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.     

Leptin effect on RANKL and OPG expression in MC3T3-E1 osteoblasts

Recent studies have suggested that leptin hormone may play a pivotal role on bone remodeling through a direct effect by modulating positively the OPG/RANKL balance. Here, we investigate the effect of leptin hormone on RANKL and OPG expression in MC3T3-E1 osteoblasts using RT-PCR and ELISA measurements. We have at first identified the expression of Ob-Rb and Ob-Ra leptin receptor isoforms in MC3T3-E1 and observed that these cells respond to mrleptin treatments. We then investigated the effect of mrleptin on RANKL and OPG expression. We show that mrleptin dose-dependently regulated the expression of RANKL mRNA with complete inhibition observed at concentrations higher than 12 ng/ml. This effect was confirmed with sRANKL protein measurements. However, the exposure of MC3T3-E1 to mrleptin had no effect on OPG mRNA. Taken together, these results suggest that leptin modulates positively OPG/RANKL balance by inhibiting the expression of RANKL gene.     

Effects of arachidonic acid, docosahexaenoic acid, prostaglandin E(2) and parathyroid hormone on osteoprotegerin and RANKL secretion by MC3T3-E1 osteoblast-like cells

Bone is continuously remodeled through resorption by osteoclasts and the subsequent synthesis of the bone matrix by osteoblasts. Cell-to-cell contact between osteoblasts and osteoclast precursors is required for osteoclast formation. RANKL (receptor activator of nuclear factor-kappaB ligand) expressed on osteoblastic cell membranes stimulates osteoclastogenesis, while osteoprotegerin (OPG) secreted by osteoblasts inhibits osteoclastogenesis. Although polyunsaturated fatty acids (PUFAs) have been implicated in bone homeostasis, the effects thereof on OPG and RANKL secretion have not been investigated. MC3T3-E1 osteoblasts were exposed to the n-6 PUFA arachidonic acid (AA) and the n-3 PUFA docosahexaenoic acid (DHA); furthermore, the bone-active hormone parathyroid hormone (PTH) and the effects thereof were tested on OPG and RANKL secretion. Prostaglandin E(2) (PGE(2)), a product of AA metabolism that was previously implicated in bone homeostasis, was included in the study. AA (5.0-20 microg/ml) inhibited OPG secretion by 25-30%, which was attenuated by pretreatment with the cyclooxygenase blocker indomethacin, suggesting that the inhibitory effect of AA on OPG could possibly be PGE(2)-mediated. MC3T3-E1 cells secreted very low basal levels of RANKL, but AA stimulated RANKL secretion, thereby decreasing the OPG/RANKL ratio. DHA suppressed OPG secretion to a smaller extent than AA. This could, however, be due to endogenous PGE(2) production. No RANKL could be detected after exposing the MC3T3-E1 cells to DHA. PTH did not affect OPG secretion, but stimulated RANKL secretion. This study demonstrates that AA and PTH reduce the OPG/RANKL ratio and may increase osteoclastogenesis. DHA, however, had no significant effect on OPG or RANKL in this model.     

Testosterone increases osteoprotegerin mRNA expression in mouse osteoblast cells.

The role that androgens play in the regulation of bone metabolism has been substantiated in animals and humans. We previously demonstrated that testosterone inhibits osteoclast differentiation stimulated by parathyroid hormone through the androgen receptor in mouse bone-cell cultures. However, the details of this mechanism are still unknown. The present study was aimed at examining whether testosterone would affect the mRNA levels of osteoprotegerin (OPG) and receptor activator of Nf kappa B ligand (RANKL) in mouse bone-cell cultures as well as mouse osteoblastic cell-line, MC3T3-E1 cells by employing semi-quantitative RT-PCR. Testosterone increased OPG mRNA expression in both mouse bone-cell cultures and MC3T3-E1 cells. 10-8 M PTH-(1-34) as well as 10-8M 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] inhibited OPG mRNA expression in mouse bone cells. 10-8 M testosterone antagonized OPG mRNA expression inhibited by 10-8 M PTH-(1-34), but failed to affect OPG mRNA expression inhibited by 10-8 M 1,25(OH)2D3. 10-8 M alpha-dehydrotestosterone, a non-aromatizable androgen, increased OPG mRNA expression. On the other hand, testosterone did not affect RANKL mRNA expression in MC3T3-E1 or mouse bone cells. In conclusion, the present study demonstrated that testosterone increased OPG mRNA expression in mouse bone-cell cultures and the osteoblastic cell line. These effects are likely to take place through the androgen receptor.     

Impact of the microgravity environment in a 3-dimensional clinostat on osteoblast- and osteoclast-like cells

Mechanical unloading conditions result in decreases in bone mineral density and quantity, which may be partly attributed to an imbalance in bone formation and resorption. To investigate the effect of mechanical unloading on osteoblast and osteoclast differentiation, and the expression of RANKL and OPG genes in osteoblasts, we used a three-dimensional (3D) clinostat system simulating microgravity to culture MC3T3-E1 and RAW264.7 cells. Long-term exposure (7 days) of MC3T3-E1 cells to microgravity in the 3D clinostat inhibited the expression of Runx2, Osterix, type I collagen alphaI chain, RANKL and OPG genes. Similarly, 3D clinostat exposure inhibited the enhancement of beta3-integrin gene expression, which normally induced by sRANKL stimulation in RAW264.7 cells. These results, taken together, demonstrate that long-term 3D clinostat exposure inhibits the differentiation of MC3T3-E1 cells together with suppression of RANKL and OPG gene expression, as well as the RANKL-dependent cellular fusion of RAW264.7 cells, suggesting that long-term mechanical unloading suppresses bone formation and resorption.     

TWEAK/Fn14 interaction regulates RANTES production, BMP-2-induced differentiation, and RANKL expression in mouse osteoblastic MC3T3-E1 cells

Tumour necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK), a member of the TNF family, is a multifunctional cytokine that regulates cell growth, migration, and survival principally through a TWEAK receptor, fibroblast growth factor-inducible 14 (Fn14). However, its physiological roles in bone are largely unknown. We herein report various effects of TWEAK on mouse osteoblastic MC3T3-E1 cells. MC3T3-E1 cells expressed Fn14 and produced RANTES (regulated upon activation, healthy T cell expressed and secreted) upon TWEAK stimulation through PI3K-Akt, but not nuclear factor-kappaB (NF-kappaB), pathway. In addition, TWEAK inhibited bone morphogenetic protein (BMP)-2-induced expression of osteoblast differentiation markers such as alkaline phosphatase through mitogen-activated protein kinase (MAPK) Erk pathway. Furthermore, TWEAK upregulated RANKL (receptor activation of NF-kappaB ligand) expression through MAPK Erk pathway in MC3T3-E1 cells. All these effects of TWEAK on MC3T3-E1 cells were abolished by mouse Fn14-Fc chimera. We also found significant TWEAK mRNA or protein expression in osteoblast- and osteoclast-lineage cell lines or the mouse bone tissue, respectively. Finally, we showed that human osteoblasts expressed Fn14 and induced RANTES and RANKL upon TWEAK stimulation. Collectively, TWEAK/Fn14 interaction regulates RANTES production, BMP-2-induced differentiation, and RANKL expression in MC3T3-E1 cells. TWEAK may thus be a novel cytokine that regulates several aspects of osteoblast function.     

Interleukin 6 and/or Interleukin 17A Modulate the OPG/RANKL System of MC3T3-E1 Murine Osteoblast Cell Line

Cytokines such as interleukin-6 (IL-6) and IL-17 which act as key regulators of the immune response have been identified to have a potential role in the bone remodeling mechanism. Receptor activator of NF-κB ligand (RANKL) has been shown to regulate osteoclast differentiation and function while the osteoprotegerin (OPG) blocks the binding of RANKL and inhibits the differentiation of osteoclasts, thus favoring osteogenesis. Alkaline phosphatase (ALP) on the other hand works as early mineralization indicator in bone regulation. The current study aims to determine the potential role of IL-6 and IL-17A in regulating the OPG/RANKL system of the murine osteoblast cell line (MC3T3-E1). Gene expression analysis showed significant up-regulation of OPG and ALP by all the treated groups (rIL-6, rIL-17A and rIL-6 + rIL-17A). In contrast, treatment of cells with rIL-6 and/or rIL-17A showed down-regulation of RANKL expression. Interestingly, the osteoblast cells treated with combinations of rIL-6 + rIL17A showed marked increased in OPG/RANKL ratio. Similar pattern of protein expression was observed in the osteoblasts treated with rIL-6 and/or rIL-17A as detected by western blotting and ELISA. These findings suggest a new mechanism of regulation by these cytokines on the expression of OPG and RANKL, which could promote osteogenesis and diminish osteoclastogenesis.     

HMGB1 expression and release by bone cells

Immune and bone cells are functionally coupled by pro-inflammatory cytokine intercellular signaling networks common to both tissues and their crosstalk may contribute to the etiologies of some immune-associated bone pathologies. For example, the receptor activator of NF-kappaB ligand (RANKL)/osteoprotegerin (OPG)/receptor activator of NF-kappaB (RANK) signaling axis plays a critical role in dendritic cell (DC) function as well as bone remodeling. The expression of RANKL by immune cells may contribute to bone loss in periodontitis, arthritis, and multiple myeloma. A recent discovery reveals that DCs release the chromatin protein high mobility group box 1 (HMGB1) as a potent immunomodulatory cytokine mediating the interaction between DCs and T-cells, via HMGB1 binding to the membrane receptor for advanced glycation end products (RAGE). To determine whether osteoblasts or osteoclasts express and/or release HMGB1 into the bone microenvironment, we analyzed tissue, cells, and culture media for the presence of this molecule. Our immunohistochemical and immunocytochemical analyses demonstrate HMGB1 expression in primary osteoblasts and osteoclasts and that both cells express RAGE. HMGB1 is recoverable in the media of primary osteoblast cultures and cultures of isolated osteoclast precursors and osteoclasts. Parathyroid hormone (PTH), a regulator of bone remodeling, attenuates HMGB1 release in cultures of primary osteoblasts and MC3T3-E1 osteoblast-like cells but augments this release in the rat osteosarcoma cell line UMR 106-01, both responses primarily via activation of adenylyl cyclase. PTH-induced HMGB1 discharge by UMR cells exhibits similar release kinetics as reported for activated macrophages. These data confirm the presence of the HMGB1/RAGE signaling axis in bone.     

Effects of cell surface α2-3 sialic acid on osteogenesis

A cell culture model of osteoblast differentiation was applied in our study of the effect of sialic acid on the osteogenesis by using the pre-osteoblast of MC3T3-E1 subclone 14 cells. Following the treatment of different concentrations of α2,3-neuraminidase, which specifically removed the α2-3 sialic acid from cell surface, a significant decrease of α2-3 sialic acid was detected with fluorescein isothiocyanate (FITC)-labeled Maackia amurensis lectin (MAL-II) by flow cytometry analysis. von Kossa staining showed that the bone mineralization decreased in MC3T3-E1 subclone 14 cells after the treatment of α2,3-neuraminidase for 2 weeks. However α2,3-neuraminidase did not affect the formation of osteoblasts in MC3T3-E1 subclone 14 cells, which was demonstrated by positive alkaline phosphatase (ALP)-staining. Characteristic biological markers and osteoblast-like cell-related factors of osteoblastic cells were also examined. Both RT-PCR and Western blot analysis demonstrated that the expression of bone sialoprotein (BSP), osteoprotegerin (OPG), and vitamin D receptor (VDR) were significantly decreased when α2-3 sialic acid expression decreased on the cell surface, while the expression of osteocalcin (OC) and osteopontin (OPN) remained unchanged. We propose a hypothesis that α2-3 sialic acid affects bone mineralization but not osteogenic differentiation.     

Activation of PKA and phosphorylation of sodium-dependent vitamin C transporter 2 by prostaglandin E2 promote osteoblast-like differentiation in MC3T3-E1 cells

Sodium-dependent vitamin C transporter (SVCT) 2-mediated L-ascorbic acid (AA) uptake is required in osteoblast-like differentiation of MC3T3-E1 cells, and prostaglandin E2 (PGE2) is among the most important local factors in bone formation, but the detailed mechanism by which PGE2 induces osteoblast differentiation remains obscure. We revealed that PGE2 induced AA uptake and osteoblast-like differential markers including alkaline phosphatase, collagen, osteocalcin expression, and mineralization in MC3T3-E1 cells. Inhibition of AA uptake by SVCT2 short isoform functioning as a dominant-negative mutant not only robustly attenuated PGE2-induced markers expression and mineralization, but also decreased their basal levels. However, upregulation of AA uptake resulted from PGE2-induced plasma membrane translocation of cytoplasm SVCT2, and this effect was abolished by pretreatment with EP4 receptor antagonist, AH-23848B or cAMP-dependent protein kinase A (PKA) inhibitor, H-89. Moreover, we showed SVCT2 physically interacted with PKA in immunoprecipitates, and PKA phosphorylated SVCT2 in vitro and in intact cells at Ser402 and Ser639 sites; however, mutation of Ser402 or/and Ser639 in SVCT2 severely diminished SVCT2 translocation in response to PGE2. Together, these results suggest that PGE2-induced SVCT2 plasma membrane translocation through EP4 receptor and subsequent phosphorylation of SVCT2 at Ser402 and Ser639 sites by PKA results in an increase of AA uptake and consequent promotion of osteoblast-like differentiation in MC3T3-E1 cells.     

Metformin stimulates osteoprotegerin and reduces RANKL expression in osteoblasts and ovariectomized rats

Anti-diabetic drug metformin has been shown to enhance osteoblasts differentiation and inhibit osteoclast differentiation in vitro and prevent bone loss in ovariectomized (OVX) rats. But the mechanisms through which metformin regulates osteoclastogensis are not known. Osteoprotegerin (OPG) and receptor activator of nuclear factor κB ligand (RANKL) are cytokines predominantly secreted by osteoblasts and play critical roles in the differentiation and function of osteoclasts. In this study, we demonstrated that metformin dose-dependently stimulated OPG and reduced RANKL mRNA and protein expression in mouse calvarial osteoblasts and osteoblastic cell line MC3T3-E1. Inhibition of AMP-activated protein kinase (AMPK) and CaM kinase kinase (CaMKK), two targets of metformin, suppressed endogenous and metformin-induced OPG secretion in osteoblasts. Moreover, supernatant of osteoblasts treated with metformin reduced formation of tartrate resistant acid phosphatase (TRAP)-positive multi-nucleated cells in Raw264.7 cells. Most importantly, metformin significantly increased total body bone mineral density, prevented bone loss and decreased TRAP-positive cells in OVX rats proximal tibiae, accompanied with an increase of OPG and decrease of RANKL expression. These in vivo and in vitro studies suggest that metformin reduces RANKL and stimulates OPG expression in osteoblasts, further inhibits osteoclast differentiation and prevents bone loss in OVX rats.     

Zinc Upregulates the Expression of Osteoprotegerin in Mouse Osteoblasts MC3T3-E1 Through PKC/MAPK Pathways

Zinc is an essential element for bone formation; however, its role in osteoblast has not been well understood. In the present study, we hypothesized that zinc could increase osteogenetic function by stimulating osteoblast proliferation and osteoprotegerin (OPG) activity. To test this hypothesis, osteoblastic MC3T3-E1 cells were cultured and treated with various concentrations of zinc (0, 10, 30, 50, 70, 110, 130, and 150 μM) for 24 and 48 h. 3-[4,5-dimethylthiazol-2-y]-2,5-diphenyltetrazolium bromide assay showed that cell proliferation was significantly stimulated with 50 μM zinc treatment. Furthermore, under the same treatment condition, OPG expression was significantly increased as evidenced by the results of RT-PCR and ELISA. However, the zinc-induced OPG expression was significantly attenuated when MC3T3-E1 cells were co-treated with either protein kinase C (PKC) inhibitor, GF109203X, or the Inhibitor of mitogen-activated extracellular signal-regulated kinase 1 (MEK1), PD98059. Moreover, OPG expression was further increased when MC3T3-E1 cells were treated with PMA (the activator of protein of kinase C) in the presence of zinc. These results suggested that zinc would increase osteogenic function by stimulating PKC and MAPK signaling pathways.     

The effect of simulated microgravity on osteoblasts is independent of the induction of apoptosis

Bone loss during spaceflight has been attributed, in part, to a reduction in osteoblast number, altered gene expression, and an increase in cell death. To test the hypothesis that microgravity induces osteoblast apoptosis and suppresses the mature phenotype, we created a novel system to simulate spaceflight microgravity combining control and experimental cells within the same in vitro environment. Cells were encapsulated into two types of alginate carriers: non-rotationally stabilized (simulated microgravity) and rotationally stabilized (normal gravity). Using these specialized carriers, we were able to culture MC3T3-E1 osteoblast-like cells for 1-14 days in simulated microgravity and normal gravity in the same rotating wall vessel (RWV). The viability of cells was not affected by simulated microgravity, nor was the reductive reserve. To determine if simulated microgravity sensitized the osteoblasts to apoptogens, cells were challenged with staurosporine or sodium nitroprusside and the cell death was measured. Simulated microgravity did not alter the sensitivity of C3H10T-1/2 stem cells, MC3T3-E1 osteoblast-like cells, or MLO-A5 osteocyte-like cells to the action of these agents. RT-PCR analysis indicated that MC3T3-E1 osteoblasts maintained expression of RUNX2, osteocalcin, and collagen type I, but alkaline phosphatase expression was decreased in cells subjected to simulated microgravity for 5 days. We conclude that osteoblast apoptosis is not induced by vector-averaged gravity, thus suggesting that microgravity does not directly induce osteoblast death.     

Inhibitory effect of 17 beta -estradiol on prostaglandin E2-induced phosphoinositide hydrolysis in osteoblast-like cells.

We examined the effect of estradiol on PGE2-induced phosphoinositide hydrolysis and cAMP production in cloned osteoblast-like MC3T3-E1 cells. 17 beta -Estradiol pretreatment significantly inhibited the formation of inositol phosphates induced by 10 microM PGE2 in a dose-dependent manner between 1 pM and 10 nM. This effect of 17 beta -estradiol was dependent on the time of pretreatment and submaximum inhibition was observed at 4 h. However, 17 beta -estradiol had little effect on the formation of inositol phosphates induced by 20 mM NaF, a GTP-binding protein activator. The cAMP production induced by PGE2 was not influenced by 17 beta -estradiol. These results suggest that 17 beta -estradiol modulates the signal transduction by PGE2 and that the effect seems to be exerted between PGE2 receptor and the GTP-binding protein coupled to phospholipase C in osteoblast-like MC3T3-E1 cells.     

Cell density and growth-dependent down-regulation of both intracellular calcium responses to agonist stimuli and expression of smooth-surfaced endoplasmic reticulum in MC3T3-E1 osteoblast-like cells

A two-dimensional intracellular Ca(2+) ([Ca(2+)](i)) imaging system was used to examine the relationship between [Ca(2+)](i) handling and the proliferation of MC3T3-E1 osteoblast-like cells. The resting [Ca(2+)](i) level in densely cultured cells was 1.5 times higher than the [Ca(2+)](i) level in sparsely cultured cells or in other cell types (mouse fibroblasts, rat vascular smooth muscle cells, and bovine endothelial cells). A high resting [Ca(2+)](i) level may be specific for MC3T3-E1 cells. MC3T3-E1 cells were stimulated with ATP (10 microM), caffeine (10 mM), thapsigargin (1 microM), or ionomycin (10 microM), and the effect on the [Ca(2+)](i) level of MC3T3-E1 cells was studied. The percentage of responding cells and the degree of [Ca(2+)](i) elevation were high in the sparsely cultured cells and low in densely cultured cells. The rank order for the percentage of responding cells and magnitude of the Ca(2+) response to the stimuli was ionomycin > thapsigargin = ATP > caffeine and suggests the existence of differences among the various [Ca(2+)](i) channels. All Ca(2+) responses in the sparsely cultured MC3T3-E1 cells, unlike in other cell types, disappeared after the cells reached confluence. Heptanol treatment of densely cultured cells restored the Ca(2+) response, suggesting that cell-cell contact is involved with the confluence-dependent disappearance of the Ca(2+) response. Immunohistological analysis of type 1 inositol trisphosphate receptors and electron microscopy showed distinct expression of inositol trisphosphate receptor proteins and smooth-surfaced endoplasmic reticulum in sparsely cultured cells but reduced levels in densely cultured cells. These results indicate that the underlying basis of confluence-dependent [Ca(2+)](i) regulation is down-regulation of smooth-surfaced endoplasmic reticulum by cell-cell contacts.     

Serial passage of MC3T3-E1 cells alters osteoblastic function and responsiveness to transforming growth factor-beta1 and bone morphogenetic protein-2.

The murine-derived clonal MC3T3-E1 cell is a well-studied osteoblast-like cell line. To understand the effects of serial passages on its cellular function, we examined changes in cell morphology, gap junctional intercellular communication (GJIC), proliferation, and osteoblastic function between early passage (<20) and late passage (>65) cells. MC3T3-E1 cells developed an elongated, spindle shape after multiple passages. Intercellular communication decreased significantly (33%) in late vs. early passage cells. Transforming growth factor-beta1 (TGF-beta1) stimulated cell proliferation in early passage cells and induced c-fos expression, while it inhibited proliferation in late passage cells. Using alkaline phosphatase (ALP) activity and osteocalcin (OC) secretion as markers for osteoblastic function and differentiation, we demonstrated that both markers were significantly reduced after multiple cell passages. Bone morphogenetic protein-2 (BMP-2) significantly enhanced ALP activity and OC secretion in early passage cells while TGF-beta1 exerted an opposite effect. Both BMP-2 and TGF-beta1 had minimal effects on late passage cells. We conclude that serial passage alters MC3T3-E1 cell morphology, and significantly diminishes GJIC, osteoblastic function, TGF-beta1-mediated cell proliferation, and responsiveness to TGF-beta1 and BMP-2. Cell passage numbers should be clearly defined in functional studies involving MC3T3-E1 cells.     

IL-6 is produced by osteoblasts and induces bone resorption

To examine the possible involvement of IL-6 in bone metabolism, a mouse osteoblastic cell line (MC3T3-E1) and primary osteoblast-like cells from fetal mouse calvaria were cultured with several systemic and local bone-resorbing agents and their expression of IL-6 mRNA was determined. Local bone-resorbing agents such as IL-1 alpha, IL-1 beta, TNF-alpha, and LPS greatly induced IL-6 mRNA expression in both MC3T3-E1 cells and primary osteoblast-like cells. Parathyroid hormone slightly increased expression of IL-6 mRNA in primary osteoblast-like cells but not in MC3T3-E1 cells. Neither IL-6 nor 1 alpha,25-dihydroxyvitamin D3 increased expression of IL-6 mRNA in either of the osteoblast-like cells. In agreement with the expression of IL-6 mRNA, biologically active IL-6 was produced in response to the treatment with IL-1 alpha, TNF-alpha, and LPS in MC3T3-E1 cells. Adding IL-6 dose dependently stimulated the release of 45Ca from prelabeled fetal mouse calvaria. Simultaneously adding suboptimal concentrations of IL-6 and IL-1 alpha induced bone resorption cooperatively. In accord with the increase in the release of 45Ca by IL-6, there were three times as many osteoclasts in the bone sections of calvaria cultured with IL-6 for 5 days as in the controls. IL-6 slightly suppressed alkaline phosphatase activity and collagen synthesis in MC3T3-E1 cells. These results indicate that IL-6 is also produced by osteoblasts, preferentially in response to local bone-resorbing agents, and it induces bone resorption both alone and in concert with other bone-resorbing agents.