ClC-3 chloride channel mediates the role of parathyroid hormone [1-34] on osteogenic differentiation of osteoblasts

Abstract

IntroductionDifferent concentrations of parathyroid hormone [1–34] (PTH [1–34]) can have totally opposite effects on osteoblasts. Intermittent stimulation with PTH can significantly increase bone mineral density in vitro, mainly through the protein kinase A (PKA) signaling pathway, which phosphorylates runt-related transcription factor 2 (Runx2). The ClC-3 chloride channel, an important anion channel, can also promote osteogenesis via the Runx2 pathway based on recent studies. The purpose of our study, therefore, is to research whether the ClC-3 chloride channel has an effect on PTH osteodifferentiation in MC3T3-E1 cells.Methods and resultsA cell counting kit (CCK-8) and real-time PCR were used to investigate the impact of different PTH stimulation modes on MC3T3-E1 cell proliferation and osteogenesis-related gene expression, respectively. We found that the minimum inhibitory concentration of PTH was 10−9 M, and the expression of alkaline phosphatase (Alpl) and Runx2 were at the highest levels when treated with 10−9 M PTH. Next, we used real-time PCR and immunofluorescence technique to detect changes in ClC-3 in MC3T3-E1 cells under PTH treatment. The results showed higher expression of the ClC-3 chloride channel at 10−9 M intermittent PTH administration than in the other groups. Finally, we used the ClC-3 siRNA technique to examine the role of the ClC-3 chloride channel in the effect of PTH on the osteogenesis of osteoblasts, and we found an obvious decrease in the expression of bone sialoprotein (Ibsp), osteocalcin (Bglap), osterix (Sp7), Alpl and Runx2, the formation of mineralization nodules as well.ConclusionsFrom the above data, we conclude that the expression of ClC-3 chloride channels in osteoblasts helps them respond to PTH stimulation, which mediates osteogenic differentiation.

Cannabidiol induces osteoblast differentiation via angiopoietin1 and p38 MAPK

“…After mineralization in bone matrix, deposition of calcium around bone tissue can be detected by calcium staining experiments, including Alizarin Red S 7 . The mineralization of bone matrix is mainly promoted by induction of collagen type I alpha 1 chain (COL1A1), 8 osteocalcin (OC), 9 osteopontin (OPN), 10 bone sialoprotein (BSP), 11 transforming growth factors‐β (TGF‐β), 12 and bone morphogenic proteins (BMPs) 13 . Moreover, Runt‐related transcription factor 2 (RUNX2), 14 osterix (OSX), 15 distal‐less homeobox 5 (DLX5), 16 and β‐catenin 17 are known to play a pivotal role in osteoblast differentiation.…”

The role of the Smad2/3/4 signaling pathway in osteogenic differentiation regulation by ClC-3 chloride channels in MC3T3-E1 cells

“…Notably, when we knocked down the expression of ClC-3 and Smad2/3/4 together, we found that ALP activity and Runx2 and Osx levels were also decreased, but the extent was less pronounced than the effects of their inhibition alone, except for the Collagen I protein. This is probably related to the fact that Collagen I is a major component of the matrix and regulates the adhesion and bone-guiding function of mature osteoblasts after differentiation,; nevertheless, the ClC-3 chloride channel is mainly involved in regulating the osteoblast differentiation process [ 7 , 37 ].…”

“…Recent studies have found that ClC-3 chloride channels are closely related to bone metabolism. Clcn3-knockout mice displayed complex genetic phenotypes, including stunting, kyphosis, and abnormalities in calcium and phosphorous metabolism [ 6 , 7 ]. Furthermore, we discovered that ClC-3 chloride channels are expressed by osteogenic lineage cells and regulate osteoblast differentiation.…”

The Mechanism of Bone Remodeling After Bone Aging

“…On the one hand, the expression of the ClC-3 chloride channel in osteoblasts is conducive to the stimulation of PTH by osteoblasts, thus mediating the differentiation of osteoblasts. 76 The ClC-3 chloride channel regulates the bone formation function of osteoblasts by regulating the estrogen a receptor. 77 , 78 The increased expression levels of ClC-3 chlorine channel proteins can upregulate the expression of the downstream Runx2 gene, thus promoting osteogenic differentiation.…”