Identification and Mapping of Keratinocyte Muscarinic Acetylcholine Receptor Subtypes in Human Epidermis11Presented at the 17th International Congress of Biochemistry and Molecular Biology in conjunction with the Annual Meeting of the American Society for Biochemistry and Molecular Biology, San Francisco, California, August 25 1997, and at the Third Tricontinental Meeting of the Society for Investigative Dermatology, the European Society for Dermatological Research and the Japanese Society for Investigativ

Abstract

Acetylcholine mediates cell-to-cell communications in the skin. Human epidermal keratinocytes respond to acetylcholine via two classes of cell-surface receptors, the nicotinic and the muscarinic cholinergic receptors. High affinity muscarinic acetylcholine receptors (mAChR) have been found on keratinocyte cell surfaces at high density. These receptors mediate effects of muscarinic drugs on keratinocyte viability, proliferation, adhesion, lateral migration, and differentiation. In this study, we investigated the molecular structure of keratinocyte mAChR and their location in human epidermis. Polymerase chain reaction amplification of cDNA sequences uniquely present within the third cytoplasmic loop of each subtype demonstrated the expression of the m1, m3, m4, and m5 mAChR subtypes. To visualize these mAChR, we raised rabbit anti-sera to synthetic peptide analogs of the carboxyl terminal regions of each subtype. The antibodies selectively bound to keratinocyte mAChR subtypes in immunoblotting membranes and epidermis, both of which could be abolished by preincubating the anti-serum with the peptide used for immunization. The immunofluorescent staining patterns produced by each antibody in the epidermis suggested that the profile of keratinocyte mAChR changes during epidermal turnover. The semiquantitative analysis of fluorescence revealed that basal cells predominantly expressed m3, prickle cells had equally high levels of m4 and m5, and granular cells mostly possessed m1. Thus, the results of this study demonstrate for the first time the presence of m1, m3, m4, and m5 mAChR in epidermal keratinocytes. Because keratinocytes express a unique combination of mAChR subtypes at each stage of their development in the epidermis, each receptor may regulate a specific cell function. Hence, a single cytotransmitter, acetylcholine, and muscarinic drugs may exert different biologic effects on keratinocytes at different stages of their maturation.

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

“…In human epidermis, M 4 receptors are predominantly expressed by the lowermost prickle cells that continuously migrate upward, whereas terminally differentiated KCs lack M 4 receptors ( Nguyen et al, 2001 ). In marked contrast, M 3 receptors are predominantly expressed by basal KCs that establish hemidesmosomal contact with the basal membrane ( Ndoye et al, 1998 ). Hence, M 3 receptor–induced downstream signaling may support the establishment and/or maintenance of stable connections of KCs with the substrate in culture, and of the epidermis to the underlying dermis in the skin.…”

“…Rabbit polyclonal antibodies to α 2 , α 3 , α 5 , α v , and β 1 integrins were purchased from Chemicon International, Inc. Anti–β-actin antibody and secondary, FITC-labeled goat anti–rabbit antibodies were from Sigma-Aldrich. The antibodies to M 3 and M 4 mAChRs that we characterized previously ( Ndoye et al, 1998 ) are commercially available from Research and Diagnostic Antibodies. Rat anti–mouse antibody to the proliferation marker Ki-67 and a secondary, FITC-conjugated antibody were purchased from DakoCytomation.…”

Cytokine-Induced Alterations of α7 Nicotinic Receptor in Colonic CD4 T Cells Mediate Dichotomous Response to Nicotine in Murine Models of Th1/Th17- versus Th2-Mediated Colitis

“…The 8 μm thick cryostat sections of mouse colons were fixed with cold acetone, blocked with 5% fetal calf serum ( FCS ), and used in the indirect immunofluorescence assays following a standard procedure (44). The V450 rat anti-mouse CD4 (BD Bioscience, San Jose, CA) and Alexa Fluor 488 anti-mouse CD25 (BioLegend, San Diego, CA) were used for double staining, and PE rat anti-mouse Foxp3 (BD Bioscience) was added for triple-staining.…”

Epidermal Growth Factor Receptor Transactivation Is Required for Mitogen-Activated Protein Kinase Activation by Muscarinic Acetylcholine Receptors in HaCaT Keratinocytes

“…The human skin utilizes non-neuronal ACh signaling to regulate cell differentiation and tissue maintenance [ 3 , 4 ], and ACh appears to play a role in wound healing [ 5 , 6 , 7 ]. Various subunits of the nAChR and distinct mAChR subtypes are expressed, depending on the skin layer and the cell type [ 8 , 9 , 10 ], and high levels of ACh are detectable in the epidermis [ 11 , 12 ]. Furthermore, the cholinergic signals are important for proliferation, differentiation and migration of keratinocytes and other cell types within the layers of the skin [ 13 , 14 , 15 , 16 ].…”