Tag Archives: in vitro

Minocycline Inhhibits Osteoclastogenesis in Mouse Cells

Abstract

Inhibitory effect of minocycline on osteoclastogenesis in mouse bone marrow cells.

To study the effects of minocycline hydrochloride (MINO) on the formation of tartrate-resistant acid phosphatase (TRAP) staining-positive multinucleated osteoclast-like cells in mouse bone marrow cells (BMCs) treated with 1α,25(OH)(2)D(3) or soluble receptor activator of nuclear factor-κB ligand (s-RANKL).
Mouse BMCs were cultured in alpha-modified minimum essential medium containing foetal calf serum (10%) and tetracyclines (2.5, 5 and 10μM), such as MINO, tetracycline hydrochloride (TC), oxytetracycline hydrochloride (OXT) or doxycycline (DOXY) in the presence of 1α,25(OH)(2)D(3) (10nM) or s-RANKL (20ng/ml) for 7 days, and the number of TRAP staining-positive osteoclast-like cells was counted. In RNA isolated from BMCs treated with 1α,25(OH)(2)D(3) or s-RANKL in the presence or absence of MINO, the expressions of osteoclast differentiation relating to mRNA were analysed by reverse transcription-polymerase chain reaction. Cell viability was examined in mouse BMCs and rabbit osteoclasts treated with MINO (0.25-20μM and 2-50μM, respectively) for 24h.
MINO, TC, OXT or DOXY inhibited 1α,25(OH)(2)D(3)-induced osteoclast-like cell formation in mouse BMCs dose dependently. MINO suppressed 1α,25(OH)(2)D(3)-induced up-regulation of mRNA expressions of TRAP, cathepsin K, carbonic anhydrase II, and calcitonin receptor, but not RANKL. MINO inhibited s-RANKL-induced osteoclast-like cell formation and up-regulation of mRNA expressions for nuclear factor of activated T-cells c1 (NFATc1), a key regulator of osteoclast differentiation; however, MINO had no effects on the viability of mouse BMCs and rabbit osteoclasts.
MINO inhibits RANKL-induced osteoclastogenesis via down-regulation of NFATc1 mRNA expression in osteoclast precursor cells.

Nagasawa T, Arai M, Togari A
Arch. Oral Biol. Sep 2011
PMID: 21377143

Minocycline Increases Bone Density in Ovariectomized Rats

Abstract

Treatment of osteoporosis with MMP inhibitors.

In the current study, we examined the effects of minocycline on the osteopenia of ovariectomized (OVX) aged rats using the marrow ablation model. This injury induces rapid bone formation followed by bone resorption in the marrow cavity. Old female rats were randomly divided into five groups: sham, OVX, OVX + minocycline (5-15 mg/day, orally), OVX + 17 beta-estradiol (25 micrograms/day, subcutaneously), and OVX + both agents. Rats were OVX, treated with minocycline and/or estrogen, followed by marrow ablation. Bone samples were collected 16 days post-marrow ablation. X-ray radiography of bones operated on showed that treatment of OVX old rats with minocycline increased bone mass in diaphyseal region. Diaphyseal bone mineral density (BMD) was measured by DEXA scan. Diaphyseal BMD of OVX rats was increased 17-25% by treatment with 5-15 mg of minocycline or 17 beta-estradiol. The effects of minocycline and estrogen treatments on the expression of osteoblast and osteoclast markers were also examined. Northern and dot blot analysis of RNA samples showed that treatment of OVX aged rats with minocycline increased the expression of type I collagen (COL I) (49%) and decreased that of interleukin-6 (IL-6) (31%). In contrast, estrogen treatment decreased the expression of interleukin-6 (IL-6) (39%), carbonic anhydrase II (CA II) (36%), and osteopontin (OP) (37%). Neither minocycline nor 17 beta-estradiol had an effect on the expression of osteocalcin (OC) and alkaline phosphatase (AP). To elucidate the mechanism by which minocycline prevented the loss of bone in OVX aged rats, we examined the colony-formation potential of bone marrow stromal cells in ex vivo cultures. Minocycline stimulated the colony-forming efficiency of marrow stromal cells derived from old animals. We have therefore concluded that the modest increase in BMD noted in OVX aged rats, in response to minocycline treatment, may be due to a change in bone remodeling that favors bone formation; and the anabolic effect of minocycline is likely due to its effect on the expression of COL I and/or the metabolism of osteoprogenitor cells.

Williams S, Barnes J, Wakisaka A, Ogasa H…
Ann. N. Y. Acad. Sci. Jun 1999
PMID: 10415730

Cranberry Inhibits Osteoclasts In Vitro

Abstract

A-type cranberry proanthocyanidins inhibit the RANKL-dependent differentiation and function of human osteoclasts.

This study investigated the effect of A-type cranberry proanthocyanidins (AC-PACs) on osteoclast formation and bone resorption activity. The differentiation of human pre-osteoclastic cells was assessed by tartrate-resistant acid phosphatase (TRAP) staining, while the secretion of interleukin-8 (IL-8) and matrix metalloproteinases (MMPs) was measured by ELISA. Bone resorption activity was investigated by using a human bone plate coupled with an immunoassay that detected the release of collagen helical peptides. AC-PACs up to 100 µg/mL were atoxic for osteoclastic cells. TRAP staining evidenced a dose-dependent inhibition of osteoclastogenesis. More specifically, AC-PACs at 50 µg/mL caused a 95% inhibition of RANKL-dependent osteoclast differentiation. This concentration of AC-PACs also significantly increased the secretion of IL-8 (6-fold) and inhibited the secretion of both MMP-2 and MMP-9. Lastly, AC-PACs (10, 25, 50 and 100 µg/ml) affected bone degradation mediated by mature osteoclasts by significantly decreasing the release of collagen helical peptides. This study suggests that AC-PACs can interfere with osteoclastic cell maturation and physiology as well as prevent bone resorption. These compounds may be considered as therapeutic agents for the prevention and treatment of periodontitis.

Tanabe S, Santos J, La VD, Howell AB…
Molecules 2011
PMID: 21399573 | Free Full Text

Propolis Inhibits IL-17

Abstract

Suppression of interleukin 17 production by Brazilian propolis in mice with collagen-induced arthritis.

Propolis is a resinous substance collected by honeybees from leaf buds and cracks in the bark of various plants. Propolis has been reported to have immunomodulatory activity. We hypothesized that propolis would be able to reduce the disease severity of rheumatoid arthritis. We evaluated the effect of Brazilian propolis ethanolic extract on the pathogenesis of collagen-induced arthritis (CIA) in mice. Mice fed propolis exhibited significant lower clinical arthritis scores than those fed the control diet. To investigate the mechanism of the effect of propolis on CIA mice, we examined interleukin-17 (IL-17) production in CIA mice fed propolis using an enzyme-linked immunospot assay and flow cytometric analysis. The numbers of IL-17-producing cells in the CIA mice fed propolis were significantly decreased. To determine direct influence of propolis on cytokine production, splenocytes were stimulated with phorbol myristate acetate in the presence of propolis extract in vitro. Concentration-dependent declines in IL-17 expression were observed by ELISA and real-time PCR methods. We further found that propolis significantly inhibited the differentiation of Th17 cells from murine splenocytes in a concentration-dependent manner. Taken together, our results may provide a new light on the potential mechanism of the immunosuppressive and anti-inflammatory effects of propolis.

Tanaka M, Okamoto Y, Fukui T, Masuzawa T
Inflammopharmacology Feb 2012
PMID: 21861090

IL-17 is implicated in osteoporosis.

EPA and DHA may Decrease, but GLA may Increase, Osteoclasts in Mouse Cells

Abstract

Long chain polyunsaturated fatty acids alter membrane-bound RANK-L expression and osteoprotegerin secretion by MC3T3-E1 osteoblast-like cells.

Inflammation triggers an increase in osteoclast (bone resorbing cell) number and activity. Osteoclastogenesis is largely controlled by a triad of proteins consisting of a receptor (RANK), a ligand (RANK-L) and a decoy receptor (osteoprotegerin, OPG). Whilst RANK is expressed by osteoclasts, RANK-L and OPG are expressed by osteoblasts. The long chain polyunsaturated fatty acid (LCPUFA) arachidonic acid (AA, 20:4n-6) and its metabolite prostaglandin E2 (PGE2), are pro-inflammatory and PGE2 is a potent stimulator of RANKL expression. Various LCPUFAs such as eicosapentaenoic acid (EPA, 20:5n-3), docosahexaenoic acid (DHA, 22:6n-3) and gamma-linolenic acid (GLA, 18:3n-6) have anti-inflammatory activity. We aimed to determine if AA itself can stimulate RANKL expression and whether EPA, DHA and GLA inhibit RANKL expression in osteoblasts. MC3T3-E1/4 osteoblast-like cells were cultured under standard conditions with each of the LCPUFAs (5microg/ml) for 48h. Membrane-bound RANKL expression was measured by flow cytometry and OPG secretion measured by ELISA. In a second experiment, RANKL expression in MC3T3-E1/4 cells was stimulated by PGE2 treatment and the effect of EPA, DHA and GLA on membrane-bound RANKL expression and OPG secretion determined. The percentage of RANKL-positive cells was higher (p<0.05) than controls following treatment with AA or GLA but not after co-treatment with the cyclooxygenase inhibitor, indomethacin. DHA and EPA had no effect on membrane-bound RANKL expression under standard cell culture conditions. Secretion of OPG was lower (p<0.05) in AA-treated cells but not significantly different from controls in GLA, EPA or DHA treated cells. Treatment with prostaglandin E2 (PGE2) resulted in an increase (p<0.05) in the percentage of RANK-L positive cells and a decrease (p<0.05) in mean OPG secretion. The percentage of RANKL positive cells was significantly lower following co-treatment with PGE2 and either DHA or EPA compared to treatment with PGE2 alone. Mean OPG secretion remained lower than controls in cells treated with PGE2 regardless of co-treatment with EPA or DHA. Results from this study suggest COX products of GLA and AA induce membrane-bound RANKL expression in MC3T3-E1/4 cells. EPA and DHA have no effect on membrane-bound RANKL expression in cells cultured under standard conditions however both EPA and DHA inhibit the PGE2-induced increase in RANKL expression in MC3T3-E1/4 cells.

Poulsen RC, Wolber FM, Moughan PJ, Kruger MC
Prostaglandins Other Lipid Mediat. Feb 2008
PMID: 18077200

Silicon as Orthosilicic Acid Decreases Osteoblast Survivability In Vitro

Abstract

Divergent effects of orthosilicic acid and dimethylsilanediol on cell survival and adhesion in human osteoblast-like cells.

Although dietary silicon (Si) is recognized to be an important factor for the growth and development of bone and connective tissue, its biochemical role has yet to be identified. The predominant Si-containing species in blood and other biofluids is orthosilicic acid, Si(OH)(4). Dimethylsilanediol, (CH(3))(2)Si(OH)(2), is an environmental contaminant that results from decomposition of silicone compounds used in personal hygiene, health care and industrial products. We examined the in vitro effects of both Si species on the survival (colony forming efficiency), proliferation (DNA content), differentiation (alkaline phosphatase activity) and adhesion (relative protein content) of the human osteoblast-like cell lines Saos-2 and hFOB 1.19. Orthosilicic acid yielded a small, dose-dependent decrease in Saos-2 cell survivability up to its 1,700 micromol/L solubility limit, by which point survival was 20% less than that of untreated cells. This negative association, although small, correlated with a reduction in the proliferation and adhesion of Saos-2 cells as well as of hFOB 1.19 and osteoclast-like GCT cells. By contrast, dimethylsilanediol treatment had no discernable influence on Saos-2 survivability at concentrations up to 50 micromol/L, and yet significantly enhanced cell survival at higher doses. Moreover, dimethylsilanediol did not affect proliferation or adhesion of any cell line. The findings show that orthosilicic acid and dimethylsilanediol affect osteoblast-like cells very differently, providing insight into the mechanism by which silicon influences bone health, although the specific site of Si activity remains unknown. There was no evidence to suggest that dimethylsilanediol is cytotoxic at environmental/physiological concentrations.

Duivenvoorden WC, Middleton A, Kinrade SD
J Trace Elem Med Biol 2008
PMID: 18755397

Taurine Leads to Bone Anabolic Action in Mouse Cells

Abstract

[Anti-osteopenic effect of taurine: possible involvement of activated MEK-ERK-Cbfa1 signaling].

Previously we first noted that taurine (TR) has anti-osteopenic effect on low Ca diet-induced osteopenia in rats (1). Employing osteoblastic MC3T3-E1 cells, the mechanism of the anti-osteopenic effect was explored in vitro. TR (1 mM) was found to promote mineralization of extracellular matrices, without affecting alkaline phosphataase activity. Gel shift assay using 32P-labeled OSE2 (osteoblast-specific cis-element 2: the consensus sequence for Cbfa1, refer to 2) indicated that TR (1 mM) increased the nuclear localization of Cbfa1, just as TPH (1-34) (3,4) and bisphosphonates did (5). In addition, TR was found to stimulate ERK phosphorylation. PD98059, a MEK inhibitor, suppressed effects of TR on both Cbfa1 transactivation and ERK activation. The results strongly suggest that TR first activates intracellular MEK-ERK-Cbfa1 signaling system thereby promoting mineralization and finally leading to its bone anabolic action.

Yasutomi C, Nakamuta H, Fujita T, Takenaga T…
Nippon Yakurigaku Zasshi Nov 2002
PMID: 12491800

Taurine Increases Markers of Bone Growth in Human and Mouse Osteoblasts

Abstract

Taurine transporter is expressed in osteoblasts.

Taurine influences bone metabolism and is taken up by cells via a specific transport system, the taurine transporter (TAUT). We report a link between taurine and bone homeostasis by demonstrating transcription and translation of TAUT in bone-forming cells. TAUT was expressed in human primary osteoblasts, the human osteosarcoma osteoblast-like cell line MG63, and the mouse osteoblastic cell line MC3T3-E1. Immunostaining with polyclonal antibodies also demonstrated the presence of TAUT in both human and murine osteoblasts. TAUT mRNA expression and [(3)H]taurine uptake increased during differentiation of MG63 cells in culture. Supplementation of culture medium with taurine enhanced alkaline phosphatase activity and osteocalcin secretion. The regulation and detailed function of taurine and TAUT in bone remain unclear, but our findings suggest a functional role for them in bone homeostasis.

Yuan LQ, Xie H, Luo XH, Wu XP…
Amino Acids Sep 2006
PMID: 16729199

Taurine Increases Osteoblasts In Vitro

Abstract

Taurine increases cell proliferation and generates an increase in [Mg2+]i accompanied by ERK 1/2 activation in human osteoblast cells.

Taurine has been reported to influence bone metabolism, and its specific transport system, the taurine transporter, is expressed in osteoblasts. The mean [Mg2+]i was 0.51+/-0.01 mM in normal culture media. Taurine caused an increase in [Mg(2+)]i by 0.72+/-0.04 mM in human osteoblast (HOB) cells. This increment in [Mg2+]i was inhibited significantly by PD98059, nifedipine, lidocaine, and imipramine. Taurine was also shown to stimulate the activation of ERK 1/2. This taurine-stimulated ERK 1/2 activation was inhibited by PD98059. In the present study, taurine was shown to increase cell proliferation and generate an increase in [Mg2+]i accompanied by ERK 1/2 activation in HOB cells.

Jeon SH, Lee MY, Kim SJ, Joe SG…
FEBS Lett. Dec 2007
PMID: 18036343

Taurine Induces Connective Tissue Growth Factor in Mouse Osteoblasts

Abstract

Taurine promotes connective tissue growth factor (CTGF) expression in osteoblasts through the ERK signal pathway.

Taurine is found in bone tissue, but its function in skeletal tissue is not fully understood. The present study was undertaken to investigate regulation of gene expression of connective tissue growth factor (CTGF), and the roles of mitogen-activated protein kinases (MAPKs) in murine osteoblast MC3T3-E1 cells treated with taurine. Western blot analysis showed taurine stimulated CTGF protein secretion in a dose- and time-dependent manner. Taurine induced activation of extracellular signal-regulated kinase (ERK), but not p38 and c-jun N-terminal Kinase (JNK), in osteoblasts. Furthermore, pretreatment of osteoblasts with the ERK inhibitor PD98059 abolished the taurine-induced CTGF production. These data indicate that taurine induces CTGF secretion in MC3T3-E1 cells mediated by the ERK pathway, and suggest that osteoblasts are direct targets of taurine.

Yuan LQ, Lu Y, Luo XH, Xie H…
Amino Acids 2007
PMID: 16937320