1. Volume 53, Issue 1, Pages 127-139 (January 2014)
Chondrocyte Proliferation Regulated by Secreted Luminal Domain of ER Stress Transducer BBF2H7/CREB3L2 
Atsushi Saito, Soshi Kanemoto, Yizhou Zhang, Rie Asada, Kenta Hino, Kazunori Imaizumi 
Molecular Cell 
Volume 53, Issue 1, Pages (January 2014)
DOI: /j.molcel
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2. 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 μm.
(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 μm (upper panels) and 10 μm (lower panels).
(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 μm.
(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 β-actin (n = 5). All error bars represent the mean ±SD; ∗p < 0.05; ∗∗p < See also Figure S1 and Tables S1 and S2.
Molecular Cell  , DOI: ( /j.molcel )
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3. 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 μm.
(E) Western blotting of the BBF2H7 C terminus in primary chondrocytes exposed to 20 μM 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 < See also Figure S2 and Table S2.
Molecular Cell  , DOI: ( /j.molcel )
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4. 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 μM 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-OASIS-HA, (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.
Molecular Cell  , DOI: ( /j.molcel )
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5. 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 μM 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 μM 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 < See also Figure S4 and Table S2.
Molecular Cell  , DOI: ( /j.molcel )
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6. 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 μM synthetic peptide fragments (aa 462–493 of the BBF2H7 C terminus).
(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 < See also Figure S4 and Table S2.
Molecular Cell  , DOI: ( /j.molcel )
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7. 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 dose-dependent 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 < See also Figures S5 and S6 and Table S2.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions