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. 2017 Feb 1;130(3):531-540.
doi: 10.1242/jcs.197285. Epub 2017 Jan 3.

Defective signaling, osteoblastogenesis and bone remodeling in a mouse model of connexin 43 C-terminal truncation

Megan C Moorer  1 , Carla Hebert  1 , Ryan E Tomlinson  2 , Shama R Iyer  1 , Max Chason  1 , Joseph P Stains  3
Affiliations

Defective signaling, osteoblastogenesis and bone remodeling in a mouse model of connexin 43 C-terminal truncation (VSports app下载)

Megan C Moorer et al. J Cell Sci. .

Abstract

In skeletal tissue, loss or mutation of the gap junction protein connexin 43 (Cx43, also known as GJA1) in cells of the osteoblast lineage leads to a profound cortical bone phenotype and defective tissue remodeling. There is mounting evidence in bone cells that the C-terminus (CT) of Cx43 is a docking platform for signaling effectors and is required for efficient downstream signaling. Here, we examined this function, using a mouse model of Cx43 CT-truncation (Gja1 K258Stop). Relative to Gja1+/- controls, male Gja1-/K258Stop mice have a cortical bone phenotype that is remarkably similar to those reported for deletion of the entire Cx43 gene in osteoblasts. Furthermore, we show that the Cx43 CT binds several signaling proteins that are required for optimal osteoblast function, including PKCδ, ERK1 and ERK2 (ERK1/2, also known as MAPK3 and MAPK1, respectively) and β-catenin. Deletion of the Cx43 CT domain affects these signaling cascades, impacting osteoblast proliferation, differentiation, and collagen processing and organization. These data imply that, at least in bone, Cx43 gap junctions not only exchange signals, but also recruit the appropriate effector molecules to the Cx43 CT in order to efficiently activate signaling cascades that affect cell function and bone acquisition VSports手机版. .

Keywords: Cx43 K258Stop; Gap junction; Intercellular communication; Osteoblast; Osteocyte; Signal transduction V体育安卓版. .

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Competing interests

The authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.
Overexpression of the Cx43 CT domain competes for binding of signaling proteins with the intact Cx43 protein. Co-immunoprecipitations (co-IPs) were performed to assess protein–protein interactions between the indicated signaling proteins and the Cx43 C-terminus. UMR106 cells were transfected with a construct encoding the CT domain of Cx43 (FLAG-tagged) or with an empty vector control. (A) Input and bead fractions are shown for co-IPs performed with anti-ERK1/2, anti-PKCδ and anti β-catenin antibodies and blotted with anti-Cx43 antibodies. A negative control co-IP with an antibody to a protein unlikely to be found at the plasma membrane (anti-Sp1) was performed and blotted with anti-Cx43 antibodies and is shown below. (B) IPs were performed for the Cx43 C-terminus (with anti-FLAG antibody), and then blotted for the indicated signaling proteins.
Fig. 2.
Fig. 2.
No apparent morphological defects are observed in mice with truncation of the Cx43 CT. (A) Digital X-rays, and (B) body length and weight measurements in 6-week-old male Gja1+/− (n=11) and Gja1−/K258Stop (n=8) mice. (B) Schematic of full-length (WT Cx43) and truncated Cx43 (Cx43 K258Stop) proteins, showing deletion of most of the C-terminus. (C) Western blot performed on tibial extracts (marrow flushed) for Cx43 (C-terminal epitope). qRT-PCR from RNA isolated from tibia (marrow flushed) of the indicated genotype was performed for Gja1 (Cx43; 3′ primers to the C-terminal encoding region) and Gjc1 (Cx45) mRNA expression. (n=7 for Gja1+/− and n=5 for Gja1−/K258Stop). Graphs depict mean±s.d. *P<0.05; n.s., not significant (two-tailed t-test).
Fig. 3.
Fig. 3.
Truncation of Cx43 results in a cortical phenotype with increased cross-sectional area, increased cortical porosity, cortical thinning and marrow cavity expansion in 6-week-old male Gja1−/K258Stop mice. (A) Top, cross section through the femoral mid-diaphysis from a representative microCT slice from Gja1+/− and Gja1−/K258Stop mice. Bottom, 3D reconstruction of intact femurs. (B) Quantification of the cortical phenotype at the femoral mid-diaphysis by microCT (n=11 for Gja1+/− and n=8 for Gja1−/K258Stop); see Materials and Methods for the definitions of the parameters. Graphs depict mean±s.d. *P<0.05 (two-tailed t-test).
Fig. 4.
Fig. 4.
Substantial periosteal bone apposition contributes to the cortical expansion of the mid-diaphysis in 6-week-old male Gja1−/K258Stop mice. Calcein and Alizarin dual labeling and quantification of the mineral apposition rate (MAR) and bone formation rate (BFR) is shown for each genotype at the (A) periosteal (Ps) and (B) endosteal (Es) surface of the femoral diaphysis (n=3 animals per genotype, 3 sections per animal). (C) Quantification of serum levels of P1NP (n=4 per genotype). (D) Static histomorphometry of osteoblast number normalized to the bone surface area (n=5 per genotype). (E) Quantification of the percentage of occupied osteocyte lacunae observed in cortical bone from the indicated genotype (n=3 animals and >1000 lacunae/genotype). A representative hematoxylin-stained image of cortical bone is shown. White arrowheads point to occupied lacunae. Graphs depict mean±s.d. *P<0.05; n.s., not significant (two-tailed t-test).
Fig. 5.
Fig. 5.
Cx43 truncation affects regulators of osteoclastogenesis, leading to increased osteoclast number and increased bone resorption. (A) qRT-PCR from RNA isolated from tibia (marrow flushed) of the indicated genotype was performed for RANKL and OPG mRNA expression (n=7 for Gja1+/− and n=5 for Gja1−/K258Stop). (B) Static histomorphometry of osteoclast number normalized to the bone surface area (OC.N/BS; n=5 per genotype). (C) Quantification of serum levels of CTX (n=3 for Gja1+/− and n=4 for Gja1−/K258Stop). Graphs depict mean±s.d. *P<0.05; n.s., not significant (two-tailed t-test).
Fig. 6.
Fig. 6.
Cx43 truncation impairs signal cascade activation and osteoblast differentiation. (A) Western blots were probed for indicated signaling proteins in tibial (marrow flushed) extracts of the indicated genotypes. The GAPDH blot shown in Fig. 6A (right panel) is the same as in Fig. 8B, as the same membrane was re-probed for the indicated factors. qRT-PCR from RNA isolated from tibia (marrow flushed) of the indicated genotype was performed for (B) Wnt/β-catenin-related genes, (C) transcriptional regulators of osteoblastogenesis, and (D) osteoblast differentiation markers (n=7 for Gja1+/− and n=5 for Gja1−/K258Stop). (C) Western blots on extracts from BMSCs cultured for 7 days in mineralization medium were probed for Runx2, Osterix and GAPDH (loading control). Graphs depict mean±s.d. *P<0.05; n.s., not significant (two-tailed t-test).
Fig. 7.
Fig. 7.
Cx43 truncation reduces mRNA expression of collagen processing factors and results in a disorganized and less mature collagen network in cortical bone. (A) qRT-PCR from RNA isolated from tibia (marrow flushed) of the indicated genotype was performed for expression of collagen-processing factors. Graphs depict mean±s.d. (n=7 for Gja1+/− and n=5 for Gja1−/K258Stop). *P<0.05 (two-tailed t-test). Western blot on extracts from BMSCs cultured for 7 days in mineralization medium and immunoblotted for Lox, Hsp47 and GAPDH (load control) are also shown. Bands for the preform (pro) and mature isoform of Lox are detected. (B) Picrosirius Red staining was performed and quantified at the mid-diaphyseal cortical bone, using a hue-range distribution method. A representative image of mid-diaphyseal cortical bone is shown. Quantification of fiber orientation and length is shown as a percentage of composition of the birefringent hue (red, orange, yellow and green).
Fig. 8.
Fig. 8.
Truncation of Cx43 leads to cell autonomous defects in osteoblast proliferation, signaling and mineralization. (A) Cell proliferation on BMSCs after 48 h in culture (n=6 wells per genotype). (B) Western blot on extracts from BMSCs cultured for 7 days in mineralization medium and immunoblotted for indicated signaling proteins. The blot for GAPDH shown in Fig. 8B is the same as in Fig. 6A (right panel), as the same membrane was re-probed for the indicated factors. (C) Alizarin Red S staining in replicate wells (n>4 wells per genotype) in BMSCs cultured for 14 days in mineralization medium. Graphs depict mean±s.d. *P<0.05 (two-tailed t-test).
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