Enhancement of osteogenic differentiation of rat adipose tissue-derived mesenchymal stem cells by zinc sulphate under electromagnetic field via the PKA, ERK1/2 and Wnt/β-catenin signaling pathways

Abstract

Zinc ion as an essential trace element and electromagnetic fields (EMFs) has been reported to be involved in the regulation of bone metabolism. The aim of this study was to elucidate the effects of zinc sulphate (ZnSO4) on the osteogenic differentiation of adipose tissue-derived mesenchymal stem cells (ADSCs) in the presence of EMF as a strategy in osteoporosis therapy. Alkaline phophatase (ALP) activity measurement, calcium assay and expression of several osteoblastic marker genes were examined to assess the effect of ZnSO4 on the osteogenic differentiation of ADSCs under EMF. The expression of cAMP and PKA was evaluated by ELISA. The expression of β-catenin, Wnt1, Wnt3a, low-density lipoprotein receptor-related protein 5 (LRP5) and reduced dickkopf1 (DKK1) genes were used to detect the Wnt/β-catenin pathway. It was found that ZnSO4, in the presence of EMF, resulted in an increase in the expression of osteogenic genes, ALP activity and calcium levels. EMF, in the presence of ZnSO4, increased the cAMP level and protein kinase A (PKA) activity. Treatment of ADSCs with (MAPK)/ERK kinase 1/2 inhibitor, or PKA inhibitor, significantly inhibited the promotion of osteogenic markers, indicating that the induction of osteogenesis was dependent on the ERK and PKA signaling pathways. Real-time PCR analysis showed that ZnSO4, in the presence of EMF, increased the mRNA expressions of β-catenin, Wnt1, Wnt3a, LRP5 and DKK1. In this study, it was shown that 0.432 μg/ml ZnSO4, in the presence of 50 Hz, 20 mT EMF, induced the osteogenic differentiation of ADSCs via PKA, ERK1/2 and Wnt/β-catenin signaling pathways.

Sinusoidal Electromagnetic Fields Increase Peak Bone Mass in Rats by Activating Wnt10b/β-Catenin in Primary Cilia of Osteoblasts

“…There have been many clinical trials and experimental studies reporting that PEMFs modulate bone metabolism through the Wnt/β‐catenin pathway . However, few researchers have investigated the Wnt ligands involved in the process, except for Wnt1 and Wnt3a . In the present study, we did not find increased expression of Wnt3a in the bone samples of rats and in vitro cultured osteoblasts treated by SEMFs, but found the significantly increased expression of Wnt10b.…”

Underlying Signaling Pathways and Therapeutic Applications of Pulsed Electromagnetic Fields in Bone Repair

“…For instance, in vitro assay studies, gene and protein expressions of canonical Wnt/β-catenin signaling pathway, including Wnt1, LRP6, and β-catenin, were all significantly enhanced after PEMF exposure at both proliferation and differentiation stages of osteoblast-like MC3T3-E1 cells [31]. In addition, except the upregulation of mRNA expressions of Wnt1, Wnt3a, LRP5 and β-catenin in tissue derived mesenchymal stem cells (ADSCs), PEMFs intervention could also reduce the expression of dickkopf1 (DKK1) which usually acts as an inhibitor of Wnt signaling pathway [32]. Furthermore, the enhanced Wnt/β-catenin signaling induced by PEMFs notably elevated the expression of proliferation phase related target genes, Ccnd 1 and Ccne 1, and differentiation phase related genes, ALP, OCN, COL1, and Runx2, in osteoblast cells, which accelerated the osteoblasts proliferation, differentiation, and mineralization, three pivotal processes of bone formation [31, 32].…”

“…In addition, except the upregulation of mRNA expressions of Wnt1, Wnt3a, LRP5 and β-catenin in tissue derived mesenchymal stem cells (ADSCs), PEMFs intervention could also reduce the expression of dickkopf1 (DKK1) which usually acts as an inhibitor of Wnt signaling pathway [32]. Furthermore, the enhanced Wnt/β-catenin signaling induced by PEMFs notably elevated the expression of proliferation phase related target genes, Ccnd 1 and Ccne 1, and differentiation phase related genes, ALP, OCN, COL1, and Runx2, in osteoblast cells, which accelerated the osteoblasts proliferation, differentiation, and mineralization, three pivotal processes of bone formation [31, 32]. On the other hand, according to in vivo assay studies, PEMFs effectively reversed the bone mass loss and deterioration of bone microarchitecture analyzed by microCT and attenuated biomechanical strength deterioration evaluated by three-point bending test in hind limb-suspended ovariectomized rats through the Wnt/Lrp5/β-catenin signal pathway [33, 34], indicating that activating this pathway by PEMF exposure is beneficial for bone disorders.…”

“…When the cells were treated with U0126, an inhibitor of the ERK1/2 signaling cascade, the positive effects of the ELF-PEMF treatment on osteoblast function were abolished [36]. Other studies also revealed that the MEK/ERK signaling pathway regulated the promoting effects of PEMF on bone marrow mesenchymal stem cell (BMSC) proliferation, expression of osteogenic genes (RUNX2, BSP, OPN), ALP activity, and calcium deposition [22, 32, 37, 38]. Additionally, one study reported that the p38 MAPK pathway is involved in the increased production of collagen synthesis in osteoblast-like cells stimulated by ELF-EMF exposure [39].…”

“…The cAMP/PKA signaling pathway is another signaling involved in the PEMF-induced bone repair. Recent studies have demonstrated that PEMFs notably increased the cAMP level and PKA activity and accelerated the osteogenic differentiation of MSCs [32, 38, 74]. (Table 1).…”