Age-related CXC chemokine receptor-4-deficiency impairs osteogenic differentiation potency of mouse bone marrow mesenchymal stromal stem cells

Authors

Liang G. Guang, Adele L. Boskey, Wei Zhu

Abstract

Cysteine (C)-X-C chemokine receptor-4 (CXCR4) is the primary transmembrane receptor for stromal cell-derived factor-1 (SDF-1). We previously reported in mouse or human bone marrow-derived mesenchymal stromal stem cells (BMSCs) that deleting or antagonizing CXCR4 inhibits bone morphogenetic protein-2 (BMP2)-induced osteogenic differentiation. The goal of this study was to determine whether CXCR4-deficiency in BMSCs is an age-related effect in association with impaired osteogenic differentiation potency of aged BMSCs. Using BMSCs derived from C57BL/6J wild type mice at ages ranging from 3 to 23 months old, we detected decreased CXCR4 mRNA and protein expression as well as SDF-1 secretion with advancing aging. Moreover, CXCR4-deficient BMSCs from elderly vs. young mice exhibited impaired osteogenic differentiation in response to BMP2 stimulation or when cultured in dexamethasone (Dex)-containing osteogenic medium, evidenced by decreased alkaline phosphatase activity, osteocalcin synthesis, and calcium deposition (markers for immature and mature osteoblasts). Mechanistically, impaired BMP2- or Dex-osteoinduction in BMSCs of elderly mice was mediated by inhibited phosphorylation of intracellular R-Smads and Erk1/2 or Erk1/2 and p38 proteins, and decreased Runx2 and Osx expression (osteogenesis “master” regulators) were also detected. Furthermore, adenovirus-mediated repair of CXCR4 expression in BMSCs of elderly mice restored their osteogenic differentiation potentials to both BMP2 treatment and osteogenic medium. Collectively, our results demonstrate for the first time that CXCR4 expression in mouse BMSCs declines with aging, and this CXCR4-deficiency impairs osteogenic differentiation potency of aged BMSCs. These findings provide novel insights into mechanisms underlying age-related changes in BMSC-osteogenesis, and will potentiate CXCR4 as a therapeutic target to improve BMSC-based bone repair and regeneration in broad orthopedic situations.

Link to Article

http://dx.doi.org/10.1016/j.biocel.2013.05.034

Neuropeptide Y modulates fracture healing through Y1 receptor signaling

Authors

Daniela M. Sousa, Michelle M. McDonald, Kathy Mikulec, Lauren Peacock, Herbert Herzog, Meriem Lamghari, David G. Little, Paul A. Baldock

Abstract

Neuropeptide Y acting via it's Y1 receptor represents a powerful pathway in the control of bone mass. The global or osteoblast-specific Y1 receptor deletion induces pronounced bone anabolic effects in mice. However, the contribution of Y1 receptor deletion in bone repair/healing remained to be clarified. Therefore, in this study we characterized the role of Y1 receptor deletion in fracture healing. Closed tibial fractures were generated in germline (Y1−/−) and osteoblastic-specific Y1 receptor knockout mice. The progression of tibial repair monitored from 1- until 6-weeks post-fracture demonstrated that in Y1−/− mice there is a delay in fracture repair, as seen by a decrease in bone callus volume and callus strength. Moreover, the histological features included elevated avascular and cartilage area and consequently delayed cartilage removal, and hence impaired union. Interestingly, this delay in bone repair was not related directly to Y1 receptors expressed by mature osteoblasts. These findings suggest that the global absence of the Y1 receptor delays fracture healing, through impairing the early phases of fracture repair to achieve bony union. The data acquired on the role of Y1 receptor signaling disruption in bone regeneration is critical for the design of future therapeutic strategies.

Link to Article

http://dx.doi.org/10.1002/jor.22400

Black Bears With Longer Disuse (Hibernation) Periods Have Lower Femoral Osteon Population Density and Greater Mineralization and Intracortical Porosity

Authors

Samantha J. Wojda, David R. Weyland, Sarah K. Gray, Meghan E. Mcgee-Lawrence, Thomas D. Drummer, Seth W. Donahue

Abstract

Intracortical bone remodeling is persistent throughout life, leading to age related increases in osteon population density (OPD). Intracortical porosity also increases with age in many mammals including humans, contributing to bone fragility and fracture risk. Unbalanced bone resorption and formation during disuse (e.g., physical inactivity) also increases intracortical porosity. In contrast, hibernating bears are a naturally occurring model for the prevention of both age-related and disuse osteoporoses. Intracortical bone remodeling is decreased during hibernation, but resorption and formation remain balanced. Black bears spend 0.25–7 months in hibernation annually depending on climate and food availability. We found longer hibernating bears demonstrate lower OPD and higher cortical bone mineralization than bears with shorter hibernation durations, but we surprisingly found longer hibernating bears had higher intracortical porosity. However, bears from three different latitudes showed age-related decreases in intracortical porosity, indicating that regardless of hibernation duration, black bears do not show the disuse- or age-related increases in intracortical porosity which is typical of other animals. This ability to prevent increases in intracortical porosity likely contributes to their ability to maintain bone strength during prolonged periods of physical inactivity and throughout life. Improving our understanding of the unique bone metabolism in hibernating bears will potentially increase our ability to develop treatments for age- and disuse-related osteoporoses in humans.

Link to Article

http://dx.doi.org/10.1002/ar.22720

Epidermal Growth Factor Receptor (EGFR) signaling promotes proliferation and survival in osteoprogenitors by increasing Early Growth Response Protein (Egr2) expression

Authors

Abhishek Chandra, Shenghui Lan, Ji Zhu, Valerie Siclari and Ling Qin

Abstract

Maintaining bone architecture requires continuous generation of osteoblasts from osteoprogenitor pool. Our previous study of mice with epidermal growth factor receptor (EGFR) specifically inactivated in osteoblast lineage cells revealed that EGFR stimulates bone formation by expanding the population of mesenchymal progenitors. EGFR ligands are potent regulators for the osteoprogenitor pool but the underlying mechanisms are largely unknown. Here we demonstrate that activation of EGFR increases the number of osteoprogenitors by promoting cell proliferation and suppressing either serum- or TNFα-induced apoptosis mainly through MAPK/Erk pathway. Mouse calvarial organ culture revealed that EGF elevated the number of proliferative cells and decreased the number of apoptotic cells, which lead to increased osteoblasts. Microarray analysis of MC3T3 cells, an osteoprogenitor cell line, revealed that EGFR signaling stimulates the expression of Mcl1, an anti-apoptotic protein, and a family of Egr transcription factors (Egr1, 2, and 3). The up-regulation of Mcl1 and Egr2 by EGF were further confirmed in osteoprogenitors close to the calvarial bone surface. Overexpression of Nab2, a co-repressor for Egrs, attenuated the EGF-induced increase in osteoprogenitor number. Interestingly, knocking down the expression of Egr2, but not Egr1 or 3, resulted in a similar effect. Using inhibitor, adenovirus overexpression, and siRNA approaches, we demonstrate that EGFR signaling activates MAPK/Erk pathway to stimulate the expression of Egr2, which in turn leads to cell growth and Mcl1-mediated cell survival. Taken together, our data clearly demonstrate that EGFR-induced Egr2 expression is critical for osteoprogenitor maintenance and new bone formation.

Link to Article

http://dx.doi.org/jbc.M112.447250

Growth Factor Directed Chondrogenic Differentiation of Porcine Bone Marrow–Derived Progenitor Cells

Authors

Harutsugi Abukawa, Brad S. Oriel, Jeremy Leaf, Joseph P. Vacanti, Leonard B. Kaban, Maria J. Troulis, Christopher J. Hartnick

Abstract

Background: Despite advances in surgical technique, reconstruction of a mandibular condyle still causes significant donor-site morbidity. The purpose of this study was to compare the effect of 3 different growth factors and define optimal cell culture conditions for bone marrow-derived progenitor cells to differentiate into chondrocytes for mandibular condyle reconstruction. Methods: Porcine bone marrow-derived progenitor cells (pBMPCs) were cultured as a pellet for 2, 3, and 4 weeks under the following conditions: group 1, TGF-β3 + standard medium; group 2, TGF-β3 + BMP-2 + standard medium; group 3, TGF-β3 + IGF-1 + standard medium; and group 4, TGF-β3 + BMP-2 + IGF-1 + standard medium. Chondrogenic differentiation was evaluated using 3 lineage differentiation markers. Results: The mean type II collagen positive area increased over weeks 2, 3, and 4 in group 4 compared to all the other groups (ANOVA; P = 0.005). At week 4, there was significantly greater type II collagen production in group 4 compared to all the other groups (ANOVA; P = 0.003). The medium in group 4 produces the greatest amount of cartilage when compared to groups 1, 2, and 3, and that 4 weeks produces the greatest amount of type II collagen. Conclusions: The results of this study indicate that the most efficacious medium for chondrogenic differentiation of pBMPCs was group 4 medium and the most type II collagen was produced at 4 weeks.

Link to Article

http://dx.doi.org/10.1097/SCS.0b013e31827ff323

Hyperactive transforming growth factor-β1 signaling potentiates skeletal defects in a neurofibromatosis type 1 mouse model

Authors

Steven D. Rhodes, Xiaohua Wu, Yongzheng He, Shi Chen, Hao Yang, Karl W. Staser, Jiapeng Wang, Ping Zhang, Chang Jiang, Hiroki Yokota, Ruizhi Dong, Xianghong Peng, Xianlin Yang, Sreemala Murthy, Mohamad Azhar, Khalid S. Mohammad, Mingjiang Xu, Theresa A. Guise, Feng-Chun Yang

Abstract

Dysregulated TGF-β signaling is associated with a spectrum of osseous defects as seen in Loeys-Dietz syndrome, Marfan syndrome, and Camurati-Engelmann disease. Intriguingly, neurofibromatosis type 1 (NF1) patients exhibit many of these characteristic skeletal features including kyphoscoliosis, osteoporosis, tibial dysplasia, and pseudarthrosis; however, the molecular mechanisms mediating these phenotypes remain unclear. Here, we provide genetic and pharmacologic evidence that hyperactive TGF-β1 signaling pivotally underpins osseous defects in Nf1flox/-;Col2.3Cre mice, a model which closely recapitulates the skeletal abnormalities found in the human disease. Compared to controls, we show that serum TGF-β1 levels are 5–6 fold increased both in Nf1flox/-;Col2.3Cre mice and in a cohort of NF1 patients. Nf1 deficient osteoblasts, the principal source of TGF-β1 in bone, overexpress TGF-β1 in a gene dosage dependent fashion. Moreover, Nf1 deficient osteoblasts and osteoclasts are hyperresponsive to TGF-β1 stimulation, potentiating osteoclast bone resorptive activity while inhibiting osteoblast differentiation. These cellular phenotypes are further accompanied by p21-Ras dependent hyperactivation of the canonical TGF-β1-Smad pathway. Re-expression of the human, full-length neurofibromin GTPase-activating protein (GAP) related domain (NF1 GRD) in primary Nf1 deficient osteoblast progenitors, attenuated TGF-β1 expression levels and reduced Smad phosphorylation in response to TGF-β1 stimulation. As an in vivo proof of principle, we demonstrate that administration of the TβRI kinase inhibitor, SD-208, can rescue bone mass deficits and prevent tibial fracture non-union in Nf1flox/-;Col2.3Cre mice. In sum, these data demonstrate a pivotal role for hyperactive TGF-β1 signaling in the pathogenesis of NF1 associated osteoporosis and pseudarthrosis, thus implicating the TGF-β signaling pathway as a potential therapeutic target in the treatment of NF1 osseous defects which are refractory to current therapies.

Link to Article

http://dx.doi.org/10.1002/jbmr.1992