The leukotrienes and peptido-leukotrienes are 5-lipoxygenase (5-LO)
metabolites of arachidonic acid that appear to have unique effects on
bone, distinct from those of the prostaglandins. Application ...of
exogenous leukotrienes in vitro and in
vivo results in increased osteoclast formation and bone
resorption. While 5-LO metabolites of arachidonic acid clearly
stimulate osteoclastic bone resorption, little is known concerning
their effects on osteoblastic bone formation. We examined the effects
of the 5-LO metabolites 5-HETE, the leukotriene LTB4 and,
as representative of the peptido-leukotrienes, LTD4 on the
formation of mineralized nodules of fetal rat calvarial cells in the
presence of dexamethasone and recombinant human bone morphogenetic
protein-2 (rhBMP-2). We also examined the effects of these 5-LO
metabolites on alkaline phosphatase activity and cell proliferation in
these cultures and the effects of 5-HETE and LTB4 on
cultured explants of neonatal murine calvariae. We found that the
bone-forming capacity of osteoblasts was impaired when cells were
cultured in the presence of 5-LO metabolites. These data indicate that
metabolites of the 5-LO pathway are negative regulators of bone
formation. The continued presence of these metabolites in the bone
environment might account, in part, for the bone loss associated with
chronic inflammatory conditions.
Matrix extracellular phosphoglycoprotein (MEPE) is expressed exclusively in osteoblasts, osteocytes and odontoblasts with markedly elevated expression found in X-linked hypophosphatemic rickets (Hyp) ...osteoblasts and in oncogenic hypophosphatemic osteomalacia (OHO) tumors. Because these syndromes are associated with abnormalities in mineralization and renal phosphate excretion, we examined the effects of insect-expressed full-length human-MEPE (Hu-MEPE) on serum and urinary phosphate in vivo,
33PO
4 uptake in renal proximal tubule cultures and mineralization of osteoblast cultures. Dose-dependent hypophosphatemia and hyperphosphaturia occurred in mice following intraperitoneal (IP) administration of Hu-MEPE (up to 400 μg kg
−1 31 h
−1), similar to mice given the phosphaturic hormone PTH (80 μg kg
−1 31 h
−1). Also the fractional excretion of phosphate (FEP) was stimulated by MEPE 65.0% (
P < 0.001) and PTH groups 53.3% (
P < 0.001) relative to the vehicle group 28.7% (SEM 3.97). In addition, Hu-MEPE significantly inhibited
33PO
4 uptake in primary human proximal tubule renal cells (RPTEC) and a human renal cell line (Hu-CL8) in vitro (
V
max 53.4% inhibition;
K
m 27.4 ng/ml, and
V
max 9.1% inhibition;
K
m 23.8 ng/ml, respectively). Moreover, Hu-MEPE dose dependently (50–800 ng/ml) inhibited BMP2-mediated mineralization of a murine osteoblast cell line (2T3) in vitro. Inhibition of mineralization was localized to a small (2 kDa) cathepsin B released carboxy-terminal MEPE peptide (protease-resistant) containing the acidic serine–aspartate-rich motif (ASARM peptide). We conclude that MEPE promotes renal phosphate excretion and modulates mineralization.
Osteoblastic bone metastases are common in prostate and breast cancer patients, but mechanisms by which tumor cells stimulate new bone formation are unclear. We identified three breast cancer cell ...lines that cause osteoblastic metastases in a mouse model and secrete endothelin-1. Tumor-produced endothelin-1 stimulates new bone formation in vitro and osteoblastic metastases in vivo via the endothelin A receptor. Treatment with an orally active endothelin A receptor antagonist dramatically decreased bone metastases and tumor burden in mice inoculated with ZR-75-1 cells. Tumor-produced endothelin-1 may have a major role in the establishment of osteoblastic bone metastases, and endothelin A receptor blockade represents effective treatment.
We have found that the ubiquitin-proteasome pathway exerts exquisite control of osteoblast differentiation and bone formation in vitro and in vivo in rodents. Structurally different inhibitors that ...bind to specific catalytic beta subunits of the 20S proteasome stimulated bone formation in bone organ cultures in concentrations as low as 10 nM. When administered systemically to mice, the proteasome inhibitors epoxomicin and proteasome inhibitor-1 increased bone volume and bone formation rates over 70% after only 5 days of treatment. Since the ubiquitin-proteasome pathway has been shown to modulate expression of the Drosophila homologue of the bone morphogenetic protein-2 and -4 (BMP-2 and BMP-4) genes, we examined the effects of noggin, an endogenous inhibitor of BMP-2 and BMP-4 on bone formation stimulated by these compounds and found that it was abrogated. These compounds increased BMP-2 but not BMP-4 or BMP-6 mRNA expression in osteoblastic cells, suggesting that BMP-2 was responsible for the observed bone formation that was inhibited by noggin. We show proteasome inhibitors regulate BMP-2 gene expression at least in part through inhibiting the proteolytic processing of Gli3 protein. Our results suggest that the ubiquitin-proteasome machinery regulates osteoblast differentiation and bone formation and that inhibition of specific components of this system may be useful therapeutically in common diseases of bone loss.
A major unmet need in the medical field today is the availability of suitable treatments for the ever-increasing incidence of osteoporosis and the treatment of bone deficit conditions. Although ...therapies exist which prevent bone loss, the options are extremely limited for patients once a substantial loss of skeletal bone mass has occurred. Patients who have reduced bone mass are predisposed to fractures and further morbidity. The FDA recently approved PTH (1-34) (Teriparatide) for the treatment of postmenopausal osteoporosis after both preclinical animal and clinical human studies indicated it induces bone formation. This is the only approved bone anabolic agent available but unfortunately it has limited use, it is relatively expensive and difficult to administer. Consequently, the discovery of low cost orally available bone anabolic agents is critical for the future treatment of bone loss conditions. The intricate process of bone formation is co-ordinated by the action of many different bone growth factors, some stored in bone matrix and others released into the bone microenvironment from surrounding cells. Although all these factors play important roles, the bone morphogenetic proteins (BMPs) clearly play a central role in both bone cartilage formation and repair. Recent research into the regulation of the BMP pathway has led to the discovery of a number of small molecular weight compounds as candidate bone anabolic agents. These agents may usher in a new wave of more innovative and versatile treatments for osteoporosis as well as orthopedic and dental indications.
Statins and bone formation Garrett, I R; Gutierrez, G; Mundy, G R
Current pharmaceutical design,
05/2001, Letnik:
7, Številka:
8
Journal Article
Recenzirano
The main therapy needed most in the bone field is an anabolic agent for the treatment of osteoporosis. Current drugs on the market, which included bisphosphonates, calcitonin, estrogen and related ...compounds, vitamin D analogues trabecular microarchitecture. Therefore, it would be desirable to have a satisfactory and universally and iprifalvone, are essentially bone resorption inhibitors that mainly act to stabilize bone mass. Patients with established osteoporosis have lost more than 50% of their bone mass at critical sites in the skeleton, and more over have marked disruption of acceptable drug that would stimulate new bone formation and correct this disturbance of trabecular microarchitecture characteristic of established osteoporosis. Recently inhibitors of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, which controls the first step in the biosynthesis of cholesterol, have been shown to stimulate bone formation in rodents both in vitro and in vivo. The effect is associated with an increased expression of the bone morphogenetic protein-2 (BMP-2) gene in bone cells. These statins drugs are widely used agents for lowering cholesterol and reducing heart attacks, however they are also known to elicit numerous pleiotropic effects including inhibition of proliferation and migration of smooth muscle cells, inhibition of tumor growth and anti-inflammatory activity. Some of these effects have been attributed to not only to the reduction of cholesterol synthesis by inhibition of the HMG-CoA reductase enzyme but also by the concurrent reduction in downstream metabolites of the mevalonate pathway such as mevalonate, farnesyl pyrophosphate and geranylgeranyl pyrophosphate. The findings that statins are capable of increasing bone formation and bone mass in rodents suggests a potential new action for the statins, which may be beneficial in patients with established osteoporosis where marked bone loss has occurred. Recent clinical data suggests that they may reduce the risk of fracture in patients taking these drugs. However, their precise role can only be determined by appropriate randomized clinical trials, which demonstrate their efficacy in this regard in patients.
Matrix Extracellular Phospho-glycoprotEin (MEPE) and proteases are elevated and PHEX is defective in HYP. PHEX prevents proteolysis of MEPE and release of a protease-resistant MEPE–ASARM peptide, an ...inhibitor of mineralization (minhibin). Thus, in HYP, mutated PHEX may contribute to increased ASARM peptide release. Moreover, binding of MEPE by PHEX may regulate this process in normal subjects. The nature of the PHEX–MEPE nonproteolytic interaction(s) (direct or indirect) is/are unknown. Our aims were to determine (1) whether PHEX binds specifically to MEPE, (2) whether the binding involves the ASARM motif region, and (3) whether free ASARM peptide affects mineralization in vivo in mice. Protein interactions between MEPE and recombinant soluble PHEX (secPHEX) were measured using surface plasmon resonance (SPR). Briefly, secPHEX, MEPE, and control protein (IgG) were immobilized on a Biacore CM5 sensor chip, and SPR experiments were performed on a Biacore 3000 high-performance research system. Pure secPHEX was then injected at different concentrations, and interactions with immobilized proteins were measured. To determine MEPE sequences interacting with secPHEX, the inhibitory effects of MEPE–ASARM peptides (phosphorylated and nonphosphorylated), control peptides, and MEPE midregion RGD peptides on secPHEX binding to chip-immobilized MEPE were measured. ASARM peptide and etidronate-mediated mineralization inhibition in vivo and in vitro were determined by quenched calcein fluorescence in hind limbs and calvariae in mice and by histological Sanderson stain. A specific, dose-dependent and Zn-dependent protein interaction between secPHEX and immobilized MEPE occurs (EC50 of 553 nM). Synthetic MEPE PO4-ASARM peptide inhibits the PHEX–MEPE interaction (KDapp = 15 uM and Bmax/inhib = 68%). In contrast, control and MEPE–RGD peptides had no effect. Subcutaneous administration of ASARM peptide resulted in marked quenching of fluorescence in calvariae and hind limbs relative to vehicle controls indicating impaired mineralization. Similar results were obtained with etidronate. Sanderson-stained calvariae also indicated a marked increase in unmineralized osteoid with ASARM peptide and etidronate groups. We conclude that PHEX and MEPE form a nonproteolytic protein interaction via the MEPE carboxy-terminal ASARM motif, and the ASARM peptide inhibits mineralization in vivo. The binding of MEPE and ASARM peptide by PHEX may explain why loss of functional osteoblast-expressed PHEX results in defective mineralization in HYP.
Statins have been shown to stimulate BMP2 transcription and bone formation. This raises the possibility that they could be useful for enhancing rates of fracture repair. Observational studies in ...patients treated with oral statins for lipid‐lowering have been controversial. The likely reason for their inconsistent effects is that the statin concentration reaching the periphery was too low after oral administration to produce a reproducible biologic effect. Thus, we examined the effects of lovastatin (LV) given transdermally in a well‐described preclinical model of fracture repair. Effects on the healing fracture callus were assessed by biomechanical strength, radiographs, and quantitative morphology. LV was administered transdermally (TD) for 5 days after fracture in several doses (0.1–5 mg/kg/d) and compared with vehicle‐treated control rats and rats treated with LV by oral gavage (PO) at 5–25 mg/kg/d for 5 days from the day of fracture. Radiological evaluation of bones treated with TD LV showed enhanced fracture repair at 2 and 6 wk. BMD in the callus area at 6 wk was also increased in the TD group compared with vehicle‐treated controls (p < 0.05). The force required to break TD‐treated bones (0.1 mg/kg/d for 5 days) was 42% greater than vehicle‐treated controls (p < 0.02), and there was a 90% increase in stiffness (p < 0.01). PO LV at much higher doses (10 and 25 mg/kg/d) showed increased stiffness but no change in other biomechanical properties. By histological examination, a significant increase was also observed in the size of the callus, surrounding proliferating cell nuclear antigen–positive cells, and osteoblast and osteoclast number in TD‐treated rats compared with controls at day 8 after fracture (n = 6). In summary, we found that TD LV in low doses accelerates fracture healing, whereas 10‐fold the lipid‐lowering dose was required to produce any effect when it was administered orally. These studies provide valuable information on the potential of statins and TD delivery as a new and effective therapeutic modality in fracture repair.
There are no universally accepted agents that will substantially increase bone mass in osteoporotic patients. A number of peptides important in normal bone formation, such as members of the ...transforming growth factor‐β superfamily, are not satisfactory for this purpose either because their beneficial effects are predominantly local or there is systemic toxicity associated with their administration. We have examined the effects of exogenous fibroblast growth factor‐1 and ‐2 (FGF‐1 and FGF‐2) on bone in vivo, since FGFs have been shown recently to be essential for normal skeletal development. FGF‐1 was injected daily (0.2 mg/kg intravenously) for 28 days into the tail vein of adult female rats immediately following and 6 months after sham operation or ovariectomy (OVX). In rats treated immediately post‐OVX, OVX produced more than a 30% decrease in tibial bone density, which was prevented by FGF‐1 and estrogen. However, FGF‐1 also had an anabolic effect. In sham‐operated rats, FGF‐1 increased bone density to 2‐fold, whereas estrogen had no effect. In rats 6 months post‐OVX, severe bone loss and disruption of trabecular microarchitecture occurred similar to that seen in patients with severe osteoporosis. In these rats, administration of FGF‐1 induced extensive new woven bone formation with new trabecular‐like structures filling much of the marrow spaces, and bone density in the tibial metaphysis increased 3‐fold. FGF‐1 and FGF‐2 were also administered subcutaneously over the calvaria of mice in doses of 2–2000 μg/day for 3 days and shown to produce substantial increases in bone formation when examined morphologically. Thus, we conclude that both local and systemic FGF‐1 increases new bone formation and bone density, and systemic FGF‐1 also appears to restore bone microarchitecture and prevent bone loss associated with estrogen‐withdrawal.
We determined the effects of the potent bisphosphonate ibandronate in a murine model of human myeloma bone disease. In this model, bone lesions typical of the human disease develop in mice following ...inoculation of myeloma cells via the tail vein. Treatment with ibandronate (4 μg per mouse per day) significantly reduced the occurrence of osteolytic bone lesions in myeloma-bearing mice. However, ibandronate did not prevent the mice from developing hindlimb paralysis and did not produce a detectable effect on survival. There was no significant effect of ibandronate on total myeloma cell burden, as assessed by morphometric measurements of myeloma cells in the bone marrow, liver, and spleen, or by measurement of serum IgG2b levels. These results support clinical findings that bisphosphonates may be useful for the treatment of myeloma-associated bone destruction, but suggest that other therapies are also required to reduce tumor growth.
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