Lynda F. Bonewald

Crosstalk Between MLO-Y4 Osteocytes and C2C12 Muscle Cells Is Mediated by the Wnt/β-Catenin Pathway: WNT/β-CATENIN PATHWAY MEDIATES OSTEOCYTES AND MUSCLE CELLS CROSSTALK

We examined the effects of osteocyte secreted factors on myogenesis and muscle function. MLO‐Y4 osteocyte‐like cell conditioned media (CM) (10%) increased ex vivo soleus muscle contractile force by ∼25%. MLO‐Y4 and primary osteocyte CM (1% to 10%) stimulated myogenic differentiation of C2C12 myoblasts, but 10% osteoblast CMs did not enhance C2C12 cell differentiation. Because WNT3a and WNT1 are secreted by osteocytes, and the expression level of Wnt3a is increased in MLO‐Y4 cells by fluid flow shear stress, both were compared, showing WNT3a more potent than WNT1 in inducing myogenesis. Treatment of C2C12 myoblasts with WNT3a at concentrations as low as 0.5 ng/mL mirrored the effects of both primary osteocyte and MLO‐Y4 CM by inducing nuclear translocation of β‐catenin with myogenic differentiation, suggesting that Wnts might be potential factors secreted by osteocytes that signal to muscle cells. Knocking down Wnt3a in MLO‐Y4 osteocytes inhibited the effect of CM on C2C12 myogenic differentiation. Sclerostin (100 ng/mL) inhibited both the effects of MLO‐Y4 CM and WNT3a on C2C12 cell differentiation. RT‐PCR array results supported the activation of the Wnt/β‐catenin pathway by MLO‐Y4 CM and WNT3a. These results were confirmed by qPCR, showing upregulation of myogenic markers and two Wnt/β‐catenin downstream genes, Numb and Flh1. We postulated that MLO‐Y4 CM/WNT3a could modulate intracellular calcium homeostasis as the trigger mechanism for the enhanced myogenesis and contractile force. MLO‐Y4 CM and WNT3a increased caffeine‐induced Ca2+ release from the sarcoplasmic reticulum (SR) of C2C12 myotubes and the expression of genes directly associated with intracellular Ca2+ signaling and homeostasis. Together, these data show that in vitro and ex vivo, osteocytes can stimulate myogenesis and enhance muscle contractile function and suggest that Wnts could be mediators of bone to muscle signaling, likely via modulation of intracellular Ca2+ signaling and the Wnt/ β‐Catenin pathway.

Bivariate genome-wide association meta-analysis of pediatric musculoskeletal traits reveals pleiotropic effects at the SREBF1/TOM1L2 locus

Bone mineral density is known to be a heritable, polygenic trait whereas genetic variants contributing to lean mass variation remain largely unknown. We estimated the shared SNP heritability and performed a bivariate GWAS meta-analysis of total-body lean mass (TB-LM) and total-body less head bone mineral density (TBLH-BMD) regions in 10,414 children. The estimated SNP heritability is 43% (95% CI: 34–52%) for TBLH-BMD, and 39% (95% CI: 30–48%) for TB-LM, with a shared genetic component of 43% (95% CI: 29–56%). We identify variants with pleiotropic effects in eight loci, including seven established bone mineral density loci: WNT4, GALNT3, MEPE, CPED1/WNT16, TNFSF11, RIN3, and PPP6R3/LRP5. Variants in the TOM1L2/SREBF1 locus exert opposing effects TB-LM and TBLH-BMD, and have a stronger association with the former trait. We show that SREBF1 is expressed in murine and human osteoblasts, as well as in human muscle tissue. This is the first bivariate GWAS meta-analysis to demonstrate genetic factors with pleiotropic effects on bone mineral density and lean mass.Author summaryBone mineral density and lean skeletal mass are heritable traits. Here, Medina-Gomez and colleagues perform bivariate GWAS analyses of total body lean mass and bone mass density in children, and show genetic loci with pleiotropic effects on both traits.

Live Imaging of Type I Collagen Assembly Dynamics in Osteoblasts Stably Expressing GFP and mCherry-Tagged Collagen Constructs: LIVE IMAGING OF TYPE I COLLAGEN ASSEMBLY

Type I collagen is the most abundant extracellular matrix protein in bone and other connective tissues and plays key roles in normal and pathological bone formation as well as in connective tissue disorders and fibrosis. Although much is known about the collagen biosynthetic pathway and its regulatory steps, the mechanisms by which it is assembled extracellularly are less clear. We have generated GFPtpz and mCherry‐tagged collagen fusion constructs for live imaging of type I collagen assembly by replacing the α2(I)‐procollagen N‐terminal propeptide with GFPtpz or mCherry. These novel imaging probes were stably transfected into MLO‐A5 osteoblast‐like cells and fibronectin‐null mouse embryonic fibroblasts (FN‐null‐MEFs) and used for imaging type I collagen assembly dynamics and its dependence on fibronectin. Both fusion proteins co‐precipitated with α1(I)‐collagen and remained intracellular without ascorbate but were assembled into α1(I) collagen‐containing extracellular fibrils in the presence of ascorbate. Immunogold‐EM confirmed their ultrastuctural localization in banded collagen fibrils. Live cell imaging in stably transfected MLO‐A5 cells revealed the highly dynamic nature of collagen assembly and showed that during assembly the fibril networks are continually stretched and contracted due to the underlying cell motion. We also observed that cell‐generated forces can physically reshape the collagen fibrils. Using co‐cultures of mCherry‐ and GFPtpz‐collagen expressing cells, we show that multiple cells contribute collagen to form collagen fiber bundles. Immuno‐EM further showed that individual collagen fibrils can receive contributions of collagen from more than one cell. Live cell imaging in FN‐null‐MEFs expressing GFPtpz‐collagen showed that collagen assembly was both dependent upon and dynamically integrated with fibronectin assembly. These GFP‐collagen fusion constructs provide a powerful tool for imaging collagen in living cells and have revealed novel and fundamental insights into the dynamic mechanisms for the extracellular assembly of collagen.

Degeneration of the osteocyte network in the C57BL/6 mouse model of aging

Age-related bone loss and associated fracture risk are major problems in musculoskeletal health. Osteocytes have emerged as key regulators of bone mass and as a therapeutic target for preventing bone loss. As aging is associated with changes in the osteocyte lacunocanalicular system, we focused on the responsible cellular mechanisms in osteocytes. Bone phenotypic analysis was performed in young-(5mo) and aged-(22mo) C57BL/6 mice and changes in bone structure/geometry correlated with alterations in osteocyte parameters determined using novel multiplexed-3D-confocal imaging techniques. Age-related bone changes analogous to those in humans were observed, including increased cortical diameter, decreased cortical thickness, reduced trabecular BV/TV and cortical porosities. This was associated with a dramatic reduction in osteocyte dendrite number and cell density, particularly in females, where osteocyte dendricity decreased linearly from 5, 12, 18 to 22mo and correlated significantly with cortical bone parameters. Reduced dendricity preceded decreased osteocyte number, suggesting dendrite loss may trigger loss of viability. Age-related degeneration of osteocyte networks may impair bone anabolic responses to loading and gender differences in osteocyte cell body and lacunar fluid volumes we observed in aged mice may lead to gender-related differences in mechanosensitivity. Therapies to preserve osteocyte dendricity and viability may be beneficial for bone health in aging.