Vu Thuong Nguyen

Desmoglein 4 in Hair Follicle Differentiation and Epidermal Adhesion: Evidence from Inherited Hypotrichosis and Acquired Pemphigus Vulgaris

Cell adhesion and communication are interdependent aspects of cell behavior that are critical for morphogenesis and tissue architecture. In the skin, epidermal adhesion is mediated in part by specialized cell-cell junctions known as desmosomes, which are characterized by the presence of desmosomal cadherins, known as desmogleins and desmocollins. We identified a cadherin family member, desmoglein 4, which is expressed in the suprabasal epidermis and hair follicle. The essential role of desmoglein 4 in skin was established by identifying mutations in families with inherited hypotrichosis, as well as in the lanceolate hair mouse. We also show that DSG4 is an autoantigen in pemphigus vulgaris. Characterization of the phenotype of naturally occurring mutant mice revealed disruption of desmosomal adhesion and perturbations in keratinocyte behavior. We provide evidence that desmoglein 4 is a key mediator of keratinocyte cell adhesion in the hair follicle, where it coordinates the transition from proliferation to differentiation.

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.

Central role of α7 nicotinic receptor in differentiation of the stratified squamous epithelium

Several ganglionic nicotinic acetylcholine receptor (nAChR) types are abundantly expressed in nonneuronal locations, but their functions remain unknown. We found that keratinocyte α7 nAChR controls homeostasis and terminal differentiation of epidermal keratinocytes required for formation of the skin barrier. The effects of functional inactivation of α7 nAChR on keratinocyte cell cycle progression, differentiation, and apoptosis were studied in cell monolayers treated with α-bungarotoxin or antisense oligonucleotides and in the skin of Acra7 homozygous mice lacking α7 nAChR channels. Elimination of the α7 signaling pathway blocked nicotine-induced influx of 45Ca2+ and also inhibited terminal differentiation of these cells at the transcriptional and/or translational level. On the other hand, inhibition of the α7 nAChR pathway favored cell cycle progression. In the epidermis of α7−/− mice, the abnormalities in keratinocyte gene expression were associated with phenotypic changes characteristic of delayed epidermal turnover. The lack of α7 was associated with up-regulated expression of the α3 containing nAChR channels that lack α5 subunit, and both homomeric α9- and heteromeric α9α10-made nAChRs. Thus, this study demonstrates that ACh signaling through α7 nAChR channels controls late stages of keratinocyte development in the epidermis by regulating expression of the cell cycle progression, apoptosis, and terminal differentiation genes and that these effects are mediated, at least in part, by alterations in transmembrane Ca2+ influx.

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.

Structure of the Plasmodium falciparum Circumsporozoite Protein, a Leading Malaria Vaccine Candidate

The Plasmodium falciparum circumsporozoite protein (CSP) is critical for sporozoite function and invasion of hepatocytes. Given its critical nature, a phase III human CSP malaria vaccine trial is ongoing. The CSP is composed of three regions as follows: an N terminus that binds heparin sulfate proteoglycans, a four amino acid repeat region (NANP), and a C terminus that contains a thrombospondin-like type I repeat (TSR) domain. Despite the importance of CSP, little is known about its structure. Therefore, recombinant forms of CSP were produced by expression in both Escherichia coli (Ec) and then refolded (EcCSP) or in the methylotrophic yeast Pichia pastoris (PpCSP) for structural analyses. To analyze the TSR domain of recombinant CSP, conformation-dependent monoclonal antibodies that recognized unfixed P. falciparum sporozoites and inhibited sporozoite invasion of HepG2 cells in vitro were identified. These monoclonal antibodies recognized all recombinant CSPs, indicating the recombinant CSPs contain a properly folded TSR domain structure. Characterization of both EcCSP and PpCSP by dynamic light scattering and velocity sedimentation demonstrated that both forms of CSP appeared as highly extended proteins (Rh 4.2 and 4.58 nm, respectively). Furthermore, high resolution atomic force microscopy revealed flexible, rod-like structures with a ribbon-like appearance. Using this information, we modeled the NANP repeat and TSR domain of CSP. Consistent with the biochemical and biophysical results, the repeat region formed a rod-like structure about 21–25 nm in length and 1.5 nm in width. Thus native CSP appears as a glycosylphosphatidylinositol-anchored, flexible rod-like protein on the sporozoite surface.

Pemphigus Vulgaris IgG and Methylprednisolone Exhibit Reciprocal Effects on Keratinocytes

Pemphigus vulgaris (PV) is a life-threatening autoimmune disease of skin adhesion associated with IgG autoantibodies against keratinocytes (KC). Treatment of PV with systemic corticosteroids is life-saving, but the mechanism of the therapeutic action has not been fully understood. We have developed an animal model that demonstrates that methylprednisolone (MP) can block PV IgG-induced acantholysis, decreasing the extent of keratinocyte detachment in the epidermis of 3–5-day-old nude mice from 77.5 ± 0.6 to 24.1 ± 1.5% (p < 0.05). We hypothesized that in addition to immunosuppression, MP may exhibit direct anti-acantholytic effects in epidermis, and we compared the effects of PV IgG and MP on KC. The use of DNA microarray showed that PV IgG down-regulated and MP up-regulated expression of the genes encoding keratinocyte adhesion molecules, antigen-processing proteins, regulators of cell cycle and apoptosis, differentiation markers, Na+,K+-ATPase, protein kinases and phosphatases, and serine proteases and their inhibitors. Overall, PV IgG decreased transcription of 198 genes and increased transcription of 31 genes. MP decreased transcription of 14 genes and increased transcription of 818 genes. Specific effects of PV IgG and MP on keratinocyte adhesion molecules were further investigated by Western blot and immunofluorescence assays. By immunoblotting, MP increased the protein levels of E-cadherin and desmogleins 1 and 3 by 300, 180, and 40%, respectively. Specific staining of KC for E-cadherin and desmogleins 1 and 3 increased by 235, 228, and 148%, respectively. In addition, PV IgG increased the level of phosphorylation of E-cadherin by 42%, β-catenin by 37%, γ-catenin by 136%, and desmoglein 3 by 300%, whereas pretreatment with 0.25 mm MP abolished phosphorylation of these adhesion molecules. These results suggested that therapeutic effects of MP in PV include both the up-regulated synthesis and post-translational modification of the keratinocyte adhesion molecules.

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.

A Receptor-Mediated Mechanism of Nicotine Toxicity in Oral Keratinocytes

Smoking and smokeless tobacco cause morbidity that originates from the epithelium lining of the skin and upper digestive tract. Oral keratinocytes (OKC) express nicotinic acetylcholine receptors (nAChRs) that bind nicotine (Nic). We studied the mechanism of the receptor-mediated toxicity of tobacco products on OKC. Preincubation of normal human OKC with Nic altered the ligand-binding kinetics of their nAChRs, suggesting that the nAChRs underwent structural changes. This hypothesis was confirmed by the finding that exposure of OKC to Nic causes transcriptional and translational changes. Through RT-PCR and immunoblotting, we found a 1.5- to 2.9-fold increase in the mRNA and protein levels of α3, α5, α7, β2, and β4 nAChR subunits. Exposure of OKC to Nic also changed the mRNA and protein levels of the cell cycle and cell differentiation markers Ki-67, PCNA, p21, cyclin D1, p53, filaggrin, loricrin, and cytokeratins 1 and 10. The nicotinic antagonist mecamylamine prevented these changes, which indicates that the Nic-induced changes in the expression of both the nAChR and the cell cycle and cell differentiation genes resulted from pharmacologic stimulation of nAChRs with Nic. To establish the relevance of these findings to the pathobiologic effects of tobacco products in vivo, we studied the above parameters in the oral tissue of rats and mice after their exposure for 3 weeks to environmental cigarette smoke or drinking water containing equivalent concentrations of Nic that are pathophysiologically relevant. The changes of the nAChRs and the cell cycle and cell differentiation genes were similar to those found in vitro. The results of indirect immunofluorescence assay of tissue specimens validated these findings. Thus, some pathobiologic effects of tobacco products in oral tissues may stem from Nic-induced alterations of the structure and function of keratinocyte nAChRs responsible for the physiologic regulation of the cell cycle by the cytotransmitter acetylcholine.

Functional role of α7 nicotinic receptor in physiological control of cutaneous homeostasis

Non-neuronal nicotinic acetylcholine receptors (nAChRs) are abundantly expressed in skin and their function remains to be elucidated. Herein, we report that cutaneous alpha7 nAChR plays a role in the physiological control of cutaneous homeostasis. We studied in vitro effects of functional inactivation of alpha7 receptor on the expression of apoptosis regulators in keratinocytes (KC) lacking alpha7 nAChR, and extracellular matrix regulators in the skin of alpha7 knockout (KO) mice. Elimination of the alpha7 component of nicotinergic signaling in KC decreased relative amounts of the pro-apoptotic Bad and Bax at both the mRNA and the protein levels, suggesting that alpha7 nAChR is coupled to stimulation of keratinocyte apoptosis. The skin of alpha7 KO mice featured decreased amounts of the extracellular matrix proteins collagen 1alpha1 and elastin as well as the metalloproteinase-1. Taken together, these results suggest an important role for alpha7 nAChR in mediating plethoric effects of non-neuronal acetylcholine on cutaneous homeostasis.

Keratinocyte acetylcholine receptors regulate cell adhesion.

We investigated the mechanism mediating cholinergic control of cell-to-cell adhesion of human epidermal keratinocytes (KC) by non-neuronal acetylcholine produced by KC themselves. We first measured cholinergic effects on the expression of desmoglein (Dsg) 1 and 3 in KC using the semi-quantitative immunofluorescence and Western blot assays. Monolayers of KC were treated overnight with 0.25 mM of the cholinergic agonist carbachol (CCh) or the acetylcholinesterase inhibitor pyridostigmine bromide (PBr). Both CCh and PBr increased the relative amounts of Dsg 1 and Dsg 3. To determine the role for cholinergic receptor-mediated phosphorylation of Dsg molecules in assembly/disassembly of keratinocyte desmosomes, we tested the effects of a cholinergic antagonist on keratinocyte adhesion and Dsg phosphorylation status in DJM-1 cell line. Atropine (Atr), 0.02 mM, induced rapid detachment of cells from each other (acantholysis), and also increased phosphorylation of Dsg 3 by 33%. The Atr-dependent phosphorylation of Dsg 3 was inhibited in the presence of 0.5 mM CCh. Thus, keratinocyte cholinergic receptors regulate desmosomal adhesion of KC by altering the level of expression of both Dsg 1 and Dsg 3 and the phosphorylation status of Dsg 3.