CYR61/CCN1 Regulates Sclerostin Levels and Bone Maintenance

Abstract

CYR61/CCN1 is a matricellular protein that resides in the extracellular matrix, but serves regulatory rather than structural roles. CYR61/CCN1 is found in mineralized tissues and has been shown to influence bone healing in vivo and osteogenic differentiation in vitro. In this study we generated Cyr61 bone-specific knockout mice to examine the physiological role of CYR61/CCN1 in bone development and maintenance in vivo. Extensive analysis of Cyr61 conditional knockout mice showed a significant decrease in both trabecular and cortical bone mass as compared to WT littermates. Our data suggest that CYR61/CCN1 exerts its effects on mature osteoblast/osteocyte function to modulate bone mass. Specifically, changes were observed in osteocyte/osteoblast expression of RankL, VegfA, and Sost. The increase in RankL expression was correlated with a significant increase in osteoclast number; decreased VegfA expression was correlated with a significant decrease in bone vasculature; increased Sost expression was associated with decreased Wnt signaling, as revealed by decreased Axin2 expression and increased adiposity in the bone marrow. Although the decreased number of vascular elements in bone likely contributes to the low bone mass phenotype in Cyr61 conditional knockout mice, this cannot explain the observed increase in osteoclasts and the decrease in Wnt signaling. We conducted in vitro assays using UMR-106 osteosarcoma cells to explore the role CYR61/CCN1 plays in modulating Sost mRNA and protein expression in osteocytes and osteoblasts. Overexpression of CYR61/CCN1 can suppress Sost expression in both control and Cyr61 knockout cells, and blocking Sost with siRNA can rescue Wnt responsiveness in Cyr61 knockout cells in vitro. Overall, our data suggest that CYR61/CCN1 modulates mature osteoblast and osteocyte function to regulate bone mass through angiogenic effects as well as by modulating Wnt signaling, at least in part through the Wnt antagonist Sost. © 2018 American Society for Bone and Mineral Research.

Keywords: ANIMAL MODELS; BONE MODELING AND REMODELING; CELL/TISSUE SIGNALING; CELLS OF BONE; GENETIC ANIMAL MODELS; MOLECULAR PATHWAYS; OSTEOCYTES; PARACRINE PATHWAYS; REMODELING; WNT/BETA-CATENIN/LRPS.

Fig. 1

Cyr61 knockdown in mature osteoblasts and osteocytes results in decreased trabecular and cortical bone. (A) eGFP immunostaining in Cyr61^eGFP mice; expression was seen in bone lining cells (white arrowheads), cuboidal osteoblasts (red arrow), trabecular osteocytes (white arrows), and cortical osteocytes (orange arrows). (B) µCT cross-sectional images and 3D reconstructions of the distal femurs of 3-month-old WT and Cyr61^OCN mice (n = 6). (C–H) µCT analysis of trabecular bone showed significantly lower BMD, BV/TV, Tb.BS, Tb.Th, and Tb.N, as well as an increase in Tb.Sp (n = 6). (I) µCT cross-sectional images of mid-shafts of the femurs of WT and Cyr61^OCN mice, (J, K) showed decreases in Ct.Th and BV in Cyr61^OCN mice (n = 6). (L) Mechanical testing of the femurs of Cyr61^OCN mice revealed significantly lower peak force as well as fail force during three-point bending tests as compared to control littermates (n = 6). BMD = bone mineral density; BV/TV = trabecular bone volume to total bone; Tb.BS = trabecular bone surface area; Tb.Th = trabecular bone thickness; Tb.N = trabecular bone number; Tb.Sp = trabecular bone spacing; Ct.Th = cortical bone thickness; BV = bone volume.

Fig. 2

Histomorphometric analysis reveals decreases in trabecular bone and the rate of cortical bone apposition. (A) Toluidine blue staining of trabecular bones of 3-month-old adult mice showing changes in osteoid between Cyr61^OCN and control (Cyr61^fx/fx) mice. (B–F) Histomorphometric analysis of trabecular bone showed significant decrease in BV/TV (B), OV/BV, OV/TV, OS/BS, and O.Th in Cyr61^OCN mice as compared to control littermates (n = 6). (G, H) Calcein labeling revealed a significant decrease in the cortical bone mineral deposition rate in Cyr61^OCN mice as compared to control littermates (n = 4). OV/BV = osteoid volume to bone volume; OV/TV = osteoid volume to total volume; OS/BS = osteoid surface to bone surface; O.Th = osteoid thickness.

Fig. 3

Increased osteoclast formation in Cyr61^OCN mice. (A) TRAP staining of the proximal tibia in 3-month-old mice. Right photomicrographs for each genotype show the chondro-osseous junction. Photomicrographs on the left for each genotype are magnified images of trabeculae. (B, C) Histomorphometric image analysis of osteoblasts revealed no significant differences in the N.Ob/BS or in Ob.S/BS in Cyr61^OCN mice compared to control (Cyr61^fx/fx) littermates (n = 6). (D) P1NP ELISA showed significant decrease in plasma P1NP in Cyr61^OCN mice as compared to control littermates (n = 6). (E, F) Image analysis of osteoclasts revealed significant increases in both the N.Oc/BS and Oc.S/BS in Cyr61^OCN mice compared to control littermates (n = 6). (G) TRAcP-5b ELISA showed significant increase in plasma TRAcP-5b in Cyr61^OCN mice as compared to control littermates (n = 7). (H–J) mRNA expression of cortical bone in 3-month-old mice showed a significant increase in RankL expression and a significant decrease in Opg expression in Cyr61^OCN mice compared to control littermates, which resulted in over 400-fold increase in the RankL/Opg ratio in Cyr61^OCN mice (n = 6). N.Ob/BS = number of osteoblasts relative to total bone surface; Ob.S/BS = bone surface area that was covered by osteoblasts relative to total surface; P1NP = procollagen I N-terminal peptide; N.Oc/BS = number of osteoclasts relative to total bone surface; Oc.S/BS = bone surface area that was covered by osteoblasts relative to total surface.

Fig. 4

Cyr61^OCN mice have decreased vasculature and VegfA expression. (A) PECAM immunostaining of 1-month-old mice demonstrating a slight decrease in vasculature at the chondro-osseous junctions in Cyr61^OCN mice compared to control (Cyr61^fx/fx) littermates. (B) VegfA mRNA expression of 3-month-old cortical bone was decreased more than twofold in Cyr61^OCN mice as compared to control littermates (n = 6). (C) Microfil-perfusion angiograms of proximal tibia in 1 month old mice showed a significant decrease in vessel volume (D) in Cyr61^OCN mice (n = 4). (E) Ex vivo fetal (E17.5) metatarsal blood vessel outgrowth assay demonstrated a considerable decrease in vascular outgrowth from Cyr61^OCN mice compared to WT (Cyr61^fx/fx) littermates. This decrease in vessel outgrowth was rescued by addition of exogenous VEGF (n = 4). (F) Deletion of Cyr61 using AdCre resulted in a significant decrease in VegfA expression in vitro (n = 3).

Fig. 5

Decreased Wnt signaling in Cyr61^OCN mice. (A) qRT-PCR analysis of expression of the Wnt responsive gene Axin2 in cortical bone of 3-month-old Cyr61^OCN mice as compared to control (Cyr61^fx/fx) littermates (n = 6). (B) Western analysis of active β-catenin (nonphosphorylated) levels in cortical bone of 3-month-old Cyr61^OCN and control littermates (n = 3). (C) H&E staining of the distal tibias of 3-month-old mice revealed abundant fat droplets in Cyr61^OCN mice, which were not present in WT (Cyr61^fx/fx) mice. (D–F) qRT-PCR analysis of bone marrow mRNAs revealed a significant increase in expression of adipogenic genes, Lpl, Cebpa, and Adiponectin in Cyr61^OCN mice compared to control littermates (n = 6). (G–I) In vitro osteogenic and adipogenic differentiation of BMSCs showed a significant decrease in mineralization and significant increase in adipogenesis in Cyr61^PRX cells as compared to WT control (n = 5).

Fig. 6

Increased Sost expression in Cyr61^OCN mice. (A–C) qRT-PCR analysis of (A) Dkk1, (B) Sost, and (C) Mef2c mRNA expression in cortical bone revealed no change in Dkk1 expression but a significant increase in Sost and Mef2c mRNA levels (n = 6). (D) Immunohistochemistry for Sost protein revealed increased staining in osteocytes in Cyr61^OCN mice. (E, F) Western blot analysis of Sost protein levels in cortical bone of 3-month-old Cyr61^OCN and control (WT; Cyr61^fx/fx) littermates (n = 3).

Fig. 7

Cyr61 inhibits Sost expression in vitro. (A) Cyr61 was significantly suppressed in UMR106 knockdown cells as compared to control (n = 3). (B, C) Cyr61 knockdown (Cyr61-kd) in UMR106 osteoblasts led to a significant decrease in Cyr61, increase in (B) Sost and (C) Mef2c expression. Rescue with AdCYR61 resulted in a significant decrease in Sost and Mef2c expression in both Cyr61-kd and control cells. *p < 0.05 AdGDF versus AdCYR61; #p < 0.05 UMR106::AdGFP versus Cyr61-kd cells (n = 3). (D) Successful knockdown of Sost using siRNA in Cyr61-kd cells and control cells (n = 3). (E) qRT-PCR analysis of Axin2 mRNA expression in UMR106 control and Cyr61-kd cells demonstrates that knockdown of Sost rescues Wnt responsiveness in Cyr61-kd cells (n = 3). AdCYR61 = Cyr61 adenovirus.

Fig. 8

Proposed mechanism of Cyr61 modulation of bone mass via regulation of Sost expression.