the nucleus (Figure 2D). HA-tagged C termini were also initially located in the ER, but moved to the Golgi apparatus and into cytosolic puncta (Figures 2D and S2D–S2F). The puncta moved to the cell surface within 4 hr and finally dissipated (Figure 2D). These puncta positive for BBF2H7 C terminus were overlapped with VAMP2-positive exocytotic vesicles, but not with LAMP2 (lysosome marker) and EEA1 (endosome marker) (Figures S2G–S2L), suggesting that BBF2H7 C terminus processed at the Golgi apparatus moves to exocytotic vesicles. The amounts of processed BBF2H7 C terminus were not changed in primary chondrocytes treated with chloroquine (Figure 2E), indicating that the processed BBF2H7 C terminus is not subjected to the lysosomal degradation system. Immunoprecipitation followed by western blotting showed high levels of the BBF2H7 C terminus in the culture medium of wild-type (WT) cells (Figure 2F). Reporter activities were also detected in the culture medium of cells expressing full-length or C-terminal BBF2H7-luciferase fusion protein (Figures 2G and 2H). These findings suggest that BBF2H7 C terminus is secreted from cells into the extracellular space (Figure S2M). BBF2H7 C Terminus Secretion Is Promoted by Physiological ER Stress Because BBF2H7 is cleaved in response to ER stress (Kondo et al., 2007), secretion of the BBF2H7 C terminus should be promoted in chondrocytes undergoing ER stress. Indeed, the amounts of secreted C terminus markedly increased during ER stress (Figure 3A). Abundant synthesis of cartilage matrix proteins causes mild ER stress in the proliferating zone of developing cartilage (Figures S3A and S3B) (Saito et al., 2009). Then, we examined whether secretion of the BBF2H7 C terminus is promoted in response to the physiological ER stress. Infection of primary immature chondrocytes with an adenovirus expressing Sox9, which is a master transcription factor for differentiation of chondrocytes and promotes synthesis of cartilage matrix proteins (Kronenberg, 2003), caused ER stress (Figure 3B) (Saito et al., 2009). At that time, the secreted C terminus was significantly increased (Figure 3C). Next, we checked the

amounts of secreted BBF2H7 C terminus during chondrocyte differentiation using micromass culture of mesenchymal stem cells prepared from WT embryonic day (E) 11.5 limbs. Increased secretion of the C terminus was synchronized with upregulation of ER stress markers (Figures 3D, 3E, S3C, and S3D). These results indicate that physiological ER stress promotes secretion of the BBF2H7 C terminus during chondrogenesis. The luminal domains of canonical ER stress transducers PERK, IRE1, and ATF6 sense the unfolded proteins accumulated in the ER and transduce signals to the cytosol or nucleus. In contrast, BBF2H7 is activated in response to ER stress in a different manner from those of the canonical ER stress transducers. BBF2H7 is an unstable protein that is easily degraded via the ubiquitin-proteasome pathway under normal conditions (Kondo et al., 2012). ER stress conditions enhanced the stability of BBF2H7. The stabilized BBF2H7 is transported from the ER to the Golgi apparatus and then is cleaved to generate N and C termini by RIP. We confirmed the increase in amounts of BBF2H7 (full-length and C terminus) and the secreted C terminus by treatment of ER stress-induced primary chondrocytes or micromass cultured cells with MG132, an inhibitor of the ubiquitinproteasome pathway (Figures S3E and S3F). Thus, secretion of BBF2H7 C terminus is dependent on its stabilization under ER stress conditions, and ER stress-sensing function is not necessary in the luminal C terminus of BBF2H7 for its activation. We further examined whether the luminal C termini of other ER stress transducers, ATF6, OASIS, CREB4, Luman, and CREBH, are secreted from cells into the extracellular space under the control of DOX (tet-off system). We found the C termini of OASIS and CREB4 in the culture medium, in addition to that of BBF2H7 (Figures 3F–3H). However, we did not detect the C termini of other ER stress transducers, including ATF6, at least under the same conditions (Figures 3F, 3I, and S3G–S3I). Promotion of Cell Proliferation by the Secreted BBF2H7 C Terminus through Activation of Hh Signaling To test the ability of the secreted BBF2H7 C terminus to promote cell proliferation, primary Bbf2h7/ cells were exposed to culture medium collected from HEK293T cells transfected with the BBF2H7 C terminus (the culture medium is abbreviated as ‘‘C-Sup.’’) (Figure 4A). The growth rate of Bbf2h7/ cells treated

Figure 2. BBF2H7 C Termini Are Secreted into the Extracellular Space after Cleavage (A) Cell numbers of Bbf2h7/ primary chondrocytes transfected with full-length BBF2H7 (full), BBF2H7 N terminus (N), or BBF2H7 C terminus (C). Mock indicates empty vector (n = 4). (B) Schema of BBF2H7 tagged with FLAG (N terminus) and HA (C terminus). (C) Western blotting of HA (top panel) and FLAG (middle panel) using MEFs expressing FLAG-BBF2H7-HA in a tet-off system. Cells were cultured in DOX-free medium (allowing expression) for the indicated times. (D) Immunostaining of FLAG (left panels, green) and HA (right panels, green) using MEFs expressing FLAG-BBF2H7-HA in a tet-off system. KDEL (red) is an ER marker. FLAG-BBF2H7-HA expression was suppressed by returning DOX to the medium for the indicated times after induction by changing to DOX-free medium for 6 hr. Bars indicate 5 mm. (E) Western blotting of the BBF2H7 C terminus in primary chondrocytes exposed to 20 mM chloroquine (lysosome inhibitor) for 24 hr. (F) Western blotting of the endogenous BBF2H7 C terminus in primary chondrocytes. The BBF2H7 C terminus or Ihh in the culture medium (right panels) was immunoprecipitated using anti-BBF2H7 C terminus or anti-Ihh antibodies, respectively. (G) Schema of each BBF2H7 construct. Blue and green boxes indicate the signal sequence of BiP and luciferase protein, respectively. Luc. indicates luciferase, BBF2H7 full-Luc. indicates BBF2H7 full-length-Luc., BBF2H7 N-Luc. indicates BBF2H7 N terminus-Luc., BBF2H7 C-Luc. indicates BBF2H7 C terminus-Luc., and S1P site indicates site-1 protease cleavage site. (H) Reporter activities in the culture medium of primary chondrocytes expressing each BBF2H7 construct shown in (G). Mock indicates empty vector. Green fluorescent protein (GFP) was used as a negative control (n = 6). All error bars represent the mean ±SD; *p < 0.05; **p < 0.01; ***p < 0.001. See also Figure S2 and Table S2.



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Figure 3. Physiological ER Stress Promotes Secretion of the BBF2H7 C Terminus (A) Western blotting of the endogenous BBF2H7 C terminus in primary chondrocytes exposed to 1 mM thapsigargin (Tg: ER stressor) for 12 hr. The BBF2H7 C terminus or Ihh in the culture medium (right panels) was immunoprecipitated using anti-BBF2H7 C terminus or anti-Ihh antibodies, respectively. (B and C) (B) RT-PCR of ER stress-related genes and (C) western blotting of BBF2H7 in primary chondrocytes infected with an adenovirus expressing Sox9 or an empty vector (mock) at the indicated times. (B) Note that expressions of ER stress-related genes were slightly upregulated, and (C, right panels) the amount of secreted BBF2H7 C terminus was increased by expression of Sox9. The BBF2H7 C terminus or Ihh in the culture medium in (C) (right panels) was immunoprecipitated using anti-BBF2H7 C terminus or anti-Ihh antibodies, respectively. Xbp1 s indicates spliced form of Xbp1, Xbp1-u indicates unspliced form of Xbp1, and Col2 indicates type II collagen. (D and E) (D) RT-PCR of ER stress-related genes and (E) western blotting of BBF2H7 in mesenchymal stem cells maintained in micromass culture. (E, right panels) Note that an increase in the amount of secreted BBF2H7 C terminus (D) coincided with upregulation of ER stress-related genes. The BBF2H7 C terminus or Ihh in the culture medium in (E, right panels) was immunoprecipitated using anti-BBF2H7 C terminus or anti-Ihh antibodies, respectively. Col10 indicates type X collagen (hypertrophic chondrocyte marker). (F) Schema of constructs of OASIS, CREB4, and ATF6 tagged with FLAG (N terminus) and HA (C terminus). (G–I) Western blotting of HA in cell lysates (left panels) and culture medium (right panels). MEFs were transfected with each expression vector, (G) FLAG-OASISHA, (H) FLAG-CREB4-HA, and (I) FLAG-ATF6-HA, in a tet-off system. Cells were cultured in DOX-free medium for the indicated times. The C termini or IL6 in the culture medium (right panels) was immunoprecipitated using anti-HA or anti-IL6 antibodies, respectively. See also Figure S3 and Tables S1 and S2.



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Figure 4. Activation of Hh Signaling by Secreted BBF2H7 C Terminus (A) Bioassay of cell proliferation using primary Bbf2h7/ cells exposed to culture medium collected from HEK293T cells transfected with the BBF2H7 N terminus (N-Sup.), C terminus (C-Sup.), or BBF2H7 C terminus-absorbed C-Sup. by immunoprecipitation using an anti-BBF2H7 C terminus antibody (C-Sup. + Ab.). The proliferative responses of Bbf2h7/ cells were measured by counting cell numbers. (B and C) Cell numbers of primary Bbf2h7/ chondrocytes treated with (B) N-Sup., C-Sup., or (C) C-Sup. + Ab. for 7 days (n = 4). (D) Western blotting of Ihh and its target proteins in primary chondrocytes treated with the indicated media for 48 hr. Note that the levels of Ihh are unchanged between lanes. (E) Western blotting of Hh target proteins in primary chondrocytes treated with the indicated media for 48 hr. Cells were exposed to 5 mM cyclopamine-KAAD (CPN-KAAD) or 200 ng/ml purmorphamine (PPN) for 24 hr. (F) Cell numbers of primary chondrocytes treated with 2, 6, or 10 mM CPN-KAAD for 7 days (n = 3). (G) Western blotting of Hh target proteins in primary chondrocytes treated with 50 ng/ml Ihh (+Ihh) for 48 hr. To absorb Ihh, Ihh in C-Sup. was immunoprecipitated with an anti-Ihh antibody (C-Sup. anti-Ihh). (H) Western blotting of Hh target proteins using primary chondrocytes treated with 0, 10, 25, 50, 75, 100, or 125 ng/ml recombinant BBF2H7C-GST and 50 ng/ml Ihh for 48 hr. BBF2H7C-GST was prepared from culture medium of HEK293T cells transfected with an expressing vector for BBF2H7C-GST. (I) Cell numbers of primary chondrocytes treated with 0, 10, 25, 50, 75, 100, or 125 ng/ml BBF2H7C-GST and 50 ng/ml Ihh for 7 days (n = 3). All error bars represent the mean ±SD; *p < 0.05; **p < 0.01. See also Figure S4 and Table S2.



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Figure 5. Direct Interactions of the BBF2H7 C Terminus with Ihh and Ptch1

(A) Schema of BBF2H7 C-terminal constructs. Black boxes indicate GST. (B) GST pull-down assays using recombinant GST-fused BBF2H7 C-terminal deletions in (A) and recombinant Ptch1 or Ihh. Each deletion series was prepared from culture medium of HEK293T cells transfected with the expressing vectors for each deletion series. Note that binding of the BBF2H7 C terminus to Ihh was competed by incubation with 125 or 250 mM synthetic peptide fragments (aa 462–493 of the BBF2H7 C terminus). D E F (C) Western blotting of Hh target proteins in primary chondrocytes treated with the culture medium of HEK293T cells expressing the indicated C-terminal constructs for 48 hr. (D) Immunoprecipitation by anti-BBF2H7 C terminus (left panels) or anti-Ihh (right panels) antibodies followed by western blotting with the indicated antibodies in mesenchymal stem cells maintained in micromass culture for 4 days. Immunoprecipitation followed by western blotting with anti-Ihh antibody (IP:Ihh-IB:Ihh) was performed using culture media. IP indicates immunoprecipitation. IB indicates immunoblotting. (E) Western blotting of Hh target proteins in mesenchymal stem cells maintained in micromass culture for 4 days. (F) Cell numbers of mesenchymal stem cells maintained in micromass culture for 7 days (n = 3). All error bars represent the mean ±SD; *p < 0.05. See also Figure S4 and Table S2.

with C-Sup. was restored to that of WT cells, but the culture medium collected from cells transfected with the BBF2H7 N terminus (the culture medium is abbreviated as ‘‘N-Sup.’’) did not have this ability (Figure 4B). The proliferative effects of C-Sup. were completely canceled by absorption of the BBF2H7 C terminus by an anti-BBF2H7 C terminus antibody (Figure 4C), indicating that the secreted BBF2H7 C terminus has the potential to promote the proliferation of neighboring cells. BBF2H7 was originally identified as the 30 partner of FUS in the fusion gene that is specific to LGFMS (Storlazzi et al., 2003). The FUS-BBF2H7 fusion protein contains the N terminus of FUS, and the bZIP domain, transmembrane domain, and full C-terminal luminal domain of BBF2H7. Interestingly, expressions of Hh signaling targets are elevated in LGFMS (Mo¨ller et al., 2011). Ligand-dependent activation of Hh signaling is associated with cell proliferation or tumorigenesis (Jiang and Hui, 2008; Mo¨ller et al., 2011). Therefore, we hypothesized that the promotion of cell proliferation by the BBF2H7 C terminus may be linked to Hh signaling. We examined the expressions of Hh targets in Bbf2h7/ chondrocytes using microarray data (GEO: GSE18052) (Saito et al., 2009). The expressions of Hh targets including Ptch1, Gli1, Foxl1, Cyclin D1, and Cyclin E1 were almost significantly reduced at the transcriptional level. Next, the expressions of Hh target proteins in primary chondrocytes and rib cartilage from Bbf2h7/ mice were analyzed by western blotting (Figures 4D and S4A). The expressions of these proteins were dramatically decreased in both primary chondrocytes and rib cartilage from Bbf2h7/ mice. Bbf2h7/ chondrocytes treated with C-Sup. were restored the expressions of Hh targets to those in WT chondrocytes. The effect was canceled by absorbing the BBF2H7 C terminus from the culture medium. By contrast, the expressions of Hh targets were not affected 134 Molecular Cell 53, 127–139, January 9, 2014 ª2014 Elsevier Inc.

by N-Sup. Treatment with cyclopamine-KAAD (Chen et al., 2002), an antagonist of Smoothened, canceled the effects of C-Sup. on the induction of Hh targets and cell proliferation (Figures 4E and 4F). Purmorphamine (Sinha and Chen, 2006), an agonist of Smoothened, restored the expressions of Hh targets in Bbf2h7/ chondrocytes. Taken together, the BBF2H7 C terminus may act upstream of Smoothened and promote chondrocyte proliferation through activation of Hh signaling. To analyze the molecular basis of Hh activation by the BBF2H7 C terminus, we examined whether the C terminus itself acts as a ligand or cofactor of the pathway (as was shown of Cdon, Boc, and Gas1) (Liu et al., 2001; Tenzen et al., 2006). Bbf2h7/ chondrocytes were treated with C-Sup. absorbed Ihh by an anti-Ihh antibody. The Hh signaling induced by C-Sup. was completely abolished in these cells (Figure 4G). Hh signaling in Bbf2h7/ chondrocytes treated with C-Sup. and recombinant Ihh was more activated than that activated by recombinant Ihh alone. Furthermore, recombinant BBF2H7 C terminus fused to GST (BBF2H7C-GST) prepared from the culture medium of HEK293T cells transfected with an expression vector for BBF2H7C-GST enhanced the expressions of Hh targets and proliferation rates of chondrocytes treated with recombinant Ihh (Figures 4H and 4I). Thus, we concluded that the BBF2H7 C terminus promotes the expressions of Hh targets and chondrocyte proliferation as a cofactor of Ihh. Binding of the BBF2H7 C Terminus to Ihh and Ptch1 Facilitates Ligand-Receptor Complex Formation To determine the precise binding sites of the BBF2H7 C terminus to Hh or its receptor Ptch1, we constructed a series of BBF2H7 C-terminal deletions fused to GST (Figure 5A). BBF2H7 431–521, 431–461, and 431–492 bound to recombinant Ptch1, and


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BBF2H7 431–521, 431–492, and 462–493 bound to recombinant Ihh and other Hh ligands, Shh and Dhh (Figures 5B and S4B). The binding of each C-terminal construct to Ihh was competed by incubation with untagged synthetic peptide fragments (aa 462–493 of BBF2H7) in a dose-dependent manner. Furthermore, the culture medium of cells transfected with the BBF2H7 C terminus containing the binding sites for Ptch1 and Ihh (aa 431–492 of BBF2H7) activated Hh signaling in Bbf2h7/ chondrocytes (Figure 5C). These results indicate that the BBF2H7 C terminus directly binds to Ptch1 at the region from aa 431 to 461, and to Ihh at the region from aa 462 to 493, to activate Hh signaling. Finally, we confirmed these interactions during chondrocyte differentiation using micromass cultures (Figure 5D). Importantly, Ihh-Ptch1 binding was minimal in Bbf2h7/ micromass cultures, and Hh signaling and chondrocyte proliferation were accordingly reduced (Figures 5D–5F). Treatment of Bbf2h7/ micromass cultures with C-Sup. restored the complex formation, the expressions of Hh targets, and chondrocyte proliferation. A prospective schema for complex formation and regulation of Hh signaling by the BBF2H7 C terminus is presented in Figure S4C. Modulation of the Ihh-PTHrP Pathway by the BBF2H7 C Terminus It is known that Ihh-dependent PTHrP induction promotes chondrocyte proliferation and inhibits hypertrophic differentiation in developing cartilage (Vortkamp et al., 1996; Lanske et al., 1996; St-Jacques et al., 1999). Thus, the Ihh-PTHrP pathway plays a crucial role in the modulation of cartilage elongation. However, the details of this regulation are still unclear. Therefore, we examined the potential roles of the BBF2H7 C terminus in

Figure 6. Modulation of the Ihh-PTHrP Pathway by the BBF2H7 C Terminus (A) Western blotting of PTHrP and Col10 in mesenchymal micromass cultures treated with 0, 25, 50, 75, 100, or 125 ng/ml BBF2H7C-GST and 50 ng/ml Ihh for 10 days. Note that the expression of Col10 was decreased in a BBF2H7C-GST dosedependent manner and restored by neutralization of PTHrP with antibodies. (B and C) Quantification of absorbance extracted from cells stained with (B) Alcian blue and (C) alizarin red in mesenchymal micromass cultures treated with 0, 25, 50, 75, 100, or 125 ng/ml BBF2H7C-GST and 50 ng/ml Ihh for 10 days. Note that the absorbance of Alcian blue staining was increased, and that of alizarin red staining was decreased, in a BBF2H7C-GST dose-dependent manner. The effects of BBF2H7C-GST were canceled by neutralization of PTHrP (n = 3). (D) Cell numbers of primary chondrocytes prepared from WT, Bbf2h7/, Bbf2h7/ Tg-N, and Bbf2h7/ Tg-C mice (n = 3). (E) Western blotting of Hh target proteins in primary chondrocytes prepared from WT, Bbf2h7/, Bbf2h7/ Tg-N, and Bbf2h7/ Tg-C mice. (F) Western blotting of PTHrP in mesenchymal stem cells maintained in micromass culture for 10 days. All error bars represent the mean ±SD; *p < 0.05; **p < 0.01; ***p < 0.001. See also Figures S5 and S6 and Table S2.

regulation of the Ihh-PTHrP pathway. WT micromass cultures were treated with Ihh and BBF2H7C-GST and maintained for 10 days. Expression of PTHrP in this model was increased in a BBF2H7C-GST dose-dependent manner (Figure 6A). Alternatively, expression of type X collagen (Col10), a hypertrophic chondrocyte marker, was decreased. The decreased Col10 was restored by neutralizing PTHrP with antibodies. Accordingly, hypertrophic differentiation was inhibited by the C terminus-dependent induction of PTHrP. We further examined ECM protein secretion and mineralization by performing Alcian blue and alizarin red staining (Figures 6B and 6C). The staining properties of Alcian blue were gradually increased, and those of alizarin red were decreased by the treatment of micromass culture with BBF2H7C-GST in dose-dependent manner. The effects of BBF2H7C-GST were canceled by neutralization of PTHrP, indicating that the BBF2H7 C terminus suppresses hypertrophic differentiation by regulating the Ihh-PTHrP pathway. Finally, we investigated the physiological functions of BBF2H7 C terminus by generating the mice that specifically expressed BBF2H7 N or C terminus in chondrocytes (Bbf2h7/ transgene [Tg]-N or Tg-C mice). The expression of the BBF2H7 Tg in the Bbf2h7/ Tg-N or Tg-C mice was targeted to chondrocytes using a chondrocyte-specific type II collagen promoter and enhancer (Figures S5A–S5C) (Zhou et al., 1995). In vitro experiments using primary Bbf2h7/ Tg-C chondrocytes showed that cell growth rates and Hh signaling were certainly recovered to levels comparable with those in WT chondrocytes (Figures 6D and 6E). PTHrP upregulation was also detected in Bbf2h7/ Tg-C micromass cultured cells (Figure 6F). These results strongly suggest that BBF2H7 C terminus promotes cell proliferation through the activation of Hh signaling and inhibits hypertrophic Molecular Cell 53, 127–139, January 9, 2014 ª2014 Elsevier Inc. 135


differentiation by activating the Ihh-PTHrP pathway. In vivo experiments showed that the expressions of Sec23a and Hh signaling-related genes in Bbf2h7/ Tg-N and Tg-C mice were restored to those in WT mice, respectively (Figure S5D). The number of proliferating chondrocytes was increased in Bbf2h7/ Tg-C mice compared with those in Bbf2h7/ mice (Figures S5E–S5I). However, it was not completely recovered to those of WT mice. Further, diffused expression of Ihh in proliferating zone of Bbf2h7/ mice (Saito et al., 2009) could not be rescued in Bbf2h7/ Tg-N and Bbf2h7/ Tg-C mice (Figure S5J). Although the exact reasons for this remain unclear, we postulate that the C-terminal function only is likely insufficient for appropriate proliferation of chondrocytes and localization of Ihh in developing cartilage. Additionally, chondrocyte proliferation and cartilage formation in Bbf2h7/ Tg-N mice were not restored to those in WT mice. Therefore, both BBF2H7 N- and C-terminal functions could be necessary for regulating chondrocyte proliferation and differentiation and appropriate localization of Ihh in developing cartilage. DISCUSSION Canonical ER stress transducers PERK, IRE1, and ATF6 sense unfolded proteins by their ER luminal domains and transduce UPR signals by their cytoplasmic domains (Bertolotti et al., 2000; Shen et al., 2002). By contrast, the ER luminal domain of BBF2H7, one of the novel types of ER stress transducers, does not act as a sensor but as a signaling molecule for cellto-cell communication by secretion into the extracellular space. This scenario has been demonstrated by the following facts: (1) the luminal domain of BBF2H7 is secreted into the extracellular space after cleavage in response to physiological ER stress, (2) culture medium containing the luminal domain promotes cell proliferation, (3) the biological activities of such culture medium are canceled by absorption of the secreted luminal domain by antibodies, (4) the secreted luminal domain interacts with both Ihh and its receptor Ptch1 on the cell surface, and (5) cell proliferation was promoted by exposure to the luminal domain through the activation of Hh signaling. It has been reported that activation of UPR signaling in one cell effects neighboring cells to modulate their biological responses, such as the proinflammatory response, apoptosis, and UPR (Burikhanov et al., 2009; Mahadevan et al., 2011). However, the molecular basis of this cell-to-cell communication has been unclear. Mahadevan et al. (2011) reported that UPR signaling in tumor cells activates UPR signaling in neighboring macrophages, and the phenomenon is termed ‘‘transmissible ER stress.’’ Their analyses showed the possibility that the transmissible factors released by tumor cells may be heat resistant and nonprotein substances, although the precise mechanisms are still unknown. The most important finding in the present study is the discovery of UPR-mediated cell-to-cell communication and identification of a transmissible factor, and further, secreted fragments of BBF2H7 may be used as a target to develop strategies that modulate cellular functions. In addition, we observed that the ER luminal domains of other ER stress transducers, OASIS and CREB4, are secreted into the extracellular space as well as that of BBF2H7. Interestingly, the luminal C-terminal domains of these three ER stress 136 Molecular Cell 53, 127–139, January 9, 2014 ª2014 Elsevier Inc.

transducers do not share homology in their aa sequences, suggesting the possibility that the secreted C termini of these ER stress transducers possess distinct physiological functions. Elucidation of each secreted C-terminal function may uncover the biological roles of UPR-dependent cell-to-cell communication mediated by partial fragments of ER stress transducers. We found that the secreted BBF2H7 C terminus promotes the proliferation of neighboring cells through activation of Hh signaling. Hh signaling is involved in the proliferation of cells, including hematopoietic and mammary stem cells (Bhardwaj et al., 2001; Liu et al., 2006), as well as chondrocytes. It is known that binding of Hh ligands to Ptch1 activates Smoothened and Gli transcription factors, followed by inducing expressions of Hh target genes, including cell cycle-related genes, and consequently, these signals promote cell proliferation (Marigo et al., 1996; Ruiz i Altaba, 1998; Denef et al., 2000; Duman-Scheel et al., 2002). The present study demonstrated that the secreted BBF2H7 C terminus directly binds to both Ihh and Ptch1, facilitating ligand-receptor complex formation as a cofactor. Actually, Smoothened and Gli transcription factors are activated, followed by inducing expressions of cell cycle-related genes downstream of binding of the BBF2H7 C terminus to the ligand-receptor complex. Therefore, we concluded that the proliferation of chondrocytes induced by the BBF2H7 C terminus is caused by activation of canonical Hh signaling. Hh signaling has been known to regulate not only cell proliferation but also differentiation (Alberta et al., 2001; Varjosalo and Taipale, 2008). One of the Hh ligands, Shh, is required for development of oligodendrocyte precursors (OLPs) but does not affect the proliferation of OLPs (Alberta et al., 2001). It is not known how the mechanisms responsible for OLP development are regulated by Hh signaling. Furthermore, it remains unclear how the variable roles of Hh signaling are determined in each cell type. One possibility of these mechanisms is that Hh ligand-receptor cofactors such as BBF2H7 C terminus may modulate biological roles including proliferation or differentiation downstream of Hh receptor activation. Previous studies have reported that Cdon, Boc, and Gas1 modulate Hh signaling to act as cofactors of the Hh ligand-receptor complex (Liu et al., 2001; Tenzen et al., 2006). It is necessary to clarify the downstream signals activated by each Hh ligand-receptor complex consisting of distinct cofactors to elucidate the multiple roles of Hh signaling in each cell type. It is well known that Ihh, which is expressed in prehypertrophic chondrocytes, induces the expression of PTHrP in the periarticular region (Vortkamp et al., 1996; Lanske et al., 1996; St-Jacques et al., 1999). In this unique regulation of PTHrP expression by Ihh, it has been unclear how the infinitesimal amounts of Ihh diffused to the periarticular region remote from prehypertrophic chondrocytes substantially activate the downstream pathway. On the basis of the evidence that the BBF2H7 C terminus is expressed in proliferating chondrocytes and binds to Ihh, we speculate the roles of the BBF2H7 C terminus in the diffusion and action of Ihh in this distinct area are as follows: a large amount of the BBF2H7 C terminus secreted from proliferating chondrocytes traps the infinitesimal amounts of Ihh released from prehypertrophic chondrocytes, and the heterodimers diffuse in the direction of the periarticular region. The heterodimers bind to Ptch1 at the cell surface of periarticular


chondrocytes, thereby inducing the expression of PTHrP (Figure S6). In this context, the BBF2H7 C terminus might support the biological functions of Ihh by efficient delivery of the infinitesimal amounts of Ihh from prehypertrophic chondrocytes to the periarticular region. Ihh at the periarticular region regulates PTHrP expression, and the pathway is involved in inhibition of differentiation from proliferating chondrocytes to hypertrophic chondrocytes. During growth of developing cartilage, exponential proliferation and formation of an ECM network are synchronized in proliferating chondrocytes. BBF2H7 is cleaved to generate N and C terminus in response to physiological ER stress. The cleaved N terminus acts as a transcription factor to promote secretion of ECM proteins, and the cleaved C terminus is involved in proliferation of chondrocytes and inhibition of its differentiation into hypertrophic chondrocytes via regulating the Ihh-PTHrP pathway. These dual functions of BBF2H7 may contribute to the efficient growth of developing cartilage. In conclusion, we found that partial fragments of ER stress transducer BBF2H7 are secreted into the extracellular space and act as signaling molecules for cell-to-cell communication, resulting in promoting the proliferation of neighboring cells. Thus far, the signaling from ER stress transducers has been restricted only to intracellular events, but our findings provide an insight into its roles in extracellular events. EXPERIMENTAL PROCEDURES Mice C57BL/6 mice and Bbf2h7/ mice (established in our lab) (Saito et al., 2009) were used in this study. BBF2H7 Tg-N and Tg-C mice were generated using a p3000i3020Col2a1 vector (Zhou et al., 1995). The linearized construct was injected into fertilized eggs derived from C57BL/6 mice by SLC, Inc. (Shizuoka). The experimental procedures and housing conditions for animals were approved by the Committee of Animal Experimentation, Hiroshima University. Cell Culture Primary chondrocytes and MEFs were prepared from rib cartilage and skin, respectively, at E18.5 WT and Bbf2h7/ mice using previously published protocols (Saito et al., 2009). Micromass culture was performed as previously described (Saito et al., 2009). HEK293T cells were grown in a-MEM containing 10% fetal calf serum (FCS) (HyClone). MG132 (Calbiochem), cyclopamineKAAD (Calbiochem), purmorphamine (Wako), thapsigargin (Sigma), recombinant Ihh (BD), and 10 mg/ml anti-PTHrP neutralizing antibody (Bachem) were used for the treatment of cells. Tet-off System Full-length mouse BBF2H7, OASIS, CREB4, ATF6, Luman, and CREBH tagged with FLAG (N terminus) and HA (C terminus) were inserted into pTRE2hyg (Clontech). For tet-off studies, MEFs were grown in Dulbecco’s modified Eagle’s medium containing 10% FCS, 100 mg/ml G418, 100 mg/ml hygromycin B, and 1 mg/ml DOX (Clontech) using previously published protocols (Kondo et al., 2012). Antibodies Anti-BBF2H7 C terminus polyclonal antibody was generated by immunizing mice against mouse recombinant BBF2H7 (aa 431–521). Anti-BBF2H7 N terminus polyclonal antibody was generated previously (Saito et al., 2009). Other antibodies were summarized in Table S2. Histological Analysis Limbs of E18.5 mice were fixed in 10% formalin and then decalcified with Morse’s solution. Hematoxylin and eosin staining (4 mm paraffin sections)

and immunohistochemistry (4 mm frozen sections) were visualized under a confocal microscope (FV1000D, Olympus). In situ hybridization was performed using previously published protocols (Saito et al., 2009). Quantitative Real-Time PCR and RT-PCR Total RNA was isolated using RNeasy kit (QIAGEN) according to manufacturer’s protocol. Primer sequences are summarized in Table S1. Bioassay of Cell Proliferation Cells were plated at 1 3 104 cells/dish (primary chondrocytes and MEFs) or 2 3 105 cells per 20 ml (mesencymal stem cells) and cultured for 24 hr. Cell number was counted on day 7 after transfection with each BBF2H7 construct or treatment with culture medium of HEK293T cells transfected with BBF2H7 N or C terminus. For BrdU-incorporation assay, primary chondrocytes were plated at 5 3 104 cells/well and treated with 10 mM BrdU (Sigma) for 24 hr. For flow cytometric analysis, primary chondrocytes plated at 1 3 106 cells/dish were treated with 2.5 mM thymidine (Sigma) for 16 hr. Cells were fixed by 100% ethanol for 2 hr at 30 C. After the centrifugation, the pellets were resuspended with 300 ml 0.1% triton and 300 ml PI/RNase Staining Buffer (BD Pharmingen). FACSCalibur apparatus (BD Biosciences) and FlowJo software (Tree Star) were used for analysis. GST Pull-Down Assay Recombinant BBF2H7 C-terminal deletions fused to GST were prepared from culture medium of HEK293T cells transfected with the expressing vectors for each BBF2H7 C-terminal deletion fused to GST. Purification of C-terminal deletions fused to GST using a glutathione Sepharose 4B column was performed using standard protocols. One hundred nanograms each BBF2H7 C terminusGST protein and 100 ng Ihh, Shh, and Dhh protein (R&D Systems) or 100 ng recombinant Ptch1 were incubated within 500 ml buffer containing 20 mM HEPES (pH 7.2), 150 mM NaCl, and 0.1% NP-40 for 90 min at room temperature. The precipitated proteins were eluted by adding 43 SDS sample buffer and detected by immunoblotting with anti-Ihh, anti-Shh, anti-Dhh, or antiPtch1 antibodies. Synthetic peptides (aa 462–493 of BBF2H7 C terminus) (GenScript) were used as a competitor. Immunoprecipitation Assay Cell lysates or supernatants were incubated with anti-BBF2H7 C terminus, anti-Ihh, anti-HA, or anti-IL6 antibodies overnight. Samples were incubated with protein G-Sepharose beads (GE Healthcare) and washed with TNE buffer (10 mM Tris, 1 mM EDTA, and 150 mM NaCl). Alcian Blue and Alizarin Red Staining Micromass cultured cells were stained as previously described (Saito et al., 2009). Each absorbance was measured using a spectrophotometer (Bio-Rad) at 595 nm (Alcian blue) and 570 nm (alizarin red). Luciferase Assay Primary chondrocytes were transfected with pcDNA3.1+ inserted BBF2H7luciferase constructs. Reporter activities of culture media were measured using Dual-Luciferase Reporter Assay System (Promega) and Glomax-Multi Detection System (Promega), according to manufacturer’s protocol. Statistical Analysis Statistical comparisons were made using unpaired Student’s t test between two samples. The statistical significance of a difference between each sample was determined on the basis of p value < 0.05. p values less than 0.05, 0.01, or 0.001 were described as *p < 0.05, **p < 0.01, or ***p < 0.001, respectively.

SUPPLEMENTAL INFORMATION Supplemental Information includes six figures and two tables and can be found with this article online at http://dx.doi.org/10.1016/j.molcel.2013. 11.008.



ACKNOWLEDGMENTS

Kaufman, R.J. (2002). Orchestrating the unfolded protein response in health and disease. J. Clin. Invest. 110, 1389–1398.

The p3000i3020Col2a1 vector was kindly gifted by Dr. R. Nishimura (University of Osaka). We thank K. Takedachi and S. Nakagawa for technical support. This work was partly supported by grants from the Japan Society for the Promotion of Science KAKENHI (#25251014, #25650069, #24659678, and #24689058), the Takeda Science Foundation, the SEI Group CSR Foundation, the Inamori Foundation, The Uehara Memorial Foundation, the Research Grant of the Princess Takamatsu Cancer Research Fund, and the Cell Science Research Foundation.

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