Phytocomponent p-hydroxycinnamic acid inhibits osteoclast-like cell formation in mouse bone marrow cultures

The phytocomponent p-hydroxycinnamic acid (HCA) has been shown to have inhibitory effects on bone-resorbing factor-stimulated bone resorption in rat femoral tissues in vitro. The effects of HCA on osteoclast-like cell formation in mouse bone marrow cultures in vitro were investigated. The bone marrow cells were cultured for 7 days in α-minimal essential medium containing a bone-resorbing agent [parathyroid hormone (1-34)] (PTH), prostaglandin E2 (PGE2), or tumor necrosis factor-α (TNF-α) in effective concentrations. Osteoclast-like cell formation was estimated by staining for tartrate-resistant acid phosphatase, a marker enzyme of osteoclasts. The presence of PTH (10−7 M), PGE2 (10−5 M), or TNF-α (10 ng/ml) induced a remarkable increase in osteoclast-like multinucleated cells. These increases were significantly inhibited in the presence of HCA (10−8-10−5 M). HCA (10−6 or 10−5 M) significantly inhibited osteoclast-like cell formation induced by dibutyryl cyclic adenosine monophosphate (10−5 M) or phorbol 12-myristate 13-acetate (10−6 M), an activator of protein kinase C. Also, HCA (10−8-10−5 M) had a significant inhibitory effect on osteoclast-like cell formation induced by the receptor activator of NF-κB ligand (RANKL) (10 ng/ml) in the presence of macrophage colony-stimulating factor (M-CSF) (10 ng/ml). The inhibitory effect of HCA (10−6 or 10−5 M) on RANKL plus M-CSF-induced osteoclast-like cell formation was not observed in the presence of cycloheximide (10−7 M), an inhibitor of protein synthesis in the transcriptional process, or 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (10−6 M), an inhibitor of transcription. This study demonstrates that HCA has a potent inhibitory effect on osteoclast-like cell formation in mouse bone marrow cultures. The inhibitory action of HCA may partly involve a newly synthesized protein component which is related to RANKL stimulation in osteoclastogenesis.