Studies On The Mechanism Of Calcium Homeostasis And The Role Of L-type Calcium Channels In Fluoride-exposed Obs

Calcium homeostasis of osteoblasts (OBs) has an important role in the physiology andpathology of bone tissue. Beides, osteoblast L-type voltage-dependent calciumchannels (VDCC) play important roles in maintaining intracellular homeostasis andinfluencing multiple cellular processes. In particular, they contribute to the activitiesand functions of osteoblasts (OBs). In order to study mechanisms of intracellularcalcium homeostasis and to study how L-type VDCC modulate calcium ion (Ca2+)homeostasis and the expression of osteogenic transcription factors in OBs exposed tofluoride, MC3T3-E1cells and Sprague Dawley rats were treated with differentconcentrations of fluoride, especially MC3T3-E1cells were exposed to a gradient ofconcentrations of fluoride (0,2.0,5.0,10.0mg/L) in combination with10μMnifedipine, a specific inhibitor of VDCC, for48h. Then we examined mRNA andprotein levels of Cav1.2, the main subunit of L-type voltage-dependent calciumchannels (VDCC), Na+/Ca2+exchange carriers (NCS), and plasma membraneCa2+-ATPase (PMCA), inositol1,4,5-triphosphate receptor (IP3R) channels,sarco/endoplasmic reticulum calcium ATPase2b (SERCA2b)/ATP2A2as well asintracellular free calcium ion ([Ca2+]i) in both MC3T3-E1cells in vitro and rat bonetissues in vivo. Additionally, we examined mRNA and protein levels of Cav1.2, themain subunit of VDCC, and c-fos, c-jun, runt-related transcription factor2(Runx2),osterix (OSX) and intracellular free Ca2+([Ca2+]i) concentrations in MC3T3-E1cells.Our results showed that [Ca2+]i of fluoride-treated OBs increased in aconcentration-dependent manner with an increase in the concentration of fluoride. Wealso found that the low dose of fluoride led to high expression levels of Cav1.2, NCS-1, PMCA and low expression levels of IP3R and SERCA2b/ATP2A2, while thehigh dose of fluoride induced an increase in SERCA2b/ATP2A2levels and decreasein Cav1.2, PMCA, NCS-1, and IP3R levels. Meantime, results indicated that lowerconcentrations of fluoride (less than5mg/L, especially2mg/L) can lead to highexpression of Cav1.2and enhance osteogenic function, while high concentration offluoride (10mg/L) can induce decreased Cav1.2and osteogenic transcriptional factorsin MC3T3E1cells exposed to fluoride. However, the levels of [Ca2+]i, Cav1.2, c-fos,c-jun, Runx2, and OSX induced by fluoride were significantly altered and evenreversed in the presence of nifedipine. These results demonstrate that fluoride affectsintracellular calcium homeostasis by regulating Cav1.2, NCS-1, PMCA, IP3R andSERCA2b/ATP2A2expression. In other words, calcium channels and calcium pumpsof plasma and endoplasmic reticulum (ER) membranes keep intracellular calciumhomeostasis by increasing or decreasing the expression levels of Cav1.2, NCS-1,PMCA, IP3R and SERCA2b/ATP2A2in response to the different concentrations offluoride. And these results show that L-type calcium channels play a crucial role inCa2+homeostasis and they affect the expression of osteogenic transcription factors influoride-treated osteoblasts.