Bmp2 in Osteoblasts of Periosteum and Trabecular Bone Links Bone
Total Page：16
File Type：pdf, Size：1020Kb
Research Article 4085 Bmp2 in osteoblasts of periosteum and trabecular bone links bone formation to vascularization and mesenchymal stem cells Wuchen Yang1,2, Dayong Guo3,4, Marie A. Harris1, Yong Cui1, Jelica Gluhak-Heinrich1, Junjie Wu5, Xiao-Dong Chen5, Charles Skinner5, Jeffry S. Nyman6, James R. Edwards6, Gregory R. Mundy6,*, Alex Lichtler7, Barbara E. Kream7, David W. Rowe7, Ivo Kalajzic7, Val David8, Darryl L. Quarles8, Demetri Villareal1, Greg Scott9, Manas Ray9, S. Liu10, James F. Martin11, Yuji Mishina12 and Stephen E. Harris1,` 1Department of Periodontics, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA 2Department of Craniofacial Sciences, University of Connecticut Health Center, Farmington, CT 06030, USA 3Department of Oral Biology, University of Missouri at Kansas City, Kansas City, MO 64108, USA 4Regeneron Inc., Tarrytown, NY 10591, USA 5Department of Comprehensive Dentistry, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA 6Department of Orthopaedic Surgery and Rehabilitation and Vanderbilt Center for Bone Biology, Vanderbilt University Medical Center, Nashville, TN 37232, USA 7Center for Regenerative Medicine and Skeletal Development, University of Connecticut Health Center, Farmington, CN 06030, USA 8Department of Medicine, University of Tennessee Health Science Center, Memphis, TN 38104, USA 9National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA 10Endocrine and Renal Science, Genzyme Co., Framingham, MA 01701, USA 11Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030, USA 12Department of Biologic and Material Sciences, School of Dentistry, University of Michigan, Ann Arbor, MI 48109, USA *Deceased `Author for correspondence ([email protected]) Accepted 10 June 2013 Journal of Cell Science 126, 4085–4098 ß 2013. Published by The Company of Biologists Ltd doi: 10.1242/jcs.118596 Summary We generated a new Bmp2 conditional-knockout allele without a neo cassette that removes the Bmp2 gene from osteoblasts (Bmp2- cKOob) using the 3.6Col1a1-Cre transgenic model. Bones of Bmp2-cKOob mice are thinner, with increased brittleness. Osteoblast activity is reduced as reflected in a reduced bone formation rate and failure to differentiate to a mature mineralizing stage. Bmp2 in osteoblasts also indirectly controls angiogenesis in the periosteum and bone marrow. VegfA production is reduced in Bmp2-cKOob Journal of Cell Science osteoblasts. Deletion of Bmp2 in osteoblasts also leads to defective mesenchymal stem cells (MSCs), which correlates with the reduced microvascular bed in the periosteum and trabecular bones. Expression of several MSC marker genes (a-SMA, CD146 and Angiopoietin- 1) in vivo, in vitro CFU assays and deletion of Bmp2 in vitro in a-SMA+ MSCs support our conclusions. Critical roles of Bmp2 in osteoblasts and MSCs are a vital link between bone formation, vascularization and mesenchymal stem cells. Key words: Bmp2, Osteoblasts, Angiogenesis, Mesenchymal stem cells, VegfA, a-SMA+ cells Introduction mesenchymal precursor cells in 10 d.p.c. embryos shows a minor The bone morphogenic protein (BMP) signaling pathway is role for Bmp2 during endochondral bone formation, but adult important for developmental and postnatal physiological mice have small bones that show an increased fracture risk and processes (Bonilla-Claudio et al., 2012; Dutko and Mullins, failure to heal (Tsuji et al., 2006). 2011; Sieber et al., 2009; Watabe and Miyazono, 2009; Yoon Vascularization and angiogenesis are linked to cartilage and et al., 2005; Zhao et al., 2002). BMP signaling pathways are bone formation and to fracture healing (Araldi and Schipani, common for BMP ligands and involve three different type I BMP 2010; Jacobsen et al., 2008; Maes et al., 2012; Schipani et al., receptors and two different type II receptors plus negative 2009). During aging or glucocorticoid treatment, bone regulators of BMP signaling, such as noggin and gremlin (Dutko angiogenesis is reduced and brittleness in bone is increased and Mullins, 2011). Selected polymorphisms in the BMP2 gene (Weinstein, 2010; Weinstein et al., 2010). VegfA overexpression are associated with increased fracture risk and osteoporosis in bone marrow leads to formation of new bone and associated (Styrkarsdottir et al., 2003). Deletion of other BMP family blood vessels (Maes et al., 2010a). BMP signaling in osteoblasts members Bmp7 and Bmp4 suggests that they play minor roles in increases VegfA through Osterix and Runx2/Hif1a (Deckers bone and cartilage development in the embryo or postnatally, et al., 2002; Kwon et al., 2011; Maes et al., 2012; Zhang et al., whereas Bmp2 is critical for chondrocyte function and 2009). Recently, Liu and colleagues (Liu et al., 2012) endochondral bone development (Bandyopadhyay et al., 2006; demonstrated that VegfA is required in the progression of Shu et al., 2011; Tsuji et al., 2008). Removal of Bmp2 in mouse osteoblast precursors (Osterix+) to mature osteoblasts. 4086 Journal of Cell Science 126 (18) Mesenchymal stem cells (MSCs) from both the periosteum and the bone osteopenic-like phenotype, with thinner bones in the bone marrow, are progenitors of osteoblasts (Bianco et al., 2010; Bmp2-cKOob became progressively advanced, as assayed by Schipani and Kronenberg, 2008) and express CD146 and a-SMA. digital radiography up to 1 year of age (Fig. 1C). We focused on MSCs are associated with microvascular structures in bone males for statistical evaluation, and phenotypes were similar in (Crisan et al., 2008; Grcevic et al., 2012; Sacchetti et al., 2007). females and males by X-ray analysis. a-SMA+ cells isolated by lineage tracing studies are a class of As analyzed by dynamic bone formation rate, both MSCs that differentiate into osteoblasts (Grcevic et al., 2012). mineralizing surface (MS/BS5active surface area) and the Osterix+ preosteoblasts, which represent a non-proliferating mineral apposition rate (MAR, rate of new mineral deposition) transient state, move into bone marrow in association with were reduced by 20% in both the bone marrow trabeculae vascular structures during bone development (Maes et al., 2010b; (Fig. 1D, top) and in the periosteum (Fig. 1D, bottom), leading to Park et al., 2012). a 40% overall decrease in bone formation rate (BFR). BFR in the Bmp2 expression is activated in MSCs (smooth muscle cells a- periosteum of femurs also showed a 40% decrease in 2-month- SMA+) on new induced blood vessels during bone formation and old Bmp2-cKOob compared with controls (data not shown). angiogenesis that is activated during distraction osteogenesis mCT analysis of vertebrae and long bones at 4 months (n55 (Matsubara et al., 2012). There is first, rapid new blood vessel per group) shows vertebrae are smaller by 35% in the Bmp2- and associated candidate MSC formation, followed by new bone cKOob with an unfused dorsal region (Fig. 2A). We observed an formation at the distraction site. From the above observations, we increase in rod-like quality of trabeculae of the Bmp2-cKOob.A hypothesize that the Bmp2 gene has multiple roles at different 40% reduction in bone volume to total volume (BV/TV) was stages of osteogenesis, ranging from a requirement for terminal found in the trabecular region, as well as a 20% decrease in differentiation to driving vascularization and formation of new MSCs. trabecular thickness. The tibia, femur and humerus showed a 30– In these studies we: (1) characterized the bone phenotype in a 40% decrease in bone volume compared with controls, but Bmp2-cKOob model in both the periosteum and in the bone similar cortical thickness (data not shown). marrow, including histomorphometry; (2) mechanically tested Apparent bone mineral density (aBMD) from 2 weeks to 8 the long bones to evaluate bone quality; (3) performed in situ months of age demonstrated a progressive decrease in aBMD hybridization and immunocytochemistry of the key genes and with age, especially in the spine (20% decrease in L4–L6 region) proteins involved in osteoblast and vascular biology; (4) created at 25 weeks and a 14–16% decrease in tibia and femur of Bmp2- 3D vascular maps of the vascular bed in both the periosteum and cKOob (Fig. 2B). At 6 and 8 months, a similar 20% decrease in bone marrow; (5) performed in vitro tests for cell autonomy of the spine aBMD was noted. MSC number and their capacity to differentiate into osteoblasts; and (6) performed in vitro testing of Bmp2 function by deletion Mechanical and material properties indicate that Bmp2- of Bmp2 in enriched MSCs. cKOob bones are more brittle By three-point bending of femurs and resulting stress–strain or Results force–displacement curves, we demonstrated a 40% decrease in Skeletal phenotype after Bmp2 gene ablation from early stiffness and 50% decrease in work to fracture (Wf) (Fig. 2C) in ob ob Journal of Cell Science osteoblasts in Bmp2-cKO mice the Bmp2-cKO bones. Overall toughness of the bones was also Fig. 1A and supplementary material Fig. S1C show that Bmp2 decreased 35% in Bmp2-cKOob bones (Fig. 2C, top right). Post- expression was reduced by 85–90% in the osteoblasts at 5 days, 1 yield toughness in Bmp2-cKOob bones was decreased by 75% month and 4 months in periosteum and trabecular bones (Yang (Fig. 2C, bottom right), demonstrating that the bones are much et al., 2012). Bmp2 protein levels were also greatly reduced in more brittle. osteoblasts (Fig. 1B). The body weight of the Bmp2-cKOob mouse changed little as assayed to 6 months (supplementary Histomorphometric and gene expression analysis material Fig. S1D, top panel). Humerus length showed a 5% demonstrates a failure in osteoblast differentiation decrease at 2 weeks with no change between control and Bmp2- There were many flat and immature osteoblasts and fewer mature cKOob at 2–4 months (supplementary material Fig. S1D, bottom cuboidal polarized osteoblasts in Bmp2-cKOob compared with panel). Alizarin Red and Alcian Blue staining of newborns control littermates, as assayed from 5 days to 6 months (Fig. 3A showed the skeleton was normal in the Bmp2-cKOob animals, and supplementary material Fig.