Assane Ndoye

Novel Human α9 Acetylcholine Receptor Regulating Keratinocyte Adhesion is Targeted by Pemphigus Vulgaris Autoimmunity

Pemphigus vulgaris (PV) is a potentially fatal autoimmune mucocutaneous blistering disease. It was assumed that PV is caused by anti-desmoglein (Dsg) 3 autoimmunity because absorption of PV sera with a chimeric baculoprotein containing the Dsg 3 and IgG1 portions, rDsg3-Ig-His, eliminated disease-causing antibodies. In this study we demonstrate that rDsg3-Ig-His adsorbs out autoantibodies to different keratinocyte antigens, including a non-Dsg 3 130-kd polypeptide. Because the pool of disease-causing PV IgGs contains antibodies against the keratinocyte acetylcholine receptor (AChR), we sought to identify the targeted receptor(s). Preincubation of monkey esophagus with PV antibodies blocked specific staining of the keratinocyte cell membrane with rabbit monoepitopic antibody to alpha9 AChR, indicating that this first of its kind AChR with dual, muscarinic and nicotinic pharmacology is targeted by PV autoimmunity. Anti-alpha9 antibody stained keratinocytes in a fishnet-like intercellular pattern, and visualized a single band at approximately 50 kd in Western blots of keratinocyte membrane proteins. Using step-by-step reverse transcription polymerase chain reactions with primers based on known alpha9 sequence regions, we identified the complete reading frame of human alpha9. Its amino acid sequence showed 85% similarity with rat alpha9. Treatment of keratinocyte monolayers with anti-alpha9 antibody induced pemphigus-like acantholysis, which could be reversed either spontaneously or by using the cholinergic agonist carbachol. We conclude that alpha9 is coupled to physiological regulation of keratinocyte adhesion, and its interaction with PV IgG may lead to blister development.

Pemphigus Vulgaris Antibody Identifies Pemphaxin A NOVEL KERATINOCYTE ANNEXIN-LIKE MOLECULE BINDING ACETYLCHOLINE

Because pemphigus vulgaris (PV) IgGs adsorbed on the rDsg3-Ig-His baculoprotein induced blisters in neonatal mice, it was proposed that anti-desmoglein 3 (Dsg 3) autoantibody causes PV. However, we found that rDsg3-Ig-His absorbs autoantibodies to different antigens, including a non-Dsg 3 keratinocyte protein of 130 kDa. This prompted our search for novel targets of PV autoimmunity. The PV IgG eluted from a 75-kDa keratinocyte protein band both stained epidermis in a pemphigus-like pattern and induced acantholysis in keratinocyte monolayers. Screening of a keratinocyte λgt11 cDNA library with this antibody identified clones carrying cDNA inserts encoding a novel molecule exhibiting ∼40% similarity with annexin-2, named pemphaxin (PX). Recombinant PX (rPX-His) was produced inEscherichia coli M15 cells, and, because annexins can act as cholinergic receptors, its conformation was tested in a cholinergic radioligand binding assay. rPX-His specifically bound [3H]acetylcholine, suggesting that PX is one of the keratinocyte cholinergic receptors known to be targeted by disease-causing PV antibodies. Preabsorption of PV sera with rPX-His eliminated acantholytic activity, and eluted antibody immunoprecipitated native PX. This antibody alone did not cause skin blisters in vivo, but its addition to the preabsorbed PV IgG fraction restored acantholytic activity, indicating that acantholysis in PV results from synergistic action of antibodies to different keratinocyte self-antigens, including both acetylcholine receptors and desmosomal cadherins.

Choline Acetyltransferase, Acetylcholinesterase, and Nicotinic Acetylcholine Receptors of Human Gingival and Esophageal Epithelia

A non-neuronal cholinergic system that includes neuronal-like nicotinic acetylcholine receptors (nAChRs) has recently been described in epithelial cells that line the skin and the upper respiratory tract. Since the use of nicotine-containing products is associated with morbidity in the upper digestive tract, and since nicotine may alter cellular functions directly via nAChRs, we sought to identify and characterize a non-neuronal cholinergic system in the gingival and esophageal epithelia. mRNA transcripts for a3, α5, α7, and β2 nAChR subunits, choline acetyltransferase, and the asymmetric and globular forms of acetylcholinesterase were amplified from gingival keratinocytes (KC) by means of polymerase chain-reactions. These proteins were visualized in the gingival and esophageal epithelia by means of specific antibodies. Variations in distribution and intensity of immunostaining were found, indicating that the repertoire of cholinergic enzymes and receptors expressed by the cells changes during epithelial maturation, and that an upward concentration gradient of free acetylcholine exists. Blocking of the nAChRs with mecamylamine resulted in reversible loss of cell-to-cell adhesion, and shrinking and rounding of cultured gingival KC. Activation of the receptors with acetylcholine or carbachol caused stretching and peripheral ruffling of the cytoplasmic aprons, and formation of new intercellular contacts. These results demonstrate that both the keratinizing epithelium of attached gingiva and the non-keratinizing epithelium lining the upper two-thirds of the esophageal mucosa possess a non-neuronal cholinergic system. The nAChRs expressed by these epithelia are coupled to regulation of cell adhesion and motility, and may provide a target for the deleterious effects of nicotine.

Central role of fibroblast α3 nicotinic acetylcholine receptor in mediating cutaneous effects of nicotine

Smoking is associated with aberrant cutaneous tissue remodeling, such as precocious skin aging and impaired wound healing. The mechanism is not fully understood. Dermal fibroblasts (DF) are the primary cellular component of the dermis and may provide a target for pathobiologic effects of tobacco products. The purpose of this study was to characterize a mechanism of nicotine (Nic) effects on the growth and tissue remodeling function of DF. We hypothesized that the effects of Nic on DF result from its binding to specific nicotinic acetylcholine receptors (nAChRs) expressed by these cells and that downstream signaling from the receptors alters normal cell functioning, leading to changes in skin homeostasis. Using RT-PCR and Western blotting, we found that a 24-hour exposure of human DF to 10 μm Nic causes a 1.9- to 28-fold increase of the mRNA and protein levels of the cell cycle regulators p21, cyclin D1, Ki-67, and PCNA and a 1.7- to 2-fold increase of the apoptosis regulators Bcl-2 and caspase 3. Nic exposure also up-regulated expression of the dermal matrix proteins collagen type Iα1 and elastin as well as matrix metalloproteinase-1. Mecamylamine (Mec), the specific antagonist of nAChRs, abolished Nic-induced alterations, indicating that they resulted from a pharmacologic stimulation of nAChRs expressed by DF. To establish the relevance of these findings to a specific nicotinergic pathway, we studied human DF transfected with anti-α3 antisense oligonucleotides and murine DF from α3 nAChR knockout mice. In both cases, lack of α3 was associated with alterations in fibroblast growth and function that were opposite to those observed in DF treated with Nic, suggesting that the nicotinic effects on DF were mostly mediated by α3 nAChR. In addition to α3, the nAChR subunits detected in human DF were α5, α7, β2, and β4. The exposure of DF to Nic altered the relative amounts of each of these subunits, leading to reciprocal changes in [3H]epibatidine-binding kinetics. Thus, some of the pathobiologic effects of tobacco products on extracellular matrix turnover in the skin may stem from Nic-induced alterations in the physiologic control of the unfolding of the genetically determined program of growth and the tissue remodeling function of DF as well as alterations in the structure and function of fibroblast nAChRs.

Human skin fibroblasts express m2, m4, and m5 subtypes of muscarinic acetylcholine receptors

Previous studies have demonstrated that muscarinic acetylcholine receptors (mAChRs) are expressed by human skin fibroblasts (HSF). We have identified the molecular subtypes of these receptors by reverse transcription‐polymerase chain reaction (RT‐PCR), using m1‐m5 subtype‐specific primers. These experiments showed that only mRNAs for m2, m4, and m5 mAChR subtypes are present in HSF. The RT‐PCR products were characterized by restriction analysis and Southern blotting. Northern blot analysis showed the presence of m2 and m4 mAChR RNA. Rabbit antibodies were raised using a synthetic peptide as immunogen corresponding to the C‐terminus of the m2 protein and were used to visualize fibroblast mAChRs. Cell membranes of HSF in cell culture and specimens of normal human skin had a unique staining pattern specific for anti‐m2 antibody, as well as for antibodies against m4 and m5. In Western blots of fibroblast proteins, the antibodies visualized the m2 receptor at 65 kDa, m4 at 70 kDa, and m5 at 95 kDa. The function of fibroblast mAChRs was examined by measuring muscarinic effects on intracellular free Ca2+ concentration ([Ca2+]i). Muscarine increased transiently [Ca2+]i in cultured HSF. This effect could be abolished by the muscarinic antagonist atropine. Thus, the results of this study showed that HSF express m2, m4, and m5 mAChR subtypes, and that fibroblast mAChRs are coupled to the regulation of [Ca2+]i. J. Cell. Biochem. 74:264–277, 1999.

Identification and characterization of muscarinic acetylcholine receptor subtypes expressed in human skin melanocytes.

The present study was designed to identify and characterize muscarinic acetylcholine receptors in normal human melanocytes. We used subtype-specific oligonucleotide primers to localize the five genetically defined mAChR mRNAs (m1 through m5) by reverse transcription-polymerase chain reaction. These experiments showed that all five mAChR subtype mRNAs are expressed in melanocytes. The PCR products were verified by restriction analysis and Southern blotting. Receptors were visualized in cultures of normal human melanocytes and specimens of normal human skin by subtype-specific rabbit anti-receptor polyclonal antibodies. Radioligand binding assays with the lipophilic drug [3H]quinuclidinyl benzilate demonstrated approximately 9000 high affinity binding sites/cell. Micromolar concentrations of muscarine or carbachol transiently increased intracellular Ca2+, which could be attenuated by atropine, demonstrating coupling of the receptors to mobilization of intracellular free Ca2+. Lower concentrations of muscarine induced spontaneous repetitive spike-like increases of intracellular Ca2+ which is characteristic for the activation of muscarinic receptors. These results indicate that normal human skin melanocytes express the m1, m2, m3, m4, and m5 subtypes of classic muscarinic acetylcholine receptors on their cell membrane and that these receptors regulate the concentration of intracellular free Ca2+, which may play an important physiologic role in melanocyte behavior and skin pigmentation.

The M4 muscarinic receptor-selective effects on keratinocyte crawling locomotion

We have investigated how the cholinergic system of epidermal keratinocytes (KC) controls migratory function of these cells. Several molecular subtypes of muscarinic acetylcholine receptors (mAChRs) have been detected in KC. Early results suggested that M(4) is the predominant mAChR regulating cell motility. To determine muscarinic effects on lateral migration of KC, we used an agarose gel keratinocyte outgrowth system (AGKOS) which provides for measurements of the response of large cell populations (> 10(4) cells). Muscarine produced a dose-dependent stimulatory effect on cell migration (p < 0.05). This activity was abolished by atropine, which decreased migration distance when given alone. To identify the mAChR subtype(s) mediating these muscarinic effects, we substituted atropine with subtype-selective antagonists. Tropicamide (M(4)-selective) was more effective at decreasing the migration distance than pirenzepine and 4-DAMP at nanomolar concentrations. We then compared lateral migration of KC obtained from M(4) mAChR knockout mice with that of wild-type murine KC, using AGKOS. In the absence of M(4) mAChR, the migration distance of KC was significantly (p < 0.05) decreased. These results indicate that the M(4) mAChR plays a central role in mediating cholinergic control of keratinocyte migration by endogenous acetylcholine produced by these cells.

MN Protein Immunolocalization in Uterine Cervix Carcinoma With Glandular Differentiation: A Clinicopathologic Study of a New Cancer-specific Biomarker

MN protein is the product of the newly described endogenous MN gene that is expressed in the tumorigenic phenotype of HeLa X fibroblast somatic cell hybrids. MN protein has carbonic anhydrase and putative DNA binding activity. With the exception of gastric mucosa, MN protein is expressed in neoplasia, particularly uterine cervix carcinoma, but not in benign tissue. This investigation examined the pathogenetic and prognostic significance of MN-protein immunoreactivity in uterine cervix carcinoma with glandular differentiation. Paraffin sections from 77 cervix carcinomas with glandular differentiations including 36 pure adenocarcinomas and 41 adenosquamous carcinomas were immunostained with anti-MN-protein (M-75 monoclonal proprietary; Ciba Corning Diagnostics, Alameda, CA). A total of 64.9% of cervix carcinomas with glandular differentiation exhibit MN-protein immunoreactivity localized to plasma membranes, cytoplasm, and some nuclei of neoplastic cells only, but not in adjacent benign tissue. The MN-protein staining intensity and distribution was as follows: 37.7% strong diffuse (≥ 50% cells positive), 19.5% strong focal (< 50% cells positive), and weak (7.8%). Immunoreactivity occurred in both squamous and glandular areas of adenosquamous carcinomas and was unrelated to histopathologic features. Follow-up information was available on 67 patients: 31 exhibited recurrent disease (7 pelvic, 14 distant, and 10 both) at 1–144 months (mean 37, median 14), and 36 were disease-free at 12–216 months (mean 67, median 44.5). MN-protein immunoreactivity (all positives, both standard diffuse and strong focal, or standard diffuse only) exhibited no association with clinical outcome. Recurrent disease was associated with nuclear grade (P < .001), lymphatic invasion (P < .005), size on clinical examination or pathologic evaluation (P < .005), pelvic lymph node involvement (P < .05), and clinical stage (P < .05). MN-protein immunoreactivity did not correlate with these features and did not help predict which patients would develop recurrence in the good prognosis groups. Our data show that expression of MN-protein is associated with cervix carcinoma with glandular differentiation carcinogenesis. MN-protein immunolocalization may have a diagnostic role in confirming cervix carcinoma with glandular differentiation in histologically challenging cases.