Inhibition of GSK-3ß rescues the impairments in bone formation and mechanical properties associated with fracture healing in osteoblast selective connexin 43 deficient mice.

Connexin 43 (Cx43) is the most abundant gap junction protein in bone and is required for osteoblastic differentiation and bone homeostasis. During fracture healing, Cx43 is abundantly expressed in osteoblasts and osteocytes, while Cx43 deficiency impairs bone formation and healing. In the present study we selectively deleted Cx43 in the osteoblastic lineage from immature osteoblasts through osteocytes and tested the hypothesis that Cx43 deficiency results in delayed osteoblastic differentiation and impaired restoration of biomechanical properties due to attenuated ß-catenin expression relative to wild type littermates. Here we show that Cx43 deficiency results in alterations in the mineralization and remodeling phases of healing. In Cx43 deficient fractures the mineralization phase is marked by delayed expression of osteogenic genes. Additionally, the decrease in the RankL/Opg ratio, osteoclast number and osteoclast size suggest decreased osteoclast bone resorption and remodeling. These changes in healing result in functional deficits as shown by a decrease in ultimate torque at failure. Consistent with these impairments in healing, ß-catenin expression is attenuated in Cx43 deficient fractures at 14 and 21 days, while Sclerostin (Sost) expression, a negative regulator of bone formation is increased in Cx43cKO fractures at 21 days, as is GSK-3ß, a key component of the ß-catenin proteasomal degradation complex. Furthermore, we show that alterations in healing in Cx43 deficient fractures can be rescued by inhibiting GSK-3ß activity using Lithium Chloride (LiCl). Treatment of Cx43 deficient mice with LiCl restores both normal bone formation and mechanical properties relative to LiCl treated WT fractures. This study suggests that Cx43 is a potential therapeutic target to enhance fracture healing and identifies a previously unknown role for Cx43 in regulating ß-catenin expression and thus bone formation during fracture repair.

Space Radiation and Bone Loss.

Exposure to ionizing radiation may negatively impact skeletal integrity during extended spaceflight missions to the moon, Mars, or near-Earth asteroids. However, our understanding of the effects of radiation on bone is limited when compared to the effects of weightlessness. In addition to microgravity, astronauts will be exposed to space radiation from solar and cosmic sources. Historically, radiation exposure has been shown to damage both osteoblast precursors and local vasculature within the irradiated volume. The resulting suppression of bone formation and a general state of low bone-turnover is thought to be the primary contributor to bone loss and eventual fracture. Recent investigations using mouse models have identified a rapid, but transient, increase in osteoclast activity immediately after irradiation with both spaceflight and clinically-relevant radiation qualities and doses. Together with a chronic suppression of bone formation after radiation exposure, this acute skeletal damage may contribute to long-term deterioration of bone quality, potentially increasing fracture risk. Direct evidence for the damaging effects of radiation on human bone are primarily demonstrated by the increased incidence of fractures at sites that absorb high doses of radiation during cancer therapy: exposures are considerably higher than what could be expected during spaceflight. However, both the rapidity of bone damage and the chronic nature of the changes appear similar between exposure scenarios. This review will outline our current knowledge of space and clinical exploration exposure to ionizing radiation on skeletal health.

Ionizing Radiation and Bone Loss: Space Exploration and Clinical Therapy Applications.

Damage to normal, nontumor bone tissue following therapeutic irradiation increases the risk of fracture among cancer patients. For example, women treated for various pelvic tumors have been shown to have a greater than 65% increased incidence of hip fracture by 5 years postradiotherapy. Another practical situation in which exposure to ionizing radiation may negatively impact skeletal integrity is during extended spaceflight missions. There is a limited understanding of how spaceflight-relevant doses and types of radiation can influence astronaut bone health, particularly when combined with the significant effects of mechanical unloading experienced in microgravity. Historically, negative effects on osteoblasts have been studied. Radiation exposure has been shown to damage osteoblast precursors. Damage to local vasculature has been observed, ranging from decreased lumen diameter to complete ablation within the irradiated volume, causing a state of hypoxia. These effects result in suppression of bone formation and a general state of low bone turnover. More recently, however, we have demonstrated in pre-clinical mouse models, a very rapid but transient increase in osteoclast activity after exposure to spaceflight and clinically relevant radiation doses. Combined with long-term suppression of bone formation, this skeletal damage may cause long-term deficits. This review will present a broad set of literature outlining our current set knowledge of both clinical therapy and space exploration exposure to ionizing radiation. Additionally, we will discuss prevention of the initial osteoclast-mediated bone loss, the need to promote normal bone turnover and long-term quality of bone tissue, and our hypothesized molecular mechanisms.

Gap Junctions and Biophysical Regulation of Bone Cells.

Communication between osteoblasts, osteoclasts, and osteocytes is integral to their ability to build and maintain the skeletal system and respond to physical signals. Various physiological mechanisms, including nerve communication, hormones, and cytokines, play an important role in this process. More recently, the important role of direct, cell-cell communication via gap junctions has been established. In this review, we demonstrate the integral role of gap junctional intercellular communication (GJIC) in skeletal physiology and bone cell mechanosensing.

Early increase in osteoclast number in mice after whole-body irradiation with 2 Gy X rays.

Bone loss is a consequence of exposure to high-dose radiotherapy. While damage to bone vasculature and reduced proliferation of bone-forming osteoblasts has been implicated in this process, the effect of radiation on the number and activity of bone-resorbing osteoclasts has not been characterized. In this study, we exposed mice to a whole-body dose of 2 Gy of X rays to quantify the early effects of radiation on osteoclasts and bone structural properties. Female C57BL/6 mice (13 weeks old) were divided into two groups: irradiated and nonirradiated controls. Animals were killed humanely 3 days after radiation exposure. Analysis of serum chemistry revealed a 14% increase in the concentration of tartrate resistant acid phosphatase (TRAP)-5b, a marker of osteoclast activity, in irradiated mice (P < 0.05). Osteoclast number (+44%; P < 0.05) and osteoclast surface (+213%; P < 0.001) were elevated in TRAP-stained histological sections of tibial metaphyses. No significant change was observed in osteoblast surface or osteocalcin concentration or in trabecular microarchitecture (i.e. bone volume fraction) as measured through microcomputed tomography (P > 0.05). This study provides definitive, quantitative evidence of an early, radiation-induced increase in osteoclast activity and number. Osteoclastic bone resorption may represent a contributor to bone atrophy observed after therapeutic irradiation.

Development of a low-dose anti-resorptive drug regimen reveals synergistic suppression of bone formation when coupled with disuse.

Safe and effective countermeasures to spaceflight-induced osteoporosis are required to mitigate the potential for mission-critical fractures and ensure long-term bone health in astronauts. Two anti-resorptive drugs, the bisphosphonate zoledronic acid (ZOL) and the anti-receptor activator of NF-kappaB ligand protein osteoprotegerin (OPG), were investigated to find the minimum, comparable doses that yield a maximal increase in bone quality, while minimizing deleterious effects on turnover and mineralization. Through a series of five trials in normally loaded female mice (n = 56/trial), analysis of trabecular volume fraction and connectivity using microcomputed tomography, along with biomechanical testing, quantitative histomorphometry, and compositional analysis, was used to select 45 microg/kg ZOL and 500 microg/kg OPG as doses that satisfy these criteria. These doses were then examined for their ability to mitigate bone loss following short-term unloading through hindlimb suspension (HLS). Seventy-two mice were prophylactically administered ZOL, OPG, or PBS and assigned to loaded control or 2-wk HLS groups (n = 12 for each of 6 groups). Both anti-resorptives were able to preserve trabecular microarchitecture and femoral elastic and maximum force in HLS mice (+30-40% ZOL/OPG vs. PBS). In HLS mice, anti-resorptive dosing reduced resorption perimeter at the femoral endocortical surface by 30% vs. PBS. In loaded control mice, anti-resorptives produced no change in bone formation rate; however, reductions in bone formation rate brought about by HLS were exacerbated by anti-resorptive treatment, suggesting synergistic inhibition of osteoblasts during disuse. Refined anti-resorptive dosing will tend to target countermeasures to the period of disuse, resulting in faster recovery and less adverse effects for astronauts.

Spaceflight-relevant types of ionizing radiation and cortical bone: Potential LET effect?

Extended exposure to microgravity conditions results in significant bone loss. Coupled with radiation exposure, this phenomenon may place astronauts at a greater risk for mission-critical fractures. In a previous study, we identified a profound and prolonged loss of trabecular bone (29-39%) in mice following exposure to an acute, 2 Gy dose of radiation simulating both solar and cosmic sources. However, because skeletal strength depends on trabecular and cortical bone, accurate assessment of strength requires analysis of both bone compartments. The objective of the present study was to examine various properties of cortical bone in mice following exposure to multiple types of spaceflight-relevant radiation. Nine-week old, female C57BL/6 mice were sacrificed 110 days after exposure to a single, whole body, 2 Gy dose of gamma, proton, carbon, or iron radiation. Femora were evaluated with biomechanical testing, microcomputed tomography, quantitative histomorphometry, percent mineral content, and micro-hardness analysis. Compared to non-irradiated controls, there were significant differences compared to carbon or iron radiation for only fracture force, medullary area and mineral content. A greater differential effect based on linear energy transfer (LET) level may be present: high-LET (carbon or iron) particle irradiation was associated with a decline in structural properties (maximum force, fracture force, medullary area, and cortical porosity) and mineral composition compared to low-LET radiation (gamma and proton). Bone loss following irradiation appears to be largely specific to trabecular bone and may indicate unique biological microenvironments and microdosimetry conditions. However, the limited time points examined and non-haversian skeletal structure of the mice employed highlight the need for further investigation.

Phylogeny of marine Gregarines (Apicomplexa)--Pterospora, Lithocystis and Lankesteria--and the origin(s) of coelomic parasitism.

Gregarines constitute a large group of apicomplexans with diverse modes of nutrition and locomotion that are associated with different host compartments (e.g. intestinal lumena and coelomic cavities). A broad molecular phylogenetic framework for gregarines is needed to infer the early evolutionary history of apicomplexans as a whole and the evolutionary relationships between the diverse ultrastructural and behavioral characteristics found in intestinal and coelomic gregarines. To this end, we sequenced the SSU rRNA gene from (1) Lankesteria abbotti from the intestines of two Pacific appendicularians, (2) Pterospora schizosoma from the coelom of a Pacific maldanid polychaete, (3) Pterospora floridiensis from the coelom of a Gulf Atlantic maldanid polychaete and (4) Lithocystis sp. from the coelom of a Pacific heart urchin. Molecular phylogenetic analyses including the new sequences demonstrated that several environmental and misattributed sequences are derived from gregarines. The analyses also demonstrated a clade of environmental sequences that was affiliated with gregarines, but as yet none of the constituent organisms have been described at the ultrastructural level (apicomplexan clade I). Lankesteria spp. (intestinal parasites of appendicularians) grouped closely with other marine intestinal eugregarines, particularly Lecudina tuzetae, from polychaetes. The sequences from all three coelomic gregarines branched within a larger clade of intestinal eugregarines and were similarly highly divergent. A close relationship between Pterospora schizosoma (Pacific) and Pterospora floridiensis (Gulf Atlantic) was strongly supported by the data. Lithocystis sp. was more closely related to a clade of marine intestinal gregarines consisting of Lankesteria spp. and Lecudina spp. than it was to the Pterospora clade. These data suggested that coelomic parasitism evolved more than once from different marine intestinal eugregarines, although a larger taxon sample is needed to further explore this inference.