Animals. C57Bl and Swiss Webster mice (Charles River Laboratories, Stone Ridge, New York, USA) were used. At 4 months of age, male Swiss Webster mice were electronically tagged (Biomedic Data System Inc., Maywood, New Jersey, USA) and kept in plastic cages (1 animal per cage) under standard laboratory conditions with a 12-hour dark/12-hour light cycle, a constant temperature of 20°C, and humidity of 48%. All mice were fed on a standard rodent diet (Agway RMH 3000; Arlington Heights, Illinois, USA) containing 22% protein, 5% fat, 5% fiber, 6% ash, 3.5 Kcal/g, 1.0 IU vitamin D3/g, 0.97% calcium, and 0.85% phosphorus with water ad libitum. The animals were weighed at the beginning and end of each experiment. The University of Arkansas for Medical Sciences (UAMS) Division of Laboratory and Animal Medicine approved the protocols.
Osteoclast survival assay. Bone marrow cells were harvested from femora of C57Bl mice (Charles River Laboratories) and cultured in αMEM media with 10% FBS (HyClone Laboratories, Logan, Utah, USA) for 2 days. Nonadherent cells were collected and aliquots of 0.2 × 106 cells were plated on 16-well chamber slides in αMEM media with 10% FBS containing 30 ng/ml human recombinant M-CSF (R & D Systems Inc., Minneapolis, Minnesota, USA) and 30 ng/ml receptor activator of NF-κB ligand (RANKL) (Amgen Inc., Thousand Oaks, California, USA) for 3–4 days. Media was then changed to fresh αMEM, M-CSF, and RANKL with 10% charcoal-stripped FBS, and the cells were incubated in triplicate for 1 hour with vehicle or 10–5 M alendronate before addition of 10–7 to 10–10 M dexamethasone for 24 hours. Floating cells were removed and the attached cells fixed with 2% paraformaldehyde for 15 minutes at room temperature. Floating cells average about 10 per well and attached cells 200 ± 40; thus, it is unlikely that exclusion of the floating cells interfered with the assessment. After washing with TBS, incubating with 3% hydrogen peroxide for 15 minutes, and blocking with 10% goat serum for 30–60 minutes, cells were incubated with active caspase-3 Ab (Santa Cruz Biotechnology Inc., Santa Cruz, California, USA) (1:50 in 2% goat serum in TBS) for 2 hours followed by biotinylated anti-rabbit Ab for 30 minutes and immunoperoxidase staining. Tartrate-resistant acid phosphatase (TRAPase) counterstaining was used to measure total osteoclast number. Apoptotic osteoclasts were identified as dark brown cells (active caspase positive) in each well and expressed as the percentage of total osteoclasts per well.
In addition, cells were incubated in triplicate for 1 hour with vehicle or 10–5 M alendronate before addition of 10–9 M dexamethasone, 10–8 M mifepristone (RU 486), or both for 24 hours, and aliquots of the supernatants from the wells were evaluated using fluorogenic substrates (Biomol Research Laboratories, Plymouth Meeting, Pennsylvania, USA) to measure the enzyme activity of caspase-3, caspase-8, and caspase-9.
Bone densitometry. Dual-energy x-ray absorptiometry (DEXA) was used to determine spinal bone mineral density (BMD) in live mice as previously described (21, 22). Over the past 3 years, the coefficient of variation of the measurement done on a plastic-embedded whole mouse skeleton was 1.8% (n = 202). BMD determinations were done at 2-week intervals to identify the peak adult bone mass of the mice, which was reached between 5 and 6 months of age (20–22). Before the experiment began, BMD measurements were repeated to allocate the animals into groups with equivalent spinal BMD values.
Glucocorticoid administration. Slow-release pellets (Innovative Research of America, Sarasota, Florida, USA) of placebo or 2.1 mg/kg/day of prednisolone were then implanted for 4, 10, or 27 days, as described previously (20). For dynamic histomorphometric measurements at the 10-day time point, tetracycline HCl (30 mg/kg body weight) was given intraperitoneally 6 and 2 days before sacrifice. At the time of sacrifice, bone marrow aspirates were obtained from the right femur for ex vivo marrow cell cultures, and the left distal femur and lumbar vertebrae (L1-L4) were prepared for histomorphometric analysis.
Detection and quantification of osteoblast and osteoclast progenitors. Femoral marrow cells were obtained as described previously (23). The number of osteoblast progenitors (CFU-OB) in the marrow isolate was determined by culturing cells at 2.0 × 106 cells per 10-cm2 well for 25–28 days with irradiated guinea pig feeder cells in phenol red–free αMEM containing 15% preselected FBS and 1 mM ascorbate-2-phosphate. One-half of the medium was replaced every 5 days. Colonies containing osteoblasts were visualized by Von Kossa staining.
CFU-OB replication in vitro was determined as described previously (23). One aliquot of cells was used to determine the number of CFU-OB per 106 marrow cells in the initial isolate, as described above. A second aliquot was used to establish replicate cultures of cells in type I collagen gels at 5 × 106 cells in 1 ml of gel, which were then maintained in the absence or presence of 10 nM prednisolone for 7 days. The cells were then dispersed using bacterial collagenase, and the number of CFU-OB within each gel was determined. To calculate the fold increase in CFU-OB during culture in the collagen gels, the number of CFU-OB obtained per gel (after 7 days of culture) was divided by the number of CFU-OB initially incorporated into the collagen gel.
The number of osteoclast progenitors within the marrow isolate was determined by coculturing 75,000 marrow cells with 8,000 UAMS-32 stromal/osteoblastic cells for 8 days in a 2-cm2 well in the presence of 10% FBS in phenol red–free αMEM supplemented with 10 nM 1,25(OH)2D3 to stimulate osteoclast formation, as described previously (24). Replicate cultures (n = 4–6) were established from each animal. Osteoclastic cells were enumerated after staining for TRAPase; both mononucleated and multinucleated cells were counted.
Bisphosphonate administration. To examine the impact of an antiresorptive agent on the loss of bone density that accompanies glucocorticoid excess, we pretreated the mice with subcutaneous injections of 0.75 mg/kg/day of alendronate (4-amino-1-hydroxybutylidene-1,1-bisphosphonate; obtained from C.W.G.M. Löwik, University Hospital, Leiden, The Netherlands) dissolved in saline or saline alone beginning 3 days before prednisolone or placebo administration and then continued as daily injections during glucocorticoid administration. That this dose of alendronate is adequate has been shown by our previous findings that administration of one-third of this amount of alendronate prevented the increase in urinary free deoxypyridinoline excretion and serum osteocalcin that occurs in mice after ovariectomy (25).
Histomorphometric analysis. The lumbar vertebrae were fixed, embedded undecalcified in methyl methacrylate, and stained as described previously (20–22). The histomorphometric examination was done with a computer and digitizer tablet (OsteoMetrics Inc., Atlanta, Georgia, USA) interfaced to a Zeiss Axioscope (Carl Zeiss Inc., Thornwood, New York, USA) with a drawing tube attachment. The identity of each specimen was concealed from the histomorphometry reader. All measurements were two-dimensional, confined to the secondary spongiosa, and made at ×400 magnification (numerical aperture 0.75). The terminology used was that recommended by the Histomorphometry Nomenclature Committee of the American Society for Bone and Mineral Research (26).
Static measurements of cancellous bone. Cancellous bone area, trabecular width, and osteoid area, perimeter, and width were measured as described previously (20). The osteoblast perimeter was expressed as a percentage of the total cancellous perimeter and also as the number of osteoblasts palisading osteoid per millimeter of cancellous perimeter. Likewise, the osteoclast perimeter was expressed as a percentage of the total cancellous perimeter covered by TRAPase-positive osteoclasts and as the number of osteoclasts per millimeter of cancellous perimeter. The ratio of osteoclasts to osteoblasts was also expressed as both the percentage and number of cells.
Dynamic measurements of cancellous bone. The rate of mineral apposition was calculated as the mean distance between the midpoints of the two tetracycline labels divided by the interdose duration (4 days). The mineralizing perimeter and rate of bone formation per cancellous perimeter (square micrometer per micrometer per day) were calculated as described previously (20, 21).
Measurement of apoptosis in undecalcified bone sections. Apoptosis was detected by in situ nick-end labeling (ISEL) using Klenow terminal deoxynucleotidyl transferase (Oncogene Research Products, Cambridge, Massachusetts, USA) as described previously (27). Sections were counterstained with 0.5–3% methyl green. Plastic-embedded sections of vertebrae taken from orchidectomized adult mice were used as a positive control. Omitting the transferase made negative controls. Apoptotic osteoblasts were identified as ISEL-positive cells lining the osteoid-covered cancellous perimeter.
Statistics. In the osteoclast apoptosis assay, drug effects were examined using one-way ANOVA. In addition, a dose-response test for a linear trend between vehicle and dexamethasone was done using linear contrast coefficients. To evaluate the changes in ex vivo marrow cell cultures and 4- and 10-day BMD measurements, we used two-way ANOVA. If variances were unequal, a Satterthwaite approximation was used to determine the degrees of freedom. Differences between group means with the CFU-OB replication assay were evaluated with Student t tests. Serial changes in BMD were analyzed using a mixed effects model of repeated measures (28). Histomorphometric data were examined by one-way ANOVA. Comparisons of interest were specified a priori and their P values adjusted with Bonferroni’s correction (29). Pearson correlation coefficients were calculated to test for an association between two independently measured variables. P values less than 0.05 were considered significant.
How do glucocorticoids affect osteoclasts? ›
Glucocorticoids may prolong the life of mature osteoclasts under selected conditions. Eventually, glucocorticoids decrease bone remodeling by depleting the population of osteoblasts. This occurs by a decrease in osteoblastogenesis, and an increase in the apoptosis of mature osteoblasts and osteocytes.Do glucocorticoids inhibit osteoclasts? ›
Glucocorticoids inhibit both osteoblastogenesis and osteoclastogenesis, yet cancellous osteoclast numbers are maintained or increased, whereas osteoblasts decrease precipitously (5).How do bisphosphonates work? ›
Bisphosphonates are drugs that target areas of higher bone turnover. The osteoclast cells, which break down old bone, absorb the bisphosphonate drug. Their activity is slowed down. This reduces bone breakdown.How does cortisol affect osteoclast? ›
It is concluded that cortisol inhibits bone resorption in vitro by limiting the ability of precur- sor cells to form osteoclasts.What are the effects of glucocorticoids on bone? ›
Glucocorticoids increase bone resorption and reduce bone formation [2-4]. The risk of bone loss is most pronounced in the first few months of use, followed by slower but steady loss of bone with continued use .Why do glucocorticoids worsen osteoporosis? ›
Glucocorticoids decrease the function of the remaining osteoblasts directly and indirectly through the inhibition of insulin-like growth factor I expression. The stimulation of bone resorption is likely responsible for the initial bone loss after glucocorticoid exposure.What is the main function of glucocorticoids? ›
Glucocorticoid hormones regulate essential body functions in mammals, control cell metabolism, growth, differentiation, and apoptosis.What is the mechanism of action of glucocorticoids? ›
Classical mechanisms of glucocorticoid action
It is generally believed that most, if not all, the effects of glucocorticoids on cells are mediated via the glucocorticoid receptor (GR). This 777 amino acid protein was cloned in humans in 1985 and is a member of the superfamily of ligand regulated nuclear receptors.
Glucocorticoid therapy is associated with an appreciable risk of bone loss, which is most pronounced in the first few months of use. In addition, glucocorticoids increase fracture risk, and fractures occur at higher bone mineral density (BMD) values than occur in postmenopausal osteoporosis.What are the risks of taking bisphosphonates? ›
- Fever and flu-like symptoms. ...
- Low levels of calcium in your blood (hypocalcaemia) ...
- Bone and joint pain. ...
- Changes in bowel movements. ...
- Tiredness and low energy levels. ...
- Feeling sick. ...
- Changes to your kidneys. ...
- Irritation of the food pipe (oesophagus)
What foods destroy bone density? ›
- Excess salt.
- Hydrogenated oil.
- Food rich in vitamin A.
- Soft drinks.
They prevent and partially even reverse the bone loss in glucocorticoid-treated patients [4, 21] and are therefore a standard therapy in patients receiving this drug over longer time. All the bisphosphonates induce a marked decrease in bone turnover when given in doses effective on BMD.What hormone is responsible for promoting osteoclast activity? ›
Hormones That Influence Osteoclasts
Two hormones that affect the osteoclasts are parathyroid hormone (PTH) and calcitonin. PTH stimulates osteoclast proliferation and activity. As a result, calcium is released from the bones into the circulation, thus increasing the calcium ion concentration in the blood.
Parathyroid hormone (PTH) stimulates bone resorption by acting directly on osteoblasts/stromal cells and then indirectly to increase differentiation and function of osteoclasts.Which hormone increases osteoclast activity? ›
Parathyroid hormone (PTH) stimulates osteoclast proliferation and resorption of bone by osteoclasts.What are 2 main effects of glucocorticoids? ›
Glucocorticoids can reduce how active immune cells are. This helps reduce the internal damage from these diseases. They suppress inflammation from autoimmune reactions. This can reduce pain, swelling, cramping, and itching.What is a common effect of glucocorticoid stimulation? ›
Glucocorticoids have been found to increase blood glucose levels as well as suppress calcium absorption through their various metabolic affects. As such, long-term anti-inflammatory therapy with glucocorticoids can often lead to swelling, skin changes, decreased immunity, and psychological changes.What are examples of glucocorticoids? ›
Glucocorticoids are powerful anti-inflammatory steroids that are utilized to treat many different health conditions. These steroids also possess properties that suppress the immune system, which is another reason doctors will prescribe them. Prednisone and dexamethasone are two examples.Why cortisol is not advised in patients with osteoporosis? ›
Cortisol may negatively affect bone density by altering bone turnover, impairing intestinal absorption and renal reabsorption of calcium, and, in premenopausal women, by inhibiting reproductive hormones .How do you manage glucocorticoid induced osteoporosis? ›
Bisphosphonates are the front-line choice for prevention of fracture in glucocorticoid-treated patients, with teriparatide as the second-line option; calcium and vitamin D supplements should be co-prescribed in the majority of individuals.
How do you treat glucocorticoid induced osteoporosis? ›
Alendronate (oral 5 or 10 mg once daily, or 70 mg once weekly), risedronate (oral 5 mg daily or 35 mg one weekly) and zoledronate (intravenous infusion 5 mg once yearly) prevent bone loss at the spine and hip in patients initiating GCs, and increase BMD in patients on long-term GCs.What hormone is responsible for glucocorticoids? ›
Activation of the hypothalamus initiates the release of corticotrophin-releasing hormone (CRH), which in turn signals to the anterior pituitary to release adrenocorticotrophin (ACTH). This then signals to the cortical layer of the adrenal gland to release glucocorticoids, which can act on peripheral tissues.Which 3 hormones are glucocorticoids? ›
Hydrocortisone, also called cortisol, corticosterone, 11-deoxycortisol, and cortisone are the types of glucocorticoids found in most vertebrates. Cortisol is the most abundant and potent glucocorticoid in humans and fish. Corticosterone is most potent in amphibians, reptiles, and birds.Where is glucocorticoids found in the body? ›
glucocorticoid, any steroid hormone that is produced by the adrenal gland and known particularly for its anti-inflammatory and immunosuppressive actions. The adrenal gland is an organ situated on top of the kidney. It consists of an outer cortex (adrenal cortex) and an inner medulla (adrenal medulla).What is one of the possible mechanisms by which glucocorticoids regulate inflammation? ›
Glucocorticoids modulate the inflammatory response by repressing the expression of pro-inflammatory cytokines by immune cells. In addition, glucocorticoids can repress the expression of adhesion molecules, which prevents rolling, adhesion and extravasation of neutrophils to the site of inflammation.What is the primary action of glucocorticoids on whole body metabolism? ›
Glucocorticoids acting through the GR regulate glucose metabolism in the liver, skeletal muscle, adipose tissue, and the pancreas, by controlling the expression of key enzymes.How do you prevent bone loss when taking prednisone? ›
Anyone using steroid medication should also aim to get plenty of calcium and vitamin D (ask your doctor before taking a supplement); practice weight-bearing exercise; avoid smoking; and limit alcohol intake.What drug is used to prevent corticosteroid induced osteoporosis? ›
The first choice for prevention of corticosteroid osteoporosis is a potent oral bisphosphonate—for example, alendronate or risedronate. Intravenous bisphosphonates should be considered for patients intolerant of the oral route.Which steroids increase bone density? ›
Anabolic steroids are currently used in the treatment of established osteoporosis. It has been demonstrated that, at least partly, anabolic steroids increase bone density by stimulating bone formation.› health › glucocorticoids ›
Glucocorticoids: List, Uses, Side Effects, and More
The use of corticosteroids and nonsteroidal antiinflammatory ...
Structures and mechanism for the design of highly potent ...
Does glucocorticoids increase osteoclast activity? ›
Glucocorticoids stimulate osteoclast proliferation by suppressing synthesis of osteoprotegerin, an inhibitor of osteoclast differentiation from hematopoietic cells of the macrophage lineage, and by stimulating production of the receptor activator of nuclear factor kappa-B (RANK), which is required for ...What role do glucocorticoids play in bone remodeling? ›
Glucocorticoids cause profound effects on bone cell replication, differentiation, and function. Glucocorticoids increase bone resorption by stimulating osteoclastogenesis by increasing the expression of RANK ligand and decreasing the expression of its decoy receptor, osteoprotegerin.What hormones inhibit osteoclasts? ›
Of the two best known hormonal inhibitors of bone resorption in vivo, calcitonin acts directly upon osteoclasts to inhibit their activity, whereas estrogen acts indirectly, via the regulation of several cytokines.Which hormone increases osteoclasts activity? ›
Parathyroid hormone (PTH) stimulates bone resorption by acting directly on osteoblasts/stromal cells and then indirectly to increase differentiation and function of osteoclasts.What are 2 main effects of glucocorticoids? ›
Glucocorticoids can reduce how active immune cells are. This helps reduce the internal damage from these diseases. They suppress inflammation from autoimmune reactions. This can reduce pain, swelling, cramping, and itching.What are the main actions of glucocorticoids? ›
Glucocorticoids inhibit many inflammation-associated molecules such as cytokines, chemokines, arachidonic acid metabolites, and adhesion molecules. In contrast, anti-inflammatory mediators often are up-regulated by glucocorticoids.What are common effects of glucocorticoid stimulation? ›
Glucocorticoid excess (due to pathology such as Cushing's syndrome or prescribed synthetic glucocorticoids) can cause immunosuppression, muscle atrophy, central adiposity, hepatosteatosis, osteoporosis, insulin resistance, hypertension, depression and insomnia.
Glucocorticoids have effects on metabolism, immune response, cell creation and turnover, and the constriction of blood vessels. Glucocorticoids can help to fight inflammation and suppress hypersensitive white blood cell responses to infection and other threats.Which hormone is responsible for bone growth? ›
The growth hormone/IGF-1 system stimulates both the bone-resorbing and bone-forming cells, but the dominant effect is on bone formation, thus resulting in an increase in bone mass. Thyroid hormones increase the energy production of all body cells, including bone cells.Which hormone is responsible for bone reduction? ›
Bones – parathyroid hormone stimulates the release of calcium from large calcium stores in the bones into the bloodstream. This increases bone destruction and decreases the formation of new bone.
What hormone breaks down bone? ›
PTH is a hormone produced by the parathyroid glands; there are four parathyroid glands, and together, they help regulate calcium levels in the body. PTH increases calcium in several ways; it breaks down bone, improves the body's ability to obtain calcium from food, and increases the kidney's ability to contain calcium.What gland increases osteoclasts? ›
The parathyroid glands produce and secrete PTH, a peptide hormone, in response to low blood calcium levels (Figure 2). PTH secretion causes the release of calcium from the bones by stimulating osteoclasts, which secrete enzymes that degrade bone and release calcium into the interstitial fluid.Does vitamin D stimulate osteoblast? ›
Vitamin D acts through the immature osteoblast to stimulate osteoclastogenesis. Transgenic elevation of VDR in mature osteoblasts was found to inhibit osteoclastogenesis associated with an altered OPG response. This inhibition was confined to cancellous bone.Which gland stimulates osteoclasts? ›
The parathyroid glands' function is to maintain serum calcium homeostasis through synthesis and release of PTH. At the bone, PTH inhibits osteoblast activity and stimulates osteoclast activity leading to bone breakdown and calcium release.