Loss of sclerostin promotes osteoarthritis in mice via β-catenin-dependent and -independent Wnt pathways

IntroductionSclerostin is a Wnt inhibitor produced by osteocytes that regulates bone formation. Because bone tissue contributes to the development of osteoarthritis (OA), we investigated the role of sclerostin in bone and cartilage in a joint instability model in mice.MethodsTen-week-old SOST-knockout (SOST-KO) and wild-type (WT) mice underwent destabilization of the medial meniscus (DMM). We measured bone volume at the medial femoral condyle and osteophyte volume and determined the OA score and expression of matrix proteins. Primary murine chondrocytes were cultured with Wnt3a and sclerostin to assess the expression of matrix proteins, proteoglycan release and glycosaminoglycan accumulation.ResultsSclerostin was expressed in calcified cartilage of WT mice with OA. In SOST-KO mice, cartilage was preserved despite high bone volume. However, SOST-KO mice with DMM had a high OA score, with increased expression of aggrecanases and type X collagen. Moreover, SOST-KO mice with OA showed disrupted anabolic–catabolic balance and cartilage damage. In primary chondrocytes, sclerostin addition abolished Wnt3a-increased expression of a disintegrin and metalloproteinase with thrombospondin motifs, matrix metalloproteinases and type X collagen by inhibiting the canonical Wnt pathway. Moreover, sclerostin inhibited Wnt-phosphorylated c-Jun N-terminal kinase (JNK) and rescued the expression of anabolic genes. Furthermore, sclerostin treatment inhibited both Wnt canonical and non-canonical JNK pathways in chondrocytes, thus preserving metabolism.ConclusionSclerostin may play an important role in maintaining cartilage integrity in OA.

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The online version of this article (doi:10.1186/s13075-015-0540-6) contains supplementary material, which is available to authorized users.     

Distinct Modes of Inhibition by Sclerostin on Bone Morphogenetic Protein and Wnt Signaling Pathways

Sclerostin is expressed by osteocytes and has catabolic effects on bone. It has been shown to antagonize bone morphogenetic protein (BMP) and/or Wnt activity, although at present the underlying mechanisms are unclear. Consistent with previous findings, Sclerostin opposed direct Wnt3a-induced but not direct BMP7-induced responses when both ligand and antagonist were provided exogenously to cells. However, we found that when both proteins are expressed in the same cell, sclerostin can antagonize BMP signaling directly by inhibiting BMP7 secretion. Sclerostin interacts with both the BMP7 mature domain and pro-domain, leading to intracellular retention and proteasomal degradation of BMP7. Analysis of sclerostin knock-out mice revealed an inhibitory action of sclerostin on Wnt signaling in both osteoblasts and osteocytes in cortical and cancellous bones. BMP7 signaling was predominantly inhibited by sclerostin in osteocytes of the calcaneus and the cortical bone of the tibia. Our results suggest that sclerostin exerts its potent bone catabolic effects by antagonizing Wnt signaling in a paracrine and autocrine manner and antagonizing BMP signaling selectively in the osteocytes that synthesize simultaneously both sclerostin and BMP7 proteins.     

Expression of sclerostin in the developing zebrafish (Danio rerio) brain and skeleton

Sclerostin is a highly conserved, secreted, cystine-knot protein which regulates osteoblast function. Humans with mutations in the sclerostin gene (SOST), manifest increased axial and appendicular skeletal bone density with attendant complications. In adult bone, sclerostin is expressed in osteocytes and osteoblasts. Danio rerio sclerostin-like protein is closely related to sea bass sclerostin, and is related to chicken and mammalian sclerostins. Little is known about the expression of sclerostin in early developing skeletal or extra-skeletal tissues. We assessed sclerostin (sost) gene expression in developing zebrafish (D. rerio) embryos with whole mount is situ hybridization methods. The earliest expression of sost mRNA was noted during 12h post-fertilization (hpf). At 15hpf, sost mRNA was detected in the developing nervous system and in Kupffer's vesicle. At 18, 20 and 22hpf, expression in rhombic lip precursors was seen. By 24hpf, expression in the upper and lower rhombic lip and developing spinal cord was noted. Expression in the rhombic lip and spinal cord persisted through 28hpf and then diminished in intensity through 44hpf. At 28hpf, sost expression was noted in developing pharyngeal cartilage; expression in pharyngeal cartilage increased with time. By 48hpf, sost mRNA was clearly detected in the developing pharyngeal arch cartilage. Sost mRNA was abundantly expressed in the pharyngeal arch cartilage, and in developing pectoral fins, 72, 96 and 120hpf. Our study is the first detailed analysis of sost gene expression in early metazoan development.     

Suppression of Autophagy in Osteocytes Mimics Skeletal Aging

Bone mass declines with age but the mechanisms responsible remain unclear. Here we demonstrate that deletion of a conditional allele for Atg7, a gene essential for autophagy, from osteocytes caused low bone mass in 6-month-old male and female mice. Cancellous bone volume and cortical thickness were decreased, and cortical porosity increased, in conditional knock-out mice compared with control littermates. These changes were associated with low osteoclast number, osteoblast number, bone formation rate, and wall width in the cancellous bone of conditional knock-out mice. In addition, oxidative stress was higher in the bones of conditional knock-out mice as measured by reactive oxygen species levels in the bone marrow and by p66^shc phosphorylation in L6 vertebra. Each of these changes has been previously demonstrated in the bones of old versus young adult mice. Thus, these results demonstrate that suppression of autophagy in osteocytes mimics, in many aspects, the impact of aging on the skeleton and suggest that a decline in autophagy with age may contribute to the low bone mass associated with aging.     

Nupr1 deficiency downregulates HtrA1, enhances SMAD1 signaling,and suppresses age-related bone loss in male mice

Nuclear protein 1 (NUPR1) is a stress-induced protein activated by various stresses, such as inflammation and oxidative stress. We previously reported that Nupr1 deficiency increased bone volume by enhancing bone formation in 11-week-old mice. Analysis of differentially expressed genes between wild-type (WT) and Nupr1-knockout (Nupr1-KO) osteocytes revealed that high temperature requirement A 1 (HTRA1), a serine protease implicated in osteogenesis and transforming growth factor-β signaling was markedly downregulated in Nupr1-KO osteocytes. Nupr1 deficiency also markedly reduced HtrA1 expression, but enhanced SMAD1 signaling in in vitro-cultured primary osteoblasts. In contrast, Nupr1 overexpression enhanced HtrA1 expression in osteoblasts, suggesting that Nupr1 regulates HtrA1 expression, thereby suppressing osteoblastogenesis. Since HtrA1 is also involved in cellular senescence and age-related diseases, we analyzed aging-related bone loss in Nupr1-KO mice. Significant spine trabecular bone loss was noted in WT male and female mice during 6−19 months of age, whereas aging-related trabecular bone loss was attenuated, especially in Nupr1-KO male mice. Moreover, cellular senescence-related markers were upregulated in the osteocytes of 6−19-month-old WT male mice but markedly downregulated in the osteocytes of 19-month-old Nupr1-KO male mice. Oxidative stress-induced cellular senescence stimulated Nupr1 and HtrA1 expression in in vitro-cultured primary osteoblasts, and Nupr1 overexpression enhanced p16^ink4a expression in osteoblasts. Finally, NUPR1 expression in osteocytes isolated from the bones of patients with osteoarthritis was correlated with age. Collectively, these results indicate that Nupr1 regulates HtrA1-mediated osteoblast differentiation and senescence. Our findings unveil a novel Nupr1/HtrA1 axis, which may play pivotal roles in bone formation and age-related bone loss.     

Spag17 Deficiency Results in Skeletal Malformations and Bone Abnormalities

Height is the result of many growth and development processes. Most of the genes associated with height are known to play a role in skeletal development. Single-nucleotide polymorphisms in the SPAG17 gene have been associated with human height. However, it is not clear how this gene influences linear growth. Here we show that a targeted mutation in Spag17 leads to skeletal malformations. Hind limb length in mutants was significantly shorter than in wild-type mice. Studies revealed differences in maturation of femur and tibia suggesting alterations in limb patterning. Morphometric studies showed increased bone formation evidenced by increased trabecular bone area and the ratio of bone area to total area, leading to reductions in the ratio of marrow area/total area in the femur. Micro-CTs and von Kossa staining demonstrated increased mineral in the femur. Moreover, osteocalcin and osterix were more highly expressed in mutant mice than in wild-type mice femurs. These data suggest that femur bone shortening may be due to premature ossification. On the other hand, tibias appear to be shorter due to a delay in cartilage and bone development. Morphometric studies showed reduction in growth plate and bone formation. These defects did not affect bone mineralization, although the volume of primary bone and levels of osteocalcin and osterix were higher. Other skeletal malformations were observed including fused sternebrae, reduced mineralization in the skull, medial and metacarpal phalanges. Primary cilia from chondrocytes, osteoblasts, and embryonic fibroblasts (MEFs) isolated from knockout mice were shorter and fewer cells had primary cilia in comparison to cells from wild-type mice. In addition, Spag17 knockdown in wild-type MEFs by Spag17 siRNA duplex reproduced the shorter primary cilia phenotype. Our findings disclosed unexpected functions for Spag17 in the regulation of skeletal growth and mineralization, perhaps because of its role in primary cilia of chondrocytes and osteoblasts.     

Skeletal Characterization of Smurf2-Deficient Mice and In Vitro Analysis of Smurf2-Deficient Chondrocytes

Overexpression of Smad ubiquitin regulatory factor 2 (Smurf2) in chondrocytes was reported to cause spontaneous osteoarthritis (OA) in mice. However, it is unclear whether Smurf2 is involved in bone and cartilage homeostasis and if it is required for OA pathogenesis. Here we characterized age-related changes in the bone and articular cartilage of Smurf2-deficient (MT) mice by microCT and histology, and examined whether reduced Smurf2 expression affected the severity of OA upon surgical destabilization of the medial meniscus (DMM). Using immature articular chondrocytes (iMAC) from MT and wild-type (WT) mice, we also examined how Smurf2 deficiency affects chondrogenic and catabolic gene expressions and Smurf2 and Smurf1 proteins upon TGF-β3 or IL-1β treatment in culture. We found no differences in cortical, subchondral and trabecular bone between WT and MT in young (4 months) and old mice (16–24 months). The articular cartilage and age-related alterations between WT and MT were also similar. However, 2 months following DMM, young MT showed milder OA compared to WT (~70% vs ~30% normal or exhibiting only mild OA cartilage phenotype). The majority of the older WT and MT mice developed moderate/severe OA 2 months after DMM, but a higher subset of aged MT cartilage (27% vs. 9% WT) remained largely normal. Chondrogenic gene expression (Sox9, Col2, Acan) trended higher in MT iMACs than WT with/without TGF-β3 treatment. IL-1β treatment suppressed chondrgenic gene expression, but Sox9 expression in MT remained significantly higher than WT. Smurf2 protein in WT iMACs increased upon TGF-β3 treatment and decreased upon IL-1β treatment in a dose-dependent manner. Smurf1 protein elevated more in MT than WT upon TGF-β3 treatment, suggesting a potential, but very mild compensatory effect. Overall, our data support a role of Smurf2 in regulating OA development but suggest that inhibiting Smurf2 alone may not be sufficient to prevent or consistently mitigate post-traumatic OA across a broad age range.     

Enhanced prostacyclin formation and Wnt signaling in sclerostin deficient osteocytes and bone

We show that prostacyclin production is increased in bone and osteocytes from sclerostin (Sost) knockout mice which have greatly increased bone mass. The addition of prostacyclin or a prostacyclin analog to bone forming osteoblasts enhances differentiation and matrix mineralization of osteoblasts. The increase in prostacyclin synthesis is linked to increases in β-catenin concentrations and activity as shown by enhanced binding of lymphoid enhancer factor, Lef1, to promoter elements within the prostacyclin synthase promoter. Blockade of Wnt signaling reduces prostacyclin production in osteocytes. Increased prostacyclin production by osteocytes from sclerostin deficient mice could potentially contribute to the increased bone formation seen in this condition.     

Osteocyte control of bone formation via sclerostin, a novel BMP antagonist

There is an unmet medical need for anabolic treatments to restore lost bone. Human genetic bone disorders provide insight into bone regulatory processes. Sclerosteosis is a disease typified by high bone mass due to the loss of SOST expression. Sclerostin, the SOST gene protein product, competed with the type I and type II bone morphogenetic protein (BMP) receptors for binding to BMPs, decreased BMP signaling and suppressed mineralization of osteoblastic cells. SOST expression was detected in cultured osteoblasts and in mineralizing areas of the skeleton, but not in osteoclasts. Strong expression in osteocytes suggested that sclerostin expressed by these central regulatory cells mediates bone homeostasis. Transgenic mice overexpressing SOST exhibited low bone mass and decreased bone strength as the result of a significant reduction in osteoblast activity and subsequently, bone formation. Modulation of this osteocyte-derived negative signal is therapeutically relevant for disorders associated with bone loss.     

Rapid loss of bone mass and strength in mice after abdominal irradiation

Localized irradiation is a common treatment modality for malignancies in the pelvic-abdominal cavity. We report here on the changes in bone mass and strength in mice 7-14 days after abdominal irradiation. Male C57BL/6 mice of 10-12 weeks of age were given a single-dose (0, 5, 10, 15 or 20 Gy) or fractionated (3 Gy × 2 per day × 7.5 days) X rays to the abdomen and monitored daily for up to 14 days. A decrease in the serum bone formation marker and ex vivo osteoblast differentiation was detected 7 days after a single dose of radiation, with little change in the serum bone resorption marker and ex vivo osteoclast formation. A single dose of radiation elicited a loss of bone mineral density (BMD) within 14 days of irradiation. The BMD loss was up to 4.1% in the whole skeleton, 7.3% in tibia, and 7.7% in the femur. Fractionated abdominal irradiation induced similar extents of BMD loss 10 days after the last fraction: 6.2% in the whole skeleton, 5.1% in tibia, and 13.8% in the femur. The loss of BMD was dependent on radiation dose and was more profound in the trabecula-rich regions of the long bones. Moreover, BMD loss in the total skeleton and the femurs progressed with time. Peak load and stiffness in the mid-shaft tibia from irradiated mice were 11.2-14.2% and 11.5-25.0% lower, respectively, than sham controls tested 7 days after a single-dose abdominal irradiation. Our data demonstrate that abdominal irradiation induces a rapid loss of BMD in the mouse skeleton. These effects are bone type- and region-specific but are independent of radiation fractionation. The radiation-induced abscopal damage to the skeleton is manifested by the deterioration of biomechanical properties of the affected bone.     

Impaired endochondral bone development and osteopenia in Gli2-deficient mice

Mice homozygous for targeted disruption of the zinc finger domain of Gli2 (Gli2(zfd/zfd)) die at birth with developmental defects in several organ systems including the skeleton. The current studies were undertaken to define the role of Gli2 in endochondral bone development by characterizing the molecular defects in the limbs and vertebrae of Gli2(zfd/zfd) mice. The bones of mutant mice removed by cesarian section at E16.5 and E18.5 demonstrated delayed endochondral ossification. This was accompanied by an increase in the length of cartilaginous growth plates, reduced bone tissue in the femur and tibia and by failure to develop the primary ossification centre in vertebral bodies. The growth plates of tibiae and vertebrae exhibited increased numbers of proliferating and hypertrophic chondrocytes with no apparent alteration in matrix mineralisation. The changes in growth plate morphology were accompanied by an increase in expression of FGF2 in proliferating chondrocytes and decreased expression of Indian hedgehog (Ihh), patched (Ptc) and parathyroid-hormone-related protein (PTHrP) in prehypertrophic cells. Furthermore, there was a reduction in expression of angiogenic molecules in hypertrophic chondrocytes, which was accompanied by a decrease in chondroclasts at the cartilage bone interface, fewer osteoblasts lining trabecular surfaces and a reduced volume of metaphyseal bone. These results indicate that functional Gli2 is necessary for normal endochondral bone development and that its absence results in increased proliferation of immature chondrocytes and decreased resorption of mineralised cartilage and bone formation.     

In vivo incidence of apoptosis evaluated with the TdT FragEL DNA fragmentation detection kit in cartilage and bone cells of the rat tibia

Previous studies have shown the occurrence of cell death by apoptosis in cartilage and bone cells, and have suggested a functional relationship between bone growth and remodelling on one hand, and numbers of apoptotic cells on the other. At present, no in vivo studies are available on the frequency of the apoptotic process measured at one time and in one place using the cartilage and bone cells of single specimens. The aim of the present investigation was to measure the in vivo incidence of apoptosis in cartilage and bone cells of the upper epiphysis and secondary ossification metaphyseal bone of the tibia in normal young adult rats. Apoptotic cells were visualized with the terminal deoxynucleotidyl transferase (TdT) FragEL DNA fragmentation detection kit, which is analogous to the TdT-mediated nick end-labelling (TUNEL) method. In the growth cartilage, only a few TUNEL-positive terminal hypertrophic chondrocytes were found; they were 1.32 +/- 0.70% of the total hypertrophic chondrocytes counted along the chondro-osseous junction. There were only a few apoptotic osteoblastic cells and osteocytes (0.22 +/- 0.22% and 0.15 +/- 0.16% of total osteoblasts and osteocytes respectively). TUNEL-positive osteoclasts were 1.03 +/- 0.57% of the total of osteoclastic cells; they usually showed only one or two apoptotic nuclei. The total number of TUNEL-positive bone marrow cells were also counted (56.78 +/- 10.29/mm2 of bone marrow spaces). Our results confirm that apoptosis does occur in hypertrophic chondrocytes and bone cells, and show that its frequency is very low. However, chiefly because of its short lifespan, the frequency of apoptosis in cartilage and bone may be higher than that shown by the TUNEL method. The static estimate that can be obtained with this method might lead to misleading conclusions on the physiological significance of such a dynamic, rapid and asynchronous process, whose precise importance in bone growth and remodelling remains to be determined.     

CCN Family Member 2/Connective Tissue Growth Factor (CCN2/CTGF) Has Anti-Aging Effects That Protect Articular Cartilage from Age-Related Degenerative Changes

To examine the role of connective tissue growth factor CCN2/CTGF (CCN2) in the maintenance of the articular cartilaginous phenotype, we analyzed knee joints from aging transgenic mice (TG) overexpressing CCN2 driven by the Col2a1 promoter. Knee joints from 3-, 14-, 40-, and 60-day-old and 5-, 12-, 18-, 21-, and 24-month-old littermates were analyzed. Ccn2-LacZ transgene expression in articular cartilage was followed by X-gal staining until 5 months of age. Overexpression of CCN2 protein was confirmed through all ages in TG articular cartilage and in growth plates. Radiographic analysis of knee joints showed a narrowing joint space and other features of osteoarthritis in 50% of WT, but not in any of the TG mice. Transgenic articular cartilage showed enhanced toluidine blue and safranin-O staining as well as chondrocyte proliferation but reduced staining for type X and I collagen and MMP-13 as compared with those parameters for WT cartilage. Staining for aggrecan neoepitope, a marker of aggrecan degradation in WT articular cartilage, increased at 5 and 12 months, but disappeared at 24 months due to loss of cartilage; whereas it was reduced in TG articular cartilage after 12 months. Expression of cartilage genes and MMPs under cyclic tension stress (CTS) was measured by using primary cultures of chondrocytes obtained from wild-type (WT) rib cartilage and TG or WT epiphyseal cartilage. CTS applied to primary cultures of mock-transfected rib chondrocytes from WT cartilage and WT epiphyseal cartilage induced expression of Col1a1, ColXa1, Mmp-13, and Mmp-9 mRNAs; however, their levels were not affected in CCN2-overexpressing chondrocytes and TG epiphyseal cartilage. In conclusion, cartilage-specific overexpression of CCN2 during the developmental and growth periods reduced age-related changes in articular cartilage. Thus CCN2 may play a role as an anti-aging factor by stabilizing articular cartilage.     

Lrp4, a Novel Receptor for Dickkopf 1 and Sclerostin,Is Expressed by Osteoblasts and Regulates Bone Growth and Turnover In Vivo

Lrp4 is a multifunctional member of the low density lipoprotein-receptor gene family and a modulator of extracellular cell signaling pathways in development. For example, Lrp4 binds Wise, a secreted Wnt modulator and BMP antagonist. Lrp4 shares structural elements within the extracellular ligand binding domain with Lrp5 and Lrp6, two established Wnt co-receptors with important roles in osteogenesis. Sclerostin is a potent osteocyte secreted inhibitor of bone formation that directly binds Lrp5 and Lrp6 and modulates both BMP and Wnt signaling. The anti-osteogenic effect of sclerostin is thought to be mediated mainly by inhibition of Wnt signaling through Lrp5/6 within osteoblasts. Dickkopf1 (Dkk1) is another potent soluble Wnt inhibitor that binds to Lrp5 and Lrp6, can displace Lrp5-bound sclerostin and is itself regulated by BMPs. In a recent genome-wide association study of bone mineral density a significant modifier locus was detected near the SOST gene at 17q21, which encodes sclerostin. In addition, nonsynonymous SNPs in the LRP4 gene were suggestively associated with bone mineral density. Here we show that Lrp4 is expressed in bone and cultured osteoblasts and binds Dkk1 and sclerostin in vitro. MicroCT analysis of Lrp4 deficient mutant mice revealed shortened total femur length, reduced cortical femoral perimeter, and reduced total femur bone mineral content (BMC) and bone mineral density (BMD). Lumbar spine trabecular bone volume per total volume (BV/TV) was significantly reduced in the mutants and the serum and urinary bone turnover markers alkaline phosphatase, osteocalcin and desoxypyridinoline were increased. We conclude that Lrp4 is a novel osteoblast expressed Dkk1 and sclerostin receptor with a physiological role in the regulation of bone growth and turnover, which is likely mediated through its function as an integrator of Wnt and BMP signaling pathways.     

Tibial loading increases osteogenic gene expression and cortical bone volume in mature and middle-aged mice

There are conflicting data on whether age reduces the response of the skeleton to mechanical stimuli. We examined this question in female BALB/c mice of different ages, ranging from young to middle-aged (2, 4, 7, 12 months). We first assessed markers of bone turnover in control (non-loaded) mice. Serum osteocalcin and CTX declined significantly from 2 to 4 months (p<0.001). There were similar age-related declines in tibial mRNA expression of osteoblast- and osteoclast-related genes, most notably in late osteoblast/matrix genes. For example, Col1a1 expression declined 90% from 2 to 7 months (p<0.001). We then assessed tibial responses to mechanical loading using age-specific forces to produce similar peak strains (-1300 με endocortical; -2350 με periosteal). Axial tibial compression was applied to the right leg for 60 cycles/day on alternate days for 1 or 6 weeks. qPCR after 1 week revealed no effect of loading in young (2-month) mice, but significant increases in osteoblast/matrix genes in older mice. For example, in 12-month old mice Col1a1 was increased 6-fold in loaded tibias vs. controls (p = 0.001). In vivo microCT after 6 weeks revealed that loaded tibias in each age group had greater cortical bone volume (BV) than contralateral control tibias (p<0.05), due to relative periosteal expansion. The loading-induced increase in cortical BV was greatest in 4-month old mice (+13%; p<0.05 vs. other ages). In summary, non-loaded female BALB/c mice exhibit an age-related decline in measures related to bone formation. Yet when subjected to tibial compression, mice from 2-12 months have an increase in cortical bone volume. Older mice respond with an upregulation of osteoblast/matrix genes, which increase to levels comparable to young mice. We conclude that mechanical loading of the tibia is anabolic for cortical bone in young and middle-aged female BALB/c mice.     

Impaired Bone Homeostasis in Amyotrophic Lateral Sclerosis Mice with Muscle Atrophy

There is an intimate relationship between muscle and bone throughout life. However, how alterations in muscle functions in disease impact bone homeostasis is poorly understood. Amyotrophic lateral sclerosis (ALS) is a neuromuscular disease characterized by progressive muscle atrophy. In this study we analyzed the effects of ALS on bone using the well established G93A transgenic mouse model, which harbors an ALS-causing mutation in the gene encoding superoxide dismutase 1. We found that 4-month-old G93A mice with severe muscle atrophy had dramatically reduced trabecular and cortical bone mass compared with their sex-matched wild type (WT) control littermates. Mechanically, we found that multiple osteoblast properties, such as the formation of osteoprogenitors, activation of Akt and Erk1/2 pathways, and osteoblast differentiation capacity, were severely impaired in primary cultures and bones from G93A relative to WT mice; this could contribute to reduced bone formation in the mutant mice. Conversely, osteoclast formation and bone resorption were strikingly enhanced in primary bone marrow cultures and bones of G93A mice compared with WT mice. Furthermore, sclerostin and RANKL expression in osteocytes embedded in the bone matrix were greatly up-regulated, and β-catenin was down-regulated in osteoblasts from G93A mice when compared with those of WT mice. Interestingly, calvarial bone that does not load and long bones from 2-month-old G93A mice without muscle atrophy displayed no detectable changes in parameters for osteoblast and osteoclast functions. Thus, for the first time to our knowledge, we have demonstrated that ALS causes abnormal bone remodeling and defined the underlying molecular and cellular mechanisms.     

Boning up on autophagy: The role of autophagy in skeletal biology

From an evolutionary perspective, the major function of bone is to provide stable sites for muscle attachment and affording protection of vital organs, especially the heart and lungs (ribs) and spinal cord (vertebrae and intervertebral discs). However, bone has a considerable number of other functions: serving as a store for mineral ions, providing a site for blood cell synthesis and participating in a complex system-wide endocrine system. Not surprisingly, bone and cartilage cell homeostasis is tightly controlled, as is the maintenance of tissue structure and mass. While a great deal of new information is accruing concerning skeletal cell homeostasis, one relatively new observation is that the cells of bone (osteoclasts osteoblasts and osteocytes) and cartilage (chondrocytes) exhibit autophagy. The focus of this review is to examine the significance of this process in terms of the functional demands of the skeleton in health and during growth and to provide evidence that dysregulation of the autophagic response is involved in the pathogenesis of diseases of bone (Paget disease of bone) and cartilage (osteoarthritis and the mucopolysaccharidoses). Delineation of molecular changes in the autophagic process is uncovering new approaches for the treatment of diseases that affect the axial and appendicular skeleton.     

Bone formation following intrarenal transplantation of isolated murine chondrocytes: chondrocyte-bone cell transdifferentiation?

Isolated syngeneic epiphyseal chondrocytes transplanted into a muscle formed cartilage in which matrix resorption and endochondral ossification began at the end of the second week after transplantation. After 56 days cartilage was converted into an ossicle. In 7-day-old intrarenal transplants, epiphyseal chondrocytes formed nodules of cartilage. In 10-day-old transplants, islands of bone appeared. Slight resorption of cartilage was first noted in 14-day-old transplants of chondrocytes. After eight weeks, transplants contained mainly bone. Intramuscularly transplanted rib chondrocytes formed cartilage which did not ossify. Nevertheless, bone islands appeared in intrarenal transplants of rib chondrocytes. Bone was not formed in allogeneic intrarenal transplants of epiphyseal or rib chondrocytes, but appeared in such transplants in animals immunosuppressed by anti-thymocyte serum and procarbazine. When spleen cells from animals immunized with allogeneic chondrocytes were transferred to immunosuppressed chondrocyte recipients two weeks after intrarenal chondrocyte transplantation, the majority of osteocytes in bone islands was dead. On the other hand, endochondral bone formed in intramuscular transplants of allogenic epiphyseal chondrocytes in immunosuppressed recipients was not damaged by sensitized spleen cells. This suggested that bone in 10- to 14-day-old intrarenal transplants of chondrocytes arose from injected cells and not by induction. To see whether bone was formed by chondrocytes or by some cells contaminating the chondrocyte suspension, the superficial layer of rib cartilage was removed by collagenase digestion and only more central chondrocytes were used for transplantation.(ABSTRACT TRUNCATED AT 250 WORDS)