GSK-3, a component of the canonical Wnt signaling pathway, is implicated in regulation of bone mass. The effect of a small molecule GSK-3 inhibitor was evaluated in pre-osteoblasts and in osteopenic rats. GSK-3 inhibitor induced osteoblast differentiation in vitro and increased markers of bone formation in vitro and in vivo with concomitant increased bone mass and strength in rats.Inactivation of glycogen synthase kinase -3 (GSK-3) leads to stabilization, accumulation, and translocation of beta-catenin into the nucleus to activate downstream Wnt target genes. To examine whether GSK-3 directly regulates bone formation and mass we evaluated the effect of 603281-31-8, a small molecule GSK-3 alpha/beta dual inhibitor in preosteoblastic cells and in osteopenic rats.Murine mesenchymal C3H10T1/2 cells were treated with GSK-3 inhibitor (603281-31-8) and assayed for beta-catenin levels, activity of Wnt-responsive promoter, expression of mRNA for bone formation, and adipogenic markers and alkaline phosphatase activity. In vivo, 6-month-old rats were ovariectomized (OVX), allowed to lose bone for 1 month, and treated with GSK-3 inhibitor at 3 mg/kg/day orally for 60 days. At the end of treatment, BMD was measured by DXA, bone formation rate by histomorphometry, vertebral strength (failure in compression), and the expression levels of osteoblast-related genes by real-time PCR.Treatment of C3H10T1/2 cells with the GSK-3 inhibitor increased the levels of beta-catenin accompanied by activation of Wnt-responsive TBE6-luciferase reporter gene. This was associated with an increased expression of mRNA for bone sialoprotein (1.4-fold), collagen alpha1 (I) (approximately 2-fold), osteocalcin (1.2-fold), collagen alpha1(V) (1.5-fold), alkaline phosphatase (approximately 160-fold), and runx2 (1.6-fold), markers of the osteoblast phenotype and bone formation activity. Alkaline phosphatase mRNA expression paralleled alkaline phosphatase activity. The mRNA levels of collagens alpha1 (I), alpha1 (V), biglycan, osteonectin, and runx-2 increased on treatment with the GSK-3 inhibitor in rat femur compared with the OVX control. DXA analyses revealed significant increases in BMC and BMD in cancellous and cortical bone of OVX rats treated with GSK-3 inhibitor. This was associated with increased strength (peak load, energy, and stiffness) assessed by lumbar vertebra load to failure in compression. Histomorphometric analyses showed that 603281-31-8 robustly increased bone formation but did not exclude a small effect on osteoclasts (resorption).An orally active, small molecule GSK-3 inhibitor induced osteoblast differentiation and increased markers of bone formation in vitro, and increased markers of bone formation, bone mass, and strength in vivo, consistent with a role for the canonical Wnt pathway in osteogenesis.
The present study compared the bone anabolic effects of graded doses of alfacalcidol in proximal femurs (hematopoietic, red marrow skeletal site) and distal tibiae (fatty, yellow marrow skeletal site). One group of 8.5-month-old female Sprague-Dawley rats were killed at baseline and 4 groups were treated 5 days on/2 days off/week for 12 weeks with 0, 0.025, 0.05 and 0.1 microg alfacalcidol/kg by oral gavage. The proximal femur, bone site with hematopoietic marrow, as well as the distal tibia bone site with fatty marrow, were processed undecalcified for quantitative bone histomorphometry. In the red marrow site of the proximal femoral metaphysis (PFM), 0.1 microg alfacalcidol/kg induced increased cancellous bone mass, improved architecture (decreased trabecular separation, increased connectivity), and stimulated local bone formation of bone 'boutons' (localized bone formation) on trabecular surfaces. There was an imbalance in bone resorption and formation, in which the magnitude of depressed bone resorption greater than depressed bone formation resulted in a positive bone balance. In addition, bone 'bouton' formation contributed to an increase in bone mass. In contrast, the yellow marrow site of the distal tibial metaphysis (DTM), the 0.1 microg alfacalcidol/kg dose induced a non-significant increased cancellous bone mass. The treatment decreased bone resorption equal to the magnitude of decreased bone formation. No bone 'bouton' formation was observed. These findings indicate that the highest dose of 0.1 microg alfacalcidol/kg for 12 weeks increased bone mass (anabolic effect) at the skeletal site with hematopoietic marrow of the proximal femoral metaphysis, but the increased bone mass was greatly attenuated at the fatty marrow site of the distal tibial metaphysis. In addition, the magnitude of the bone gain induced by alfacalcidol treatment in red marrow cancellous bone sites of the proximal femoral metaphysis was half that of the lumbar vertebral body. The latter data were from a previous report from the same animal and protocol. These findings indicated that alfacalcidol as an osteoporosis therapy is less efficacious as a positive bone balance agent that increased trabecular bone mass in a non-vertebral skeletal site where bone marrow is less hematopoietic.
Cyclic AMP (cAMP) is a continually produced nucleotide inactivated by hydrolysis to 5'AMP via phosphodiesterase (PDE) enzymes. Rolipram is a selective PDE4 inhibitor reported to have anti-inflammatory effects and used in the treatment of asthma and chronic obstructive pulmonary disease (COPD). The current study was designed to determine whether Rolipram could prevent and restore bone loss in ovariectomized (OVX) rats. Six-month-old Sprague Dawley rats underwent either sham-operated or bilateral ovariectomy, and were left untreated for 60 days to develop osteopenia. Then they were treated with vehicle, 6 mg/kg PGE(2), 3 microg/kg Alendronate or 0.1-1.0 mg/kg Rolipram for 60 days. At sacrifice, the right tibiae were processed for quantitative bone histomorphometric measurements. The right femurs were measured by dual energy A-ray absorptiometry and the 5th lumbar vertebrae were subjected to micro-computed tomography to access bone mass and architecture changes. Our results indicated that OVX induced negative bone balance in all five bone sites we tested, with bone resorption exceeding bone formation. Rolipram at 0.1-0.6 mg/kg dose levels prevented while at 1 mg/kg restored ovariectomy-induced cancellous and cortical bone loss in the tibia, femur and lumbar vertebra. Dynamic bone histomorphometry suggested that these beneficial effects were achieved by partially maintaining the elevated bone formation at the trabecular bone surface and increasing bone formation at the periosteal bone surface of the cortex. Furthermore, it reduced bone turnover at the trabecular and the endocortical bone surfaces. The prevention of further bone loss effects were comparable to those of an anti-resorption agent (Alendronate) but were not as great as those of an anabolic agent (PGE(2)). In addition, Rolipram treatment increased body and muscle weights compared to the vehicle-treated OVX rats. In conclusion, our study in an osteopenic rat model suggested that a selective PDE4 inhibitor may be used for the treatment of established osteoporosis.
To evaluate the type I collagen cross-linked N-telopeptide (NTx) levels in human urine as an indicator of bone resorption rate in different ages, sex and in bone metabolic diseases.Urinary NTx was determined by immunoassay in 591 Beijing healthy subjects aged from 0 to 86 years and 379 patients with bone metabolic diseases.The levels of urinary NTx were significantly higher in children than in adults (P < 0.001) and higher in boys than in girls (P < 0.01) and increased 1.4-2.2 times in postmenopausal females than in men and premenopausal women. Urine NTx had a positive linear correlation with urine HOP/Cr (r = 0.778, P < 0.01) and Ca/Cr ratio (r = 0.320, P < 0.01), and a negative linear correlation with age (r = -0.523, P < 0.01) and lumbar spine BMD (r = -0.426, P < 0.01). The levels of urine NTx increased for 3.6 times in pregnancy, 1.5 times in osteoporosis, 1.9 times in fragility fracture, 3.6 times in chronic renal failure, 2.1 times in rickets and 7.2 times in multiple myeloma compared to age matched controls.NTx in urine is a specific and sensitive indicator of bone resorption and is able to distinguish normal premenopause from late osteoporotic patients. NTx could be used as diagnostic information about metabolic bone diseases, and to monitor antiresorptive therapy.
The Utah Paradigm of Skeletal Physiology with its key component, the mechanostat hypothesis, suggest plausible explanations of some of the tissue-level changes occurring from combining selected non-mechanical agents (anabolic and anti-resorptive/( re)modeling agents) with mechanical loading (osteogenic exercise) to increase bone mass and strength. The evidence for combining selected anabolic agents like parathyroid hormone, prostaglandin E(2), growth hormone, etc. with mechanical loading can increase bone mass is strong. Anabolic agents influence loading-related bone formation changes in a permissive manner and modulate (increase) the responsiveness of bone tissue to mechanical loading by changing thresholds for bone formation and resorption. However, any beneficial effect of combining selected anti-resorptive/(re)modeling agents like estrogen with loading is marginal, especially in adult skeletons. Postulated changes in modeling and remodeling thresholds (set points) and known direct effects on bone cells by non-mechanical agents may explain the observed tissue-level changes associated with large and minor increases in bone mass. Although the pharmaceutical industry has avoided considering osteogenic loading in the treatment of osteoporosis, a methodical dose-response study of anabolic agents combined with loading should: (1) provide opportunities for therapeutic intervention to imitate or enhance the osteogenic response to loading in order to correct osteopenias; (2) provide the potential to diminish the dosage of drugs required to induce bone formation in ways that enhanced efficacy and reduced any side effects; and (3) improve the quality of life and reduce the risk of falls by improving balance, gait speed and muscle strength with a non-mechanical agent like GH that could improve both muscle and bone mass and strength. Lastly, more studies are needed which determine bone strength instead of only "mass" in aged skeletons so one can assess how effective such treatments would reduce the risk of fracture in the clinic.
'/%3e%3cuse xlink:href='%23a' transform='rotate(23.036 .093 25.536)'/%3e%3cuse xlink:href='%23a' transform='rotate(45.87 1.273 16.18)'/%3e%3cuse xlink:href='%23a' transform='rotate(69.945 .996 12.078)'/%3e%3cuse xlink:href='%23a' transform='rotate(20.66 -19.689 31.932)'/%3e%3c/svg%3e)