Abstract
It has been suggested that mechanical strain may elicit cell differentiation in adult somatic cells through activation of epithelial sodium channels (ENaC). However, such phenomenon has not been previously demonstrated in mesenchymal stromal cells (MSCs). The present study was thus conducted to investigate the role of ENaC in human bone marrow-derived MSCs (hMSCs) tenogenic differentiation during uniaxial tensile loading. Passaged-2 hMSCs were seeded onto silicone chambers coated with collagen I and subjected to stretching at 1 Hz frequency and 8% strain for 6, 24, 48, and 72 hours. Analyses at these time points included cell morphology and alignment observation, immunocytochemistry and immunofluorescence staining (collagen I, collagen III, fibronectin, and N-cadherin), and gene expression (ENaC subunits, and tenogenic markers). Unstrained cells at similar time points served as the control group. To demonstrate the involvement of ENaC in the differentiation process, an ENaC blocker (benzamil) was used and the results were compared to the noninhibited hMSCs. ENaC subunits' (α, β, γ, and δ) expression was observed in hMSCs, although only α subunit was significantly increased during stretching. An increase in tenogenic genes' (collagen1, collagen3, decorin, tenascin-c, scleraxis, and tenomodulin) and proteins' (collagen I, collagen III, fibronectin, and N-cadherin) expression suggests that hMSCs underwent tenogenic differentiation when subjected to uniaxial loading. Inhibition of ENaC function resulted in decreased expression of these markers, thereby suggesting that ENaC plays a vital role in tenogenic differentiation of hMSCs during mechanical loading.
Highlights
Ion channels have been regarded as an important mediator for a multitude of physiological processes including muscle contraction, synaptic transmission, immune regulation, and many others [1, 2]
Several studies have reported that ion channels residing in the plasma membrane of chondrocytes and osteoblasts are involved in the transduction of mechanical signals [7,8,9]
Semiquantitative PCR was performed to identify the presence of α, β, γ, and δ subunits of ENaC, and it was found that all four subunits are expressed in human bone marrow-derived MSCs (hMSCs) (see Figure 2(a))
Summary
Ion channels have been regarded as an important mediator for a multitude of physiological processes including muscle contraction, synaptic transmission, immune regulation, and many others [1, 2]. Whilst many of the common ion channels have been extensively investigated, the role of the less common ones has been underrated. This has led to the lack of understanding of the mechanism regulating specific cellular function involving these channels such as the signalling process in response to mechanical stimuli. Several studies have reported that ion channels residing in the plasma membrane of chondrocytes and osteoblasts are involved in the transduction of mechanical signals [7,8,9]. The existence of ENaC in load-bearing cells suggests that ENaC has a mechanoactive role in cellular signalling. Such signalling processes are deemed important for cellular
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