Novel Function of Tetrahydrobiopterin in Osteoclast Formation in RAW264.7 Cells via the NFκB Pathway

Abstract

Tetrahydrobiopterin (BH4) serves as a cofactor for the activation of tryptophan hydroxylase and nitric oxide (NO) synthase (NOS), which facilitate serotonin synthesis and NO production, respectively. BH4 is rapidly generated from dihydrobiopterin (BH2) and sepiapterin (SP) via intricate enzymatic reactions in the cytoplasm. Recent studies have suggested that serotonin and NO are involved in osteoclast-related bone resorption; however, the precise role of BH4 in osteoclastogenesis remains elusive. To elucidate the effects of BH4, BH2, and SPon RANKL-induced osteoclastogenesis, a comparative study was conducted using macrophage-like RAW264.7 cells. Tartrate-resistant acid phosphatase staining revealed that BH4, BH2, and SP enhanced RANKL-induced multinucleated osteoclast formation. Notably, the intracellular BH4 concentration was significantly elevated in cells treated with SP compared with those treated with BH2 and BH4. Furthermore, N-acetylserotonin, an inhibitor of the conversion of SP to BH4, was used to reduce SP-induced osteoclast formation. Additionally, BH4, BH2, and SP increased the mRNA expression levels of osteoclast markers, including OC- and DC-STAMPs, Cath K, CTR, RANK, Siglec-15, NFκB, and NFATc1, in RANKL-treated cells. However, three distinct NOS inhibitors demonstrated insufficient efficacy in preventing BH4-induced osteoclast differentiation in RANKL-treated cells. Given that BH4, BH2, and SP upregulated the expression of p65, a component of NFκB, but not p52, their potential roles in promoting osteoclast formation in RANKL-treated 264.7 cells may be attributed to the NFκB pathway rather than the autocrine action of BH4-induced serotonin and NO.