RANKL Treatment Research Articles

Critical roles of c-Jun signaling in regulation of NFAT family and RANKL-regulated osteoclast differentiation

Receptor activator of NF-κB ligand (RANKL) plays an essential role in osteoclast formation and bone resorption. Although genetic and biochemical studies indicate that RANKL regulates osteoclast differentiation by activating receptor activator of NF-κB and associated signaling molecules, the molecular mechanisms of RANKL-regulated osteoclast differentiation have not yet been fully established. We investigated the role of the transcription factor c-Jun, which is activated by RANKL, in osteoclastogenesis using transgenic mice expressing dominant-negative c-Jun specifically in the osteoclast lineage. We found that the transgenic mice manifested severe osteopetrosis due to impaired osteoclastogenesis. Blockade of c-Jun signaling also markedly inhibited soluble RANKL-induced osteoclast differentiation in vitro. Overexpression of nuclear factor of activated T cells 1 (NFAT1) (NFATc2/NFATp) or NFAT2 (NFATc1/NFATc) promoted differentiation of osteoclast precursor cells into tartrate-resistant acid phosphatase–positive (TRAP–positive) multinucleated osteoclast-like cells even in the absence of RANKL. Overexpression of NFAT1 also markedly transactivated the TRAP gene promoter. These osteoclastogenic activities of NFAT were abrogated by overexpression of dominant-negative c-Jun. Importantly, osteoclast differentiation and induction of NFAT2 expression by NFAT1 overexpression or soluble RANKL treatment were profoundly diminished in spleen cells of the transgenic mice. Collectively, these results indicate that c-Jun signaling in cooperation with NFAT is crucial for RANKL-regulated osteoclast differentiation.

Vitamin D antagonist, TEI-9647, inhibits osteoclast formation induced by 1α,25-dihydroxyvitamin D 3 from pagetic bone marrow cells

(23 S)-25-Dehydro-1α-hydroxyvitamin D 3-26,23-lactone (TEI-9647) functions an antagonist of the 1α,25-dihydroxyvitamin D 3 (1α,25-(OH) 2D 3) nuclear receptor (VDR)-mediated differentiation of human leukemia (HL-60) cells [J. Biol. Chem. 274 (1999) 16392]. We examined the effect of vitamin D antagonist, TEI-9647, on osteoclast formation induced by 1α,25-(OH) 2D 3 from bone marrow cells of patients with Paget’s disease. TEI-9647 itself never induced osteoclast formation even at 10 −6 M, but dose-dependently (10 −10 to 10 −6 M) inhibited osteoclast formation induced by physiologic concentrations of 1α,25-(OH) 2D 3 (41 pg/ml, 10 −10 M) from bone marrow cells of patients with Paget’s disease. At the same time, 10 −8 M of TEI-9647 alone did not cause 1α,25-(OH) 2D 3 dependent gene expression, but almost completely suppressed TAF II-17, a potential coactivator of VDR and 25-hydroxyvitamin D 3-24-hydroxylase (25-OH-D 3-24-hydroxylase) gene expression induced by 10 −10 M 1α,25-(OH) 2D 3 in bone marrow cells of patients with Paget’s disease. Moreover, TEI-9647 dose-dependently inhibited bone resorption induced by 10 −9 M 1α,25-(OH) 2D 3 by osteoclasts produced by RANKL and M-CSF treatment of measles virus nucleocapsid gene transduced bone marrow cells. These results suggest that TEI-9647 acts directly on osteoclast precursors and osteoclasts, and that TEI-9647 may be a novel agent to suppress the excessive bone resorption and osteoclast formation in patients with Paget’s disease.

Phosphatidylinositol 3-kinase coordinately activates the MEK/ERK and AKT/NFkappaB pathways to maintain osteoclast survival.

We have examined highly purified osteoclasts that were generated in vitro from murine co-culture of marrow precursors with stromal support cells and have found evidence of activation of the MEK/ERK and AKT/NFkappaB survival pathways. Many mature marrow-derived osteoclasts survived for at least 48 h in culture whether or not they are maintained with stromal cells. Moreover, supplementing purified osteoclasts with RANKL and/or M-CSF had no impact on their survival pattern. In addition, spleen-derived osteoclasts generated with RANKL and M-CSF treatment exhibited a similar survival pattern. Blocking MEK, AKT, or NFkappaB activity resulted in apoptosis of many, but not all, of the osteoclasts in purified marrow-derived osteoclasts, marrow-derived osteoclasts co-cultured with stromal cells, and spleen-derived osteoclasts maintained with RANKL and M-CSF. These data support that both the MEK/ERK and AKT/NFkappaB pathways contribute to osteoclast survival. Since PI3K has been shown to activate either of these pathways, we have examined its role in osteoclast survival. PI3K inhibition caused apoptosis of nearly all osteoclasts in purified and co-cultured marrow-derived osteoclasts and spleen-derived osteoclasts maintained with RANKL and M-CSF. Interestingly, in marrow-derived co-cultures, the apoptotic response was restricted to osteoclasts as there was no evidence of stromal support cell apoptosis. PI3K inhibition also blocked MEK1/2, ERK1/2, and AKT phosphorylation and NFkappaB activation in purified osteoclasts. Simultaneous blockage of both AKT and MEK1/2 caused rapid apoptosis of nearly all osteoclasts, mimicking the response to PI3K inhibition. These data reveal that PI3K coordinately activates two distinct survival pathways that are both important in osteoclast survival.

Transforming growth factor-β1 (TGF-β) stimulates the osteoclast-forming potential of peripheral blood hematopoietic precursors in a lymphocyte-rich microenvironment

Osteoclasts are generated from peripheral blood mononuclear cells (PBMNCs) in the presence of soluble receptor activator of NFκB ligand (sRANKL) and macrophage colony-stimulating factor (M-CSF). We show that human osteoclast formation is enhanced when PBMNCs are cultured in the presence of transforming growth factor (TGF)-β and M-CSF prior to the addition of sRANKL. The effect was only observed in the presence of a nonadherent lymphocyte PBMNC fraction. Osteoclast formation was enhanced to a level equivalent to that induced by TGF-β when nonadherent PBMNC fraction was removed from the cultures, prior to RANKL treatment. These data suggest that TGF-β enhances osteoclast formation by abrogating the suppressive effect of the nonadherent PBMNCs, thereby maintaining the osteoclast-forming potential of the osteoclast precursor population. TGF-β was without effect on proliferation of the adherent PBMNCs and did not stimulate osteoclast size or modify their immunophenotype. The effect was not mediated through prostaglandin synthesis. These results indicate that the microenvironment encountered by the osteoclast precursor prior to RANKL exposure contributes significantly to the regulation of osteoclast formation. Furthermore, the data emphasize that the effect of TGF-β is determined by the cytokine milieu of the microenvironment and/or the state of activation of the cell being targeted by TGF-β; thus, the effect of TGF-β is context-dependent.

Bone matrix inhibits csf-1 induced osteoclast spreading

Prostate cancer metastases to bone are observed in around 80% of prostate cancer patients and represent the most critical complication of advanced prostate cancer, frequently resulting in significant morbidity and mortality. As the underlying mechanisms are not fully characterized, understanding the biological mechanisms that govern prostate cancer metastases to bone at the molecular level should lead to the determination of new potential therapeutic targets. Receptor activator of NFκB ligand (RANKL)/RANK/Osteoprotegerin (OPG) are the key regulators of bone metabolism both in normal and pathological condition, including prostate cancer bone metastases. In the present study, we demonstrated that human prostate cancer cell lines, DU145 and PC3 express biologically functional RANK. Indeed, soluble human RANKL (shRANKL, 100 ng/ml) treatment induced ERK 1/2, p38 and IκB phosphorylations in these cells. shRANKL administration also promoted DU145 and PC3 prostate cancer cell invasion in vitro. Whereas human OPG (hOPG) administration alone (100 ng/ml) had no marked effect, combined association of both agents abolished the RANKL-induced DU145 cell invasion. As RANKL had no direct effect on DU145 cell proliferation, the observed effects were indeed related to RANKL-induced cell migration. DU145 human prostate cancer cells promoted osteoclastogenesis of osteoclast precursors generated from mouse bone marrow. Moreover, DU145 cells produced soluble factor(s) that up-regulate the proliferation of MC3T3-E1 pre-osteoblasts through the activation of the ERK 1/2 and STAT3 signal transduction pathways. This stimulation of pre-osteoblast proliferation resulted in an increased local RANKL expression that can activate both osteoclasts/osteoclast precursors and prostate cancer cells, thus facilitating prostate cancer metastasis development in bone. We confirm that RANKL is a factor that facilitates metastasis to bone by acting as an activator of both osteoclasts and RANK-positive prostate cancer cells in our model. Furthermore, the present study provides the evidence that blocking RANKL–RANK interaction offer new therapeutic approach not only at the level of bone resorbing cells, but also by interfering with RANK-positive prostate cancer cells in the prostate cancer bone metastasis development.