Juan Arredondo

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.

Desmoglein Versus Non-desmoglein Signaling in Pemphigus Acantholysis CHARACTERIZATION OF NOVEL SIGNALING PATHWAYS DOWNSTREAM OF PEMPHIGUS VULGARIS ANTIGENS

Although it is accepted that pemphigus antibody binding to keratinocytes (KCs) evokes an array of intracellular biochemical events resulting in cell detachment and death, the triggering events remain obscure. It has been postulated that the binding of pemphigus vulgaris IgG (PVIgG) to KCs induces “desmosomal” signaling. Because in contrast to integrins and classical cadherins, desmoglein (Dsg) molecules are not known to elicit intracellular signaling, and because PV patients also produce non-Dsg autoantibodies, we investigated the roles of both Dsg and non-desmoglein PV antigens. The time course studies of KCs treated with PVIgG demonstrated that the activity of Src peaked at 30 min, EGF receptor kinase (EGFRK) at 60 min, and p38 MAPK at 240 min. The Src inhibitor PP2 decreased EGFRK and p38 activities by ∼45 and 30%, respectively, indicating that in addition to Src, PVIgG evokes other triggering events. The shrinkage of KCs (cell volume reduction) became significant at 120 min, keratin aggregation at 240 min, and an increase of TUNEL positivity at 360 min. Pretreatment of KCs with PP2 blocked PVIgG-dependent cell shrinkage and keratin aggregation by ∼50% and TUNEL positivity by ∼25%. The p38 MAPK inhibitor PD169316 inhibited these effects by ∼15, 20, and 70%, respectively. Transfection of KCs with small interfering RNAs that silenced expression of Dsg1 and/or Dsg3 proteins, blocked ∼50% of p38 MAPK activity but did not significantly alter the PVIgG-dependent rise in Src and EGFRK activities. These results indicate that activation of p38 MAPK is a late signaling step associated with collapse of the cytoskeleton and disassembly of desmosomes caused by upstream events involving Src and EGFRK. Therefore, the early acantholytic events are triggered by non-Dsg antibodies.

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.

M3 muscarinic receptor antagonists inhibit small cell lung carcinoma growth and mitogen-activated protein kinase phosphorylation induced by acetylcholine secretion

The importance of acetylcholine as a neurotransmitter in the nervous system is well established, but little is yet known about its recently described role as an autocrine and paracrine hormone in a wide variety of nonneuronal cells. Consistent with the expression of acetylcholine in normal lung, small cell lung carcinoma (SCLC) synthesize and secrete acetylcholine, which acts as an autocrine growth factor through both nicotinic and muscarinic cholinergic mechanisms. The purpose of this study was to determine if interruption of autocrine muscarinic cholinergic signaling has potential to inhibit SCLC growth. Muscarinic receptor (mAChR) agonists caused concentration-dependent increases in intracellular calcium and mitogen-activated protein kinase (MAPK) and Akt phosphorylation in SCLC cell lines. The inhibitory potency of mAChR subtype-selective antagonists and small interfering RNAs (siRNAs) on acetylcholine-increased intracellular calcium and MAPK and Akt phosphorylation was consistent with mediation by M3 mAChR (M3R). Consistent with autocrine acetylcholine secretion stimulating MAPK and Akt phosphorylation, M3R antagonists and M3R siRNAs alone also caused a decrease in basal levels of MAPK and Akt phosphorylation in SCLC cell lines. Treatment of SCLC cells with M3R antagonists inhibited cell growth both in vitro and in vivo and also decreased MAPK phosphorylation in tumors in nude mice in vivo. Immunohistochemical staining of SCLC and additional cancer types showed frequent coexpression of acetylcholine and M3R. These findings suggest that M3R antagonists may be useful adjuvants for treatment of SCLC and, potentially, other cancers.

Differential regulation of keratinocyte chemokinesis and chemotaxis through distinct nicotinic receptor subtypes

Nicotinergic agents can act as both chemokines and chemoattractants for cell migration. Epidermal keratinocytes both synthesize acetylcholine and use it as a paracrine and autocrine regulator of cell motility. To gain a mechanistic insight into nicotinergic control of keratinocyte motility, we determined types of nicotinic acetylcholine receptors and signaling pathways regulating keratinocyte chemokinesis and chemotaxis, using respective modifications of the agarose gel keratinocyte outgrowth assay. Random migration of keratinocytes was significantly (P<0.05) inhibited by hemicholinum-3, a metabolic inhibitor of acetylcholine synthesis, as well as by the α-conotoxins MII and AuIB, preferentially blocking α3-containing nicotinic acetylcholine receptors. The use of antisense oligonucleotides specific for nicotinic-acetylcholine-receptor subunits and knockout mice demonstrated pivotal role for the α3β2 channel in mediating acetylcholine-dependent chemokinesis. Signaling pathways downstream of α3β2 included activation of the protein-kinase-C isoform δ and RhoA-dependent events. The nicotinergic chemotaxis of keratinocytes was most pronounced towards the concentration gradient of choline, a potent agonist of α7 nicotinic acetylcholine receptor. The α7-preferring antagonist α-bungarotoxin significantly (P<0.05) diminished keratinocyte chemotaxis, further suggesting a central role for the α7 nicotinic acetylcholine receptor. This hypothesis was confirmed in experiments with anti-α7 antisense oligonucleotides and α7-knockout mice. The signaling pathway mediating α7-dependent keratinocyte chemotaxis included intracellular calcium, activation of calcium/calmodulin-dependent protein-kinase II, conventional isoforms of protein-kinase C, phosphatidylinositol-3-kinase and engagement of Rac/Cdc42. Redistribution of α7 immunoreactivity to the leading edge of keratinocytes upon exposure to a chemoattractant preceded crescent shape formation and directional migration. Application of high-resolution deconvolution microscopy demonstrated that, on the cell membrane of keratinocytes, the nicotinic acetylcholine receptor subunits localize with the integrin β1. The obtained results demonstrate for the first time that α3 and α7 nicotinic acetylcholine receptors regulate keratinocyte chemokinesis and chemotaxis, respectively, and identify signaling pathways mediating these functions, which has clinical implications for wound healing and control of cancer metastases.

Mutations in LAMB2 causing a severe form of synaptic congenital myasthenic syndrome

Background: We describe a severe form of congenital myasthenic syndrome (CMS) associated with congenital nephrosis and ocular malformations caused by two truncating mutations in the gene encoding the laminin β2 subunit (LAMB2). Methods and results: Mutational analysis in the affected patient, who has a history of a serious untoward reaction to treatment with acetylcholinesterase inhibition, revealed two frame-shifting heteroallelic mutations, a maternally inherited 1478delG and a paternally inherited 4804delC. An anconeus muscle biopsy demonstrated a profound distortion of the architecture and function of the neuromuscular junction, which was strikingly similar to that seen in mice lacking laminin β2 subunit. The findings included: pronounced reduction of the axon terminal size with encasement of the nerve endings by Schwann cells, severe widening of the primary synaptic cleft and invasion of the synaptic space by the processes of Schwann cells, and moderate simplification of postsynaptic folds and intact expression of the endplate acetylcholinesterase. The endplate potential quantal content was notably reduced, while the frequencies and amplitudes of miniature endplate potentials were only moderately diminished and the decay phases of miniature endplate potentials were normal. Western blot analysis of muscle and kidney tissue and immunohistochemistry of kidney tissue showed no laminin β2 expression. Conclusion: This case, which represents a new type of synaptic CMS, exemplifies the wide variability of phenotypes associated with LAMB2 mutations and underscores the fundamental role that laminin β2 plays in the development of the human neuromuscular junction.

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.

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.