Loss of Runx2 in Committed Osteoblasts Impairs Postnatal Skeletogenesis

Authors

Mitra D. Adhami BS, Harunur Rashid MSc, Haiyan Chen PhD, John C. Clarke BS, Yang Yang MD and Amjad Javed PhD

Abstract

The Runx2 transcription factor is critical for commitment to the osteoblast lineage. However, its role in committed osteoblasts and its functions during postnatal skeletogenesis remain unclear. We established a Runx2-floxed line with insertion of loxP sites around exon 8 of the Runx2 gene. Runx2 protein lacking the region encoded by exon 8 is imported into the nucleus and binds target DNA, but exhibits diminished transcriptional activity. We specifically deleted the Runx2 gene in committed osteoblasts using 2.3kb col1a-Cre transgenic mice. Surprisingly, the homozygous Runx2 mutant mice were born alive. The Runx2 heterozygous and homozygous null were grossly indistinguishable from wild-type littermates at birth. Runx2 deficiency did not alter proliferative capacity of osteoblasts during embryonic development (E18). Chondrocyte differentiation and cartilage growth in mutants was similar to wild-type mice from birth to 3 months of age. Analysis of the embryonic skeleton revealed poor calcification in homozygous mutants, which was more evident in bones formed by intramembranous ossification. Runx2 mutants showed progressive retardation in postnatal growth and exhibited significantly low bone mass by 1 month of age. Decreased bone formation was associated with decreased gene expression of osteoblast markers and impaired collagen assembly in the extracellular matrix. Consequently, Runx2 mutant bones exhibited decreased stiffness and structural integrity. By 3 months of age, bone acquisition in mutant mice was roughly half that of wild-type littermates. In addition to impaired osteoblast function, mutant mice showed markedly decreased osteoclast number and postnatal bone resorption. Taken together, functional deficiency of Runx2 in osteoblasts does not result in failed embryonic skeletogenesis, but disrupts postnatal bone formation.

Link To Article

http://dx.doi.org/10.1002/jbmr.2321

Genetic manipulation of the ghrelin signaling system in male mice reveals bone compartment specificity of acylated and unacylated ghrelin in the regulation of bone remodeling

Authors

Patric JD Delhanty, Martijn van der Velde, Bram CJ van der Eerden, Yuxiang Sun, Julia MM Geminn, Aart-Jan van der Lely, Roy G Smith, and Johannes PTM van Leeuwen

Abstract

Ghrelin receptor deficient (Ghsr-/-) mice, which lack acylated ghrelin (AG) signaling, retain a metabolic response to unacylated ghrelin (UAG). Recently, we showed that Ghsr-deficiency affects bone metabolism. The aim of this study was to further establish the impact of AG and UAG on bone metabolism. We compared bone metabolism in Ghsr-/- (lacking only AG signaling) and ghrelin deficient (Ghrl-/-; both AG and UAG deficient) male mice. Ghrl-/- mice had lower cortical bone mass, while Ghsr-/- mice had lower trabecular bone mass. This demonstrates bone compartment-specific effects of AG and a role for UAG in bone metabolism. Also, Ghrl-/- but not Ghsr-/- mice had increased bone formation rate and increased osteogenic stem cell numbers in their bone marrow. In ex vivo bone marrow cultures both AG and UAG inhibited osteoblast differentiation. This indicated that bone resorption must be increased in these mice. Accordingly, osteoclastogenesis rate was faster in bone marrow cultures from Ghsr-/- and Ghrl-/- mice, and osteoclast formation was inhibited by AG signaling and partially suppressed by UAG. In osteoblast cultures, AG markedly induced osteoprotegerin gene expression and both peptides reduced RANKL/osteoprotegerin ratio. These data describe unique cell-type specific effects of AG and UAG within a single tissue, supporting a tight and complex control of bone formation and resorption as well as a link between nutrition and bone metabolism. The balance between AG and UAG actions in the bone marrow may lead to bone compartmental specific effects.

Link To Article

http://dx.doi.org/10.1210/en.2013-2055

Immobilization contributes to exaggerated neuropeptide signaling, inflammatory changes, and nociceptive sensitization after fracture in rats

Authors

Tian-Zhi Guo, Tzuping Wei, Wen-Wu Li, Xiang-Qi Li, J. David Clark, Wade S. Kingery

Abstract

A tibia fracture cast immobilized for 4 weeks can induce exaggerated substance P (SP) and CGRP signaling and neuropeptide-dependent nociceptive and inflammatory changes in the hindlimbs of rats similar to those seen in complex regional pain syndrome (CRPS). Four weeks of hindlimb cast immobilization can also induce nociceptive and vascular changes resembling CRPS. To test our hypothesis that immobilization alone could cause exaggerated neuropeptide signaling and inflammatory changes we tested 5 cohorts of rats; 1) controls, 2) tibia fracture and hindlimb casted, 3) hindlimb casted, no fracture, 4) tibia fracture with intrameduallary pinning, no cast, and 5) tibia fracture with intrameduallary pinning and hindlimb casting. After 4 weeks the casts were removed and hindlimb allodynia, unweighting, warmth, edema, sciatic nerve neuropeptide content, cutaneous and spinal cord inflammatory mediator levels, and spinal c-Fos activation were measured. After fracture with casting there was allodynia, unweighting, warmth, edema, increased sciatic nerve SP and CGRP, increased skin NK1 receptors and keratinocyte proliferation, increased in inflammatory mediator expression in the hindpaw skin (TNF-α, IL-1β, IL-6, NGF) and cord (IL-1β, NGF), and increased spinal c-Fos activation. These same changes were observed after cast immobilization alone, except spinal IL-1β levels were not increased. Treating cast only rats with an NK1 receptor antagonist inhibited development of nociceptive and inflammatory changes. Four weeks after fracture with pinning all nociceptive and vascular changes had resolved and there were no increases in neuropeptide signaling or inflammatory mediator expression.

Link To Article

http://dx.doi.org/10.1016/j.jpain.2014.07.004

Porphyromonas gingivalis infection increases osteoclastic bone resorption and osteoblastic bone formation in a periodontitis mouse model

Authors

Wenjian Zhang, Jun Ju, Todd Rigney and Gena Tribble

Abstract

Background Porphyromonas gingivalis has been shown to invade osteoblasts and inhibit their differentiation and mineralization in vitro. However, it is unclear if P. gingivalis can invade osteoblasts in vivo and how this would affect alveolar osteoblast/osteoclast dynamics. This study aims to answer these questions using a periodontitis mouse model under repetitive P. gingivalis inoculations.

Methods For 3-month-old BALB/cByJ female mice, 109 CFU of P. gingivalis were inoculated onto the gingival margin of maxillary molars 4 times at 2-day intervals. After 2 weeks, another 4 inoculations at 2-day intervals were applied. Calcein was injected 7 and 2 days before sacrificing animals to label the newly formed bone. Four weeks after final inoculation, mice were sacrificed and maxilla collected. Immunohistochemistry, micro-CT, and bone histomorphometry were performed on the specimens. Sham infection with only vehicle was the control.

Results P. gingivalis was found to invade gingival epithelia, periodontal ligament fibroblasts, and alveolar osteoblasts. Micro-CT showed alveolar bone resorption and significant reduction of bone mineral density and content in the infected mice compared to the controls. Bone histomorphometry showed a decrease in osteoblasts, an increase in osteoclasts and bone resorption, and a surprisingly increased osteoblastic bone formation in the infected mice compared to the controls.

Conclusions P. gingivalis invades alveolar osteoblasts in the periodontitis mouse model and cause alveolar bone loss. Although P. gingivalis appears to suppress osteoblast pool and enhance osteoclastic bone resorption, the bone formation capacity is temporarily elevated in the infected mice, possibly via some anti-microbial compensational mechanisms.

Link To Article

http://dx.doi.org/10.1186/1472-6831-14-89

A Combination of rhBMP-2 (Recombinant Human Bone Morphogenetic Protein-2) and MEK (MAP Kinase/ERK Kinase) Inhibitor...

Title

A Combination of rhBMP-2 (Recombinant Human Bone Morphogenetic Protein-2) and MEK (MAP Kinase/ERK Kinase) Inhibitor PD0325901 Increases Bone Formation in a Murine Model of Neurofibromatosis Type I Pseudarthrosis

Authors

J. El-Hoss, PhD; T. Cheng, BE/BMedSc; E.C. Carpenter, MRCS; K. Sullivan, PhD; N. Deo, BSc(Hons); K. Mikulec ; D.G. Little, MBBS, FRACS(Orth), PhD; A. Schindeler, PhD

Abstract

Background: Congenital tibial dysplasia is a severe pediatric condition that classically results in a persistent pseudarthrosis. A majority of these cases are associated with neurofibromatosis type I (NF1), a genetic disorder in which inactivation of the NF1 gene leads to overactivity of the Ras-MEK-MAPK (mitogen-activated protein kinase) signaling pathway. We therefore hypothesized that pharmaceutical inhibition of MEK-MAPK may be a beneficial therapeutic strategy.

Methods: In vitro methods were used to demonstrate a role for the MEK inhibitor PD0325901 in promoting osteogenic differentiation in Nf1−/− calvarial osteoblasts. Local applications of rhBMP-2 and/or PD0325901 were then tested in a mouse model of NF1 tibial pseudarthrosis featuring localized double inactivation of the Nf1 gene in a fracture. Mice received no treatment, PD0325901 (10 mg/kg/day from two days before fracture to ten days after fracture), rhBMP-2 (10 μg), or a combination of rhBMP-2 and PD0325901.

Results: Animals treated with the delivery vehicle alone, PD0325901, rhBMP-2, or the PD0325901 + rhBMP-2 combination showed union rates of 0%, 8%, 69% (p < 0.01), or 80% (p < 0.01), respectively, at twenty-one days after fracture. Mice treated with the rhBMP-2 + PD0325901 combination displayed a callus volume sixfold greater than the vehicle controls and twofold greater than the group receiving rhBMP-2 alone. Although MEK inhibition combined with rhBMP-2 led to increases in bone formation and union, the proportion of fibrous tissue in the callus was not significantly reduced.

Conclusions: The data suggest that MEK inhibition can promote bone formation in combination with rhBMP-2 in the context of an NF1 pseudarthrosis. However, PD0325901 did not promote substantive bone anabolism in the absence of an exogenous anabolic stimulus and did not suppress fibrosis.

Clinical Relevance: This study examines a signaling pathway-based approach to treating poor bone healing in a model of NF1 pseudarthrosis.

Link To Article

http://dx.doi.org/10.2106/JBJS.M.00862

Estrogen via Estrogen Receptor Beta Partially Inhibits Mandibular Condylar Cartilage Growth

Authors

J. Chen, Y. Kamiya, I. Polur, M. Xu, T. Choi, Z. Kalajzic, H. Drissi, S. Wadhwa

Abstract

Objective Temporomandibular joint diseases predominantly afflict women, suggesting a role for female hormones in the disease process. However, little is known about the role of estrogen receptor (ER) signaling in regulating mandibular condylar cartilage growth. Therefore, the goal of this study was to examine the effects of altered estrogen levels on the mandibular condylar cartilage in WT and ER beta KO mice.

Materials and Methods 21-day-old female WT (n=37) and ER beta KO mice (n=36) were either sham operated or ovariectomized, and treated with either placebo or estradiol. The mandibular condylar cartilage was evaluated by histomorphometry, proliferation was analyzed by double EdU/BrdU labeling, and assays on gene and protein expression of chondrocyte maturation markers were performed.

Results In WT mice, ovariectomy caused a significant increase in mandibular condylar cartilage cell numbers, a significant increase in Sox9 expression and a significant increase in proliferation compared with sham operated WT mice. In contrast, ovariectomy did not cause any of these effects in the ER beta KO mice. Estrogen replacement treatment in ovariectomized WT mice caused a significant decrease in ER alpha expression and a significant increase in Sost expression compared with ovariectomized mice treated with placebo. Estrogen replacement treatment in ovariectomized ER beta KO mice caused a significant increase in Col2 expression, no change in ER alpha expression, and a significant increase in Sost expression.

Conclusion Estrogen via ER beta inhibits proliferation and ER alpha expression while estrogen independent of ER beta induces Col2 and Sost expression.

Link To Article

http://dx.doi.org/10.1016/j.joca.2014.07.003