Analysis of Signal Transduction Pathways Downstream M2 Receptor Activation: Effects on Schwann Cell Migration and Morphology

Abstract

Background: Schwann cells (SCs) express cholinergic receptors, suggesting a role of cholinergic signaling in the control of SC proliferation, differentiation and/or myelination. Our previous studies largely demonstrated that the pharmacological activation of the M2 muscarinic receptor subtype caused an inhibition of cell proliferation and promoted the expression of pro-myelinating differentiation genes. In order to elucidate the molecular signaling activated downstream the M2 receptor activation, in the present study we investigated the signal transduction pathways activated by the M2 orthosteric agonist arecaidine propargyl ester (APE) in SCs.

Methods: Using Western blot we analyzed some components of the noncanonical pathways involving β1-arrestin and PI3K/AKT/mTORC1 signaling. A wound healing assay was used to evaluate SC migration.

Results: Our results demonstrated that M2 receptor activation negatively modulated the PI3K/Akt/mTORC1 axis, possibly through β1-arrestin downregulation. The involvement of the mTORC1 complex was also supported by the decreased expression of its specific target p-p70 S6KThr389. Then, we also analyzed the expression of p-AMPKαthr172, a negative regulator of myelination that resulted in reduced levels after M2 agonist treatment. The analysis of cell migration and morphology allowed us to demonstrate that M2 receptor activation caused an arrest of SC migration and modified cell morphology probably by the modulation of β1-arrestin/cofilin-1 and PKCα expression, respectively.

Conclusions: The data obtained demonstrated that M2 receptor activation in addition to the canonical Gi protein-coupled pathway modulates noncanonical pathways involving the mTORC1 complex and other kinases whose activation may contribute to the inhibition of SC proliferation and migration and address SC differentiation.

Keywords: AMPK; M2 muscarinic receptor; Schwann cells; cell migration; mTOR pathway; β1-arrestin.

Figure 1

Western blot analysis of several signal transduction effectors upon 48 h of 100 μM APE treatment. (A) ${\mathsf{\beta }}_1$-arrestin expression. β-Actin was used as reference protein. The graph shows the densitometric analysis of the bands normalized with the reference protein β-Actin. (B) ${\mathrm{PI}3\mathrm{Kinase}}^{p85}$ expression. β-Actin was used as reference protein. The graph shows the densitometric analysis of the bands normalized with the bands of the reference protein β-Actin. (C) $AK{T}^{Thr 308}$ expression. AKT (pan) and β-Actin were used as reference protein. The graph shows the densitometric analysis of the bands normalized with the bands of the reference protein AKT (pan). (D) $\mathrm{p-p}70\mathrm{S}6{\mathrm{K}}^{Thr389}$ expression. β-Actin was used as reference protein. The graph shows the densitometric analysis of the bands normalized with the bands of the reference protein β-Actin. (E) $AK{T}^{ser473}$ expression. AKT (pan) and β-Actin were used as reference proteins. The graph shows the densitometric analysis of the bands of Western blot analysis for $AK{T}^{ser473}$ normalized with the bands of the reference protein AKT (pan). (F) $\mathrm{p-}{\mathrm{AMPK}\mathsf{\alpha }}^{thr172}$ expression. β-Actin was used as reference protein. The graph shows the densitometric analysis of the bands of Western blot analysis for $\mathrm{p-}{\mathrm{AMPK}\mathsf{\alpha }}^{thr172}$ normalized with the bands of the reference protein β-Actin. All the data are the average ± SEM of three independent experiments. Student’s t-test was used (* p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 2

Analysis of SC migration. (A) The distance of the gap between two fronts was measured at two different time points (t0–t6); (scale bar = 100 µm); (B) M2 activation by the selective agonist APE 100 μM was able to reduce SC migration; this effect was counteracted by the M2 antagonist methoctramine 0.1 μM (**** p < 0.0001); (C) treatment with Muscarine 100 μM, a nonselective muscarinic receptor agonist, promoted cell migration compared to the control (**** p < 0.0001 Ctrl vs. Muscaine); treatment with Muscarine 100 μM + Pirenzepine 0.1 μM (M1 muscarinic receptor antagonist) + 4-DAMP 0.01 μM (M3 muscarinic receptor antagonist) caused a decrease in cell migration, (**** p < 0.0001 Muscarine 100 μM vs. Muscarine 100 μM + Pirenzepine 0.1 μM +4-DAMP 0.01 μM); treatment with Muscarine 100 μM + Methoctramine 0.1 μM (M2 muscarinic receptor antagonist) was able to promote cell migration, inhibiting the M2 effect, (* p < 0.05 Muscarine 100 μM vs. Muscarine 100 μM + Methoctramine 0.1 μM). The data obtained are the average ± SEM of at least three independent experiments carried out in triplicate. (D) α-cofilin-1 expression by Western blot analysis in Schwann cells after 48 h of 100 μM APE treatment. β-Actin was used as the internal reference protein. The graph shows the densitometric analysis of the bands of Western blot analysis for α-cofilin-1 normalized with β-Actin. The data are the average ± SEM of three independent experiments. Student’s t-test was used * p < 0.05.

Figure 3

(A) PKCα kinase expression by Western blot analysis in Schwann cells after 48 h of 100 μM APE treatment. β-Actin was used as reference protein. The graph shows the densitometric analysis of the bands of Western blot analysis for PKCα kinase normalized with the bands of β-Actin used as the reference protein. The data are the average ± SEM of three independent experiments. Student’s t-test was used, * p < 0.05. (B) Immunocytochemistry for α-tubulin and GFAP in control condition (10% FBS + 2 μM fsk and 10 ng/mL Neuregulin-1), after 100 μM APE; 1 μM Chelerythrine Chloride; 100 μM APE plus 1 μM Chelerythrine Chloride (scale bar = 20 μm). SCs in control condition showed a bipolar elongated morphology, compatible with the immature phenotype; SCs after APE 100 μM showed a rounded morphology; SCs after co-treatment with APE 100 μM and Chelerythrine Chloride 1 μM showed an intermediate morphology between ctrl and APE treatment.

Figure 4

Schematic representation of signaling pathways downstream the M2 muscarinic receptor activation modulating SC proliferation/differentiation. In addition to the canonical pathway mediated by Gαi, M2 receptors may modulate the PI3K/Akt pathway via β1-arrestin or/and βγ-subunits of Gi-proteins, causing downregulation of mTORC1 pathway activity. This complex regulation mediated by M2 receptor activation may explain the decreased SC proliferation and the upregulation of the differentiation factors. β1-arrestin downregulation may also contribute to the modulation of the SC morphological changes, influencing the cofilin phosphorylation and actin de-polymerization with possible consequence on SC migration.