Levo-Tryptophan Promotes Osteogenesis Through Calcium-Sensing Receptor

Abstract

Previous studies reported the pro-osteogenic ability of L-Tryptophan (L-Trp) and Calcium-Sensing RCeceptor (CaSR) respectively. Recent researchers found L-Trp could activate CaSR. Therefore, this study investigated the osteogenic mechanisms of L-Trp through CaSR activation. Using in vivo and in vitro models, we evaluated L-Trp's effects on bone formation and osteoblast activity. Levo-Trp solution was injected into the temporomandibular joint of 3-week-old mice, and the mandibular development was observed by Micro-CT at 6 weeks of age. The pre-osteoblast cell line MC3T3-E1 cells were stimulated by L-Trp in vitro, and their proliferation, migration, and osteogenic ability were detected by CCK8 assay, alizarin red staining, etc. Transcriptome sequencing was used to investigate the underlying mechanism of L-Trp stimulation and validated by qPCR and Western blot analyses. Local injection of 0.5% L-Trp in juvenile mice significantly increased mandibular bone mineral density. In vitro, L-Trp enhanced MC3T3-E1 pre-osteoblast proliferation, migration, and differentiation, with upregulated osteogenic markers (Runx2, Sp7, Alp) and mineralization. CaSR antagonism (NPS-2143) abolished these effects, confirming CaSR's pivotal role. Transcriptome sequencing revealed L-Trp activation of the focal adhesion pathway, characterized by increased Ptk2, Rhoa, Itga11, and Clec11a expression. These findings established L-Trp as a CaSR-dependent osteogenic enhancer, mediated via the focal adhesion pathway.