Inhibitory effect of β-cryptoxanthin on osteoclast-like cell formation in mouse marrow cultures

Abstract

The carotenoid β-cryptoxanthin has been shown to have an inhibitory effect on bone-resorping factor-stimulated bone resorption in rat bone tissues in vitro. The effect of β-cryptoxanthin on osteoclast-like cell formation in mouse marrow culture in vitro was 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 E 2, 1,25-dihydroxyvitamin D 3, lipopolysaccharide, or tumor necrosis factor-α (TNFα)] with an effective concentration. 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), prostaglandin E 2 (10 −5 M), 1,25-dihydroxyvitamin D 3 (10 −7 M), lipopolysaccharide (10 μg/mL), or TNFα (10 ng/mL) induced a remarkable increase in osteoclast-like multinucleated cells. These increases were significantly inhibited in the presence of β-cryptoxanthin (10 −8 to 10 −6 M). β-Cryptoxanthin (10 −7 and 10 −6 M) significantly inhibited dibutyryl cyclic adenosine monophosphate (DcAMP) (10 −5 M) or phorbol 12-myristate 13-acetate (PMA) (10 −5 M), an activator of protein kinase C, induced osteoclast-like cell formation. Also, β-cryptoxanthin (10 −7 and 10 −6 M) had a significant inhibitory effect on osteoclast-like formation induced by receptor activator of NF-κB ligand (RANKL) (10 and 20 ng/mL) in the presence of macrophage colony-stimulating factor (M-CSF) (10 and 20 ng/mL). The stimulatory effect of RANKL and M-CSF on osteoclast-like cell formation was significantly enhanced in the presence of PMA, while such an effect was not seen by DcAMP. β-Cryptoxanthin (10 −6 M) significantly inhibited osteoclast-like cell formation induced by RANKL and M-CSF in the presence of PMA or DcAMP. Moreover, the inhibitory effect of β-cryptoxanthin on RANKL plus M-CSF-, PTH-, or TNFα-induced osteoclast-like cell formation was not observed in the presence of cycloheximide (10 −7 M), an inhibitor of protein synthesis at translational process, or 5,6-dichloro-1-β- d-ribofuranosylbenzimidazole (10 −6 M), an inhibitor of transcription. This study demonstrates that β-cryptoxanthin has a potent inhibitory effect on osteoclast-like cell formation in mouse marrow culture. The inhibitory action of β-cryptoxanthin may partly involve in a newly synthesized protein component which is related to RANKL stimulation in osteoclastogenesis.