Acetylcholine induces mesenchymal stem cell migration via Ca2+ /PKC/ERK1/2 signal pathway

Abstract

Acetylcholine (ACh) plays an important role in neural and non-neural function, but its role in mesenchymal stem cell (MSC) migration remains to be determined. In the present study, we have found that ACh induces MSC migration via muscarinic acetylcholine receptors (mAChRs). Among several mAChRs, MSCs express mAChR subtype 1 (m1AChR). ACh induces MSC migration via interaction with mAChR1. MEK1/2 inhibitor PD98059 blocks ERK1/2 phosphorylation while partially inhibiting the ACh-induced MSC migration. InsP3Rs inhibitor 2-APB that inhibits MAPK/ERK phosphorylation completely blocks ACh-mediated MSC migration. Interestingly, intracellular Ca(2+) ATPase-specific inhibitor thapsigargin also completely blocks ACh-induced MSC migration through the depletion of intracellular Ca(2+) storage. PKCα or PKCβ inhibitor or their siRNAs only partially inhibit ACh-induced MSC migration, but PKC-ζ siRNA completely inhibits ACh-induced MSC migration via blocking ERK1/2 phosphorylation. These results indicate that ACh induces MSC migration via Ca(2+), PKC, and ERK1/2 signal pathways.

Fig 1. MSC phenotype characterization

Flow cytometric analysis showed that MSC expressed CD34 (0.74%) (A), CD45 (0.19%) (B), CD90 (92%) (B), and CD29 (91%) (D). (E–F) MSC differentiation in special culture conditions in vitro. (E) Osteocyte differentiation as shown by von Kossa staining. (F) Adipogenic differentiation as shown by Oil-O-Red staining.

Fig 2. mAChR expression in MSC

(A) m1AChR mRNA expression in MSC was detected by RT-PCR. (B) m1AChR expression was detected by immunostaining of MSC. (C) Flow cytometric analysis of m1AChR expression in MSCs. (D) L-Ca²⁺ channel, N-Ca²⁺ channel, and InsP3Rs expression in MSC was detected by RT-PCR. (E) RYRs expression in MSC was detected by RT-PCR.

Fig 3. The effect of ACh on MSC proliferation and migration

(A, B) ACh had no effect on MSC proliferation, * p>0.05 vs Ctrl. (C) Ach induced MSC migration in a dose-dependent manner, * p<0.05 vs Ctrl. (D) ACh induced MSC migration in a time-dependent manner, ^* p<0.05 vs Ctrl in 12 h, ^# p<0.05 vs Ctrl in 24 h. (E). ACh-induced MSC migration was inhibited by atropine, a mAChR blocker, ^# p<0.05 vs ACh (10⁻⁶ M).

Fig 4. MSC migration determined by wound healing assay

(A) 2×10⁵ cells were seeded into 24-well plates coated with 10 mg/ml fibronectin. Monolayer cell wounds were produced by CytoSelect^™ insert. 10⁻⁵~10⁻⁹ M of ACh was added to 15% FCS-containing DMEM. MSC migration was photographed at the times indicated. * p<0.05 vs ACh (0, 10⁻⁷, 10⁻⁸, or, 10⁻⁹ M). ^# p<0.05 vs ACh (10⁻⁶ M). (B) MSCs were treated with ACh (10⁻⁶ M) for 0, 6, 12, and 24 h, and scrape wound healing assays were performed as described in A. * p<0.05 vs Ctrl in 6 h, ^& p<0.05 vs Ctrl in 12 h, ^# p<0.05 vs Ctrl in 24 h. (C) MSCs were treated with ACh (10⁻⁶M) with or without atropine treatment (10⁻⁴ M) for 12 h. Scrape wound healing assays were performed. ^*p<0.05. vs. ACh (10⁻⁶ M).

Fig 5. ACh promoted MSC migration via activation of ERK1/2

(A) ACh Activated ERK1/2 via mAChR. Upper panel: western blot detection of ERK1/2 phosphorylation (pERK1/2) and ERK1/2 expression. α-tubulin served as an internal control. Lower panel: Quantification of phosphorylated ERK1/2. *^{, #}P<0.05 vs ACh (10⁻⁶ M) (n=5). (B) ERK1/2 is essential for ACh-induced MSC migration. *p<0.05 vs ACh (10⁻⁶ M), n=5.

Fig 6. ACh promoted MSC migration via activation of PKC signaling

(A) ACh Activated PKCα via mAChR. PKC phosphorylation and expression were determined by western blot. α-tubulin served as an internal control. (B) Quantification of phosphorylated PKCα. *^{, #} P < 0.05 vs ACh (10⁻⁶ M), n=5. (C) PKCα/PKCβ selective inhibitor Gö-6976 (6 nM) blocked ACh-induced MSC migration. *^{, #} P<0.05 vs ACh, n=5. (D) siRNA blockade of PKCα, PKCβ, or PKCζ inhibited ACh-induced MSC migration. *^{, #, &} P<0.05 vs ACh (10⁻⁶ M), n=5.

Fig 7. Role of Calcium in ACh-induced MSC migration and PKC and ERK1/2 phosphorylation

(A) Extracellular Ca²⁺ in ACh-induced MSC migration, *P<0.05 vs PBS; ^#P<0.05 vs PBS+ACh. (B) ACh-induced MSC migration required Ca²⁺ release mediated by InsP3Rs. *P> 0.05 vs ACh (10⁻⁶ M); ^{#, &, $, @}P<0.05 vs ACh (10⁻⁶ M). (C–D) Role of Calcium in ACh-induced PKC and ERK1/2 phosphorylation. Upper panel: phosphorylated and total PKC (C) or ERK1/2 (D) was detected by western blot. α-tubulin served as an internal control. Lower panel: Quantification of phosphorylated PKC or ERK1/2. *^{, #}P<0.05 vs ACh (10⁻⁶ M), n=5.

Fig 8. Role of PKC in ACh-induced ERK1/2 phosphorylation

(A–D) Western blot of ERK1/2 phosphorylation (pERK1/2) and expression (tERK1/2), α-tubulin served as an internal control. *^{, #, &}P < 0.05 vs ACh (10⁻⁶ M). in each panel, n = 5.