Fibroblast growth factor (FGF)-2 directly stimulates mature osteoclast function through activation of FGF receptor 1 and p42/p44 MAP kinase

We previously reported that fibroblast growth factor-2 (FGF-2) acts not only on osteoblasts to stimulate osteoclastic bone resorption indirectly but also on mature osteoclasts directly. In this study, we investigated the mechanism of this direct action of FGF-2 on mature osteoclasts using mouse and rabbit osteoclast culture systems. FGF-2 stimulated pit formation resorbed by isolated rabbit osteoclasts moderately from low concentrations (>/=10(-12) m), whereas at high concentrations (>/=10(-9) m) it showed stimulation on pit formation resorbed by unfractionated bone cells very potently. FGF-2 (>/=10(-12) m) also increased cathepsin K and MMP-9 mRNA levels in mouse and rabbit osteoclasts. Among FGF receptors (FGFR1 to 4) only FGFR1 was detected on isolated mouse osteoclasts, whereas all FGFRs were identified on mouse osteoblasts. FGF-2 (>/=10(-12) m) up-regulated the phosphorylation of cellular proteins, including p42/p44 mitogen-activated protein (MAP) kinase, and increased the kinase activity of immunoprecipitated FGFR1 in mouse osteoclasts. The stimulation of FGF-2 on mouse and rabbit osteoclast functions was abrogated by PD-98059, a specific inhibitor of p42/p44 MAP kinase. These results strongly suggest that FGF-2 acts directly on mature osteoclasts through activation of FGFR1 and p42/p44 MAP kinase, causing the stimulation of bone resorption at physiological or pathological concentrations.     

Vascular endothelial growth factor (VEGF) directly enhances osteoclastic bone resorption and survival of mature osteoclasts

In bone development and regeneration, angiogenesis and bone/cartilage resorption are essential processes and are closely associated with each other, suggesting a common mediator for these two biological events. To address this interrelationship, we examined the effect of vascular endothelial growth factor (VEGF), the most critical growth factor for angiogenesis, on osteoclastic bone-resorbing activity in a culture of highly purified rabbit mature osteoclasts. VEGF caused a dose- and time-dependent increase in the area of bone resorption pits excavated by the isolated osteoclasts, partially by enhancing the survival of the cells. Two distinct VEGF receptors, KDR/Flk-1 and Flt-1, were detectable in osteoclasts at the gene and protein levels, and VEGF induced tyrosine phosphorylation of proteins in osteoclasts. Thus, osteoclastic function and angiogenesis are up-regulated by a common mediator such as VEGF.     

Cross-phosphorylation, signaling and proliferative functions of the Tyro3 and Axl receptors in Rat2 cells

The dysregulation of receptor protein tyrosine kinase (RPTK) function can result in changes in cell proliferation, cell growth and metastasis leading to malignant transformation. Among RPTKs, the TAM receptor family composed of three members Tyro3, Axl, and Mer has been recognized to have a prominent role in cell transformation. In this study we analyzed the consequences of Tyro3 overexpression on cell proliferation, activation of signaling pathways and its functional interactions with Axl. Overexpression of Tyro3 in the Rat2 cell line that expresses Axl, but not Mer or Tyro3, resulted in a 5 fold increase in cell proliferation. This increase was partially blocked by inhibitors of the mitogen-activated protein kinase (MAPK) signaling pathway but not by inhibitors of the phosphatidylinositol 3-kinase (PI(3)K) signaling pathway. Consistent with these findings, an increase in ERK1/2 phosphorylation was detected with Tyro3 but not with Axl overexpression. In contrast, activation of Axl stimulated the PI(3)K pathway, which was mitigated by co-expression of Tyro3. The overexpression of Tyro3 enhanced Gas6-mediated Axl phosphorylation, which was not detected upon overexpression of a "kinase dead" form of Tyro3 (kdTyro3). In addition, the overexpression of Axl induced kdTyro3 phosphorylation. Co-immunoprecipitation experiments confirmed that the Axl and Tyro3 receptors are closely associated. These findings show that overexpression of Tyro3 in the presence of Axl promotes cell proliferation, and that co-expression of Axl and Tyro3 can affect the outcome of Gas6-initiated signaling. Furthermore, they demonstrate a functional interaction between the members of the TAM receptor family which can shed light on the molecular mechanisms underlying the functional consequences of TAM receptor activation in cell transformation, neural function, immune function, and reproductive function among others.     

Cloning and function of rabbit peroxisome proliferator-activated receptor delta/beta in mature osteoclasts

Osteoclasts modulate bone resorption under physiological and pathological conditions. Previously, we showed that both estrogens and retinoids regulated osteoclastic bone resorption and postulated that such regulation was directly mediated through their cognate receptors expressed in mature osteoclasts. In this study, we searched for expression of other members of the nuclear hormone receptor superfamily in osteoclasts. Using the low stringency homologous hybridization method, we isolated the peroxisome proliferator-activated receptor delta/beta (PPARdelta/beta) cDNA from mature rabbit osteoclasts. Northern blot analysis showed that PPARdelta/beta mRNA was highly expressed in highly enriched rabbit osteoclasts. Carbaprostacyclin, a prostacyclin analogue known to be a ligand for PPARdelta/beta, significantly induced both bone-resorbing activities of isolated mature rabbit osteoclasts and mRNA expression of the cathepsin K, carbonic anhydrase type II, and tartrate-resistant acid phosphatase genes in these cells. Moreover, the carbaprostacyclin-induced bone resorption was completely blocked by an antisense phosphothiorate oligodeoxynucleotide of PPARdelta/beta but not by the sense phosphothiorate oligodeoxynucleotide of the same DNA sequence. Our results suggest that PPARdelta/beta may be involved in direct modulation of osteoclastic bone resorption.     

Furosin, an ellagitannin, suppresses RANKL-induced osteoclast differentiation and function through inhibition of MAP kinase activation and actin ring formation

Phenolic compounds including tannins and flavonoids have been implicated in suppression of osteoclast differentiation/function and prevention of bone diseases. However, the effects of hydrolysable tannins on bone metabolism remain to be elucidated. In this study, we found that furosin, a hydrolysable tannin, markedly decreased the differentiation of both murine bone marrow mononuclear cells and Raw264.7 cells into osteoclasts, as revealed by the reduced number of tartrate resistant acid phosphatase (TRAP)-positive multinucleated cells and decreased TRAP activity. Furosin appears to target at the early stage of osteoclastic differentiation while having no cytotoxic effect on osteoclast precursors. Analysis of the inhibitory mechanisms of furosin revealed that it inhibited the receptor activator of nuclear factor-kappaB ligand (RANKL)-induced activation of p38 mitogen-activated protein kinase (p38MAPK) and c-Jun N-terminal kinase (JNK)/activating protein-1 (AP-1). Furthermore, furosin reduced resorption pit formation in osteoclasts, which was accompanied by disruption of the actin rings. Taken together, these results demonstrate that naturally occurring furosin has an inhibitory activity on both osteoclast differentiation and function through mechanisms involving inhibition of the RANKL-induced p38MAPK and JNK/AP-1 activation as well as actin ring formation.     

Non-enzymatic glycation of bone collagen modifies osteoclastic activity and differentiation

Type I collagen, the major organic component of bone matrix, undergoes a series of post-translational modifications that occur with aging, such as the non-enzymatic glycation. This spontaneous reaction leads to the formation of advanced glycation end products (AGEs), which accumulate in bone tissue and affect its structural and mechanical properties. We have investigated the role of matrix AGEs on bone resorption mediated by mature osteoclasts and the effects of exogenous AGEs on osteoclastogenesis. Using in vitro resorption assays performed on control- and AGE-modified bone and ivory slices, we showed that the resorption process was markedly inhibited when mature osteoclasts were seeded on slices containing matrix pentosidine, a well characterized AGE. More specifically, the total area resorbed per slice, and the area degraded per resorption lacuna created by osteoclasts, were significantly decreased in AGE-containing slices. This inhibition of bone resorption was confirmed by a marked reduction of the release of type I collagen fragments generated by the collagenolytic enzymes secreted by osteoclasts in the culture medium of AGE-modified mineralized matrices. This effect is likely to result from decreased solubility of collagen molecules in the presence of AGEs, as documented by the reduction of pepsin-mediated digestion of AGE-containing collagen. We found that AGE-modified BSA totally inhibited osteoclastogenesis in vitro, most likely by impairing the commitment of osteoclast progenitors into pre-osteoclastic cells. Although the mechanisms remain unknown, AGEs might interfere with osteoclastic differentiation and activity through their interaction with specific cell-surface receptors, because we showed that both osteoclast progenitors and mature osteoclasts expressed different AGEs receptors, including receptor for AGEs (RAGEs). These results suggest that AGEs decreased osteoclast-induced bone resorption, by altering not only the structural integrity of bone matrix proteins but also the osteoclastic differentiation process. We suggest that AGEs may play a role in the alterations of bone remodeling associated with aging and diabetes.     

Estrogen prevents bone loss via estrogen receptor alpha and induction of Fas ligand in osteoclasts

Estrogen prevents osteoporotic bone loss by attenuating bone resorption; however, the molecular basis for this is unknown. Here, we report a critical role for the osteoclastic estrogen receptor alpha (ERalpha) in mediating estrogen-dependent bone maintenance in female mice. We selectively ablated ERalpha in differentiated osteoclasts (ERalpha(DeltaOc/DeltaOc)) and found that ERalpha(DeltaOc/DeltaOc) females, but not males, exhibited trabecular bone loss, similar to the osteoporotic bone phenotype in postmenopausal women. Further, we show that estrogen induced apoptosis and upregulation of Fas ligand (FasL) expression in osteoclasts of the trabecular bones of WT but not ERalpha(DeltaOc/DeltaOc) mice. The expression of ERalpha was also required for the induction of apoptosis by tamoxifen and estrogen in cultured osteoclasts. Our results support a model in which estrogen regulates the life span of mature osteoclasts via the induction of the Fas/FasL system, thereby providing an explanation for the osteoprotective function of estrogen as well as SERMs.     

Targeted overexpression of osteoactivin in cells of osteoclastic lineage promotes osteoclastic resorption and bone loss in mice

This study sought to test whether targeted overexpression of osteoactivin (OA) in cells of osteoclastic lineage, using the tartrate-resistant acid phosphase (TRAP) exon 1B/C promoter to drive OA expression, would increase bone resorption and bone loss in vivo. OA transgenic osteoclasts showed ～2-fold increases in OA mRNA and proteins compared wild-type (WT) osteoclasts. However, the OA expression in transgenic osteoblasts was not different. At 4, 8, and 15.3 week-old, transgenic mice showed significant bone loss determined by pQCT and confirmed by μ-CT. In vitro, transgenic osteoclasts were twice as large, had twice as much TRAP activity, resorbed twice as much bone matrix, and expressed twice as much osteoclastic genes (MMP9, calciton receptor, and ADAM12), as WT osteoclasts. The siRNA-mediated suppression of OA expression in RAW264.7-derived osteoclasts reduced cell size and osteoclastic gene expression. Bone histomorphometry revealed that transgenic mice had more osteoclasts and osteoclast surface. Plasma c-telopeptide (a resorption biomarker) measurements confirmed an increase in bone resorption in transgenic mice in vivo. In contrast, histomorphometric bone formation parameters and plasma levels of bone formation biomarkers (osteocalcin and pro-collagen type I N-terminal peptide) were not different between transgenic mice and WT littermates, indicating the lack of bone formation effects. In conclusion, this study provides compelling in vivo evidence that osteoclast-derived OA is a novel stimulator of osteoclast activity and bone resorption.     

Characterization of interferon gamma receptors on osteoclasts: effect of interferon gamma on osteoclastic superoxide generation

Osteoclasts are the primary cells responsible for bone resorption. Osteoclast formation and bone resorption activities involve processes tightly controlled by a network of cytokines. The presence of interferon gamma (IFN-gamma) receptors on osteoclasts is a necessary prerequisite for IFN-gamma to directly affect osteoclastic activity. To date, the presence of the IFN-gamma receptor on osteoclasts has not been established. This study provides evidence that osteoclasts express the IFN-gamma receptor. Specific binding of IFN-gamma to the osteoclastic receptor stimulates osteoclastic superoxide generation. The p91 and p47 components of the NADPH oxidase increase after IFN-gamma stimulation and may account for the enhanced superoxide generation. Antisense experiments targeting p91 and p47 subunits abrogate the increased osteoclastic superoxide production stimulated by IFN-gamma. Thus, superoxide generation by osteoclasts is stimulated by activation of a functional IFN-gamma receptor on the osteoclast.     

Vav3 regulates osteoclast function and bone mass

Osteoporosis, a leading cause of morbidity in the elderly, is characterized by progressive loss of bone mass resulting from excess osteoclastic bone resorption relative to osteoblastic bone formation. Here we identify Vav3, a Rho family guanine nucleotide exchange factor, as essential for stimulated osteoclast activation and bone density in vivo. Vav3-deficient osteoclasts show defective actin cytoskeleton organization, polarization, spreading and resorptive activity resulting from impaired signaling downstream of the M-CSF receptor and alpha(v)beta3 integrin. Vav3-deficient mice have increased bone mass and are protected from bone loss induced by systemic bone resorption stimuli such as parathyroid hormone or RANKL. Moreover, we provide genetic and biochemical evidence for the role of Syk tyrosine kinase as a crucial upstream regulator of Vav3 in osteoclasts. Thus, Vav3 is a potential new target for antiosteoporosis therapy.     

Vanadium salts stimulate mitogen-activated protein (MAP) kinases and ribosomal S6 kinases

Effect of several vanadium salts, sodium orthovanadate, vanadyl sulfate and sodium metavanadate on protein tyrosine phosphorylation and serine/threonine kinases in chinese hamster ovary (CHO) cells overexpressing a normal human insulin receptor was examined. All the compounds stimulated protein tyrosine phosphorylation of two major proteins with molecular masses of 42 kDa (p42) and 44 kDa (p44). The phosphorylation of p42 and p44 was associated with an activation of mitogen activated protein (MAP) kinase as well as increased protein tyrosine phosphorylation of p42^mapk and p44^mapk. Vanadinm salts also activated the 90 kDa ribosomal s6 kinase (p90^rsk) and 70 kDa ribosomal s6 kinase (p70^s6k). Among the three vanadium salts tested, vanadyl sulfate appeared to be slightly more potent than others in stimulating MAP kinases and p70^s6k activity. It is suggested that vanadium-induced activation of MAP kinases and ribosomal s6 kinases may be one of the mechanisms by which insulin like effects of this trace element are mediated.Abbreviations eIF-4 eukaryotic protein synthesis initiation factor-4 - GRB-2 growth factor receptor bound protein-2 - GSK-3 Glycogen Synthase Kinase-3 - IRS-1 insulin receptor substrate-1 - ISPK insulin stimulated protein kinase - MAPK mitogen activated protein kinase, also known as - ERK extracellular signal regulated kinase - MAPKK mitogen activated protein kinase kinase, also known as-MEK, MAPK or ERK kinase - PHAS-1 phosphorylated heat and acid stable protein regulated by insulin - PI3K phosphatidyl inositol 3-kinase - PP1-G protein phosphatase-glycogen bound form - PTK protein tyrosine kinase - PTPase protein tyrosine phosphatase - rsk ribosomal s6 kinases - shc src homology domain containing protein - SOS son of sevenless     

Stimulation of catecholamine synthesis through unique estrogen receptors in the bovine adrenomedullary plasma membrane by 17beta-estradiol

Incubation of cultured bovine adrenal medullary cells with 17beta-estradiol (E(2)) (0.3-100nM) or membrane-impermeable E(2)-bovine serum albumin (100nM) acutely increased (14)C-catecholamine synthesis from [(14)C]tyrosine. The stimulatory effect of E(2) was not inhibited by ICI182,780, a nuclear estrogen receptor inhibitor. E(2) also increased tyrosine hydroxylase activity and p44/42MAPK phosphorylation, the former of which was attenuated by U0126, an inhibitor of p44/42MAPK kinase. The plasma membrane isolated from the gland showed two classes of specific binding sites of [(3)H]E(2) with apparent K(d)s of 3.2 and 106nM, and B(max)s of 0.44 and 8.5pmol/mg protein, respectively. The high-affinity binding of [(3)H]E(2) was most strongly inhibited by E(2) and phytoestrogens, and to lesser extents by other steroid hormones, while it was enhanced by ICI182,780 and environmental estrogenic pollutants. These findings suggest that E(2) acutely stimulates catecholamine synthesis via activation of p44/42MAPK through unique estrogen receptors in the plasma membrane of bovine adrenal medulla.     

Decoy receptor 3 (DcR3) induces osteoclast formation from monocyte/macrophage lineage precursor cells

Recent evidence indicates that the decoy receptor 3 (DcR3) of the TNF receptor superfamily, which initially though prevents cytokine responses of FasL, LIGHT and TL1A by binding and neutralization, can modulate monocyte function through reverse signaling. We show in this work that DcR3 can induce osteoclast formation from human monocytes, murine RAW264.7 macrophages, and bone marrow cells. DcR3-differentiated cells exhibit characteristics unique for osteoclasts, including polynuclear giant morphology, bone resorption, TRAP, CD51/61, and MMP-9 expression. Consistent with the abrogation of osteoclastogenic effect of DcR3 by TNFR-Fc, DcR3 treatment can induce osteoclastogenic cytokine TNF-alpha release through ERK and p38 MAPK signaling pathways. We conclude that DcR3 via coupling reverse signaling of ERK and p38 MAPK and stimulating TNF-alpha synthesis is a critical regulator of osteoclast formation. This action of DcR3 might play an important role in significant osteoclastic activity in osteolytic bone metastases.     

Regulation of osteoblast, chondrocyte, and osteoclast functions by fibroblast growth factor (FGF)-18 in comparison with FGF-2 and FGF-10.

This study investigated the actions of fibroblast growth factor (FGF)-18, a novel member of the FGF family, on osteoblasts, chondrocytes, and osteoclasts and compared them with those of FGF-2 and FGF-10. FGF-18 stimulated the proliferation of cultured mouse primary osteoblasts, osteoblastic MC3T3-E1 cells, primary chondrocytes, and prechondrocytic ATDC5 cells, although it inhibited the differentiation and matrix synthesis of these cells. FGF-18 up-regulated the phosphorylation of extracellular signal-regulated kinase in both osteoblasts and chondrocytes and up-regulated the phosphorylation of p38 mitogen-activated protein kinase only in chondrocytes. FGF-18 mitogenic actions were blocked by a specific inhibitor of extracellular signal-regulated kinase in both osteoblasts and chondrocytes and by a specific inhibitor of p38 mitogen-activated protein kinase in chondrocytes. With regard to the action of FGF-18 on bone resorption, FGF-18 not only induced osteoclast formation through receptor activator of nuclear factor-kappaB ligand and cyclooxygenase-2 but also stimulated osteoclast function to form resorbed pits on a dentine slice in the mouse coculture system. All these effects of FGF-18 bore a close resemblance to those of FGF-2, whereas FGF-10 affects none of these cells. FGF-18 may therefore compensate for the action of FGF-2 on bone and cartilage.     

Oxidative stress causes mucin synthesis via transactivation of epidermal growth factor receptor: role of neutrophils

Oxidative stress has been implicated in the pathogenesis of inflammatory diseases of airways. Here we show that oxidative stress causes ligand-independent activation of epidermal growth factor receptors (EGFR) and subsequent activation of mitogen-activated protein kinase kinase (MEK)-p44/42 mitogen-activated protein kinase (p44/42mapk), resulting in mucin synthesis in NCI-H292 cells. Exogenous hydrogen peroxide and neutrophils activated by IL-8, FMLP, or TNF-alpha increased EGFR tyrosine phosphorylation and subsequent activation of p44/42mapk and up-regulated the expression of MUC5AC at both mRNA and protein levels in NCI-H292 cells. These effects were blocked by selective EGFR tyrosine kinase inhibitors (AG1478, BIBX1522) and by a selective MEK inhibitor (PD98059), whereas a selective platelet-derived growth factor receptor tyrosine kinase inhibitor (AG1295), a selective p38 MAPK inhibitor (SB203580), and a negative compound of tyrosine kinase inhibitors (A1) were without effect. Neutrophil supernatant-induced EGFR tyrosine phosphorylation, activation of p44/42mapk, and MUC5AC synthesis were inhibited by antioxidants (N-acetyl-cysteine, DMSO, dimethyl thiourea, or superoxide dismutase); neutralizing Abs to EGFR ligands (EGF and TGF-alpha) were without effect, and no TGF-alpha protein was found in the neutrophil supernatant. In contrast, the EGFR ligand, TGF-alpha, increased EGFR tyrosine phosphorylation, activation of p44/42mapk, and subsequent MUC5AC synthesis, but these effects were not inhibited by antioxidants. These results implicate oxidative stress in stimulating mucin synthesis in airways and provide new therapeutic approaches in airway hypersecretory diseases.     

Lipoteichoic acid-stimulated p42/p44 MAPK activation via Toll-like receptor 2 in tracheal smooth muscle cells

Lipoteichoic acid (LTA), the principal component of the cell wall of gram-positive bacteria, triggers several inflammatory responses. However, the mechanisms underlying its action on human tracheal smooth muscle cells (HTSMCs) were largely unknown. This study was to investigate the mechanisms underlying LTA-stimulated p42/p44 mitogen-activated protein kinase (MAPK) using Western blotting assay. LTA stimulated phosphorylation of p42/p44 MAPK via a Toll-like receptor 2 (TLR2). Pretreatment with pertussis toxin attenuated the LTA-induced responses. LTA-stimulated phosphorylation of p42/p44 MAPK was attenuated by inhibitors of tyrosine kinase (genistein), phosphatidylcholine-phospholipase C (PLC; D609), phosphatidylinositol (PI)-PLC (U-73122), PKC (staurosporine, G?-6976, rottlerin, or Ro-318220), MEK1/2 (U-0126), PI 3-kinase (LY-294002 and wortmannin), and an intracellular Ca(2+) chelator (BAPTA-AM). LTA directly evoked initial transient peak of [Ca(2+)](i), supporting the involvement of Ca(2+) mobilization in LTA-induced responses. These results suggest that in HTSMCs, LTA-stimulated p42/p44 MAPK phosphorylation is mediated through a TLR2 receptor and involves tyrosine kinase, PLC, PKC, Ca(2+), MEK, and PI 3-kinase.     

Gas6 negatively regulates the Staphylococcus aureus-induced inflammatory response via TLR signaling in the mouse mammary gland

Staphylococcus aureus (S. aureus)-induced mastitis is the most frequent, pathogenic, and prevalent infection of the mammary gland. The ligand growth arrest-specific 6 (Gas6) is a secretory protein that binds to and activates Tyro3, Axl, and MerTK receptors. This study explored the role of Gas6 in S. aureus-induced mastitis. Our results revealed that TLR receptors initiate the innate immune response in mammary gland tissues and epithelial cells and that introducing S. aureus activates TLR2 and TLR6 to drive multiple intracellular mitogen-activated protein kinase (MAPK) and nuclear factor kappa-B (NF-κB) pathways. Moreover, S. aureus also induces Gas6, which then activates the TAM receptor kinase pathway, which is related to the inhibition of TLR2- and TLR6-mediated inflammatory pathways through SOCS1 and SOCS3 induction. Gas6 absence alone was found to be involved in the downregulation of TAM receptor-mediated anti-inflammatory effects by inducing significantly prominent expression of TRAF6 and low protein and messenger RNA expression of SOCS1 and SOCS3. S. aureus-induced MAPK and NF-ĸB p65 phosphorylation were also dependent on Gas6, which negatively regulated the production of Pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) in S. aureus-treated mammary tissues and mammary epithelial cells. Our in vivo and in vitro study uncovered the Gas6-mediated negative feedback mechanism, which inhibits TLR2- and TLR6-mediated MAPK and NF-ĸB signaling by activating TAM receptor kinase (MerTK, Axl, and Tyro3) through the induction of SOCS1/SOCS3 proteins.     

Interactions of myeloma cells with osteoclasts promote tumour expansion and bone degradation through activation of a complex signalling network and upregulation of cathepsin K, matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA)

Bone destruction is one of the most debilitating manifestations of multiple myeloma (MM) and results from the interaction of myeloma cells with the bone marrow microenvironment. Within the bone marrow, the disturbed balance between osteoclasts and osteoblasts is important for the development of lytic lesions. However, the mechanisms behind myeloma-mediated bone destruction are not completely understood. In order to address the importance of myeloma cell-osteoclast interactions in MM pathogenesis, we have developed a functional coculture system. We found that myeloma-osteoclast interactions resulted in stimulation of myeloma cell growth and osteoclastic activity through activation of major signalling pathways and upregulation of proteases. Signals from osteoclasts activated the p44/p42 MAPK, STAT3 and PI3K/Akt pathways in myeloma cells. In turn, myeloma cells triggered p38 MAPK and NF-kappaB signalling in osteoclasts. Myeloma-osteoclast interactions stimulated the production of TRAP, cathepsin K, matrix metalloproteinase (MMP)-1, -9, and urokinase plasminogen activator (uPA). Consistent data with myeloma cell lines and primary myeloma cells underlined the biological relevance of these findings. In conclusion, we demonstrated the critical role of myeloma cell-osteoclast interactions in the existing interdependence between tumour expansion and bone disease. The identified molecular events might provide the rationale for novel treatment strategies.     

Estrogen directly attenuates human osteoclastogenesis, but has no effect on resorption by mature osteoclasts

Estrogen deficiency arising with the menopause promotes marked acceleration of bone resorption, which can be restored by hormone replacement therapy. The inhibitory effects of estrogen seem to involve indirect cytokine- mediated effects via supporting bone marrow cells, but direct estrogen-receptor mediated effects on the bone-resorbing osteoclasts have also been proposed. Little information is available on whether estrogens modulate human osteoclastogenesis or merely inhibit the functional activity of osteoclasts. To clarify whether estrogens directly modulate osteoclastic activities human CD14+ monocytes were cultured in the presence of M-CSF and RANKL to induce osteoclast differentiation. Addition of 0.1-10 nM 17beta-estradiol to differentiating osteoclasts resulted in a dose-dependent reduction in tartrate resistant acid phosphatase (TRACP) activity reaching 60% at 0.1 nM. In addition, 17beta-estradiol inhibited bone resorption, as measured by the release of the C-terminal crosslinked telopeptide (CTX), by 60% at 0.1 nM, but had no effect on the overall cell viability. In contrast to the results obtained with differentiating osteoclasts, addition of 17beta-estradiol (0.001-10 nM) to mature osteoclasts did not affect bone resorption or TRACP activity. We investigated expression of the estrogen receptors, using immunocytochemistry and Western blotting. We found that ER-alpha is expressed in osteoclast precursors, whereas ER- beta is expressed at all stages, indicating that the inhibitory effect of estrogen on osteoclastogenesis is mediated by ER-alpha for the major part. In conclusion, these results suggest that the in vivo effects of estrogen are mediated by reduction of osteoclastogenesis rather than direct inhibition of the resorptive activity of mature osteoclasts.