Effect of inward rectifier potassium 2.1 channel on the osteogenic differentiation of human dental follicle cells and its mechanism

doi:10.7518/hxkq.2022.02.003

Abstract

Abstract: Objective

This study aims to explore the effect of inward rectifier potassium (Kir) 2.1 channel on the osteogenic differentiation of human dental follicle cells (hDFCs) and its mechanism.

Methods

hDFCs were isolated and cultured, and their source was verified by flow cytometry. Osteogenic differentiation ability of hDFCs was evaluated by osteogenic induction. Reverse-transcription polymerase chain reaction (RT-PCR) was performed to detect the gene expression of Kir2.1 gene (KCNJ2) in hDFCs. Real-time quantitative PCR (RT-qPCR) was performed to detect the expression of the Kir2.1 gene (KCNJ2) in hDFCs before and after osteogenic induction. Patch clamp technique was conducted to record the membrane potential changes of hDFCs before and after osteogenic induction. Moreover, the effect on the osteogenic differentiation of hDFCs was confirmed by increasing the concentration of extracellular potassium ions (50 mmol·L^-1). Kir2.1 channel blockers cesium chloride (CsCl) and C19H20CINO (ML133) were applied to determine the effect of the Kir2.1 potassium channel on the osteogenic differentiation of hDFCs. At the same time, RT-qPCR was used to observe the expression changes of osteogenic differentiation related genes Runx related transcription factor 2 (Runx2) and osteocalcin (OCN) before and after the two intervention measures. Calcium imaging was performed to observe the effect of membrane potential hyperpolarization caused by decreased extracellular potassium level (2 mmol·L^-1) on intracellular calcium concentration.

Results

RT-PCR results showed that hDFCs expressed the Kir2.1 channel gene (KCNJ2). The RT-qPCR results showed that the KCNJ2 gene expression in hDFCs was upregulated 7 days after osteogenic induction. The patch clamp results showed that the membrane potential of hDFCs hyperpolarized to (-47±5.2) mV from (-12±3.2) mV. Alizarin red and alkaline phosphatase staining results showed that increasing the concentration of the extracellular potassium or blocking the function of the Kir2.1 channel significantly inhibited the osteogenic mineralization ability of hDFCs. The membrane potential hyperpolarization increased the intracellular calcium concentration in hDFCs.

Conclusion

Membrane potential hyperpolarization mediated by the Kir2.1 channel plays an important role in the osteogenic differentiation of hDFCs.

Key words: human dental follicle cell, inward rectifier potassium 2.1 channel, osteogenic differentiation, membrane potential hyperpolarization

CLC Number:

R 781.4

Zhang Peng, Zuo Dongchuan, Mou Siyu, Zhong Yutong, Yuan Xiaoping, Zeng Jin. Effect of inward rectifier potassium 2.1 channel on the osteogenic differentiation of human dental follicle cells and its mechanism[J]. West China Journal of Stomatology, 2022, 40(2): 139-147.

Figures/Tables 11

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References 23

1	Ercal P, Pekozer GG, Kose GT. Dental stem cells in bone tissue engineering: current overview and challenges[J]. Adv Exp Med Biol, 2018, 1107: 113-127.
2	Dave JR, Tomar GB. Dental tissue-derived mesenchymal stem cells: applications in tissue engineering[J]. Crit Rev Biomed Eng, 2018, 46(5): 429-468.
3	Matichescu A, Ardelean LC, Rusu LC, et al. Advanced biomaterials and techniques for oral tissue engineering and regeneration-a review[J]. Materials (Basel), 2020, 13(22): 5303.
4	Zhou T, Pan JH, Wu PY, et al. Dental follicle cells: roles in development and beyond[J]. Stem Cells Int, 2019, 2019: 9159605.
5	Morsczeck C, Reichert TE. Dental stem cells in tooth regeneration and repair in the future[J]. Expert Opin Biol Ther, 2018, 18(2): 187-196.
6	Hibino H, Inanobe A, Furutani K, et al. Inwardly rectifying potassium channels: their structure, function, and physiological roles[J]. Physiol Rev, 2010, 90(1): 291-366.
7	Bouchard R, Clark RB, Juhasz AE, et al. Changes in extracellular K⁺ concentration modulate contractility of rat and rabbit cardiac myocytes via the inward rectifier K⁺ current IK₁ [J]. J Physiol, 2004, 556(Pt 3): 773-790.
8	Zhang XY, Cui XD, Li X, et al. Inhibition of Kir2.1 channel-induced depolarization promotes cell biological activity and differentiation by modulating autophagy in late endothelial progenitor cells[J]. J Mol Cell Cardiol, 2019, 127: 57-66.
9	Komarova SV, Dixon SJ, Sims SM. Osteoclast ion channels: potential targets for antiresorptive drugs[J]. Curr Pharm Des, 2001, 7(8): 637-654.
10	Weidema AF, Dixon SJ, Sims SM. Electrophysiological characterization of ion channels in osteoclasts isolated from human deciduous teeth[J]. Bone, 2000, 27(1): 5-11.
11	Wang SP, Wang JA, Luo RH, et al. Potassium channel currents in rat mesenchymal stem cells and their possible roles in cell proliferation[J]. Clin Exp Pharmacol Ph-ysiol, 2008, 35(9): 1077-1184.
12	Park KS, Jung KH, Kim SH, et al. Functional expression of ion channels in mesenchymal stem cells derived from umbilical cord vein[J]. Stem Cells, 2007, 25(8): 2044-2052.
13	Morsczeck C, Götz W, Schierholz J, et al. Isolation of precursor cells (PCs) from human dental follicle of wisdom teeth[J]. Matrix Biol, 2005, 24(2): 155-165.
14	Huang YY, Wu S, Zhou Y, et al. The effects of extracts from “Red Complex” pathogens on human dental follicle cells[J]. J Biomater Tis Eng, 2020, 10(8): 1128-1134.
15	Kirkham GR, Elliot KJ, Keramane A, et al. Hyperpolarization of human mesenchymal stem cells in response to magnetic force[J]. IEEE Trans Nanobioscience, 2010, 9(1): 71-74.
16	Sundelacruz S, Levin M, Kaplan DL. Membrane potential controls adipogenic and osteogenic differentiation of mesenchymal stem cells[J]. PLoS One, 2008, 3(11): e-3737.
17	Bhavsar MB, Cato G, Hauschild A, et al. Membrane potential (V_mem) measurements during mesenchymal stem cell (MSC) proliferation and osteogenic differentiation[J]. PeerJ, 2019, 7: e6341.
18	Kito H, Yamazaki D, Ohya S, et al. Up-regulation of K_ir2.1 by ER stress facilitates cell death of brain capillary endothelial cells[J]. Biochem Biophys Res Commun, 2011, 411(2): 293-298.
19	Gattlen C, Deftu AF, Tonello R, et al. The inhibition of Kir2.1 potassium channels depolarizes spinal microglial cells, reduces their proliferation, and attenuates neuropathic pain[J]. Glia, 2020, 68(10): 2119-2135.
20	Pini J, Giuliano S, Matonti J, et al. Osteogenic and chondrogenic master genes expression is dependent on the Kir2.1 potassium channel through the bone morphogenetic protein pathway[J]. J Bone Miner Res, 2018, 33(10): 1826-1841.
21	Pchelintseva E, Djamgoz MBA. Mesenchymal stem cell differentiation: control by calcium-activated potassium channels[J]. J Cell Physiol, 2018, 233(5): 3755-3768.
22	Aydin S, Şahin F. Stem cells derived from dental tissues[J]. Adv Exp Med Biol, 2019, 1144: 123-132.
23	Sacco S, Giuliano S, Sacconi S, et al. The inward rectifier potassium channel Kir2.1 is required for osteoblastogenesis[J]. Hum Mol Genet, 2015, 24(2): 471-479.

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