Osteoclast formation and resorption.
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Animals↗
Biomedical subjects
U H Lerner
Publications and source records attributed to U H Lerner.
A peptidyl derivative structurally based on the inhibitory center of cystatin C inhibits bone resorption in vitro.
Human cystatin C is a cysteine proteinase inhibitor belonging to the cystatin superfamily, which previously has been shown to inhibit bone resorption in bone organ culture. The aminoterminal segment, Arg(8)-Leu(9)-Val(10)-Gly(11) (RLVG), of the single polypeptide chain of cystatin C constitutes an essential part of its inhibitory center. In the present study, the effect of benzyloxycarbonyl-Arg(8)-Leu(9)-Val(10)-Gly(11)-diazomethane (Z-RLVG-CHN(2)) on bone resorption in vitro was compared with the effects of cystatin C and calcitonin. Bone resorption was assessed by the release of (45)Ca and (3)H from mouse calvarial bones prelabeled with [(45)Ca]CaCl(2) and [(3)H]-proline, respectively. Z-RLVG-CHN(2) concentration-dependently inhibited the release of (45)Ca and (3)H in bones stimulated by parathyroid hormone (PTH), with half-maximal inhibition obtained at 1 micromol/L. The inhibitory actions of Z-RLVG-CHN(2) and cystatin C were persistent, whereas action induced initially by calcitonin was lost with time. The inhibition caused by Z-RLVG-CHN(2) and cystatin C on PTH-stimulated (45)Ca release was observed after 6 h, whereas inhibition by calcitonin was seen already after 2 h. In contrast, the inhibitory effects of Z-RLVG-CHN(2) and cystatin C, as well as that of calcitonin, on (3)H release was seen already after 2 h. Z-RLVG-CHN(2), in which the reactive carboxyterminal diazomethane was substituted by nonreactive groups [-OH, -NH(2), or -N(CH(3))(2)], resulted in peptidyl derivatives, which, in contrast to Z-RLVG-CHN(2) and cystatin C, inhibited neither cysteine proteinases nor bone resorption. In contrast to wild-type cystatin C, recombinant human cystatin C with Gly substitutions for residues Arg(8), Leu(9), Val(10), and Trp(106), and with low or nonexistent affinity for cysteine proteinases, did not display any inhibitory effect on bone resorption. These data strongly indicate that Z-RLVG-CHN(2) inhibits bone resorption in vitro by a mechanism that seems primarily to be due to an inhibition of bone matrix degradation via cysteine proteinases. The data also corroborate the hypothesis that cystatin C inhibits bone resorption by virtue of its cysteine proteinase inhibitory capacity.
Animals↗
Vasoactive intestinal peptide regulates osteoclast activity via specific binding sites on both osteoclasts and osteoblasts.
Clinical and experimental observations, together with immunohistochemical findings, suggest that neuro-osteogenic interactions may occur in the skeleton. In this study, we have examined the effect of vasoactive intestinal peptide (VIP), one of the neuropeptides present in bone, on the activity of the bone-resorbing osteoclast. Effects on bone resorption were assessed by counting the number of pits formed by rat osteoclasts incubated on devitalized slices of bovine cortical bone. Under conditions with an initially sparse density of stromal cells/osteoblasts, VIP caused a rapid cytoplasmic contraction and decreased motility of osteoclasts. This was coupled with a decrease in the number of resorption lacunae and a decrease in the total area resorbed by the osteoclasts in 48-h cultures. Time-course experiments revealed that the inhibitory effects on contraction and motility were transient and that the cells gradually regained their activity, such that, when culture time was prolonged to 120 h, a stimulatory effect by VIP on bone resorption was observed. When osteoclasts were incubated on bone slices, in the presence of an initially large number of stromal cells/osteoblasts, VIP treatment increased the number of resorption pits and total bone area resorbed in 48-h cultures. Using atomic force microscopy, we provide direct evidence that both osteoclasts and stromal cells/osteoblasts bind VIP. Also, VIP was shown to cause a rapid rise of intracellular calcium in osteoclasts and in a proportion (20%) of stromal cells/osteoblasts. Taken together, these data suggest that differentiated osteoclasts are equipped with receptors for VIP that are linked to a transient inhibition of osteoclast activity and, in addition, that stromal cells/osteoblasts have VIP receptors coupled to a delayed stimulation of osteoclastic resorption.
Animals↗
Characterization of bone resorbing activity in gingival crevicular fluid from patients with periodontitis.
BACKGROUND: In attempts to elucidate factors stimulating bone resorption in patients with different inflammatory diseases in the vicinity of the skeleton, e.g., peridontal disease and rheumatoid arthritis, we are investigating the presence of bone-resorbing activity in a variety of inflammatory exudates. The aim of the present study was to characterize the bone-resorbing activity present in patients with periodontitis. METHODS: Bone-resorbing activity was assessed in gingival crevicular fluids (GCFs) collected from patients with periodontitis and from patients with no signs of gingivitis. Bone-resorbing activity was evaluated by analyzing the capacity of GCFs to stimulate the release of minerals and the breakdown of bone matrix proteins in cultured neonatal mouse calvariae. The concentrations of IL-1alpha, IL-1beta and PGE2 were determined with ELISA and RIA techniques, respectively. RESULTS: GCF eluates from 24 different healthy sites caused a 1.23+/-0.05 fold stimulation of 45Ca release, whereas GCF eluates from 45 different diseased (periodontitis) sites caused a 2.46+/-0.10 fold stimulation. The effect on 45Ca release was time- and concentration-dependent, inhibited by 3 different osteoclast inhibitors and associated with enhanced release of 3H from [3H]-proline-labelled bones. The activity in GCF causing enhanced 45Ca release was unaffected, or in some samples partially reduced, by ultrafiltration using a filter with a molecular weight cut-off of 3000 Daltons. The bone-resorbing activity was temperature sensitive (+90degrees C, 10 min). The concentrations of prostaglandin E2 (PGE2) in the diluted GCF eluates, used in the bone resorption bioassay, were too low to be responsible for the release of 45Ca. Antisera specifically neutralizing human IL-1a inhibited the stimulatory effect of GCF pooled from several diseased sites. The specific, recombinant human IL-1 receptor antagonist completely inhibited the effect of pooled GCFs. GCF eluates from diseased sites contained human IL-1alpha and IL-1beta at concentrations of 1838+/-294 pg/ml and 512+/-91 pg/ml, respectively. CONCLUSIONS: These data show that GCF contains activity(ies) stimulating osteoclastic bone resorption in vitro. The factor primarily responsible for this activity seems to be IL-1alpha, but IL-1alpha is not the sole activator of bone resorption in GCF.
Adult↗
The bone resorbing activity released by gingival fibroblasts isolated from patients with periodontitis is independent of interleukin-1.
Supernatants from gingival fibroblast cultures obtained from 14 patients with periodontal disease contained factor(s) capable of stimulating bone resorption in vitro, as assessed by the release of 45Ca from neonatal mouse calvariae. The possibility that the factor(s) was interleukin-1 alpha (IL-1 alpha), IL-1 beta or prostaglandin E2 (PGE2) was next investigated. The human fibroblast conditioned media (HFCM) stimulated PGE2 biosynthesis in bone. The stimulatory effect by HFCM on 45Ca release, however, was not affected by blocking prostaglandin biosynthesis with indomethacin. In contrast, 45Ca release induced by IL-1 alpha, IL-1 beta, thrombin and bradykinin was significantly reduced by indomethacin, whereas the effects of PTH and PTHrP were unaffected by indomethacin. The concentration of PGE2 in HFCM was too low to be solely responsible for the 45Ca release response. In addition, the amount of bone resorbing activity produced by the gingival fibroblasts was unaffected by cyclo-oxygenase inhibitors. Similar to IL-1 alpha and IL-1 beta, the stimulatory effect of HFCM was inhibited by gamma-interferon. HFCM did not stimulate cyclic AMP formation in the mouse calvarial bones. Antisera which specifically blocked human IL-1 alpha or IL-1 beta induced 45Ca release, and the specific IL-1 receptor antagonistic protein, did not inhibit the stimulatory effect of HFCM. These data show that gingival fibroblasts secrete bone resorbing factor(s) which is not due to IL-1 and which stimulates bone resorption by a prostaglandin- and cyclic AMP-independent mechanism.
Animals↗
Functional characterization of osteoblasts and osteoclasts from alkaline phosphatase knockout mice.
Tissue nonspecific alkaline phosphatase (TNAP) knockout (ko) mice manifest defects in bone mineralization that mimic the phenotypic abnormalities of infantile hypophosphatasia. In this article, we have searched for phenotypic differences between calvarial osteoblasts and osteoclasts in wild-type (wt), heterozygous and homozygous TNAP null mice. In vitro release of 45Ca from calvarial bones, with and without stimulation with parathyroid hormone (PTH), revealed no functional difference between osteoclasts from the three TNAP genotypes. Studies of primary cultures of TNAP+/+, TNAP+/-, and TNAP-/- calvarial osteoblasts revealed no differences in the rate of protein synthesis or in the expression levels of messenger RNAs (mRNAs) for osteopontin (OP), osteocalcin (OC), collagen type I, core binding factor alpha1 (Cbfa 1), N-cadherin, Smad 5, and Smad 7. Release of interleukin-6 (IL-6) from calvarial osteoblasts under basal conditions and after stimulation with PTH, tumor necrosis factor alpha (TNF-alpha) or IL-1beta was similar in all genotypes. The amount of cyclic adenosine monophosphate (cAMP) accumulation also was comparable. However, although cultures of primary TNAP-/- osteoblasts were able to form cellular nodules as well as TNAP positive osteoblasts do, they lacked the ability to mineralize these nodules in vitro. Mineralization also was delayed in TNAP+/- osteoblast cultures compared with cultures of wt osteoblasts. Incubation with media supplemented with recombinant TNAP, but not with enzymatically inactive TNAP, restored mineralization in ko osteoblast cultures. Our data provide evidence that osteoblasts in TNAP null mice differentiate normally but are unable to initiate mineralization in vitro. The fact that even heterozygous osteoblasts show delayed mineralization provides a rationale for the presence of bone disease in carriers of hypophosphatasia.
Alkaline Phosphatase↗
Neuro-hormonal control of bone metabolism: vasoactive intestinal peptide stimulates alkaline phosphatase activity and mRNA expression in mouse calvarial osteoblasts as well as calcium accumulation mineralized bone nodules.
Based upon the immunohistochemical demonstration of neuropeptides in the skeleton, including vasoactive intestinal peptide (VIP), we have addressed the question of whether neuropeptides may exert regulatory roles on bone tissue metabolism or not. In the present communication, we have investigated if VIP can affect anabolic processes in osteoblasts. Osteoblasts were isolated from neonatal mouse calvariae by time sequential enzyme-digestion and subsequently cultured for 2-28 days in the presence of VIP and other modulators of cyclic AMP formation. VIP (10(-6) M) stimulated ALP activity and calcium content. The cyclic AMP phosphodiesterase inhibitors ZK 62 711 (10(-4) M) and isobutyl-methylxanthine (10(-4) M) stimulated ALP activity and synergistically potentiated the effect of VIP. Neither VIP, nor isobutyl-methylxanthine or ZK 62 711, in the absence or presence of VIP, affected cell number. The stimulatory effect of VIP on ALP activity, in the presence of ZK 62 711, was dependent on time and concentration of VIP. The stimulatory effects of VIP and ZK 62 711 on ALP activity was seen also in cells stained for ALP. VIP (10(-6) M), in the presence of ZK 62 711 (10(-6) M), significantly enhanced mRNA for tissue non-specific ALP. VIP (10(-6) M), in the presence of ZK 62 711, stimulated cyclic AMP production. Forskolin and choleratoxin stimulated ALP activity and cyclic AMP formation in a concentration-dependent manner, without affecting cell number. VIP (10(-6) M) and ZK 62 711 (10(-5) M) stimulated, and their combination synergistically enhanced, calcium content in bone noduli. These data show that VIP, without affecting cell proliferation, can stimulate osteoblastic ALP biosynthesis and bone noduli formation by a mechanism mediated by cyclic AMP. Our observations suggest a possibility that anabolic processes in bone are under neurohormonal control.
Alkaline Phosphatase↗
Bone resorbing activity released from zymosan-activated mouse peritoneal macrophages--the role of prostanoids and interleukin-1.
OBJECTIVE: To study the effect of zymosan on the release of osteoclast stimulating activity from macrophages. MATERIALS: Calvarial bones from neonatal mice and peritoneal macrophages were incubated in the absence and presence of zymosan for 72 h and supernatants harvested for subsequent analysis of bone resorbing activities and prostaglandin concentration. METHODS: Bone resorption was assessed in vitro by analysing the release of 45Ca and 3H from neonatal mouse calvarial bones prelabelled in vivo by injections of [45Ca]CaCl2 or [3H]-proline. Prostaglandin E2 (PGE2) and I2 (PGI2) were analyzed using radioimmunoassays. RESULTS: Supernatants from macrophages treated with zymosan stimulated the release of 45Ca and 3H. The amount of bone resorbing activity present in the macrophage supernatants was dependent on the concentration of zymosan (0.1-100 microg/ml), as well as the number of macrophages present. The 45Ca release induced by zymosan treated macrophages was inhibited by three different inhibitors of osteoclastic bone resorption (calcitonin, acetazolamide, amino bisphosphonate). The bone resorbing activity released by the zymosan-activated macrophages was lost after ultrafiltration using a filter with a molecular weight cut off of 30,000 Daltons, but retained when using a filter with a cut off of 3000 Daltons. Time-course studies of the production of bone resorbing activity in macrophages showed that activity increased during the first hour of exposure to zymosan and then reached a plateau for 96 h. PGE2 and PGI2 release from macrophages was increased during the first three hours of exposure to zymosan. This prostanoid production, together with bone resorbing activity, was abolished by indomethacin. The bone resorbing activity present 3-72 h after zymosan exposure, however, was not inhibited by indomethacin. Bone resorption stimulated by conditioned media from zymosan treated macrophages after 3 h was inhibited by 60-75% in the presence of anti IL-1alpha, 0-20% by anti IL-1beta, and completely by antisera neutralizing both IL-1alpha and IL-1beta. In addition, an IL-1 receptor antagonist abolished the stimulatory effect of conditioned media from zymosan treated macrophages. CONCLUSIONS: These data indicate that treatment of mouse peritoneal macrophages with zymosan results in production of activities capable of stimulating bone resorption in vitro. The activity released initially appears to be due to a zymosan induced burst of prostanoid production, while the activity released during prolonged exposure to zymosan is due primarily to IL-1alpha.
Animals↗
Synergistic interactions of bradykinin, thrombin, interleukin 1 and tumor necrosis factor on prostanoid biosynthesis in human periodontal-ligament cells.
Prostaglandins are involved in force-induced orthodontic tooth movement. Bradykinin (BK) and thrombin are known to cause a significant time- and concentration-dependent burst of prostanoid biosynthesis in cultured human periodontal-ligament (PDL) cells. The aim now was to investigate interactive effects between interleukin 1 alpha, -beta (IL-1 alpha, -1 beta), tumour necrosis factor-alpha,-beta (TNF-alpha, -beta) and BK or thrombin on prostaglandin biosynthesis in human PDL cells. IL-1 alpha and -1 beta produced time- and concentration-dependent stimulation of prostanoid biosynthesis [prostaglandin (PG)E2 and 6-keto-PGF1alpha]. Synergistic stimulation of prostanoid biosynthesis was demonstrated when BK or thrombin were added together with IL-1 alpha or -1 beta. BK and IL-1 beta both significantly stimulated the release of [3H]arachidonic acid. No synergistic effect on [3H]arachidonic acid release was seen when BK and IL-1 beta were added simultaneously. These data suggest that the synergistic effect of BK and IL-1 beta on prostanoid biosynthesis is not due to interactions at the receptor level nor to enhanced release of arachidonic acid, but may be due to increased activity of cyclo-oxygenase. Also, TNF-alpha and -beta produced a concentration-dependent stimulation of PGE2 formation in cultured human PDL cells. Synergistic effects of BK and thrombin were demonstrated when PGE2 production was stimulated in combination with TNF-beta. In addition, a synergistic effect on the PGE2 response to IL-1 alpha or -1 beta was demonstrated when added in combination with TNF-alpha. These experiments demonstrate synergistic interactions between BK, thrombin, IL-1 and TNF on prostaglandin biosynthesis in cultured human PDL cells. The findings suggest that inflammatory mediators may act in concert in stimulating prostanoid production in response to pro-inflammatory stimuli. As an inflammatory reaction is seen in the periodontal ligament when teeth are orthodontically treated, this synergistic interaction may be of importance in force-induced tooth movement.
6-Ketoprostaglandin F1 alpha↗
Gingival crevicular fluid from patients with periodontitis contains bone resorbing activity.
Inflammatory processes occurring in the vicinity of bone tissue often result in stimulation of osteoclast activity and loss of skeletal mass. The aim of the current study was to determine if inflammatory exudates collected from gingival pockets in patients with periodontitis contain factors capable of stimulating resorptive activity. The degree of bone mineral mobilization and bone matrix degradation was assessed by analysis of the release of 45Ca and 3H from bones prelabelled with 45CaCl2 and [3H]proline, respectively. Gingival crevicular washings from six patients with signs of periodontitis stimulated 45Ca or 3H release from the calvarial bones. The stimulatory effect of the gingival crevicular washings on 45Ca release was concentration- and time-dependent, and reduced by calcitonin, a specific osteoclast inhibitor. These data demonstrate that crevicular fluid contains factor(s) which can stimulate osteoclastic degradation of bone in vitro. The bone resorbing activity was partially retained after extensive dialysis. Analysis of the concentrations of prostaglandin E2, interleukin-1alpha and interleukin-1beta in the crevicular fluids, and comparisons of these agents as stimulators of 45Ca release in the mouse calvarial assay, suggest that prostaglandin E2 is not the sole factor responsible for the bone resorbing activity of the exudates. The data indicate that other factors, such as IL-1, may play key roles in the stimulation of osteoclastic activity by gingival crevicular washings.
Adolescent↗
Gutta-percha-stimulated mouse macrophages release factors that activate the bone resorptive system of mouse calvarial bone.
This study investigated the capacity of macrophages exposed to gutta-percha particles to produce factors affecting bone metabolism. Peritoneal mouse macrophages were isolated and incubated with and without gutta-percha particles, and the supernatants were assessed for bone resorbing activity by adding macrophage-conditioned media to cultures of neonatal mouse calvarial bones. Bone resorption was measured by mineral mobilization (45Ca release) and matrix degradation (3H from [3H]proline labelled bones). The results showed that supernatant from gutta-percha-stimulated macrophages enhanced bone resorption. This effect was related to the amount of gutta-percha, and the concentration and time of exposure to the conditioned media. Stimulated macrophages released enhanced amounts of prostaglandins E2 and I2; however, indomethacin, which inhibits the prostanoid response, had no effect on bone resorbing activity. The stimulatory effect on bone resorption was inhibited by calcitonin, interleukin-1 receptor antagonistic protein, and by antiserum neutralizing mouse interleukin-1alpha(IL-1alpha), but not by anti-IL-1beta. Filtration experiments revealed that the molecules involved in the resorption activity had an apparent molecular weight between 3000 and 30,000 Da. These experiments show that mouse peritoneal macrophages, when exposed to gutta-percha particles, release factors which have a bone resorbing activity that is primarily due to enhanced production of IL-1alpha.
Animals↗
Prostaglandin-independent stimulation of bone resorption in mouse calvariae and in isolated rat osteoclasts by thyroid hormones (T4, and T3).
The thyroid hormones, thyroxine (T4) and triiodothyronine (T3), were found to enhance both neonatal mouse calvarial bone resorption and pit formation on bovine slices by isolated rat osteoclasts. Dosage-dependent release of 45Ca from mouse calvarial bones was observed after 120 hr of culture with 10(-6)-10(-8) MT4 and 10(-6)-10(-10) M T3. Maximum treatment/control ratios of 45Ca release were recorded for 10(-7) M T4 and 10(-8) MT3. Inhibition of 45Ca release stimulated by 10(-8) M T3 was observed in the presence of 30 nM salmon calcitonin at 48 hr and 120 hr of culture with no indication of "escape" by T3-treated bones. In contrast, stimulation of 45Ca release from mouse calvarial bones by 10(-7) MT4 and 10(-8) MT3 was not inhibited by 10(-6) M indomethacin. Formation of PGE2 and PGI2 (evaluated by measuring 6-keto-PGF1alpha) by mouse calvariae was also not increased by 10(-8) MT3 after 120 hr of culture. Furthermore, no increases in cAMP formation were observed in calvarial bone cultures after either 10 min or 24 hr of exposure to 10(-8) MT3. However, significant inhibition of 45Ca release stimulated by 10(-8) M T3 was found at 120 hr in the presence of 10(-3) M hydroxyurea. When isolated rat osteoclasts were cultured in the presence of 10(-7) MT3, a 1.4-fold stimulation of pit number was observed. Pit formation was not affected by addition of 10(-6) M indomethacin to either the control or T3-treated cultures. These data suggest that the stimulation of bone resorption in neonatal mouse calvariae and activation of isolated rat osteoclasts by the thyroid hormones is not related to either prostaglandin or cAMP formation. In mouse calvariae, the effect on bone resorption of the thyroid hormones is dependent on increased cellular replication, perhaps of osteoclast precursors, or other bone cells involved in the resorptive process.
6-Ketoprostaglandin F1 alpha↗
Bacteria inhibit biosynthesis of bone matrix proteins in human osteoblasts.
The effect of extracts from Staphylococcus aureus and Staphylococcus epidermidis on bone matrix production were assessed by analyzing the biosynthesis of osteocalcin and Type I collagen in a human osteoblastic osteosarcoma cell line (MG-63). In MG-63 cells, extracts from Staphylococcus aureus and Staphylococcus epidermidis decreased 1,25(OH)2-vitamin D3 stimulated osteocalcin biosynthesis, and insulin-like growth factor I induced production of Type I collagen in a concentration dependent manner. The basal rate of osteocalcin and Type I collagen formation was unaffected by the bacterial extracts. The inhibitory effect of the bacteria on osteocalcin biosynthesis was seen after 24 hours of treatment and was maintained for at least 96 hours. The extracts of Staphylococcus aureus and Staphylococcus epidermidis enhanced prostaglandin E2 formation in the MG-63 cells. Abolition of the prostaglandin E2 response by treatment with indomethacin and flurbiprofen did not affect bacteria induced inhibition of osteocalcin production. Stimulation of osteocalcin biosynthesis by 1,25(OH)2-vitamin D3 was associated with a decreased rate of cell proliferation. The inhibitory action of the bacterial extracts was not linked to any inhibition of [3H]-thymidine incorporation into deoxyribonucleic acid. These data show that extracts of Staphylococcus aureus and Staphylococcus epidermidis have the ability to inhibit the biosynthesis of bone matrix proteins by a nonprostaglandin and noncytotoxic dependent mechanism and suggest that bone loss in inflammatory processes containing Staphylococcus aureus or Staphylococcus epidermidis may not be caused only by enhanced bone resorption but also by decreased bone formation.
Calcitriol↗
Cystatin C, and inhibitor of bone resorption produced by osteoblasts.
The effects of human cystatin C on bone resorption, enzyme release, osteoclast generation, bone cell proliferation and bone matrix protein biosynthesis have been examined in different in vitro systems. The effects of cystatin C were compared with those of calcitonin and E 64 (trans-Epoxysuccinyl-L-leucyl-amido-(4-guanidino)butane). Recombinant human cystatin C and E 64 dose dependently inhibited the mobilization of 45Ca and the release of 3H (from [3H]-proline-labelled bones) in mouse calvariae stimulated to resorb by parathyroid hormone (PTH) or 1,25(OH)2-vitamin D3. Cystatin C and E 64 also inhibited the release of 45Ca from bones stimulated by thrombin, interleukin-1 and prostaglandin E2. In PTH-stimulated bones, the inhibitory action of cystatin C and E 64 on 45Ca release was observed after 6-9 h, whereas the inhibitory effect on 3H release was seen after just 2 h. In contrast, calcitonin caused an inhibition of both 45Ca and 3H release which was seen after 2 h. The PTH-stimulated release of the lysosomal enzymes was not affected by cystatin C and E 64, whereas calcitonin caused a significant inhibition. In contrast to calcitonin, cystatin C did not affect PTH-stimulated enhancement of osteoclast generation in the mouse calvariae. Using Western blot analysis and radioimmunoassay, we demonstrated that mouse calvarial bones and MC3T3-E1 cells produce cystatin C. These data show that cystatin C is synthesized by bone cells and that recombinant human cystatin C inhibits bone resorption in vitro without affecting bone cell proliferation, bone matrix formation or osteoclast generation. The mechanism seems to be due primarily to inhibition of the activity of osteoclastic proteolytic enzymes released into the resorption lacunae.
Acetylglucosaminidase↗
Differential effects of glucocorticoids on bone resorption in neonatal mouse calvariae stimulated by peptide and steroid-like hormones.
Differential effects on in vitro bone resorption were observed when the glucocorticoids, hydrocortisone and dexamethasone, were added to neonatal mouse calvariae treated with either parathyroid hormone (PTH), 1,25(OH)2-vitamin D3, all trans-retinoic acid (t-RA), or prostaglandin E2 (PGE2). Bone resorption was assessed by analyzing either the release of 45Ca from [45Ca]CaCl2 prelabeled calvarial bones or the release of 3H from [3H]proline prelabeled calvariae. At PGE2 concentrations of 3 x 10(-8) and 3 x 10(-7) mol/l, co-treatment with either 10(-6) mol/l dexamethasone or 10(-6) mol/l hydrocortisone caused additive 45Ca release from neonatal mouse calvariae. In contrast, synergistic release from mouse calvarial bones of both 45Ca and 3H was found after either 10(-6) mol/l hydrocortisone or 10(-6) mol/l dexamethasone was combined with 3 x 10(-11) mol/l PTH treatment for 120 h. Dose-response studies indicated that the synergistic stimulation of 45Ca release from neonatal mouse calvariae by glucocorticoids and PTH could be elicited at glucocorticoid concentrations of 10(-8) to 10(-6) mol/l and at PTH concentrations of 10(-11) to 10(-9) mol/l. Progesterone and RU 38486 (a derivative of 19-nortestosterone with antiglucocorticoid activity) blocked the synergism noted with glucocorticoid and PTH co-treatment, suggesting that interaction between the steroids and PTH was dependent on glucocorticoid receptor interaction. Addition of either 10(-6) mol/l hydrocortisone or 10(-6) mol/l dexamethasone to neonatal mouse calvariae treated with 1,25(OH)2-vitamin D3 (10(-11) and 10(-10) mol/l) also resulted in synergistic stimulation of 45Ca release. In contrast to these observations, the stimulatory effect of t-RA (10(-8) mol/l) on 45Ca release from calvarial bones was abolished in the presence of 10(-6) mol/l dexamethasone. These results suggest that an important role of glucocorticoids may be to synergistically potentiate bone resorption stimulated by PTH and 1,25(OH)2-vitamin D3, but indicate an opposing interaction between the glucocorticoids and bone resorptive retinoids.
Animals↗
In situ microdialysis in bone tissue. Stimulation of prostaglandin E2 release by weight-bearing mechanical loading.
In this study we describe, to our knowledge, the first experiment using the microdialysis technique for studying the release of prostaglandin E2 (PGE2) in the proximal tibia metaphysis secondary to mechanical loading. Nine healthy females, six in the loading group and three controls, mean age 34+/-2 (years+/-SEM), participated. A standard microdialysis catheter was inserted into the tibia metaphyseal bone under local anesthesia. Samplings were done every 15 min under a 2-h equilibration period. Thereafter, heel-drops (one impact per second) with as hard impact of the heels into the floor as possible, were done for 5 min by the subjects in the loading group. The control group performed no exercise. Sampling continued after this for another 2-h period. Basal levels of PGE2 in the proximal tibial metaphysis were determined to a mean of 45+/-10 pg/ml (mean+/-SEM, n = 6). The major finding was a 2.5-3.5-fold increase in the release of PGE2 after mechanical loading. The increase was statistically significant (P < 0.05 compared with basal levels) 1 h after the mechanical loading and persisted for the rest of the experimental period. No major alterations were observed in the control group. In conclusion, our data demonstrate that in situ microdialysis is a useful method for studying the PGE2 production in human bone. Furthermore, a rapid and significant increase in PGE2 levels was noticed in response to dynamic mechanical loading.
Adult↗
Stimulation of neonatal mouse calvarial bone resorption by the glucocorticoids hydrocortisone and dexamethasone.
In vitro stimulation of bone resorption was observed with the glucocorticoids hydrocortisone and dexamethasone. Dosage-dependent release of 45Ca from neonatal mouse calvarial bones was found for both steroids, with half-maximal responses for hydrocortisone and dexamethasone of 0.3 and 0.08 microM, respectively. Significant release of stable calcium (Ca2+), inorganic phosphate (Pi), and the lysosomal enzyme beta-N-acetylglucosaminidase was noted following treatment of mouse calvariae with either 1 microM hydrocortisone or 1 microM dexamethasone. Additionally, both 1 microM hydrocortisone and 1 microM dexamethasone elicited release of 3H from calvarial bones prelabeled with [3H]proline. The stimulation of bone resorption by the glucocorticoids, as assessed by 45Ca release, was sustained over 120 h of culture. Inhibition of 45Ca release from calvariae treated with either 1 microM hydrocortisone or 0.1 microM dexamethasone was observed with 0.01-30 nM salmon calcitonin (sCT), 0.1 mM acetazolamide, and 0.1 mM of the bisphosphonate AHPrBP. Inhibition of glucocorticoid-induced bone resorption by sCT occurred without "escape from calcitonin-induced inhibition." The 45Ca release stimulated by 1 microM hydrocortisone and 0.1 microM dexamethasone was also inhibited by 10 microM progesterone in a competitive manner and by 1 microM of the antiglucocorticoid RU38486, both of which are modulators of glucocorticoid binding. Prostaglandin E2 (PGE2) formation by 10 nM parathyroid hormone (PTH) in neonatal mouse calvarial bones was inhibited by both 1 microM hydrocortisone and 1 microM dexamethasone, but neither compound altered basal PGE2 formation. Exposure of calvarial bones to the mitotic inhibitors hydroxyurea and mitomycin C inhibited 45Ca release stimulated by 1 microM hydrocortisone and 1 microM dexamethasone. In contrast, addition of 1 ng/ml of recombinant murine granulocyte macrophage colony stimulating factor (rmGM-CSF) had no effect on 45Ca release elicited by the glucocorticoids. These results suggest that hydrocortisone and dexamethasone stimulate osteoclastic resorption in neonatal mouse calvariae by a receptor-mediated mechanism that is dependent on cellular replication.
Acetazolamide↗
Transforming growth factor-beta stimulates bone resorption in neonatal mouse calvariae by a prostaglandin-unrelated but cell proliferation-dependent pathway.
The relationships between bone resorption, prostanoid formation, and cell proliferation in cultured neonatal mouse calvariae stimulated with transforming growth factor-beta (TGF-beta) have been examined. Bone resorption was assessed by analyzing the mobilization of minerals (45Ca, Ca2+., Pi) and the release of 3H from bones prelabeled with [3H]proline. Prostanoid formation was determined by analyzing the amounts of prostaglandin E2 (PGE2) and 6-keto-prostaglandin F1 alpha (the stable breakdown product of PGI2) in culture media. Purified porcine TGF-beta 1 and recombinant human TGF-beta 2 stimulated the release of 45Ca and the formation of prostanoids. The effects were time and dose dependent. The concentrations of TGF-beta 1 and TGF-beta 2 causing half maximal stimulation of 45Ca release were 1 and 0.1 ng/ml, respectively. TGF-beta 1 also enhanced the release of 3H from [3H]proline labeled bones and the mobilization of Ca2+ and Pi from unlabeled bones, as well as the release of lysosomal enzymes (beta-N-acetylglucosaminidase). The degree of stimulation of mineral mobilization and matrix degradation was less than that obtained in bones stimulated with parathyroid hormone or 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). TGF-beta 1-induced stimulation of 45Ca release was inhibited by calcitonin, acetazolamide, and the biphosphonate AHPrBP, three different osteoclast inhibitors. In contrast to the escape from calcitonin-induced inhibition seen in parathyroid hormone (PTH)-stimulated bones, the inhibitory effect of calcitonin in TGF-beta 1-stimulated bones persisted in long-term cultures (144 h). The stimulatory effect of TGF-beta 1 was inhibited by anti-TGF-beta 1 and by gamma-interferon (1000 U/ml). Indomethacin (1 microM), flurbiprofen (1 microM), and meclofenamic acid (1 microM) completely abolished the stimulatory effect of TGF-beta 1 on PGE2 and 6-keto-PGF 1 alpha formation without affecting TGF-beta 1-induced stimulation of 45Ca release. Similarly, the stimulatory effect of TGF-beta 2 on 45Ca release was unaffected by indomethacin. In bones in which prostaglandin formation was abolished by indomethacin, a 45Ca release response to TGF-beta 1 was obtained at 12 h. The mitotic inhibitor hydroxyurea inhibited TGF-beta 1 but not PTH-induced 45Ca release. These data demonstrate that TGF-beta 1 and TGF-beta 2 have the capacity to stimulate bone resorption and prostanoid formation in neonatal mouse calvariae, but that the effect of TGF-beta on bone resorption is unrelated to prostanoid formation. In addition, it is shown that bone resorption stimulated by TGF-beta is dependent on cell replication.
Acetylglucosaminidase↗