Toll like receptor 2 and Toll like receptor 4 activation and vitamin D effects in mouse osteoblasts

The effects of vitamin D on bone health have been widely described, but the precise direct actions of vitamin D on bone forming osteoblasts have yet to be fully elucidated. Vitamin D can act to promote innate immune responses. Non-classical vitamin D functions are described in monocytes and macrophages. Stress and damage signals, as well as infection, may influence bone function and fracture healing. The current PhD project hypothesizes that innate immune response to vitamin D contributes to its effects on bone. Mouse osteoblasts obtained from primary calvaria and MC3T3-E1 cell lines were used and differentiated for 5 days, 14 days and 21 days to represent different stages of osteoblast differentiation. Cells were treated with vitamin D metabolites 25(OH)D 3 and 1,25(OH)2D3 to observe vitamin D effects on differentiated osteoblasts. Vitamin D metabolites did not alter vitamin D metabolism genes Vdr and Cyp27b1 in differentiation osteoblasts. In addition, LPS as Toll-like receptor 4 agonist and Pam3CysK as Toll-like receptor 2 agonist were stimulated in differentiated osteoblasts to evaluate osteoblastic responses. Western blot and RT-PCR analyses confirmed expression of TLR2 and TLR4 and TLR4-specific signaling factors TRIF at all stages of differentiation. Mouse osteoblasts displayed divergent responses to LPS and Pam3CysK stimulations in osteoblast differentiation markers and osteoclastogenesis markers. Although both LPS and Pam3CysK promote inflammatory cytokines, only Pam3CysK stimulation demonstrated potent modulation to promote osteoclastogenesis through increased expression of Rankl and suppression of Ocn. TLR2 and TLR4 share an MyD88 signaling pathway to induce inflammation and osteoclastogeneis, but only TLR4 can signal via the intracellular TRIF/TRAM pathway. TrifsiRNA was introduced to 14 days differentiated MC3T3-E1 cells. RT-PCR analysis showed suppression of Vdr, Alp, Opg and Il-6 by Trif siRNA. In addition, TLRs signaling activates MAPK phosphorylation, Pam3CysK induced higher phosphorylation of p38 MAPK and inhibition of p38 by TLCK (NF-kappaB inhibitor) neutralized suppression of osteoblast differentiation markers by Pam3CysK. In conclusion, TLR2 and TLR4 stimulation showed differential effects in osteoblasts possibly due to distinct MAPK phosphorylation pattern.