Morphology of epiphyseal apparatus of a ranid frog (Rana Esculenta)

Morphological, histochemical and ultrastructural investigations on epiphyseal apparatus of Rana Esculenta were made. The most important findings were the following: 1) metaphyseal cartilage is localized inside proximal diaphyseal compact bone as a plug; 2) metaphyseal cartilage do not reduce in thickness during ageing; 3) metaphyseal cartilage do not show vascular invasion and do not mineralize in degenerative zone; 4) trabecular bone was not at all evident in this animal; 5) external periosteum is well vascularized and proliferates in correspondence to marginal epiphyseal end of the diaphyseal. From these results the hypothesis that the ranid frog bone growth is not due to metaphyseal metabolism (as in avian and mammals) but to bone periosteal marginal mineralization is reached.     

Parathyroid Hormone (PTH)/PTH-related Peptide Type 1 Receptor (PPR) Signaling in Osteocytes Regulates Anabolic and Catabolic Skeletal Responses to PTH

Parathyroid hormone (PTH) is the only Food and Drug Administration-approved anabolic agent to treat osteoporosis; however, the cellular targets of PTH action in bone remain controversial. PTH modulates bone turnover by binding to the PTH/PTH-related peptide (PTHrP) type 1 receptor (PPR), a G-protein-coupled receptor highly expressed in bone and kidneys. Osteocytes, the most abundant cells in adult bone, also express PPR. However, the physiological relevance of PPR signaling in osteocytes remains to be elucidated. Toward this goal, we generated mice with PPR deletion in osteocytes (Ocy-PPRKO). Skeletal analysis of these mice revealed a significant increase in bone mineral density and trabecular and cortical bone parameters. Osteoblast activities were reduced in these animals, as demonstrated by decreased collagen type I α1 mRNA and receptor activator of NF-κB ligand (RANKL) expression. Importantly, when subjected to an anabolic or catabolic PTH regimen, Ocy-PPRKO animals demonstrated blunted skeletal responses. PTH failed to suppress SOST/Sclerostin or induce RANKL expression in Ocy-PPRKO animals compared with controls. In vitro, osteoclastogenesis was significantly impaired in Ocy-PPRKO upon PTH administration, indicating that osteocytes control osteoclast formation through a PPR-mediated mechanism. Taken together, these data indicate that PPR signaling in osteocytes is required for bone remodeling, and receptor signaling in osteocytes is needed for anabolic and catabolic skeletal responses.     

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.     

A sclerostin-based theory for strain-induced bone formation

Bone formation responds to mechanical loading, which is believed to be mediated by osteocytes. Previous theories assumed that loading stimulates osteocytes to secrete signals that stimulate bone formation. In computer simulations this 'stimulatory' theory successfully produced load-aligned trabecular structures. In recent years, however, it was discovered that osteocytes inhibit bone formation via the protein sclerostin. To reconcile this with strain-induced bone formation, one must assume that sclerostin secretion decreases with mechanical loading. This leads to a new 'inhibitory' theory in which loading inhibits osteocytes from inhibiting bone formation. Here we used computer simulations to show that a sclerostin-based model is able to produce a load-aligned trabecular architecture. An important difference appeared when we compared the response of the stimulatory and inhibitory models to loss of osteocytes, and found that the inhibitory pathway prevents the loss of trabeculae that is seen with the stimulatory model. Further, we demonstrated with combined stimulatory/inhibitory models that the two pathways can work side-by-side to achieve a load-adapted bone architecture.     

Heavy Metal Ion Regulation of Gene Expression: MECHANISMS BY WHICH LEAD INHIBITS OSTEOBLASTIC BONE-FORMING ACTIVITY THROUGH MODULATION OF THE Wnt/β-CATENIN SIGNALING PATHWAY*

Exposure to lead (Pb) from environmental sources remains an overlooked and serious public health risk. Starting in childhood, Pb in the skeleton can disrupt epiphyseal plate function, constrain the growth of long bones, and prevent attainment of a high peak bone mass, all of which will increase susceptibility to osteoporosis later in life. We hypothesize that the effects of Pb on bone mass, in part, come from depression of Wnt/β-catenin signaling, a critical anabolic pathway for osteoblastic bone formation. In this study, we show that depression of Wnt signaling by Pb is due to increased sclerostin levels in vitro and in vivo. Downstream activation of the β-catenin pathway using a pharmacological inhibitor of GSK-3β ameliorates the Pb inhibition of Wnt signaling activity in the TOPGAL reporter mouse. The effect of Pb was determined to be dependent on sclerostin expression through use of the SOST gene knock-out mice, which are resistant to Pb-induced trabecular bone loss and maintain their mechanical bone strength. Moreover, isolated bone marrow cells from the sclerostin null mice show improved bone formation potential even after exposure to Pb. Also, our data suggest that the TGFβ canonical signaling pathway is the mechanism by which Pb controls sclerostin production. Taken together these results support our hypothesis that the osteoporotic-like phenotype observed after Pb exposure is, in part, regulated through modulation of the Wnt/β-catenin pathway.     

Reduction of SOST gene promotes bone formation through the Wnt/β-catenin signalling pathway and compensates particle-induced osteolysis

The increase in bone resorption and/or the inhibition of bone regeneration caused by wear particles are the main causes of periprosthetic osteolysis. The SOST gene and Sclerostin, a protein synthesized by the SOST gene, are the characteristic marker of osteocytes and regulate bone formation and resorption. We aimed to verify whether the SOST gene was involved in osteolysis induced by titanium (Ti) particles and to investigate the effects of SOST reduction on osteolysis. The results showed osteolysis on the skull surface with an increase of sclerostin levels after treated with Ti particles. Similarly, sclerostin expression in MLO-Y4 osteocytes increased when treated with Ti particles in vitro. After reduction of SOST, local bone mineral density and bone volume increased, while number of lytic pores on the skull surface decreased and the erodibility of the skull surface was compensated. Histological analyses revealed that SOST reduction increased significantly alkaline phosphatase- (ALP) and osterix-positive expression on the skull surface which promoted bone formation. ALP activity and mineralization of MC3T3-E1 cells also increased in vitro when SOST was silenced, even if treated with Ti particles. In addition, Ti particles decreased β-catenin expression with an increase in sclerostin levels, in vivo and in vitro. Inversely, reduction of SOST expression increased β-catenin expression. In summary, our results suggested that reduction of SOST gene can activate the Wnt/β-catenin signalling pathway, promoting bone formation and compensated for bone loss induced by Ti particles. Thus, this study provided new perspectives in understanding the mechanisms of periprosthetic osteolysis.     

Resorption Controls Bone Anabolism Driven by Parathyroid Hormone (PTH) Receptor Signaling in Osteocytes

The contribution of remodeling-based bone formation coupled to osteoclast activity versus modeling-based bone formation that occurs independently of resorption, to the anabolic effect of PTH remains unclear. We addressed this question using transgenic mice with activated PTH receptor signaling in osteocytes that exhibit increased bone mass and remodeling, recognized skeletal effects of PTH elevation. Direct inhibition of bone formation was accomplished genetically by overexpressing the Wnt antagonist Sost/sclerostin; and resorption-dependent bone formation was inhibited pharmacologically with the bisphosphonate alendronate. We found that bone formation induced by osteocytic PTH receptor signaling on the periosteal surface depends on Wnt signaling but not on resorption. In contrast, bone formation on the endocortical surface results from a combination of Wnt-driven increased osteoblast number and resorption-dependent osteoblast activity. Moreover, elevated osteoclasts and intracortical/calvarial porosity is exacerbated by overexpressing Sost and reversed by blocking resorption. Furthermore, increased cancellous bone is abolished by Wnt inhibition but further increased by blocking resorption. Thus, resorption induced by PTH receptor signaling in osteocytes is critical for full anabolism in cortical bone, but tempers bone gain in cancellous bone. Dissecting underlying mechanisms of PTH receptor signaling would allow targeting actions in different bone compartments, enhancing the therapeutic potential of the pathway.     

Identification of the disease-causing gene in sclerosteosis--discovery of a novel bone anabolic target?

Genetic studies recently unraveled the genetic cause of sclerosteosis, a rare skeletal dysplasia characterized by a generalized increase in bone mass. Different loss-of-function mutations were identified in SOST, a gene with no homology to any known gene. This SOST gene is also involved in the pathogenesis of van Buchem disease, a disorder closely resembling sclerosteosis, since a 52-kb deletion located downstream of SOST is found in patients diagnosed with this condition. Molecular studies showed a very restricted expression pattern of SOST and its gene product, sclerostin, with areas in the bone tissue, more precisely in cells of the osteoblast lineage, being the major sites of expression. Sclerostin is a secreted protein with a cysteine knot motif. In vitro studies demonstrated that sclerostin acts as a modulator of BMP signaling by binding to different members of the BMP growth factor family and acting on downstream BMP signal transduction events. The important function of sclerostin in bone metabolism has also been proven in vivo by the osteopenic phenotype of transgenic mice overexpressing SOST in bone. The identification of sclerostin as an important protein in bone metabolism opens new perspectives for the development of anabolic therapeutics to prevent and treat osteoporosis.     

Decreased Serum Wnt Antagonist Levels in Patients With Active Acromegaly

ObjectiveThe Wnt signaling pathway is an important modulator of bone metabolism. This study aims to clarify the changes in Wnt antagonists in active and biochemically controlled acromegalic patients.MethodsWe recruited 77 patients recently diagnosed with acromegaly. Of those, 41 patients with complete follow-up data were included. Thirty healthy patients matched for age, sex, and body mass index served as controls. At baseline and posttreatment, Wnt antagonists (sclerostin [SOST], dickkopf-related protein 1 [DKK-1], and Wnt inhibitory factor 1 [WIF-1]), bone turnover markers (osteocalcin, procollagen type 1 N-terminal propeptide [P1NP], and C-terminal telopeptide of type 1 collagen [CTX]) and the bone remodeling index were investigated.ResultsAcromegalic patients had higher serum osteocalcin, P1NP, and CTX and a higher bone remodeling index than controls (P < .01). Serum SOST, DKK-1, and WIF-1 levels were significantly decreased in patients compared to controls (all P < .01). Serum SOST and WIF-1 levels were negatively correlated with growth hormone levels; SOST levels were positively correlated with WIF-1. After treatment, serum bone turnover markers and the bone remodeling index decreased, while SOST and WIF-1 significantly increased (P < .05). DKK-1 levels did not change compared to baseline (P > .05). In biochemically controlled patients, SOST and WIF-1 levels and bone turnover markers were restored and did not differ from those of the control participants (all P > .05).ConclusionPatients with active acromegaly exhibited significantly decreased Wnt antagonist levels. The reduction in Wnt antagonists is a compensatory mechanism to counteract increased bone fragility in active acromegaly.     

β-Criptoxanthin stimulates bone formation and inhibits bone resorption in tissue culture in vitro

The effect of beta-cryptoxanthin, which is greatly present in fruits, has not been clarified so far on bone metabolism. The effect of beta-cryptoxanthin on bone formation and bone resorption was investigated in tissue culture in vitro. Rat femoral-diaphyseal (cortical bone) and -metaphyseal (trabecular bone) tissues were cultured for 48 h in Dulbecco's modified Eagle's medium (high glucose, 4.5%) supplemented with antibiotics and bovine serum albumin. The experimental cultures contained 10(-8)-10(-5) M beta-cryptoxanthin. The presence of beta-cryptoxanthin (10(-6) or 10(-5) M) caused a significant increase in calcium content, alkaline phosphatase activity and deoxyribonucleic acid (DNA) content in the diaphyseal and metaphyseal tissues. These increases were completely prevented in the presence of cycloheximide (10(-6) M), an inhibitor of protein synthesis. beta-Carotene (10(-6) or 10(-5) M) or xantine (10(-6) or 10(-5) M) had no effect on the diaphyseal and metaphyseal calcium contents. The bone-resorbing factors parathyroid hormone (1-34) (PTH; 10(-7) M) or prostaglandin E2 (PGE2; 10(-5) M) caused a significant decrease in calcium content in the diaphyseal and metaphyseal tissues. The decrease in bone calcium content induced by PTH or PGE2 was completely inhibited by beta-cryptoxanthin (10(-8)-10(-6) M). In addition, beta-cryptoxanthin (10(-8)-10(-6) M) completely inhibited the PTH (10(-7) M)- or PGE, (10(-5) M)-induced increase in medium glucose consumption and lactic acid production by diaphyseal and metaphyseal tissues. The inhibitory effect of beta-cryptoxanthin (10(-7) M) on PTH (10(-7) M)- or PGE2 (10(-5) M)-stimulated decrease in the diaphyseal calcium content was significantly prevented in the presence of 10(-3) M vanadate, an inhibitor of protein tyrosine phosphatase. Vanadate (10(-3) M) did not have a significant effect on calcium content and lactic acid production in control bone tissues. The present study demonstrates that beta-cryptoxanthin has a direct stimulatory effect on bone formation and an inhibitory effect on bone resorption in tissue culture in vitro.     

Simulated resistance training, but not alendronate, increases cortical bone formation and suppresses sclerostin during disuse

Mechanical loading modulates the osteocyte-derived protein sclerostin, a potent inhibitor of bone formation. We hypothesized that simulated resistance training (SRT), combined with alendronate (ALEN) treatment, during hindlimb unloading (HU) would most effectively mitigate disuse-induced decrements in cortical bone geometry and formation rate (BFR). Sixty male, Sprague-Dawley rats (6-mo-old) were randomly assigned to either cage control (CC), HU, HU plus either ALEN (HU+ALEN), or SRT (HU+SRT), or combined ALEN and SRT (HU+SRT/ALEN) for 28 days. Computed tomography scans on days -1 and 28 were taken at the middiaphyseal tibia. HU+SRT and HU+SRT/ALEN rats were subjected to muscle contractions once every 3 days during HU (4 sets of 5 repetitions; 1,000 ms isometric + 1,000 ms eccentric). The HU+ALEN and HU+SRT/ALEN rats received 10 μg/kg ALEN 3 times/wk. Compared with the CC animals, HU suppressed the normal slow growth-induced increases of cortical bone mineral content, cortical bone area, and polar cross-sectional moment of inertia; however, SRT during HU restored cortical bone growth. HU suppressed middiaphyseal tibia periosteal BFR by 56% vs. CC (P < 0.05). However, SRT during HU restored BFR at both periosteal (to 2.6-fold higher than CC) and endocortical (14-fold higher than CC) surfaces (P < 0.01). ALEN attenuated the SRT-induced BFR gains during HU. The proportion of sclerostin-positive osteocytes in cortical bone was significantly higher (+121% vs. CC) in the HU group; SRT during HU effectively suppressed the higher proportion of sclerostin-positive osteocytes. In conclusion, a minimum number of high-intensity muscle contractions, performed during disuse, restores cortical BFR and suppress unloading-induced increases in sclerostin-positive osteocytes.     

SOST基因的表达调控

硬化蛋白(Sclerostin, SOST)主要由骨细胞特异性表达, 是骨形成的负性调节因子。甲状旁腺激素和雌激素抑制SOST基因表达, 转录因子Osterix、Runx2和Mef2c促进SOST基因表达, 而转录因子Sirt1负调控SOST表达。此外, SOST基因表达还受DNA甲基化和microRNA等表观遗传学调控。SOST基因突变可引起骨硬缩症和Van Buchem病, 与骨质疏松症相关联。Wnt和BMP是骨代谢调节的两个重要信号途径, SOST可通过结合BMP的Ⅰ型或Ⅱ型受体和Wnt的共受体LRP5/6分别抑制BMP和Wnt信号途径来调控成骨细胞分化和骨形成。抑制SOST为骨质疏松症的治疗提供了新的途径。文章综述了SOST基因的结构、功能、表达调控、与人类疾病的关系、调节骨代谢的机制及其临床应用前景。     

Effects of Dietary Phosphate on Adynamic Bone Disease in Rats with Chronic Kidney Disease – Role of Sclerostin?

High phosphate intake is known to aggravate renal osteodystrophy along various pathogenetic pathways. Recent studies have raised the possibility that dysregulation of the osteocyte Wnt/β-catenin signaling pathway is also involved in chronic kidney disease (CKD)-related bone disease. We investigated the role of dietary phosphate and its possible interaction with this pathway in an experimental model of adynamic bone disease (ABD) in association with CKD and hypoparathyroidism. Partial nephrectomy (Nx) and total parathyroidectomy (PTx) were performed in male Wistar rats. Control rats with normal kidney and parathyroid function underwent sham operations. Rats were divided into three groups and underwent pair-feeding for 8 weeks with diets containing either 0.6% or 1.2% phosphate: sham 0.6%, Nx+PTx 0.6%, and Nx+PTx 1.2%. In the two Nx+PTx groups, serum creatinine increased and blood ionized calcium decreased compared with sham control group. They also presented hyperphosphatemia and reduced serum parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23) levels. Fractional urinary excretion of phosphate increased in Nx+PTx 1.2% rats despite lower PTH and FGF23 levels than in sham group. These biochemical changes were accompanied by a decrease in bone formation rates. The Nx+PTx 1.2% group had lower bone volume (BV/TV), higher osteoblast and osteocyte apoptosis, and higher SOST and Dickkopf-1 gene expression than the Nx+PTx 0.6% group. Nx+PTx 0.6% rat had very low serum sclerostin levels, and Nx+PTx 1.2% had intermediate sclerostin levels compared with sham group. Finally, there was a negative correlation between BV/TV and serum sclerostin. These results suggest that high dietary phosphate intake decreases bone volume in an experimental model of CKD-ABD, possibly via changes in SOST expression through a PTH-independent mechanism. These findings could have relevance for the clinical setting of CKD-ABD in patients who low turnover bone disease might be attenuated by optimal control of phosphate intake and/or absorption.     

Parathyroid hormone induces bone formation in phosphorylation-deficient PTHR1 knockin mice

Activation of G protein-coupled receptors by agonists leads to receptor phosphorylation, internalization of ligand receptor complexes, and desensitization of hormonal response. The role of parathyroid hormone (PTH) receptor 1, PTHR1, is well characterized and known to regulate cellular responsiveness in vitro. However, the role of PTHR1 phosphorylation in bone formation is yet to be investigated. We have previously demonstrated that impaired internalization and sustained cAMP stimulation of phosphorylation-deficient (PD) PTHR1 leads to exaggerated cAMP response to subcutaneous PTH infusion in a PD knockin mouse model. To understand the physiological role of receptor internalization on PTH bone anabolic action, we examined bone parameters of wild-type (WT) and PD knockin female and male mice following PTH treatment. We found a decrease in total and diaphyseal bone mineral density in female but not in male PD mice compared with WT controls at 3-6 mo of age. This effect was attenuated at older age groups. PTH administration displayed increased bone volume and trabecular thickness in the vertebrae and distal femora of both WT and PD animals. These results suggest that PTHR1 phosphorylation does not play a major role in the anabolic action of PTH.     

The contribution of cancellous bone to long bone strength and rigidity

A recent article (Burr and Piotrowski, 1982) suggested that structural analyses of long bone cross-sectional geometry will be inaccurate and should be considered inappropriate when cancellous bone accounts for 10-15% or more of the cross-sectional area. Consideration of material property differences between compact and cancellous bone, however, indicates that even significant proportions of cancellous bone (10-40% of total cross-sectional area) will very likely have negligible effects on bone strength and rigidity, and can be effectively ignored in geometrical analyses of diaphyseal sections. In metaphyseal and epiphyseal regions, however, geometric analyses of section properties such as area moments of inertia are inappropriate, both because of significant trabecular bone effects, and because of the inherent constraints of mechanical beam models.     

Loss of Gsα in the Postnatal Skeleton Leads to Low Bone Mass and a Blunted Response to Anabolic Parathyroid Hormone Therapy

Parathyroid hormone (PTH) is an important regulator of osteoblast function and is the only anabolic therapy currently approved for treatment of osteoporosis. The PTH receptor (PTH1R) is a G protein-coupled receptor that signals via multiple G proteins including G_sα. Mice expressing a constitutively active mutant PTH1R exhibited a dramatic increase in trabecular bone that was dependent upon expression of G_sα in the osteoblast lineage. Postnatal removal of G_sα in the osteoblast lineage (P-G_sα^OsxKO mice) yielded markedly reduced trabecular and cortical bone mass. Treatment with anabolic PTH(1–34) (80 μg/kg/day) for 4 weeks failed to increase trabecular bone volume or cortical thickness in male and female P-G_sα^OsxKO mice. Surprisingly, in both male and female mice, PTH administration significantly increased osteoblast numbers and bone formation rate in both control and P-G_sα^OsxKO mice. In mice that express a mutated PTH1R that activates adenylyl cyclase and protein kinase A (PKA) via G_sα but not phospholipase C via G_q/11 (D/D mice), PTH significantly enhanced bone formation, indicating that phospholipase C activation is not required for increased bone turnover in response to PTH. Therefore, although the anabolic effect of intermittent PTH treatment on trabecular bone volume is blunted by deletion of G_sα in osteoblasts, PTH can stimulate osteoblast differentiation and bone formation. Together these findings suggest that alternative signaling pathways beyond G_sα and G_q/11 act downstream of PTH on osteoblast differentiation.