Radiation‑induced cytochrome c release and the neuroprotective effects of the pan-caspase inhibitor z‑VAD‑fmk in the hypoglossal nucleus

Authors

Jianguo Li, Yan Wang, Liqing Du, Chang Xu, Jia Cao, Qin Wang, Qiang Liu, Feiyue Fan

Abstract

Numerous studies have demonstrated that neuronal cell death occurs via extrinsic (death receptors) and intrinsic (mitochondria) pathways. Radiation induces caspase activation fundamentally via the mitochondrial pathway. To investigate the role of caspase, a cell permeable pan-caspase inhibitor, z-VAD-fmk [N-benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone], was used to investigate the effects of caspase blockade in vivo following irradiation. Adult male Sprague‑Dawley rats (weight, 250‑300 g) underwent irradiation at room temperature with a 4‑Gy dose of radiation. Since z-VAD-fmk does not penetrate the blood‑brain barrier, it was applied intracerebroventricularly via a bolus injection (0.2 µg/h for 1 h). Terminal deoxynucleotidyl transferase dUTP nick end‑labeling (TUNEL) demonstrated that z-VAD‑fmk reduced the numbers of TUNEL-positive cells within the hypoglossal nucleus, suggesting that intervention in the caspase cascade following radiation may have therapeutic applications. The caspase inhibitor z-VAD-fmk reduced the expression and activation of caspase-3, caspase-8 and caspase‑9 in the irradiated rats, indicating that caspase may be a potential therapeutic target in the treatment of brain radiation injury. Treatment with z-VAD-fmk also reduced the appearance of cytochrome c within the cytosolic fraction following radiation. The hypoglossal nucleus may be used as a model of radiation‑induced injury in the central nervous system, providing visual information and displaying apoptotic nuclear morphology.

Link To Article

http://dx.doi.org/10.3892/etm.2013.1419

Streptozotocin-induced diabetes in rats diminishes the size of the osteoprogenitor pool in bone marrow

Authors

E. Weinberg, T. Maymon, O. Moses, M. Weinre

Abstract

Aims

Bone formation is reduced in animals and humans with type 1 diabetes, leading to lower bone mass and inferior osseous healing. Since bone formation greatly depends on the recruitment of osteoblasts from their bone marrow precursors, we tested whether experimental type 1 diabetes in rats diminishes the number of bone marrow osteoprogenitors.

Methods

Diabetes was induced by 65 mg/kg streptozotocin and after 4 weeks, femoral bone marrow cells were extracted and cultured. Tibia and femur were frozen for further analysis.

Results

The size of the osteoprogenitor pool in bone marrow of diabetic rats was significantly reduced, as evidenced by 1) lower (∼35%) fraction of adherent stromal cells (at 24 h of culture); 2) lower (20-25%) alkaline phosphatase activity at 10 days of culture; and 3) lower (∼40%) mineralized nodule formation at 21 days of culture. Administration of insulin to hyperglycemic rats normalized glycemia and abrogated most of the decline in ex-vivo mineralized nodule formation. Apoptotic cells in tibial bone marrow were more numerous in hyperglycemic rats. Also, the levels of malondialdehyde (indicator of oxidative stress) were significantly elevated in bone marrow of diabetic animals.

Conclusions

Experimental type 1 diabetes diminishes the osteoprogenitor population in bone marrow, possibly due to increased apoptosis via Oxidative Stress. Reduced number of osteoprogenitors is likely to impair osteoblastogenesis, bone formation, and bone healing in diabetic animals.

Link to Article

http://dx.doi.org/10.1016/j.diabres.2013.11.015

Osteoblast CFTR Inactivation Reduces Differentiation and Osteoprotegerin Expression in a Mouse Model of Cystic Fibrosis-Related Bone Disease

Authors

Michael S. Stalvey, Katrina L. Clines, Viktoria Havasi, Christopher R. McKibbin, Lauren K. Dunn, W. Joon Chung, Gregory A. Clines

Abstract

Low bone mass and increased fracture risk are recognized complications of cystic fibrosis (CF). CF-related bone disease (CFBD) is characterized by uncoupled bone turnover—impaired osteoblastic bone formation and enhanced osteoclastic bone resorption. Intestinal malabsorption, vitamin D deficiency and inflammatory cytokines contribute to CFBD. However, epidemiological investigations and animal models also support a direct causal link between inactivation of skeletal cystic fibrosis transmembrane regulator (CFTR), the gene that when mutated causes CF, and CFBD. The objective of this study was to examine the direct actions of CFTR on bone. Expression analyses revealed that CFTR mRNA and protein were expressed in murine osteoblasts, but not in osteoclasts. Functional studies were then performed to investigate the direct actions of CFTR on osteoblasts using a CFTR knockout (Cftr−/−) mouse model. In the murine calvarial organ culture assay, Cftr−/− calvariae displayed significantly less bone formation and osteoblast numbers than calvariae harvested from wildtype (Cftr+/+) littermates. CFTR inactivation also reduced alkaline phosphatase expression in cultured murine calvarial osteoblasts. Although CFTR was not expressed in murine osteoclasts, significantly more osteoclasts formed in Cftr−/− compared to Cftr+/+ bone marrow cultures. Indirect regulation of osteoclastogenesis by the osteoblast through RANK/RANKL/OPG signaling was next examined. Although no difference in receptor activator of NF-κB ligand (Rankl) mRNA was detected, significantly less osteoprotegerin (Opg) was expressed in Cftr−/− compared to Cftr+/+ osteoblasts. Together, the Rankl:Opg ratio was significantly higher in Cftr−/− murine calvarial osteoblasts contributing to a higher osteoclastogenesis potential. The combined findings of reduced osteoblast differentiation and lower Opg expression suggested a possible defect in canonical Wnt signaling. In fact, Wnt3a and PTH-stimulated canonical Wnt signaling was defective in Cftr−/− murine calvarial osteoblasts. These results support that genetic inactivation of CFTR in osteoblasts contributes to low bone mass and that targeting osteoblasts may represent an effective strategy to treat CFBD.

Link To Article

http://dx.doi.org/10.1371/journal.pone.0080098

Sclerostin Inhibition Reverses Skeletal Fragility in an Lrp5-Deficient Mouse Model of OPPG Syndrome

Authors

Rajendra Kedlaya, Shreya Veera, Daniel J. Horan, Rachel E. Moss, Ugur M. Ayturk, Christina M. Jacobsen, Margot E. Bowen, Chris Paszty, Matthew L. Warman, and Alexander G. Robling

Abstract

Osteoporosis pseudoglioma syndrome (OPPG) is a rare genetic disease that produces debilitating effects in the skeleton. OPPG is caused by mutations in LRP5, a WNT co-receptor that mediates osteoblast activity. WNT signaling through LRP5, and also through the closely related receptor LRP6, is inhibited by the protein sclerostin (SOST). It is unclear whether OPPG patients might benefit from the anabolic action of sclerostin neutralization therapy (an approach currently being pursued in clinical trials for postmenopausal osteoporosis) in light of their LRP5 deficiency and consequent osteoblast impairment. To assess whether loss of sclerostin is anabolic in OPPG, we measured bone properties in a mouse model of OPPG (Lrp5−/−), a mouse model of sclerosteosis (Sost−/−), and in mice with both genes knocked out (Lrp5−/−;Sost−/−). Lrp5−/−;Sost−/− mice have larger, denser, and stronger bones than do Lrp5−/− mice, indicating that SOST deficiency can improve bone properties via pathways that do not require LRP5. Next, we determined whether the anabolic effects of sclerostin depletion in Lrp5−/− mice are retained in adult mice by treating 17-week-old Lrp5−/− mice with a sclerostin antibody for 3 weeks. Lrp5+/+ and Lrp5−/− mice each exhibited osteoanabolic responses to antibody therapy, as indicated by increased bone mineral density, content, and formation rates. Collectively, our data show that inhibiting sclerostin can improve bone mass whether LRP5 is present or not. In the absence of LRP5, the anabolic effects of SOST depletion can occur via other receptors (such as LRP4/6). Regardless of the mechanism, our results suggest that humans with OPPG might benefit from sclerostin neutralization therapies.

Link To Article

http://dx.doi.org/10.1126/scitranslmed.3006627

Inhibition of GSK-3β Rescues the Impairments in Bone Formation and Mechanical Properties Associated with Fracture Healing in Osteoblast Selective Connexin 43 Deficient Mice

Authors

Alayna E. Loiselle, Shane A. J. Lloyd, Emmanuel M. Paul, Gregory S. Lewis, Henry J. Donahue

Abstract

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 β-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, β-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-3β, a key component of the β-catenin proteasomal degradation complex. Furthermore, we show that alterations in healing in Cx43 deficient fractures can be rescued by inhibiting GSK-3β 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 β-catenin expression and thus bone formation during fracture repair.

Link To Article

http://dx.doi.org/10.1371/journal.pone.0081399

Sclerostin deficient mice rapidly heal bone defects by activating β-catenin and increasing intramembranous ossification

Authors

Meghan E. McGee-Lawrence, Zachary C. Ryan, Lomeli R. Carpioc, Sanjeev Kakar, Jennifer J. Westendorf, Rajiv Kumar

Abstract

We investigated the influence of the osteocyte protein, sclerostin, on fracture healing by examining the dynamics and mechanisms of repair of single-cortex, stabilized femoral defects in sclerostin knockout (Sost−/−; KO) and sclerostin wild-type (Sost+/+; WT) mice. Fourteen days following generation of bone defects, Sost KO mice had significantly more bone in the healing defect than WT mice. The increase in regenerating bone was due to an increase in the thickness of trabecularized spicules, osteoblast numbers and surfaces within the defect. Enhanced healing of bone defects in Sost KO mice was associated with significantly more activated β-catenin expression than observed in WT mice. The findings were similar to those observed in Axin2−/− mice, in which β-catenin signaling is known to be enhanced to facilitate bone regeneration. Taken together, these data indicate that enhanced β-catenin signaling is present in Sost−/− mice that demonstrate accelerated healing of bone defects, suggesting that modulation of β-catenin signaling in bone could be used to promote fracture repair.

Link to Article

http://dx.doi.org/10.1016/j.bbrc.2013.10.155