A critical role of the KCa 3.1 channel in mechanical stretch-induced proliferation of rat bone marrow-derived mesenchymal stem cells

Abstract

Mechanical stimulation is an important factor regulating mesenchymal stem cell (MSC) functions such as proliferation. The Ca²⁺ -activated K⁺ channel, K_Ca 3.1, is critically engaged in MSC proliferation but its role in mechanical regulation of MSC proliferation remains unknown. Here, we examined the K_Ca 3.1 channel expression and its role in rat bone marrow-derived MSC (BMSC) proliferation in response to mechanical stretch. Application of mechanical stretch stimulated BMSC proliferation via promoting cell cycle progression. Such mechanical stimulation up-regulated the K_Ca 3.1 channel expression and pharmacological or genetic inhibition of the K_Ca 3.1 channel strongly suppressed stretch-induced increase in cell proliferation and cell cycle progression. These results support that the K_Ca 3.1 channel plays an important role in transducing mechanical forces to MSC proliferation. Our finding provides new mechanistic insights into how mechanical stimuli regulate MSC proliferation and also a viable bioengineering approach to improve MSC proliferation.

Keywords: KCa3.1 channel; bone marrow-derived mesenchymal stem cells; cell proliferation; mechanical stretch.

Figure 1

Effects of mechanical stretch on BMSC proliferation. A, summary of the effects of exposing BMSC to 2.5%‐15% mechanical stretch for 6, 12 and 24 h on cell proliferation relative to static control (SC). The mean data are from five independent experiments. *P < .05 and **P < .01 compared to SC using one‐way ANOVA and post hoc Fisher's test. B‐C, representative analysis of cell cycle distribution in cells under indicated conditions (B), and summary of the mean data from 4 independent experiments (C). Cells were fixed with 70% ethanol overnight, incubated in PBS staining solution (20 μg/mL propidium iodide, 100 μg/mL RNase A, and 0.1% Triton X‐100) at 37°C for 30 min and analysed by FACS on FL‐2 channel. The data were analysed using ModFit software

Figure 2

Effects of mechanical stretch on K_Ca3.1 expression and activity and the role of K_Ca3.1 channel in mechanical stimulation of BMSC proliferation. A‐D, effects of exposing BMSC to 2.5%‐15% mechanical stretch for 24 h on the K_Ca3.1 expression levels. A and C, representative results showing the K_Ca3.1 mRNA expression using RT‐PCR and K_Ca3.1 cell surface protein expression using flow cytometry. B and D, summary of the mean data as shown in (A) and (C), respectively, from six independent experiments. *P < .05 and **P < .01, using one‐way ANOVA and post hoc Fisher's test. E, summary of the I‐V relationship curves of the mean TRAM‐34 sensitive K⁺ current densities recorded from seven cells for each condition. Control, isotonic solution; Stretch, hypotonic solution. *P < .05 and **P < .01. Student's t test was used to compare the current density between control and stretch at the same potential. F‐K, summary of BMSC proliferation and cell cycle under indicated conditions after treatment with 100 nmol/L TRAM34 (F, H, J) or siRNA‐mediated knockdown of the K_Ca3.1 expression (G, I, K), from four independent experiments. *P < .05 and **P < .01, using one‐way ANOVA and post hoc Fisher's test