Novel signaling pathways mediating reciprocal control of keratinocyte migration and wound epithelialization through M3 and M4 muscarinic receptors

Abstract

To test the hypothesis that keratinocyte (KC) migration is modulated by distinct muscarinic acetylcholine (ACh) receptor subtypes, we inactivated signaling through specific receptors in in vitro and in vivo models of reepithelialization by subtype-selective antagonists, small interfering RNA, and gene knockout in mice. KC migration and wound reepithelialization were facilitated by M4 and inhibited by M3. Additional studies showed that M4 increases expression of “migratory” integrins α5β1, αVβ5, and αVβ6, whereas M3 up-regulates “sedentary” integrins α2β1 and α3β1. Inhibition of migration by M3 was mediated through Ca2+-dependent guanylyl cyclase–cyclic GMP–protein kinase G signaling pathway. The M4 effects resulted from inhibition of the inhibitory pathway involving the adenylyl cyclase–cyclic AMP–protein kinase A pathway. Both signaling pathways intersected at Rho, indicating that Rho kinase provides a common effector for M3 and M4 regulation of cell migration. These findings offer novel insights into the mechanisms of ACh-mediated modulation of KC migration and wound reepithelialization, and may aid the development of novel methods to promote wound healing.

The P2Y2 Nucleotide Receptor Interacts with αv Integrins to Activate Go and Induce Cell Migration

“…5, A and B). These observations are consistent with other reports indicating that integrin activity is required for both GPCR-mediated (34,35) and growth factor receptor-mediated cell migration (36,37).…”

The Ras/Raf-1/MEK1/ERK Signaling Pathway Coupled to Integrin Expression Mediates Cholinergic Regulation of Keratinocyte Directional Migration

“…Both coupling of M 1 to the Ras/Raf/MAP kinase pathway and coupling of this pathway to up-regulation of integrin ␣ 2 have been demonstrated in other cell types (30,55). In a previous study we demonstrated that activation of keratinocyte M 3 mAChR leads to an up-regulated expression of the sedentary integrin receptors ␣ 2 ␤ 1 and ␣ 3 ␤ 1 and arrested migration (11). It is well known that the odd-numbered mAChRs, M 1 , M 3 , and M 5 , all can couple the same intracellular signaling pathways (56).…”

“…The muscarinic ACh receptor (mAChR) subtypes M 3 and M 4 also exhibit reciprocal regulation of the keratinocyte migratory function by inhibiting or stimulating it via the signaling pathways coupled to preferential expression of either sedentary (␣ 2 and ␣ 3 ) or migratory (␣ 5 , ␣ v , and ␤ 5 ) integrins, respectively (11). We next sought to gain a mechanistic insight into the cholinergic regulation of the directionality of keratinocyte migration.…”

Stimulation of Gαq-coupled M1 muscarinic receptor causes reversible spectrin redistribution mediated by PLC, PKC and ROCK

“…Inhibition of RhoA and ROCK causes parallel effects on M3 mAChR signaling (Chernyavsky et al, 2004;Murthy et al, 2003), whereas ROCK and RhoA activations are independent of Ins(1,4,5)P 3 -induced [Ca 2+ ]i rise and PKC (Ueda et al, 2001;Vogt et al, 2003), which suggests that RhoA, but not PLC and PKC, is upstream of ROCK, linking G␣ q/11 -coupled receptor signaling to reorganization of the cytoskeleton. However, two lines of evidence in our study argue against this activation mechanism.…”

“…It has been shown that the RhoA-ROCK pathway links G␣ q/11 -coupled M3 mAChR signaling to cytoskeleton responses involved in muscle-cell contraction (Murthy et al, 2003) and cell migration (Chernyavsky et al, 2004). Also, overexpression of both constitutively active ROCK-II and RhoA V14 caused cell rounding and membrane blebbing in PC12 cells (Frantz et al, 2002), suggesting that RhoA was upstream of ROCK activation.…”