Aged male rats regenerate cortical bone with reduced osteocyte density and reduced secretion of nitric oxide after mechanical stimulation.

Mechanical loading is integral to the repair of bone damage. Osteocytes are mechanosensors in bone and participate in signaling through gap junction channels, which are primarily comprised of connexin 43 (Cx43). Nitric oxide (NO) and prostaglandin E2 (PGE2) have anabolic and catabolic effects on bone, and the secretion of these molecules occurs after mechanical stimulation. The effect of age on the repair of bone tissue after damage and on the ability of regenerated bone to transduce mechanical stimulation into a cellular response is unexplored. The goal of this study was to examine (1) osteocytes and their mineralized matrix within regenerated bone from aged and mature animals and (2) the ability of regenerated bone explants from aged and mature animals to transduce cyclic mechanical loading into a cellular response through NO and PGE2 secretion. Bilateral cortical defects were created in the diaphysis of aged (21-month-old) or mature (6-month-old) male rats, and new bone tissue was allowed to grow into a custom implant of controlled geometry. Mineralization and mineral-to-matrix ratio were significantly higher in regenerated bone from aged animals, while lacunar and osteocyte density and phosphorylated (pCx43) and total Cx43 protein were significantly lower, relative to mature animals. Regenerated bone from mature rats had increased pCx43 protein and PGE2 secretion with loading and greater NO secretion relative to aged animals. Reduced osteocyte density and Cx43 in regenerated bone in aged animals could limit the establishment of gap junctions as well as NO and PGE2 secretion after loading, thereby altering bone formation and resorption in vivo.

Bone marrow stromal cells from aged male rats have delayed mineralization and reduced response to mechanical stimulation through nitric oxide and ERK1/2 signaling during osteogenic differentiation.

Bone marrow stromal cells (MSCs) are a source of osteoblast precursors that can be recruited during bone remodeling or injury, both important processes in aging populations. With advancing age, alterations in bone structure and mineralization are often associated with an increase in osteoporosis and fracture risk. Changes in the number of osteoprogenitor cells and their osteogenic potential may occur with advancing age; however few studies have considered the influence of mechanical conditions. Here, we investigated the ability of bone MSCs from mature and aged rats to differentiate into osteoblasts and to respond to short and long periods of mechanical stimulation through signaling by ERK1/2, nitric oxide (NO), and prostaglandin E(2) (PGE(2)) during differentiation. Mineralization was delayed and reduced, but extracellular matrix production appeared less affected by increased age. Differentiating MSCs from aged animals had a decreased response to short and long periods of mechanical stimulation through ERK1/2 signaling, and to long periods of mechanical loading through NO signaling early and late during differentiation. Increases in relative PGE(2) signaling were higher in MSCs from aged animals, which could compensate for reduced ERK1/2 and NO signaling. The decreased mineralization may decrease the ability of cells from aged animals to respond to mechanical stimulation through ERK1/2 and NO signaling, with increased impairment over differentiation time. Decreasing the delay in mineralization of MSCs from aging animals might improve their ability to respond to mechanical stimulation during bone remodeling and injury, suggesting therapies for bone fragility diseases and tissue engineering treatments in elderly populations.

Controversy of physiological vs. pharmacological effects of BMP signaling: Constitutive activation of BMP type IA receptor-dependent signaling in osteoblast lineage enhances bone formation and resorption, not affecting net bone mass.

Bone morphogenetic proteins (BMPs) were first described over 50 years ago as potent inducers of ectopic bone formation when administrated subcutaneously. Preclinical studies have extensively examined the osteoinductive properties of BMPs in vitro and new bone formation in vivo. BMPs (BMP-2, BMP-7) have been used in orthopedics over 15 years. While osteogenic function of BMPs has been widely accepted, our previous studies demonstrated that loss-of-function of BMP receptor type IA (BMPR1A), a potent receptor for BMP-2, increased net bone mass by significantly inhibiting bone resorption in mice, indicating a positive role of BMP signaling in bone resorption. The physiological role of BMPs (i.e. osteogenic vs. osteoclastogenic) is still largely unknown. The purpose of this study was to investigate the physiological role of BMP signaling in endogenous long bones during adult stages. For this purpose, we conditionally and constitutively activated the Smad-dependent canonical BMP signaling thorough BMPR1A in osteoblast lineage cells using the mutant mice (Col1CreER™:caBmpr1a). Because trabecular bones were largely increased in the loss-of-function mouse study for BMPR1A, we hypothesized that the augmented BMP signaling would affect endogenous trabecular bones. In the mutant bones, the Smad phosphorylation was enhanced within physiological level three-fold while the resulting gross morphology, bodyweights, bone mass/shape/length, serum calcium/phosphorus levels, collagen cross-link patterns, and healing capability were all unchanged. Interestingly, we found; 1) increased expressions of both bone formation and resorption markers in femoral bones, 2) increased osteoblast and osteoclast numbers together with dynamic bone formation parameters by trabecular bone histomorphometry, 3) modest bone architectural phenotype with reduced bone quality (i.e. reduced trabecular bone connectivity, larger diametric size but reduced cortical bone thickness, and reduced bone mechanical strength), and 4) increased expression of SOST, a downstream target of the Smad-dependent BMPR1A signaling, in the mutant bones. This study is clinically insightful because gain-of-function of BMP signaling within a physiological window does not increase bone mass while it alters molecular and cellular aspects of osteoblast and osteoclast functions as predicted. These findings help explain the high-doses of BMPs (i.e. pharmacological level) in clinical settings required to substantially induce a bone formation, concurrent with potential unexpected side effects (i.e. bone resorption, inflammation) presumably due to a broader population of cell-types exposed to the high-dose BMPs rather than osteoblastic lineage cells.

Loss of BMP signaling mediated by BMPR1A in osteoblasts leads to differential bone phenotypes in mice depending on anatomical location of the bones.

Bone morphogenetic protein (BMP) signaling in osteoblasts plays critical roles in skeletal development and bone homeostasis. Our previous studies showed loss of function of BMPR1A, one of the type 1 receptors for BMPs, in osteoblasts results in increased trabecular bone mass in long bones due to an imbalance between bone formation and bone resorption. Decreased bone resorption was associated with an increased mature-to-immature collagen cross-link ratio and mineral-matrix ratios in the trabecular compartments, and increased tissue-level biomechanical properties. Here, we investigated the bone mass, bone composition and biomechanical properties of ribs and spines in the same genetically altered mouse line to compare outcomes by loss of BMPR1A functions in bones from different anatomic sites and developmental origins. Bone mass was significantly increased in both cortical and trabecular compartments of ribs with minimal to modest changes in compositions. While tissue-levels of biomechanical properties were not changed between control and mutant animals, whole bone levels of biomechanical properties were significantly increased in association with increased bone mass in the mutant ribs. For spines, mutant bones showed increased bone mass in both cortical and trabecular compartments with an increase of mineral content. These results emphasize the differential role of BMP signaling in osteoblasts in bones depending on their anatomical locations, functional loading requirements and developmental origin.

Bone mass is inversely proportional to Dkk1 levels in mice.

The Wnt/beta-catenin signaling pathway has emerged as a key regulator in bone development and bone homeostasis. Loss-of-function mutations in the Wnt co-receptor LRP5 result in osteoporosis and "activating" mutations in LRP5 result in high bone mass. Dickkopf-1 (DKK1) is a secreted Wnt inhibitor that binds LRP5 and LRP6 during embryonic development, therefore it is expected that a decrease in DKK1 will result in an increase in Wnt activity and a high bone mass phenotype. Dkk1-/- knockout mice are embryonic lethal, but mice with hypomorphic Dkk1d (doubleridge) alleles that express low amounts of Dkk1 are viable. In this study we generated an allelic series by crossing Dkk1+/- and Dkk1+/d mice resulting in the following genotypes with decreasing Dkk1 expression levels: +/+, +/d, +/- and d/-. Using muCT imaging we scanned dissected left femora and calvariae from 8-week-old mice (n=60). We analyzed the distal femur to represent trabecular bone and the femur diaphysis for cortical endochondral bone. A region of the parietal bones was used to analyze intramembranous bone of the calvaria. We found that trabecular bone volume is increased in Dkk1 mutant mice in a manner that is inversely proportional to the level of Dkk1 expression. Trabeculae number and thickness were significantly higher in the low Dkk1 expressing genotypes from both female and male mice. Similar results were found in cortical bone with an increase in cortical thickness and cross sectional area of the femur diaphysis that correlated with lower Dkk1 expression. No consistent differences were found in the calvaria measurements. Our results indicate that the progressive Dkk1 reduction increases trabecular and cortical bone mass and that even a 25% reduction in Dkk1 expression could produce significant increases in trabecular bone volume fraction. Thus DKK1 is a negative regulator of normal bone homeostasis in vivo. Our study suggests that manipulation of DKK1 function or expression may have therapeutic significance for the treatment of low bone mass disorders.

Characterization of genetically engineered mouse models carrying Col2a1-cre-induced deletions of Lrp5 and/or Lrp6.

Mice carrying Collagen2a1-cre-mediated deletions of Lrp5 and/or Lrp6 were created and characterized. Mice lacking either gene alone were viable and fertile with normal knee morphology. Mice in which both Lrp5 and Lrp6 were conditionally ablated via Collagen2a1-cre-mediated deletion displayed severe defects in skeletal development during embryogenesis. In addition, adult mice carrying Collagen2a1-cre-mediated deletions of Lrp5 and/or Lrp6 displayed low bone mass suggesting that the Collagen2a1-cre transgene was active in cells that subsequently differentiated into osteoblasts. In both embryonic skeletal development and establishment of adult bone mass, Lrp5 and Lrp6 carry out redundant functions.

Accelerated and increased joint damage in young mice with global inactivation of mitogen-inducible gene 6 after ligament and meniscus injury.

INTRODUCTION: Ligament and meniscal damage can cause joint disease. Arthritic joints contain increased amounts of epidermal growth factor receptor (EGFR) protein, and polymorphisms in EGFR are associated with arthritis risk. The role of endogenous EGFR regulation during joint disease due to ligament and meniscal trauma is unknown. Mitogen-inducible gene 6 (MIG-6) can reduce EGFR phosphorylation and downstream signaling. We examined the effect of EGFR modulation by MIG-6 on joint disease development after ligament and meniscus injury. METHODS: Knee ligament transection and meniscus removal were performed surgically on mice homozygous for a global inactivating mutation in MIG-6 (Mig-6⁻/⁻) and in wild-type (WT) animals. RESULTS: Two weeks after surgery, Mig-6⁻/⁻mice had bone erosion as well as greater fibrous tissue area and serum RANKL concentration than WT mice. Four weeks after surgery, Mig-6⁻/⁻mice had less cartilage and increased cell proliferation relative to contralateral control and WT knees. Increased apoptotic cells and growth outside the articulating region occurred in Mig-6⁻/⁻mice. Tibia trabecular bone mineral density (BMD) and the number of trabeculae were lower in surgically treated knees relative to the respective control knees for both groups. BMD, as well as trabecular thickness and number, were lower in surgically treated knees from Mig-6⁻/⁻mice relative to WT surgically treated knees. Phosphorylated EGFR staining in surgically treated knees decreased for WT mice and increased for Mig-6⁻/⁻mice. Fewer inflammatory cells were present in the knees of WT mice. CONCLUSION: Mig-6⁻/⁻mice have rapid and increased joint damage after ligament and meniscal trauma. Mig-6 modification could lessen degenerative disease development after this type of injury.

LRP5 and LRP6 in development and disease.

Low-density lipoprotein-related receptors 5 and 6 (LRP5/6) are highly homologous proteins with key functions in canonical Wnt signaling. Alterations in the genes encoding these receptors or their interacting proteins are linked to human diseases, and as such they have been a major focus of drug development efforts to treat several human conditions including osteoporosis, cancer, and metabolic disease. Here, we discuss the links between alterations in LRP5/6 and disease, proteins that interact with them, and insights gained into their function from mouse models. We also highlight current drug development related to LRP5/6 as well as how the recent elucidation of their crystal structures may allow further refinement of our ability to target them for therapeutic benefit.