Alexander I. Chernyavsky

Receptor-mediated tobacco toxicity: cooperation of the Ras/Raf-1/MEK1/ERK and JAK-2/STAT-3 pathways downstream of α7 nicotinic receptor in oral keratinocytes

The use of tobacco products is associated with an increased incidence of periodontal disease, poor response to periodontal therapy, and a high risk for developing head and neck cancer. Nicotine and tobacco‐derived nitrosamines have been shown to exhibit their pathobiologic effects due in part to activation of the nicotinic acetylcholine (ACh) receptors (nAChRs), mainly α7 nAChR, expressed by oral keratinocytes (KCs). This study was designed to gain mechanistic insight into α7‐mediated morbidity of tobacco products in the oral cavity. We investigated the signaling pathways downstream of α7 nAChR in monolayers of oral KCs exposed for 24 h to aged and diluted sidestream cigarette smoke (ADSS) or an equivalent concentration of pure nicotine. By both real‐time polymerase chain reaction (PCR) and In‐cell Western, the KCs stimulated with ADSS or nicotine showed multifold increases of STAT‐3. These effects could be completely blocked or significantly P < <0.05) diminished if the cells were pretreated with the α7 antagonist ‐bungarotoxin (BTX) or transfected with anti‐α7 small interfering RNA (siRNA‐α7). The use of pathway inhibitors revealed that signaling through the Ras/Raf‐1/MEK1/ ERK steps mediated 7‐dependent up‐regulation of STAT‐3. Targeted mutation of the α7 gene prevented ERK1/2 activation by nicotine. Using the gel mobility shift assay, we demonstrated that an increased protein binding activity of STAT‐3 caused by ADSS or pure nicotine was mediated by janus‐activated kinase (JAK)‐2. Activation of JAK‐2/STAT‐3 pathway could be prevented by BTX or siRNA‐α7. Thus, nuclear transactivation of STAT‐3 in KCs exposed to tobacco products is mediated via intracellular signaling downstream from α7, which proceeds via two complementary pathways. The Ras/Raf‐1/MEK1/ERK cascade culminates in up‐regulated expression of the gene encoding STAT‐3, whereas recruitment and activation of tyrosine kinase JAK‐2 phosphorylates it. Elucidation of this novel mechanism of nicotine‐dependent nuclear transactivation of STAT‐3 identifies oral α7 nAChR as a promising molecular target to prevent, reverse, or retard tobacco‐related periodontal disease and progression of head and neck cancer by receptor inhibitors.—Arredondo, J., Chernyavsky, A. I., Jolkovsky, D. L., Pinkerton, K. E., Grando, S. A. Receptor‐mediated tobacco toxicity: cooperation of the Ras/Raf‐1/MEK1/ ERK and JAK‐2/STAT‐3 pathways downstream of α7 nicotinic receptor in oral keratinocytes. FASEB J. 20, 2093–2101 (2006)

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.

Novel mechanisms of target cell death and survival and of therapeutic action of IVIg in Pemphigus.

Pemphigus vulgaris (PV) is a potentially lethal mucocutaneous blistering disease characterized by cell-cell detachment within the stratified epithelium (acantholysis) caused by IgG autoantibodies. Intravenous immunoglobulin (IVIg) therapy effectively treats PV, but the mechanism is not fully understood. To further understand acantholysis and the efficacy of IVIg, we measured effects of IgG fractions from PV patients on keratinocyte death processes. Using IgGs from representative PV patients who improved with IVIg, we identified apoptotic and oncotic signaling pathways in in vitro and in vivo PV models. We identified two groups of PV patients, each producing autoantibodies activating predominantly either apoptotic or oncotic cell death pathway. Experimental treatments with caspase 3 or calpain inhibitors demonstrated that PV IgGs induced acantholysis through both pathways. Upstream, the apoptotic signaling involved activation of caspases 8 and 3 and up-regulation of Fas ligand mRNA, whereas calpain-mediated cell death depended on elevated intracellular free Ca(2+). IVIg reduced PV IgG-mediated acantholysis and cell death and up-regulated the caspase inhibitor FLIP and the calpain inhibitor calpastatin. These results indicate that in different PV patients, IgG-induced acantholysis proceeds predominantly via distinct, yet complementary, pathways of programmed cell death differentially mediated by apoptosis and oncosis effectors, with IVIg protecting target cells by up-regulating endogenous caspase and calpain inhibitors.

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.

Endogenous Galectin-3 Is Localized in Membrane Lipid Rafts and Regulates Migration of Dendritic Cells

This study reveals a function of endogenous galectin-3, an animal lectin recognizing beta-galactosides, in regulating dendritic cell motility both in vitro and in vivo, which to our knowledge is unreported. First, galectin-3-deficient (gal3(-/-)) bone marrow-derived dendritic cells exhibited defective chemotaxis compared to gal3(+/+) cells. Second, cutaneous dendritic cells in gal3(-/-) mice displayed reduced migration to draining lymph nodes upon hapten stimulation compared to gal3(+/+) mice. Moreover, gal3(-/-) mice were impaired in the development of contact hypersensitivity relative to gal3(+/+) mice in response to a hapten, a process in which dendritic cell trafficking to lymph nodes is critical. In addition, defective signaling was detected in gal3(-/-) cells upon chemokine receptor activation. By immunofluorescence microscopy, we observed that galectin-3 is localized in membrane ruffles and lamellipodia in stimulated dendritic cells and macrophages. Furthermore, galectin-3 was enriched in lipid raft domains under these conditions. Finally, we determined that ruffles on gal3(-/-) cells contained structures with lower complexity compared to gal3(+/+) cells. In view of the participation of membrane ruffles in signal transduction and cell motility, we conclude that galectin-3 regulates cell migration by functioning at these structures.

Apoptolysis: a novel mechanism of skin blistering in pemphigus vulgaris linking the apoptotic pathways to basal cell shrinkage and suprabasal acantholysis

Abstract:  Understanding the acantholytic pathways leading to blistering in pemphigus vulgaris (PV) is a key to development of novel treatments. A novel paradigm of keratinocyte damage in PV, termed apoptolysis, links the suprabasal acantholytic and cell death pathways to basal cell shrinkage rendering a ‘tombstone’ appearance to PV lesions. In contrast to apoptolysis, the classic keratinocyte apoptosis mediating toxic epidermal necrolysis causes death and subsequent sloughing of the entire epidermis. Apoptolysis includes five consecutive steps. (1) Binding of autoantibodies to PV antigens. (2) Activation of EGF receptor, Src, mTOR, p38 MAPK and other signalling elements downstream of ligated antigens, elevation of intracellular calcium and launching of the cell death cascades. (3) Basal cell shrinkage due to: (i) collapse and retraction of the tonofilaments cleaved by executioner caspases; and (ii) dissociation of interdesmosomal adhesion complexes caused by phosphorylation of adhesion molecules. (4) Massive cleavage of cellular proteins by activated cell death enzymes leading to cell collapse, and tearing off desmosomes from the cell membrane stimulating secondary autoantibody production. (5) Rounding up and death of acantholytic cells. Thus, the structural damage (acantholysis) and death (apoptosis) of keratinocytes are mediated by the same cell death enzymes. Appreciation of the unifying concept of apoptolysis have several important implications: (i) linking together a number of seemingly unrelated events surrounding acantholysis; (ii) opening new avenues of investigation into the pathomechanism of pemphigus; and (iii) creating new approaches to the treatment of pemphigus based on blocking the signalling pathways and enzymatic processes that lead to blistering.

Receptor-mediated tobacco toxicity: acceleration of sequential expression of α5 and α7 nicotinic receptor subunits in oral keratinocytes exposed to cigarette smoke

Tobacco products and nicotine alter the cell cycle and lead to squamatization of oral keratinocytes (KCs) and squamous cell carcinoma. Activation of nicotinic acetylcholine receptors (nAChRs) elicits Ca2+ influx that varies in magnitude between different nAChR subtypes. Normal differentiation of KCs is associated with sequential expression of the nAChR subtypes with increasing Ca2+ permeability, such as α5‐containing α3 nAChR and α7 nAChR. Exposure to environmental tobacco smoke (ETS) or an equivalent concentration of nicotine accelerated by severalfold the α5 and α 7 expression in KCs, which could be abolished by mecamylamine and α‐bungarotoxin with different efficacies, suggesting the following sequence of auto regulation of the expression of nAChR subtypes: α3(β2/β4) > α3(β2/β4)α5 > α7 > α7. This conjecture was corroborated by results of quantitative assays of subunit mRNA and protein levels, using nAChR‐ specific pharmacologic antagonists and small interfering RNAs. The genomic effects of ETS and nicotine involved the transcription factor GATA‐2 that showed a multifold increase in quantity and activity in exposed KCs. Using protein kinase inhibitors and dominant negative and constitutively active constructs, we charac terized the principal signaling cascades mediating a switch in the nAChR subtype. Cumulative results indi cated that the α3(β2/β4) to α3(β2/α4)α5 nAChR tran sition predominantly involved protein kinase C, α3(β2/ β4)α5 to α7 nAChR transition—Ca2+/calmodulin‐ dependent protein kinase II and p38 MAPK, and α7 self‐up‐regulatio—the p38 MAPK/Akt pathway, and JAK‐2. These results provide a mechanistic insight into the genomic effects of ETS and nicotine on KCs and characterize signaling pathways mediating autoregulation of stepwise overexpression of nAChR subtypes with increasing Ca2+ permeability in exposed cells. These observations have salient clinical implications, because a switch in the nAChR subunit composition can bring about a corresponding switch in receptor function, leading to profound pathobiologic effects ob served in KCs exposed to tobacco products.—Arredondo, J., Chernyavsky, A. I., Jolkovsky, D. L., Pinkerton, K. E., Grando, S. A. Receptor‐mediated tobacco toxicity: acceleration of sequential expression of α5 and α7 nicotinic receptor subunits in oral keratinocytes exposed to cigarette smoke. FASEB J. 22, 1356–1368 (2008)

SLURP-2: A novel cholinergic signaling peptide in human mucocutaneous epithelium.

The biologic role of novel cholinergic toxin‐like signaling peptides termed SLURP (secreted mammalian Ly‐6/uPAR‐related protein) in the mucocutaneous epithelium is a subject of intense research. Previous studies demonstrated that SLURP‐1 activates the α7 subtype of keratinocyte nicotinic acetylcholine receptors (nAChRs) and facilitates keratinization and programmed cell death, and that the level of SLURP‐2 was found to be upregulated several fold in the hyperproliferative skin of patients with psoriasis. In this study, we demonstrated for the first time that human epidermal and oral keratinocytes secrete SLURP‐2. We cloned human SLURP‐2 and produced the mouse monoclonal antibody 341F10‐1F12 that visualized SLURP‐2 in the cytoplasm of normal human epidermal and oral keratinocytes grown in culture. In epidermis, SLURP‐2 was found predominantly in the suprabasal compartment, whereas in the attached gingiva—in the lowermost epithelial layers. Recombinant SLURP‐2 (rSLURP‐2) competed with nicotinic radioligands for binding to keratinocytes, showing a higher affinity to the [3H]epibatidine‐ than [3H]nicotine‐labeled sites. Treatment with rSLURP‐2 significantly (P < 0.05) increased the number of keratinocytes in culture and their resistance to apoptosis, which could be abolished by mecamylamine more efficiently than α‐bungarotoxin. By real‐time PCR and in‐cell western, rSLURP‐2 significantly (P < 0.05) downregulated gene expression of the differentiation markers loricrin, filaggrin, and cytokeratins 1 and 10, and pro‐apoptotic Bax, Bad, and caspase 3 which were elevated by high extracellular calcium, and rSLURP‐2 also abolished activation of caspases 3 and 8 caused by camptothecin. These results indicated that SLURP‐2 competes with acetylcholine predominantly at the α3 nAChR, and that receptor ligation with SLURP‐2 delays keratinocyte differentiation and prevents apoptosis. Thus, the different effects observed for SLURP‐1 and ‐2 can be explained by their differential binding to the nAChR subtypes expressed in keratinocytes. These findings present a novel paradigm of the physiologic regulation of mucocutaneous epithelial cells by locally produced small hormone‐like peptide molecules, and open novel directions toward better understanding and treating of skin and mucosal diseases. J. Cell. Physiol.

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.

Receptor-Mediated Tobacco Toxicity: Regulation of Gene Expression through α3β2 Nicotinic Receptor in Oral Epithelial Cells

Tobacco is a known cause of oral disease but the mechanism remains elusive. Nicotine (Nic) is a likely culprit of pathobiological effects because it displaces the local cytotransmitter acetylcholine from the nicotinic receptors (nAChRs) expressed by oral keratinocytes (KCs). To gain a mechanistic insight into tobacco-induced morbidity in the oral cavity, we studied effects of exposures to environmental tobacco smoke (ETS) versus equivalent concentration of pure Nic on human and murine KCs. Both ETS and Nic up-regulated expression of cell cycle and apoptosis regulators, differentiation marker filaggrin, and signal transduction factors at both the mRNA and protein levels. These changes could be abolished in cultured human oral KCs transfected with anti-α3 small interfering RNA or treated with the α3β2-preferring antagonist α-conotoxin MII. Functional inactivation of α3-mediated signaling in α3−/− mutant KCs prevented most of the ETS/Nic-dependent changes in gene expression. To determine relevance of the in vitro findings to the in vivo situation, we studied gene expression in oral mucosa of neonatal α3+/+ and α3−/− littermates delivered by heterozygous mice soon after their exposures to ETS or equivalent concentration of pure Nic in drinking water. In addition to reverse transcriptase-polymerase chain reaction and Western blot, the ETS/Nic-dependent alterations in gene expression were also detected by semiquantitative immunofluorescence assay directly in KCs comprising murine oral mucosa. Only wild-type mice consistently developed significant ( P 2 and M 3 was also altered. This novel mechanism offers innovative solutions to ameliorate the tobacco-related cell damage and intercede in disease pathways, and may shed light on general mechanisms regulating and driving tobacco-related morbidity in human cells.