Juan Arredondo - Academia.edu (original) (raw)
Papers by Juan Arredondo
Life Sciences, 2003
We investigated the mechanism mediating cholinergic control of cell-to-cell adhesion of human epi... more 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.
Life Sciences, 2003
We have investigated how the cholinergic system of epidermal keratinocytes (KC) controls migrator... more 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 dosedependent 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.
Journal of Cell Biology, 2002
Several ganglionic nicotinic acetylcholine receptor (nAChR) types are abundantly expressed in non... more 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 charact...
The FASEB Journal, 2006
The use of tobacco products is associated with an increased incidence of periodontal disease, poo... more 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 ␣7 nAChR, expressed by oral keratinocytes (KCs). This study was designed to gain mechanistic insight into ␣7-mediated morbidity of tobacco products in the oral cavity. We investigated the signaling pathways downstream of ␣7 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 ␣7 antagonist ␣-bungarotoxin (␣BTX) or transfected with anti-␣7 small interfering RNA (siRNA-␣7). 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 ␣7 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-␣7. Thus, nuclear transactivation of STAT-3 in KCs exposed to tobacco products is mediated via intracellular signaling downstream from ␣7, 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 ␣7 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 ␣7 nicotinic receptor in oral keratinocytes.
The FASEB Journal, 2007
Tobacco products and nicotine alter the cell cycle and lead to squamatization of oral keratinocyt... more 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 Ca 2؉ influx that varies in magnitude between different nAChR subtypes. Normal differentiation of KCs is associated with sequential expression of the nAChR subtypes with increasing Ca 2؉ permeability, such as ␣5-containing ␣3 nAChR and ␣7 nAChR. Exposure to environmental tobacco smoke (ETS) or an equivalent concentration of nicotine accelerated by severalfold the ␣5 and ␣7 expression in KCs, which could be abolished by mecamylamine and ␣-bungarotoxin with different efficacies, suggesting the following sequence of autoregulation of the expression of nAChR subtypes: ␣3(2/4) > ␣3(2/4)␣5 > ␣7 > ␣7. This conjecture was corroborated by results of quantitative assays of subunit mRNA and protein levels, using nAChRspecific 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 characterized the principal signaling cascades mediating a switch in the nAChR subtype. Cumulative results indicated that the ␣3(2/4) to ␣3(2/4)␣5 nAChR transition predominantly involved protein kinase C, ␣3(2/ 4)␣5 to ␣7 nAChR transition-Ca 2؉ /calmodulindependent protein kinase II and p38 MAPK, and ␣7 self-up-regulation-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 Ca 2؉ 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 observed in KCs exposed to tobacco products. Arre
Laboratory Investigation, 2001
Smoking and smokeless tobacco cause morbidity that originates from the epithelium lining of the s... more 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 ␣3, ␣5, ␣7, 2, and 4 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.
Journal of Cellular Physiology, 2006
The biologic role of novel cholinergic toxin-like signaling peptides termed SLURP (secreted mamma... more 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 alpha7 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 &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05) increased the number of keratinocytes in culture and their resistance to apoptosis, which could be abolished by mecamylamine more efficiently than alpha-bungarotoxin. By real-time PCR and in-cell western, rSLURP-2 significantly (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 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 alpha3 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.
Journal of Cancer Research and Clinical Oncology, 2006
Identification of the mechanisms leading to malignant transformation of respiratory cells may pro... more Identification of the mechanisms leading to malignant transformation of respiratory cells may prove useful in the prevention and treatment of tobacco-related lung cancer. Nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-nitrosonornicotine (NNN) can induce tumors both locally and systemically. In addition to the genotoxic effect, they have been shown to affect lung cells due to ligating the nicotinic acetylcholine receptors (nAChRs) expressed on the plasma membrane. In this study, we sought to establish the role for nAChRs in malignant transformation caused by NNK and NNN. We used the BEP2D cells that represent a suitable model for studying the various stages of human bronchial carcinogenesis. We found that these cells express alpha1, alpha3, alpha5, alpha7, alpha9, alpha10, beta1, beta2, and beta4 nAChR subunits that can form high-affinity binding sites for NNK and NNN. Exposure of BEP2D cells to either NNK or NNN in both cases increased their proliferative potential which could be abolished in the presence of nAChR antagonists alpha-bungarotoxin, which worked most effectively against NNK, or mecamylamine, which was most efficient against NNN. The BEP2D cells stimulated with the nitrosamines showed multifold increases of the transcription of the PCNA and Bcl-2 genes by both real-time polymerase chain reaction and in-cell western assays. To gain a mechanistic insight into NNK- and NNN-initiated signaling, we investigated the expression of genes encoding the signal transduction effectors GATA-3, nuclear factor-kappaB, and STAT-1. Experimental results indicated that stimulation of nAChRs with NNK led to activation of all three signal transduction effectors under consideration, whereas NNN predominantly activated GATA-3 and STAT-1. The GATA-3 protein-binding activity induced by NNK and NNN correlated with elevated gene expression. The obtained results support the novel concept of receptor-mediated action of NNK and NNN placing cellular nAChRs in the center of the pathophysiologic loop, and suggest that an nAChR antagonist may serve as a chemopreventive agent.
Journal of Biological Chemistry, 2009
A loss of epidermal cohesion in pemphigus vulgaris (PV) results from autoantibody action on kerat... more A loss of epidermal cohesion in pemphigus vulgaris (PV) results from autoantibody action on keratinocytes (KCs) activating the signaling kinases and executioner caspases that damage KCs, causing their shrinkage, detachment from neighboring cells, and rounding up (apoptolysis). In this study, we found that PV antibody binding leads to activation of epidermal growth factor receptor kinase, Src, p38 MAPK, and JNK in KCs with time pattern variations from patient to patient. Both extrinsic and intrinsic apoptotic pathways were also activated. Although Fas ligand neutralizing antibody could inhibit the former pathway, the mechanism of activation of the latter remained unknown. PV antibodies increased cytochrome c release, suggesting damage to mitochondria. The immunoblotting experiments revealed penetration of PVIgG into the subcellular mitochondrial fraction. The antimitochondrial antibodies from different PV patients recognized distinct combinations of antigens with apparent molecular sizes of 25, 30, 35, 57, 60, and 100 kDa. Antimitochondrial antibodies were pathogenic because their absorption abolished the ability of PVIgG to cause keratinocyte detachment both in vitro and in vivo. The downstream signaling of antimitochondrial antibodies involved JNK and late p38 MAPK activation, whereas the signaling of anti-desmoglein 3 (Dsg3) antibody involved JNK and biphasic p38 MAPK activation. Using KCs grown from Dsg3 ؊/؊ mice, we determined that Dsg3 did not serve as a surrogate antigen allowing antimitochondrial antibodies to enter KCs. The PVIgG-induced activation of epidermal growth factor receptor and Src was affected neither in Dsg3 ؊/؊ KCs nor due to absorption of antimitochondrial antibodies. These results demonstrated that apoptolysis in PV is a complex process initiated by at least three classes of autoantibodies directed against desmosomal, mitochondrial, and other keratinocyte self-antigens. These autoantibodies synergize with the proapoptotic serum and tissue factors to trigger both extrinsic and intrinsic pathways of cell death and break the epidermal cohesion, leading to blisters. Further elucidation of the primary signaling events downstream of PV autoantigens will be crucial for the development of a more successful therapy for PV patients.
Journal of Biological Chemistry, 2009
Defining the signaling mechanisms and effector proteins mediating phenotypic and mechanical plast... more Defining the signaling mechanisms and effector proteins mediating phenotypic and mechanical plasticity of keratinocytes (KCs) during wound epithelialization is one of the major goals in epithelial cell biology. The acetylcholine (ACh)-gated ion channels, or nicotinic ACh receptors (nAChRs), mediate the nicotinergic signaling that controls crawling locomotion of KCs. To elucidate relative contributions of the ionic and protein kinase-mediated events elicited due to activation of ␣7 nAChRs, we quantitated expression of ␣ 2-integrin gene at the mRNA and protein levels and also measured Rho kinase activity in KCs stimulated with the ␣7 agonist AR-R17779 while blocking the Na ؉ or Ca 2؉ entry and/or inhibiting signaling kinases. The results demonstrated the existence of the two-component signaling systems coupling the ionic events and protein kinase signaling cascades downstream of ␣7 nAChR to simultaneous upregulation of ␣ 2-integrin expression and activation of Rho kinase. The Raf/MEK1/ERK1/2 cascade up-regulating ␣ 2-integrin was activated due to both Ca 2؉-dependent recruitment of Ca 2؉ /calmodulin-dependent protein kinase II and protein kinase C and Ca 2؉-independent activation of Ras. Likewise the phosphatidylinositol 3-kinase-mediated activation of Rho kinase was elicited due to both Ca 2؉ entry-dependent involvement of Ca 2؉ /calmodulin-dependent protein kinase II and Ca 2؉-independent activation of Jak2. Thus, although the initial signals emanating from activated ␣7 nAChR are different in nature the pathways intersect at common effector molecules providing for a common end point effect. This novel paradigm of nAChR-mediated coordination of the ionic and metabolic signaling events can allow an auto/paracrine ACh to simultaneously alter gene expression and induce reciprocal changes in the cytoskeleton and contractile system of KCs required to compete a particular step of wound epithelialization.
Journal of Biological Chemistry, 2007
Although it is accepted that pemphigus antibody binding to keratinocytes (KCs) evokes an array of... more 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 ϳ45 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 ϳ50% and TUNEL positivity by ϳ25%. The p38 MAPK inhibitor PD169316 inhibited these effects by ϳ15, 20, and 70%, respectively. Transfection of KCs with small interfering RNAs that silenced expression of Dsg1 and/or Dsg3 proteins, blocked ϳ50% 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.
Journal of Biological Chemistry, 2007
The mechanisms mediating and regulating assembly and disassembly of intercellular junctions is a ... more The mechanisms mediating and regulating assembly and disassembly of intercellular junctions is a subject of intensive research. The IgG autoantibodies produced in patients with the immunoblistering skin disease pemphigus vulgaris (PV) can induce keratinocyte (KC) dyshesion (acantholysis) via mechanisms that involve signaling kinases targeting intercellular adhesion molecules, thus providing a useful model to study the physiologic regulation of KC cohesion. Previous studies showed that activation of Src and protein kinase C are the earliest events in the PV IgG-induced intracellular phosphorylation cascades and that cholinergic agonists are effective for treating patients with pemphigus. In this study, we sought to elucidate the molecular mechanisms allowing cholinergic agonists to inhibit PV IgG-induced acantholysis and phosphorylation of KC adhesion molecules. The extent of acantholysis in KC monolayers correlated closely with the degree of PV IgG-induced phosphorylation of p120-and -catenins, with classic isoforms of protein kinase C mediating serine phosphorylation of -catenin and Src-tyrosine phosphorylation of p120-catenin. The M 1 muscarinic agonist pilocarpine blocked phosphorylation of both catenins, which could be abolised by the M 1 antagonist MT7. The ␣7 nicotinic agonist AR-R17779 inhibited phosphorylation of P120-cateinin. The ␣7 antagonist methyllycaconitine abolished the effect of AR-R17779. Okadaic acid abrogated protective effects of agonists on phosphorylation of -catenin, and pervanadate, on that of p120-catenin. Stimulation of KCs with pilocarpine significantly (p < 0.05) elevated both serine/threonine and tyrosine phosphatase activities in KCs. AR-R17779 both stimulated tyrosine phosphatase and decreased PV IgGinduced Src activity. Methyllycaconitine released Src activity in intact KCs and caused acantholysis. Thus, downstream signaling from M 1 abolished PV IgG-dependent catenin phosphorylation due to activation of both serine/threonine and tyrosine phosphatases, whereas ␣7 action involved both activation of tyrosine phosphatase and inhibition of Src. These findings identified novel paradigm of regulation of signaling kinases associated with cholinergic receptors and provided mechanistic explanation of therapeutic activity of cholinomimetics in PV patients.
Journal of Biological Chemistry, 2005
The physiologic mechanisms that determine directionality of lateral migration are a subject of in... more The physiologic mechanisms that determine directionality of lateral migration are a subject of intense research. Galvanotropism in a direct current (DC) electric field represents a natural model of cell reorientation toward the direction of future migration. Keratinocyte migration is regulated through both the nicotinic and muscarinic classes of acetylcholine (ACh) receptors. We sought to identify the signaling pathway mediating the cholinergic regulation of chemotaxis and galvanotropism. The pharmacologic and molecular modifiers of the Ras/Raf-1/MEK1/ERK signaling pathway altered both chemotaxis toward choline and galvanotropism toward the cathode in a similar way, indicating that the same signaling steps were involved. The galvanotropism was abrogated due to inhibition of ACh production by hemicholinium-3 and restored by exogenously added carbachol. The concentration gradients of ACh and choline toward the cathode in a DC field were established by highperformance liquid chromatographic measurements. This suggested that keratinocyte galvanotaxis is, in effect, chemotaxis toward the concentration gradient of ACh, which it creates in a DC field due to its highly positive charge. A time-course immunofluorescence study of the membrane redistribution of ACh receptors in keratinocytes exposed to a DC field revealed rapid relocation to and clustering at the leading edge of ␣7 nicotinic and M 1 muscarinic receptors. Their inactivation with selective antagonists or small interfering RNAs inhibited galvanotropism, which could be prevented by transfecting the cells with constitutively active MEK1. The end-point effect of the cooperative signaling downstream from ␣7 and M 1 through the MEK1/ERK was an up-regulated expression of ␣ 2 and ␣ 3 integrins, as judged from the results of real-time PCR and quantitative immunoblotting. Thus, ␣7 works together with M 1 to orient a keratinocyte toward direction of its future migration. Both ␣7 and M 1 apparently engage the Ras/Raf/MEK/ERK pathway to up-regulate expression of the "sedentary" integrins required for stabilization of the lamellipodium at the keratinocyte leading edge.
International Immunopharmacology, 2010
International Immunopharmacology, 2010
Although acetylcholine (ACh) is well known for its neurotransmitter function, recent studies have... more Although acetylcholine (ACh) is well known for its neurotransmitter function, recent studies have indicated that it also functions as an immune cytokine that prevents macrophage activation through a 'cholinergic (nicotinic) anti-inflammatory pathway'. In this study, we used the macrophage-like U937 cells to elucidate the mechanisms of the physiologic control of cytokine production by auto/ paracrine ACh through the nicotinic class of ACh receptors (nAChRs) expressed in these cells. Stimulation of cells with lipopolysaccharide upregulated expression of α1, α4, α5, α7, α10, β1 and β3 subunits, downregulated α6 and β2 subunits, and did not alter the relative quantity of α9 and β4 mRNAs. Distinct nAChR subtypes showed differential regulation of the production of pro-and antiinflammatory cytokines. While inhibition of the expression of the TNF-α gene was mediated predominantly by the α-bungarotoxin sensitive nAChRs, that of the IL-6 and IL-18 genes-by the mecamylamine-sensitive nAChRs. Both the Mec-and αBtx-sensitive nAChRs regulated expression of the IL-1β gene equally efficiently. Upregulation of IL-10 production by auto/paracrine ACh was mediated predominantly through α7 nAChR. These findings offer a new insight on how nicotinic agonists control inflammation, thus laying a groundwork for the development of novel immunomodulatory therapies based on the nAChR subtype selectivity of nicotinic agonists.
Experimental Cell Research, 2004
The biological mechanisms involved in initiating, coordinating, and ultimately terminating cell-c... more The biological mechanisms involved in initiating, coordinating, and ultimately terminating cell-cell adhesion in the stratified epithelium are not well understood at present. This study was designed to elucidate the roles of the muscarinic M 3 , the nicotinic a3, and the mixed muscarinic-nicotinic a9 acetylcholine receptors in physiologic control of keratinocyte adhesion. Both muscarinic and nicotinic antagonists caused keratinocyte detachment and reversibly increased the permeability of keratinocyte monolayers, indicative of the involvement of both muscarinic and nicotinic pathways in the cholinergic control of keratinocyte adhesion. Since phosphorylation of adhesion proteins plays an important role in rapid assembly and disassembly of intercellular junctions, we measured muscarinic and nicotinic effects on phosphorylation of keratinocyte adhesion molecules. The phosphorylation levels of E-cadherin, h-catenin, and g-catenin increased following pharmacological blockage of muscarinic receptors. Long-term blocking of a3, a9, and M 3 receptor signaling pathways with antisense oligonucleotides resulted in cell-cell detachment and changes in the expression levels of E-cadherin, h-catenin, and g-catenin in cultured human keratinocytes. Simultaneous inhibition of several receptor subtypes with a mixture of antisense oligonucleotides produced intensified abnormalities with cell adhesion. Moreover, altered cell-cell adhesion was found in the stratified epithelium of a3, a9, and M 3 receptor knockout mice. Keratinocytes from these mice exhibited abnormal expression of adhesion molecules at both the protein and the mRNA levels. Thus, our data indicate that the a3, a9, and M 3 acetylcholine receptors play key roles in regulating in a synergistic mode keratinocyte adhesion, most probably by modulating cadherin and catenin levels and activities. These findings may aid in the development of novel methods useful for the treatment of skin adhesion diseases and tumor metastasis.
Experimental Cell Research, 2007
Epithelialization, a major component of wound healing, depends on keratinocyte adhesion and migra... more Epithelialization, a major component of wound healing, depends on keratinocyte adhesion and migration. Initiation of migration relies upon the ability of keratinocytes to free themselves from neighboring cells and basement membrane. The local cytotransmitter acetylcholine (ACh) controls keratinocyte adhesion and locomotion through different classes of ACh receptors (AChR). In this study, we explored signaling pathways downstream of the α9 AChR subtype that had been shown to control cell shape and cytoplasm mobility. Inactivation of α9 signaling by pharmacologic antagonism and RNA interference in keratinocyte cultures and null mutation in knockout mice delayed wound re-epithelization in vitro and in vivo, respectively, and diminished the extent of colony scattering and cell outgrowth from the megacolony. Although keratinocytes at the leading edge elongated, produced filopodia and moved out, most of them remained anchored to the substrate by long cytoplasmic processes that stretched during their migration instead of retracting the uropod. Since the velocity of keratinocyte migration was not altered, we investigated the role of α9 in assembly/disassembly of the cell-cell and cell-matrix adhesion complexes. Stimulation of α9 upregulated in a time-dependent fashion phosphorylation of the adhesion molecules comprising focal adhesions (FAK, paxillin) and intercellular junctions (β-catenin, desmoglein 3) as well as cytokeratins. Stimulation of α9 was associated with activation of phospholipase C, Src, EGF receptor kinase, protein kinase C, Rac and Rho, whereas inhibition of this receptor interfered with phosphorylation of adhesion and cytoskeletal proteins, and also altered cell-cell cohesion. We conclude that signaling through α9 AChR is critical for completion of the very early stages of epithelialization. By activating α9 AChR, ACh can control the dynamics and strength of cell-cell cohesion, disabling of a trailing uropod and disassembly and re-assembly of focal adhesions, thus facilitating crawling locomotion.
Cancer Biology & Therapy, 2006
ACh acetylcholine αBtx α-bungarotoxin GAPDH glyceraldehyde-3-phosphate dehydrogenase Mec mecamyla... more ACh acetylcholine αBtx α-bungarotoxin GAPDH glyceraldehyde-3-phosphate dehydrogenase Mec mecamylamine nAChRs nicotinic acetylcholine receptors NNK 4-(methylnitrosamino)-1-(3-pyridyl)-1butanone NNN N'-nitrosonornicotine RT-PCR reverse-transcription polymerase chain reaction TBD trypan blue dye ACKNOWLEDGEMENTS
Autoimmunity, 2006
This study tested a recently proposed &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;am... more This study tested a recently proposed &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;Basal Cell Shrinkage&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot; hypothesis of pemphigus acantholysis through a quantitative analysis of individual and cooperative effects of pemphigus vulgaris (PV) IgG, Fas-ligand (Fas-L) and tumor necrosis factor-alpha (TNFalpha) on keratinocyte (KC) volume (i.e. cell size) and adhesive properties. Exposure of KC monolayers and MatTek EpiDermFT tissues cultures to the physiologic concentrations of Fas-L, TNFalpha or IgGs from two PV patients resulted in various degrees of reversible changes, which were not observed in control cultures either exposed to normal IgG or left intact. Within 12-24 h of exposure, basal cells in experimental cultures lost their ability to form stress fibers, retracted cytoplasmic aprons and formed keratin aggregates, indicating that their cytoskeleton collapsed. The cell volume decreased significantly (p &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05) as the polygonal cell shape changed to a round one. The shrunk cells detached from their neighbors and the substrate, resulting in a reciprocal increase of both the areas of acantholysis and the number of detached KCs, respectively. Since in the skin of PV patients, KCs are targeted by autoantibodies concomitantly with being exposed to autocrine and paracrine pro-apoptotic and pro-inflammatory cytokines, we combined PV IgG with Fas-L and/or TNFalpha in the cell culture experiments. This amplified several fold an ability of PV IgG to cause basal cell shrinkage and detachment. The obtained results demonstrated for the first time that PV IgG works together with Fas-L and TNFalpha to induce acantholysis via basal cell shrinkage, which provides a novel mechanism explaining successful treatment of PV patients with TNFalpha inhibitors.
American Journal of Physiology-Cell Physiology, 2010
SLURP-1 (secreted mammalian Ly-6/urokinase plasminogen activator receptor-related protein-1) is a... more SLURP-1 (secreted mammalian Ly-6/urokinase plasminogen activator receptor-related protein-1) is a novel auto/paracrine cholinergic peptide that can bind to α7-nicotinic acetylcholine receptor (nAChR), a high Ca2+-permeable ion channel coupled to regulation of nuclear factor-κB (NF-κB) expression. Elucidation of intracellular signaling events elicited by SLURP-1 is crucial for understanding the molecular mechanism of functioning of this novel hormone-like peptide that alters vital cell functions and can protect from tumorigenic transformation. In this study, we sought to dissect out the role of α7-nAChR in mediating the biologic effects of recombinant SLURP-1 on the immortalized line of human oral keratinocytes Het-1A. A multifold upregulation of the NF-κB expression at the mRNA and protein levels by SLURP-1 was only slightly diminished due to elimination of Na+, whereas in Ca2+-free medium the effect of SLURP-1 was inhibited by >50%. Both in the absence of extracellular Ca2+and i...
Life Sciences, 2003
We investigated the mechanism mediating cholinergic control of cell-to-cell adhesion of human epi... more 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.
Life Sciences, 2003
We have investigated how the cholinergic system of epidermal keratinocytes (KC) controls migrator... more 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 dosedependent 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.
Journal of Cell Biology, 2002
Several ganglionic nicotinic acetylcholine receptor (nAChR) types are abundantly expressed in non... more 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 charact...
The FASEB Journal, 2006
The use of tobacco products is associated with an increased incidence of periodontal disease, poo... more 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 ␣7 nAChR, expressed by oral keratinocytes (KCs). This study was designed to gain mechanistic insight into ␣7-mediated morbidity of tobacco products in the oral cavity. We investigated the signaling pathways downstream of ␣7 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 ␣7 antagonist ␣-bungarotoxin (␣BTX) or transfected with anti-␣7 small interfering RNA (siRNA-␣7). 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 ␣7 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-␣7. Thus, nuclear transactivation of STAT-3 in KCs exposed to tobacco products is mediated via intracellular signaling downstream from ␣7, 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 ␣7 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 ␣7 nicotinic receptor in oral keratinocytes.
The FASEB Journal, 2007
Tobacco products and nicotine alter the cell cycle and lead to squamatization of oral keratinocyt... more 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 Ca 2؉ influx that varies in magnitude between different nAChR subtypes. Normal differentiation of KCs is associated with sequential expression of the nAChR subtypes with increasing Ca 2؉ permeability, such as ␣5-containing ␣3 nAChR and ␣7 nAChR. Exposure to environmental tobacco smoke (ETS) or an equivalent concentration of nicotine accelerated by severalfold the ␣5 and ␣7 expression in KCs, which could be abolished by mecamylamine and ␣-bungarotoxin with different efficacies, suggesting the following sequence of autoregulation of the expression of nAChR subtypes: ␣3(2/4) > ␣3(2/4)␣5 > ␣7 > ␣7. This conjecture was corroborated by results of quantitative assays of subunit mRNA and protein levels, using nAChRspecific 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 characterized the principal signaling cascades mediating a switch in the nAChR subtype. Cumulative results indicated that the ␣3(2/4) to ␣3(2/4)␣5 nAChR transition predominantly involved protein kinase C, ␣3(2/ 4)␣5 to ␣7 nAChR transition-Ca 2؉ /calmodulindependent protein kinase II and p38 MAPK, and ␣7 self-up-regulation-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 Ca 2؉ 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 observed in KCs exposed to tobacco products. Arre
Laboratory Investigation, 2001
Smoking and smokeless tobacco cause morbidity that originates from the epithelium lining of the s... more 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 ␣3, ␣5, ␣7, 2, and 4 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.
Journal of Cellular Physiology, 2006
The biologic role of novel cholinergic toxin-like signaling peptides termed SLURP (secreted mamma... more 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 alpha7 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 &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05) increased the number of keratinocytes in culture and their resistance to apoptosis, which could be abolished by mecamylamine more efficiently than alpha-bungarotoxin. By real-time PCR and in-cell western, rSLURP-2 significantly (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 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 alpha3 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.
Journal of Cancer Research and Clinical Oncology, 2006
Identification of the mechanisms leading to malignant transformation of respiratory cells may pro... more Identification of the mechanisms leading to malignant transformation of respiratory cells may prove useful in the prevention and treatment of tobacco-related lung cancer. Nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-nitrosonornicotine (NNN) can induce tumors both locally and systemically. In addition to the genotoxic effect, they have been shown to affect lung cells due to ligating the nicotinic acetylcholine receptors (nAChRs) expressed on the plasma membrane. In this study, we sought to establish the role for nAChRs in malignant transformation caused by NNK and NNN. We used the BEP2D cells that represent a suitable model for studying the various stages of human bronchial carcinogenesis. We found that these cells express alpha1, alpha3, alpha5, alpha7, alpha9, alpha10, beta1, beta2, and beta4 nAChR subunits that can form high-affinity binding sites for NNK and NNN. Exposure of BEP2D cells to either NNK or NNN in both cases increased their proliferative potential which could be abolished in the presence of nAChR antagonists alpha-bungarotoxin, which worked most effectively against NNK, or mecamylamine, which was most efficient against NNN. The BEP2D cells stimulated with the nitrosamines showed multifold increases of the transcription of the PCNA and Bcl-2 genes by both real-time polymerase chain reaction and in-cell western assays. To gain a mechanistic insight into NNK- and NNN-initiated signaling, we investigated the expression of genes encoding the signal transduction effectors GATA-3, nuclear factor-kappaB, and STAT-1. Experimental results indicated that stimulation of nAChRs with NNK led to activation of all three signal transduction effectors under consideration, whereas NNN predominantly activated GATA-3 and STAT-1. The GATA-3 protein-binding activity induced by NNK and NNN correlated with elevated gene expression. The obtained results support the novel concept of receptor-mediated action of NNK and NNN placing cellular nAChRs in the center of the pathophysiologic loop, and suggest that an nAChR antagonist may serve as a chemopreventive agent.
Journal of Biological Chemistry, 2009
A loss of epidermal cohesion in pemphigus vulgaris (PV) results from autoantibody action on kerat... more A loss of epidermal cohesion in pemphigus vulgaris (PV) results from autoantibody action on keratinocytes (KCs) activating the signaling kinases and executioner caspases that damage KCs, causing their shrinkage, detachment from neighboring cells, and rounding up (apoptolysis). In this study, we found that PV antibody binding leads to activation of epidermal growth factor receptor kinase, Src, p38 MAPK, and JNK in KCs with time pattern variations from patient to patient. Both extrinsic and intrinsic apoptotic pathways were also activated. Although Fas ligand neutralizing antibody could inhibit the former pathway, the mechanism of activation of the latter remained unknown. PV antibodies increased cytochrome c release, suggesting damage to mitochondria. The immunoblotting experiments revealed penetration of PVIgG into the subcellular mitochondrial fraction. The antimitochondrial antibodies from different PV patients recognized distinct combinations of antigens with apparent molecular sizes of 25, 30, 35, 57, 60, and 100 kDa. Antimitochondrial antibodies were pathogenic because their absorption abolished the ability of PVIgG to cause keratinocyte detachment both in vitro and in vivo. The downstream signaling of antimitochondrial antibodies involved JNK and late p38 MAPK activation, whereas the signaling of anti-desmoglein 3 (Dsg3) antibody involved JNK and biphasic p38 MAPK activation. Using KCs grown from Dsg3 ؊/؊ mice, we determined that Dsg3 did not serve as a surrogate antigen allowing antimitochondrial antibodies to enter KCs. The PVIgG-induced activation of epidermal growth factor receptor and Src was affected neither in Dsg3 ؊/؊ KCs nor due to absorption of antimitochondrial antibodies. These results demonstrated that apoptolysis in PV is a complex process initiated by at least three classes of autoantibodies directed against desmosomal, mitochondrial, and other keratinocyte self-antigens. These autoantibodies synergize with the proapoptotic serum and tissue factors to trigger both extrinsic and intrinsic pathways of cell death and break the epidermal cohesion, leading to blisters. Further elucidation of the primary signaling events downstream of PV autoantigens will be crucial for the development of a more successful therapy for PV patients.
Journal of Biological Chemistry, 2009
Defining the signaling mechanisms and effector proteins mediating phenotypic and mechanical plast... more Defining the signaling mechanisms and effector proteins mediating phenotypic and mechanical plasticity of keratinocytes (KCs) during wound epithelialization is one of the major goals in epithelial cell biology. The acetylcholine (ACh)-gated ion channels, or nicotinic ACh receptors (nAChRs), mediate the nicotinergic signaling that controls crawling locomotion of KCs. To elucidate relative contributions of the ionic and protein kinase-mediated events elicited due to activation of ␣7 nAChRs, we quantitated expression of ␣ 2-integrin gene at the mRNA and protein levels and also measured Rho kinase activity in KCs stimulated with the ␣7 agonist AR-R17779 while blocking the Na ؉ or Ca 2؉ entry and/or inhibiting signaling kinases. The results demonstrated the existence of the two-component signaling systems coupling the ionic events and protein kinase signaling cascades downstream of ␣7 nAChR to simultaneous upregulation of ␣ 2-integrin expression and activation of Rho kinase. The Raf/MEK1/ERK1/2 cascade up-regulating ␣ 2-integrin was activated due to both Ca 2؉-dependent recruitment of Ca 2؉ /calmodulin-dependent protein kinase II and protein kinase C and Ca 2؉-independent activation of Ras. Likewise the phosphatidylinositol 3-kinase-mediated activation of Rho kinase was elicited due to both Ca 2؉ entry-dependent involvement of Ca 2؉ /calmodulin-dependent protein kinase II and Ca 2؉-independent activation of Jak2. Thus, although the initial signals emanating from activated ␣7 nAChR are different in nature the pathways intersect at common effector molecules providing for a common end point effect. This novel paradigm of nAChR-mediated coordination of the ionic and metabolic signaling events can allow an auto/paracrine ACh to simultaneously alter gene expression and induce reciprocal changes in the cytoskeleton and contractile system of KCs required to compete a particular step of wound epithelialization.
Journal of Biological Chemistry, 2007
Although it is accepted that pemphigus antibody binding to keratinocytes (KCs) evokes an array of... more 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 ϳ45 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 ϳ50% and TUNEL positivity by ϳ25%. The p38 MAPK inhibitor PD169316 inhibited these effects by ϳ15, 20, and 70%, respectively. Transfection of KCs with small interfering RNAs that silenced expression of Dsg1 and/or Dsg3 proteins, blocked ϳ50% 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.
Journal of Biological Chemistry, 2007
The mechanisms mediating and regulating assembly and disassembly of intercellular junctions is a ... more The mechanisms mediating and regulating assembly and disassembly of intercellular junctions is a subject of intensive research. The IgG autoantibodies produced in patients with the immunoblistering skin disease pemphigus vulgaris (PV) can induce keratinocyte (KC) dyshesion (acantholysis) via mechanisms that involve signaling kinases targeting intercellular adhesion molecules, thus providing a useful model to study the physiologic regulation of KC cohesion. Previous studies showed that activation of Src and protein kinase C are the earliest events in the PV IgG-induced intracellular phosphorylation cascades and that cholinergic agonists are effective for treating patients with pemphigus. In this study, we sought to elucidate the molecular mechanisms allowing cholinergic agonists to inhibit PV IgG-induced acantholysis and phosphorylation of KC adhesion molecules. The extent of acantholysis in KC monolayers correlated closely with the degree of PV IgG-induced phosphorylation of p120-and -catenins, with classic isoforms of protein kinase C mediating serine phosphorylation of -catenin and Src-tyrosine phosphorylation of p120-catenin. The M 1 muscarinic agonist pilocarpine blocked phosphorylation of both catenins, which could be abolised by the M 1 antagonist MT7. The ␣7 nicotinic agonist AR-R17779 inhibited phosphorylation of P120-cateinin. The ␣7 antagonist methyllycaconitine abolished the effect of AR-R17779. Okadaic acid abrogated protective effects of agonists on phosphorylation of -catenin, and pervanadate, on that of p120-catenin. Stimulation of KCs with pilocarpine significantly (p < 0.05) elevated both serine/threonine and tyrosine phosphatase activities in KCs. AR-R17779 both stimulated tyrosine phosphatase and decreased PV IgGinduced Src activity. Methyllycaconitine released Src activity in intact KCs and caused acantholysis. Thus, downstream signaling from M 1 abolished PV IgG-dependent catenin phosphorylation due to activation of both serine/threonine and tyrosine phosphatases, whereas ␣7 action involved both activation of tyrosine phosphatase and inhibition of Src. These findings identified novel paradigm of regulation of signaling kinases associated with cholinergic receptors and provided mechanistic explanation of therapeutic activity of cholinomimetics in PV patients.
Journal of Biological Chemistry, 2005
The physiologic mechanisms that determine directionality of lateral migration are a subject of in... more The physiologic mechanisms that determine directionality of lateral migration are a subject of intense research. Galvanotropism in a direct current (DC) electric field represents a natural model of cell reorientation toward the direction of future migration. Keratinocyte migration is regulated through both the nicotinic and muscarinic classes of acetylcholine (ACh) receptors. We sought to identify the signaling pathway mediating the cholinergic regulation of chemotaxis and galvanotropism. The pharmacologic and molecular modifiers of the Ras/Raf-1/MEK1/ERK signaling pathway altered both chemotaxis toward choline and galvanotropism toward the cathode in a similar way, indicating that the same signaling steps were involved. The galvanotropism was abrogated due to inhibition of ACh production by hemicholinium-3 and restored by exogenously added carbachol. The concentration gradients of ACh and choline toward the cathode in a DC field were established by highperformance liquid chromatographic measurements. This suggested that keratinocyte galvanotaxis is, in effect, chemotaxis toward the concentration gradient of ACh, which it creates in a DC field due to its highly positive charge. A time-course immunofluorescence study of the membrane redistribution of ACh receptors in keratinocytes exposed to a DC field revealed rapid relocation to and clustering at the leading edge of ␣7 nicotinic and M 1 muscarinic receptors. Their inactivation with selective antagonists or small interfering RNAs inhibited galvanotropism, which could be prevented by transfecting the cells with constitutively active MEK1. The end-point effect of the cooperative signaling downstream from ␣7 and M 1 through the MEK1/ERK was an up-regulated expression of ␣ 2 and ␣ 3 integrins, as judged from the results of real-time PCR and quantitative immunoblotting. Thus, ␣7 works together with M 1 to orient a keratinocyte toward direction of its future migration. Both ␣7 and M 1 apparently engage the Ras/Raf/MEK/ERK pathway to up-regulate expression of the "sedentary" integrins required for stabilization of the lamellipodium at the keratinocyte leading edge.
International Immunopharmacology, 2010
International Immunopharmacology, 2010
Although acetylcholine (ACh) is well known for its neurotransmitter function, recent studies have... more Although acetylcholine (ACh) is well known for its neurotransmitter function, recent studies have indicated that it also functions as an immune cytokine that prevents macrophage activation through a 'cholinergic (nicotinic) anti-inflammatory pathway'. In this study, we used the macrophage-like U937 cells to elucidate the mechanisms of the physiologic control of cytokine production by auto/ paracrine ACh through the nicotinic class of ACh receptors (nAChRs) expressed in these cells. Stimulation of cells with lipopolysaccharide upregulated expression of α1, α4, α5, α7, α10, β1 and β3 subunits, downregulated α6 and β2 subunits, and did not alter the relative quantity of α9 and β4 mRNAs. Distinct nAChR subtypes showed differential regulation of the production of pro-and antiinflammatory cytokines. While inhibition of the expression of the TNF-α gene was mediated predominantly by the α-bungarotoxin sensitive nAChRs, that of the IL-6 and IL-18 genes-by the mecamylamine-sensitive nAChRs. Both the Mec-and αBtx-sensitive nAChRs regulated expression of the IL-1β gene equally efficiently. Upregulation of IL-10 production by auto/paracrine ACh was mediated predominantly through α7 nAChR. These findings offer a new insight on how nicotinic agonists control inflammation, thus laying a groundwork for the development of novel immunomodulatory therapies based on the nAChR subtype selectivity of nicotinic agonists.
Experimental Cell Research, 2004
The biological mechanisms involved in initiating, coordinating, and ultimately terminating cell-c... more The biological mechanisms involved in initiating, coordinating, and ultimately terminating cell-cell adhesion in the stratified epithelium are not well understood at present. This study was designed to elucidate the roles of the muscarinic M 3 , the nicotinic a3, and the mixed muscarinic-nicotinic a9 acetylcholine receptors in physiologic control of keratinocyte adhesion. Both muscarinic and nicotinic antagonists caused keratinocyte detachment and reversibly increased the permeability of keratinocyte monolayers, indicative of the involvement of both muscarinic and nicotinic pathways in the cholinergic control of keratinocyte adhesion. Since phosphorylation of adhesion proteins plays an important role in rapid assembly and disassembly of intercellular junctions, we measured muscarinic and nicotinic effects on phosphorylation of keratinocyte adhesion molecules. The phosphorylation levels of E-cadherin, h-catenin, and g-catenin increased following pharmacological blockage of muscarinic receptors. Long-term blocking of a3, a9, and M 3 receptor signaling pathways with antisense oligonucleotides resulted in cell-cell detachment and changes in the expression levels of E-cadherin, h-catenin, and g-catenin in cultured human keratinocytes. Simultaneous inhibition of several receptor subtypes with a mixture of antisense oligonucleotides produced intensified abnormalities with cell adhesion. Moreover, altered cell-cell adhesion was found in the stratified epithelium of a3, a9, and M 3 receptor knockout mice. Keratinocytes from these mice exhibited abnormal expression of adhesion molecules at both the protein and the mRNA levels. Thus, our data indicate that the a3, a9, and M 3 acetylcholine receptors play key roles in regulating in a synergistic mode keratinocyte adhesion, most probably by modulating cadherin and catenin levels and activities. These findings may aid in the development of novel methods useful for the treatment of skin adhesion diseases and tumor metastasis.
Experimental Cell Research, 2007
Epithelialization, a major component of wound healing, depends on keratinocyte adhesion and migra... more Epithelialization, a major component of wound healing, depends on keratinocyte adhesion and migration. Initiation of migration relies upon the ability of keratinocytes to free themselves from neighboring cells and basement membrane. The local cytotransmitter acetylcholine (ACh) controls keratinocyte adhesion and locomotion through different classes of ACh receptors (AChR). In this study, we explored signaling pathways downstream of the α9 AChR subtype that had been shown to control cell shape and cytoplasm mobility. Inactivation of α9 signaling by pharmacologic antagonism and RNA interference in keratinocyte cultures and null mutation in knockout mice delayed wound re-epithelization in vitro and in vivo, respectively, and diminished the extent of colony scattering and cell outgrowth from the megacolony. Although keratinocytes at the leading edge elongated, produced filopodia and moved out, most of them remained anchored to the substrate by long cytoplasmic processes that stretched during their migration instead of retracting the uropod. Since the velocity of keratinocyte migration was not altered, we investigated the role of α9 in assembly/disassembly of the cell-cell and cell-matrix adhesion complexes. Stimulation of α9 upregulated in a time-dependent fashion phosphorylation of the adhesion molecules comprising focal adhesions (FAK, paxillin) and intercellular junctions (β-catenin, desmoglein 3) as well as cytokeratins. Stimulation of α9 was associated with activation of phospholipase C, Src, EGF receptor kinase, protein kinase C, Rac and Rho, whereas inhibition of this receptor interfered with phosphorylation of adhesion and cytoskeletal proteins, and also altered cell-cell cohesion. We conclude that signaling through α9 AChR is critical for completion of the very early stages of epithelialization. By activating α9 AChR, ACh can control the dynamics and strength of cell-cell cohesion, disabling of a trailing uropod and disassembly and re-assembly of focal adhesions, thus facilitating crawling locomotion.
Cancer Biology & Therapy, 2006
ACh acetylcholine αBtx α-bungarotoxin GAPDH glyceraldehyde-3-phosphate dehydrogenase Mec mecamyla... more ACh acetylcholine αBtx α-bungarotoxin GAPDH glyceraldehyde-3-phosphate dehydrogenase Mec mecamylamine nAChRs nicotinic acetylcholine receptors NNK 4-(methylnitrosamino)-1-(3-pyridyl)-1butanone NNN N'-nitrosonornicotine RT-PCR reverse-transcription polymerase chain reaction TBD trypan blue dye ACKNOWLEDGEMENTS
Autoimmunity, 2006
This study tested a recently proposed &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;am... more This study tested a recently proposed &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;Basal Cell Shrinkage&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot; hypothesis of pemphigus acantholysis through a quantitative analysis of individual and cooperative effects of pemphigus vulgaris (PV) IgG, Fas-ligand (Fas-L) and tumor necrosis factor-alpha (TNFalpha) on keratinocyte (KC) volume (i.e. cell size) and adhesive properties. Exposure of KC monolayers and MatTek EpiDermFT tissues cultures to the physiologic concentrations of Fas-L, TNFalpha or IgGs from two PV patients resulted in various degrees of reversible changes, which were not observed in control cultures either exposed to normal IgG or left intact. Within 12-24 h of exposure, basal cells in experimental cultures lost their ability to form stress fibers, retracted cytoplasmic aprons and formed keratin aggregates, indicating that their cytoskeleton collapsed. The cell volume decreased significantly (p &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05) as the polygonal cell shape changed to a round one. The shrunk cells detached from their neighbors and the substrate, resulting in a reciprocal increase of both the areas of acantholysis and the number of detached KCs, respectively. Since in the skin of PV patients, KCs are targeted by autoantibodies concomitantly with being exposed to autocrine and paracrine pro-apoptotic and pro-inflammatory cytokines, we combined PV IgG with Fas-L and/or TNFalpha in the cell culture experiments. This amplified several fold an ability of PV IgG to cause basal cell shrinkage and detachment. The obtained results demonstrated for the first time that PV IgG works together with Fas-L and TNFalpha to induce acantholysis via basal cell shrinkage, which provides a novel mechanism explaining successful treatment of PV patients with TNFalpha inhibitors.
American Journal of Physiology-Cell Physiology, 2010
SLURP-1 (secreted mammalian Ly-6/urokinase plasminogen activator receptor-related protein-1) is a... more SLURP-1 (secreted mammalian Ly-6/urokinase plasminogen activator receptor-related protein-1) is a novel auto/paracrine cholinergic peptide that can bind to α7-nicotinic acetylcholine receptor (nAChR), a high Ca2+-permeable ion channel coupled to regulation of nuclear factor-κB (NF-κB) expression. Elucidation of intracellular signaling events elicited by SLURP-1 is crucial for understanding the molecular mechanism of functioning of this novel hormone-like peptide that alters vital cell functions and can protect from tumorigenic transformation. In this study, we sought to dissect out the role of α7-nAChR in mediating the biologic effects of recombinant SLURP-1 on the immortalized line of human oral keratinocytes Het-1A. A multifold upregulation of the NF-κB expression at the mRNA and protein levels by SLURP-1 was only slightly diminished due to elimination of Na+, whereas in Ca2+-free medium the effect of SLURP-1 was inhibited by >50%. Both in the absence of extracellular Ca2+and i...