The Role Of RANKL, PTHrP And P38 MAP Kinase On Osteoclastogenesis In Vitro And Bone Resorption In Vivo

Various bone resorptive disease, such as osteoporosis, inflammatory bone disease and bone metastasis, results from imbalance between bone resorption and bone formation: bone destruction exceeds bone formation. RANKL(Receptor activator of NF-B Ligand, or OPGL/ODF, osteoprotegerin ligand/osteoclast differentiation factor), a membran-bound cytokine by osteoblast/bone marrow stromal cell, plays a key role in differentiation and functional activity of osteoclast. RNAKL induce osteoclast differentiation and function by binding to its receptor RANK (Receptor activator of NF- K B) on the osteoclast precursor and mature osteoclast. P38 MAP Kinase (P38 Mitogen-Activated-Protein Kinase) mediated-signals were essential for RANKL-induced osteoclast differentiation. OPG (Osteoprotegerin) is a 'decoy receptor' of RANKL and act as antagonist of RANKL. In bone metastasis, PTHrP secreted by tumor cells upregulate the RANKL expression level by osteoblast/bone marrow stromal cell, which in turn stimulated osteoclast differentiation and function, leading to bone destruction.We tried a novel water-soluble P38 MAPK inhibitor, Fr 167653, to study: 1 whether it can inhibit osteoclastogenesis induced by RANKL and PTHrP in vitro. 2 Whether P38 MAPK-mediated signals were involved in RANKL and OPG expression by bone marrow stromal cells. 3 Established local bone resorption animal models induced by PTHrP and studied characteristic of hypercalcemia. 4 whether inhibition of P38 MAP Kinase would block local bone resorption and hypercalcemia induced by PTHrP in vivo. Due to the key role of RANKL in osteoclast differentiation and function, we cloned, expressed and purified the recombinant RANKL.1.Fr 167653 on osteoclast formation induced by RANKLIn the presence of M-CSF, RANKL stimulates osteoclast formation by direct acting on the osteoclast precursor. After culturing bone marrow cells for 3 days, nonadherent and loosely attached bone marrow cells were stimulated by RANKL for another 3 days, large number of osteoclast were formed in the presence of M-CSF. After adding various dose of Frl67653, even at the dose of 0.1M, osteoclast formation were inhibited. When increasing the dose of Frl67653 to 10M, the number of osteoclast decreased dramatically.2. Fr 167653 on osteoclast formation induced by PTHrPStimulating bone marrow cells by PTHrP directly leaded to osteoclast formation in vitro. After 6-day culture of bone marrow cells, large numbers of TRAP positive osteoclasts were formed. After adding different dose of Frl 67653, osteoclast formation was inhibited in a various degree. Osteoclast formation was inhibited at dose as low as 0.1M. At dose of 10M, osteoclast formation were almost abrogated.3. Frl67653 on OPG and RANKL gene expression by adherent bone marrow stromal cellsBone marrow cells were cultured for 10 days by change medium every two days. Nonadherent hematopoitic cells were removed by gently pipetting every time when changing medium. Adherent bone marrow cells were almost bone marrow stromal cells. RNA was extracted from bone marrow stromal cells, which were stimulated by PTHrP for 3 or 6 days. RNA was reverse- transcripted into cDNA and subjected to real-time PCR reaction as template. OPG and RANKL expression level were detected by real-time PCR. After stimulated by PTHrP for 3 and 6 days, OPG expression level were decreased in bone marrow stromal cells while RANKL expression level were upregulated. However, Frl 6765 3, even at dose of 10 u M, had no effect on OPG and RANKL expression. The results suggested that P38 MAPK-mediated-signals were not involved in the regulation of bone resorption-related functions of bone marrow stromal cells such as OPG and RANKL expression.4. Establishment of local bone resorption animal model induced by PTHrP9g PTHrP were injected 2 times daily on the calvarias of mice and mice were sacrificed at 8, 12, 24 hours after last injection at day 3 and 12 hours after last injection at day 5. Bone resorption occurred radiographically and histologically. However, bone reso...