L-carnitine Affects The Osteoblastic Differentiation In NIH3T3 Fibroblasts

Ectopic calcification is defined as inappropriate deposition of calcium/phosphate complexes in connective tissues in aberrant locations. Pseudoxanthoma elasticum(PXE) is a prototype of multisystem ectopic mineralization disorders characterized by calcium phosphate deposition in various tissues. PXE is caused by mutations in the ABCC6 gene which encodes a putative transmembrane transporter protein, ABCC6. ABCC6-/- mouse which recapitulates the features of PXE, is a mouse model of PXE. Despite the molecular mechanisms underlying the regulation of ectopic calcification are unclear, however, some evidence had emerged in support of the concept that ectopic calcification is a cell regulated process. Fibroblasts are present in all the connective tissue, which are the main component of dense connective tissue. Studies have confirmed that when there was an appropriate stimulus conditions, fibroblasts could be transformed into osteoblasts, and finally formed mineralized nodules.Cellular metabolic activity plays an important role in regulating cell survival, differentiation and tissue growth. L-carnitine(LC) is an essential cofactor for the transport of long-chain fatty acids across the inner mitochondrial membrane into the mitochondrial matrix for their subsequent β-oxidation. Studies indicated that LC could affect osteoblastic activity. Our understanding of the effects of LC on the osteoblastic differentiation and mineralization has been advanced by our previous study on the metabonomics analysis of ABCC6-/- knock-out mice whose LC concentration was decreased in plasma. Therefore, we hypothesized that LC may have a negative impact on osteoblastic differentiation and mineralization. However, the mechanism by which LC may inhibit osteoblasts mineralization is not clear. Moreover, studies in animals and humans have shown that supplementation of LC increased plasma concentrations of IGF-1. Further studies in vivo and in vitro have suggested that proliferating and differentiating factors which affect osteoblastic activity exert their roles through the involvement of insulin-like growth factor(IGF) expression. IGF-1 is produced and stored in the bone matrix which stimulates proliferation and differentiation of osteoblasts. IGF-1 plays an important role in the activation of the IGF-1/PI3K/Akt signaling pathway. Considerable evidence collected in vitro and in vivo substantiated that the activation of IGF-1/PI3K/Akt pathway could effectively increase the osteoblasts differentiation and calcification. Although LC plays important roles in mineralization, the regulatory mechanism of LC function remains poorly understood.Objective: L-carnitine(LC) a trimethylated amino acids, is an essential cofactor for long-chain fatty acids for β-oxidation and affects osteoblastic activity. However, the molecular mechanisms of this process remain unclear. This study aimed to investigate the effects of LC on the proliferation and osteoblastic differentiation of NIH3T3 cells.Methods:(1) The study utilized the mouse embryonic fibroblast cells NIH3T3 to establish a cell model of ectopic calcification. NIH3T3 cells were cultured in Pi-inducing medium(growth medium supplemented with 2 mM Na3PO4) for 12 days, and then the mineralization was evaluated by alizarin red staining and calcium deposition analysis.(2) To observe the effects of LC on the Pi-induced osteogenic differentiation of NIH3T3 cells, NIH3T3 cells were cultured in Pi-inducing medium consisting of 10 μM and 100 μM LC. Cell proliferation was analyzed by MTT analysis.(3) The osteoblastic differentiation was analyzed at the m RNA level of osteogeenic marker genes(Runx2, ALP and OCN) by real-time PCR. The effects of LC on the osteogenic activities in NIH3T3 cells were determined by the ALP activity using ALP kit and the protein level by western blot analysis for Runx2. Finally, alizarin red staining and calcium deposition analysis for mineralization were measured.(4) The effects of LC on the IGF-1/PI3K/Akt signal pathway in NIH3T3 cells were determined by the mRNA expression of IGF-1 and the protein expression of phospho-Akt using real-time PCR and western blot analysis.Results:(1) Alizarin red staining showed that NIH3T3 cells induced by 2m M Na3PO4(Pi-induced group) produced mineralized nodules, while the control group had no mineralized nodules. To evaluated calcium deposition in the NIH3T3 cells matrix, quantitative assays of mineralization were carried out by cetylpyridium chloride analysis and calcium assay kit. The results showed that the Pi-induced group was much higher than the control.(2) The MTT assay showed that 10 μM LC increased the proliferation of NIH3T3 cells, but 100 μM LC slightly inhibited cell proliferation.(3) Compared with the control, the m RNA expression level of Runx2, ALP and OCN which regulate osteoblast differentiation were slightly upregulated in the 10 μM LC group, and downregulated in the 100 μM LC group. The ALP activity and the protein expression of Runx2 were also increased in 10 μM LC treated cells and decreased in 100 μM LC group. Moreover, the results of mineralized nodule formation and calcium deposition were consistent with the ALP activity and the protein expression of Runx2 showed above.(4) Compared with the control, 10 μM LC increased the mRNA expression of IGF-1, but the expression of p-Akt protein had no significant difference. However, 100 μM LC downregulated the mRNA expression of IGF-1 and significantly decreased the protein expression of p-Akt.Conclusions: Na3PO4 induced osteogenic differentiation and mineralization of NIH3T3 fibroblasts, so we successfully established a cell model of ectopic calcification. Low concentration of LC played a positive role in proliferation, osteoblast differentiation and mineralization, while the high concentration had an opposite effect. The inhibition of LC on the osteogenic differentiation and mineralization were consistent with our previous study on the metabonomics analysis of ABCC6 knock-out mice, so it indited that LC is a potential calcification or mineralization inhibitor. Moreover, the high concentration of LC downregulated the mRNA expression of IGF-1 and p-Akt protein which suggested that LC maybe exert its inhibitory effect through IGF-1/PI3K/Akt signal pathway.