Emerging roles of planar cell polarity proteins in glutamatergic synapse formation, maintenance and function in health and disease.

The local signaling mechanism which directly assembles and maintains glutamatergic synapses has not been well understood. Glutamatergic synapses are made of presynaptic and postsynaptic compartments with distinct sets of proteins. The planar cell polarity (PCP) pathway is highly conserved and responsible for establishing and maintaining the cell and tissue polarity along the tissue plane. The six core PCP proteins form antagonizing complexes within the cells and asymmetric intercellular complexes across neighboring cells which regulate cell-cell interactions during planar polarity signaling. Accumulating evidence suggests that the PCP proteins play essential roles in glutamatergic synapse assembly, maintenance and function in the brain. This review summarizes the key evidence that PCP proteins may be responsible for the formation and stability of the vast majority of the glutamatergic synapses in hippocampus and medial prefrontal cortex, the progress in understanding the mechanisms of how PCP proteins assemble and maintain glutamatergic synapses and initial insights on how disruption of the function of the PCP proteins can lead to neurodegenerative, neurodevelopmental and neuropsychiatric disorders. The PCP proteins may be the missing pieces of a long-standing puzzle and filling this gap of knowledge may provide the basis for understanding many unsolved questions in synapse biology.

Inter-growth cone communication mediated by planar cell polarity pathway in axon guidance.

The emergence of exquisitely organized axonal projections is one of the greatest wonders of nervous system development. In addition to growing along stereotyped directions, axons join one another as they extend. It is well known that axonal growth cones recognize cell surface guidance cues on axons and either grow along the axons or away from the axons. However, it is less well understood whether and how the growth cones communicate with each other and, if so, what do these interactions mean. Recent studies from our lab provided direct evidence that the growth cones do interact with each other during axon pathfinding. And this interaction is regulated by highly regulated protein-protein interactions among components of the planar cell polarity pathway. The disruption of these interactions lead to guidance defects and disorganization of axons. We propose that this local inter-growth cone PCP-like signaling mechanism reinforces and increases the sensitivity of the growth cone response to shallow Wnt gradients to turn in a precise and organized fashion.

LRRK2 mediates axon development by regulating Frizzled3 phosphorylation and growth cone-growth cone communication.

Axon-axon interactions are essential for axon guidance during nervous system wiring. However, it is unknown whether and how the growth cones communicate with each other while sensing and responding to guidance cues. We found that the Parkinson's disease gene, leucine-rich repeat kinase 2 (LRRK2), has an unexpected role in growth cone-growth cone communication. The LRRK2 protein acts as a scaffold and induces Frizzled3 hyperphosphorylation indirectly by recruiting other kinases and also directly phosphorylates Frizzled3 on threonine 598 (T598). In LRRK1 or LRRK2 single knockout, LRRK1/2 double knockout, and LRRK2 G2019S knockin, the postcrossing spinal cord commissural axons are disorganized and showed anterior-posterior guidance errors after midline crossing. Growth cones from either LRRK2 knockout or G2019S knockin mice showed altered interactions, suggesting impaired communication. Intercellular interaction between Frizzled3 and Vangl2 is essential for planar cell polarity signaling. We show here that this interaction is regulated by phosphorylation of Frizzled3 at T598 and can be regulated by LRRK2 in a kinase activity-dependent way. In the LRRK1/2 double knockout or LRRK2 G2019S knockin, the dopaminergic axon bundle in the midbrain was significantly widened and appeared disorganized, showing aberrant posterior-directed growth. Our findings demonstrate that LRRK2 regulates growth cone-growth cone communication in axon guidance and that both loss-of-function mutation and a gain-of-function mutation (G2019S) cause axon guidance defects in development.

Molecular-level insights into the surface-induced assembly of functional bacterial amyloid.

Protein coating material is important in many technological fields. The interaction between carbon nanomaterial and protein is especially interesting since it makes the development of novel hybrid materials possible. Functional bacterial amyloid (FuBA) is promising as a coating material because of its desirable features, such as well-defined molecular structure, robustness against harsh conditions, and easily engineerable functionality. Here, we report the systematic assembly of the functional amyloid protein, CsgA, from Escherichia coli (E. coli) on graphite. We characterize the assemblies using scanning tunneling microscopy (STM) and show that CsgA forms assemblies according to systematic patterns, dictated by the graphite lattice. In addition, we show that graphite flakes induce the fibrillization of CsgA, in vitro, suggesting a surface-induced conformational change of CsgA facilitated by the graphite lattice. Using coarse-grained molecular dynamics simulations, we model the adhesion and lamellar formation of a CsgA-derived peptide and conclude that peptides are adsorbed both as monomers and smaller aggregates leading initially to unordered graphite-bound aggregates, which are followed by rearrangement into lamellar structures. Finally, we show that CsgA-derived peptides can be immobilized in very systematic assemblies and their molecular orientation can be tuned using a small chaperone-like molecule. Our findings have implications for the development of FuBA-based biosensors, catalysts, and other technologies requiring well-defined protein assemblies on graphite.

Composition-dependent multivalency of peptide-peptide interactions revealed by tryptophan-scanning mutagenesis.

We have examined in this contribution the composition dependence of binding characteristics in peptide-peptide interactions between an oligopeptide octa-glycine and a series of tryptophan-containing octapeptides. The binding energy associated with tryptophan-glycine interactions manifests pronounced stepwise binding characteristics as the number of tryptophan increases from 0 to 8 in the octapeptides consisting only of glycine and can be attributed to mono-, di-, and tri-valent peptide-peptide interactions. At the same time, only weak fluctuations in binding energy were observed as the number of tryptophan increases from 2 to 7. Such distinctive nonlinearity of composition-dependent tryptophan-glycine binding energy characteristics due to continuously varying tryptophan compositions in the octapeptides could be considered as a reflection of combinatorial contributions due to the hydrogen bonds originated from the indole moieties of tryptophan with the main chains of octapeptide of glycine containing N-H and C=O moieties and the van der Waals interactions (including π-π and π-CH interactions) between peptides.

Position-coded multivalent peptide-peptide interactions revealed by tryptophan-scanning mutagenesis.

We demonstrate in this contribution the evidence that significant cooperative binding effect can be identified for the amino acid sites that are determinant to the binding characteristics in peptide-peptide interactions. The analysis of tryptophan-scanning mutagenesis of the 14-mer peptide consisting only of glycine provides a mapping of position-dependent contributions to the binding energy. The pronounced tryptophan-associated peptide-peptide interactions are originated from the indole moieties with the main chains of 14-mer glycines containing N-H and CO moieties. Specifically, with the presence of two tryptophans as determinant amino acids, cooperative binding can be observed, which are dependent on relative positions of the two tryptophans with a "volcano"-like characteristics. An optimal separation of 6-10 amino acids between two adjacent binding sites can be identified to achieve maximal binding interactions.

Evidence for opposing roles of Celsr3 and Vangl2 in glutamatergic synapse formation.

The signaling mechanisms that choreograph the assembly of the highly asymmetric pre- and postsynaptic structures are still poorly defined. Using synaptosome fractionation, immunostaining, and coimmunoprecipitation, we found that Celsr3 and Vangl2, core components of the planar cell polarity (PCP) pathway, are localized at developing glutamatergic synapses and interact with key synaptic proteins. Pyramidal neurons from the hippocampus of Celsr3 knockout mice exhibit loss of ∼50% of glutamatergic synapses, but not inhibitory synapses, in culture. Wnts are known regulators of synapse formation, and our data reveal that Wnt5a inhibits glutamatergic synapses formed via Celsr3. To avoid affecting earlier developmental processes, such as axon guidance, we conditionally knocked out Celsr3 in the hippocampus 1 week after birth. The CA1 neurons that lost Celsr3 also showed a loss of ∼50% of glutamatergic synapses in vivo without affecting the inhibitory synapses assessed by miniature excitatory postsynaptic current (mEPSC) and electron microscopy. These animals displayed deficits in hippocampus-dependent behaviors in adulthood, including spatial learning and memory and fear conditioning. In contrast to Celsr3 conditional knockouts, we found that the conditional knockout of Vangl2 in the hippocampus 1 week after birth led to a large increase in synaptic density, as evaluated by mEPSC frequency and spine density. PCP signaling is mediated by multiple core components with antagonizing functions. Our results document the opposing roles of Celsr3 and Vangl2 in glutamatergic synapse formation.

Steric Dependence of Chirality Effect in Surface-Mediated Peptide Assemblies Identified with Scanning Tunneling Microscopy.

Amino acid chirality has been recognized as an important driving force in constructing peptide architectures, via interactions such as chirality-induced stereochemical effect. The introduction of site-specific chiral conversion of l- and d-amino acids in peptide sequences could enable the pursuit of the chirality effects in peptide assembly. In this work, we characterized the assemblies of heptapeptides with various side chain moieties and their chiral variants using STM. Specifically, two pairs of amino acids, Gln (Q) and Asn (N), Glu (E) and Asp (D), having one methylene difference in their side chains, are selected to elucidate the steric dependence of amino acid chiral effects on surface-bound peptide assemblies. The observed heptapeptide assembly structures reveal that chirality switching of a single amino acid is able to destabilize the surface-mediated peptide assemblies, and this disturbance effect can be positively correlated with the steric hindrance of amino acid side chains. Furthermore, the strength of the impact due to chiral conversion on heptapeptide assembly structure is noticeably dependent on the mutation sites, indicative of structural heterogeneity of chiral effects. These results could contribute to the molecular insights of chirality-induced stereochemical interactions in peptide assembly.

PKD1 Promotes Functional Synapse Formation Coordinated with N-Cadherin in Hippocampus.

Functional synapse formation is critical for the wiring of neural circuits in the developing brain. The cell adhesion molecule N-cadherin plays important roles in target recognition and synaptogenesis. However, the molecular mechanisms that regulate the localization of N-cadherin and the subsequent effects remain poorly understood. Here, we show that protein kinase D1 (PKD1) directly binds to N-cadherin at amino acid residues 836-871 and phosphorylates it at Ser 869, 871, and 872, thereby increasing the surface localization of N-cadherin and promoting functional synapse formation in primary cultured hippocampal neurons obtained from embryonic day 18 rat embryos of either sex. Intriguingly, neuronal activity enhances the interactions between N-cadherin and PKD1, which are critical for the activity-dependent growth of dendritic spines. Accordingly, either disruption the binding between N-cadherin and PKD1 or preventing the phosphorylation of N-cadherin by PKD1 in the hippocampal CA1 region of male rat leads to the reduction in synapse number and impairment of LTP. Together, this study demonstrates a novel mechanism of PKD1 regulating the surface localization of N-cadherin and suggests that the PKD1-N-cadherin interaction is critical for synapse formation and function.SIGNIFICANCE STATEMENT Defects in synapse formation and function lead to various neurological diseases, although the mechanisms underlying the regulation of synapse development are far from clear. Our results suggest that protein kinase D1 (PKD1) functions upstream of N-cadherin, a classical synaptic adhesion molecule, to promote functional synapse formation. Notably, we identified a crucial binding fragment to PKD1 at C terminus of N-cadherin, and this fragment also contains PKD1 phosphorylation sites. Through this interaction, PKD1 enhances the stability of N-cadherin on cell membrane and promotes synapse morphogenesis and synaptic plasticity in an activity-dependent manner. Our study reveals the role of PKD1 and the potential downstream mechanism in synapse development, and contributes to the research for neurodevelopment and the therapy for neurological diseases.

Identifying Terminal Assembly Propensity of Amyloidal Peptides by Scanning Tunneling Microscopy.

The abnormal accumulation of beta-amyloids (Aß) in brain is considered as a key initiating cause for Alzheimer's disease (AD) due to their richness in plaques and self-aggregate propensity. In recent studies, N-terminally extended Aß peptides (NTE-Aß) with the N-terminus originating prior to the canonical ß-secretase cleavage site were found in humans and suggested to have possible relevance to AD. However, the effects of the extended N-terminus on the amyloidegenic structure and aggregation propensity have not been fully elucidated. Herein, we characterized the assembly structures of Aß1-42, Aß(-5)-42, Aß(-10)-42 and Aß(-15)-42 with both normal and reversed sequences on highly oriented pyrolytic graphite (HOPG) surfaces with scanning tunneling microscopy (STM). The molecularly resolved surface-mediated peptide assemblies enable identification of amyloidegenic fragments. The observations reveal that the assembly propensity of the C-terminal strand of Aß1-42 is highly conserved and insensitive to N-terminal extensions. In contrast, different assembly structures of the N-terminal strand of Aß variants can be observed with possible assignment of varied amyloidegenic fragments in the extended N-termini, which may contribute to the varied aggregation propensities of Aß42 species.

High Density Individually Addressable Nanowire Arrays Record Intracellular Activity from Primary Rodent and Human Stem Cell Derived Neurons.

We report a new hybrid integration scheme that offers for the first time a nanowire-on-lead approach, which enables independent electrical addressability, is scalable, and has superior spatial resolution in vertical nanowire arrays. The fabrication of these nanowire arrays is demonstrated to be scalable down to submicrometer site-to-site spacing and can be combined with standard integrated circuit fabrication technologies. We utilize these arrays to perform electrophysiological recordings from mouse and rat primary neurons and human induced pluripotent stem cell (hiPSC)-derived neurons, which revealed high signal-to-noise ratios and sensitivity to subthreshold postsynaptic potentials (PSPs). We measured electrical activity from rodent neurons from 8 days in vitro (DIV) to 14 DIV and from hiPSC-derived neurons at 6 weeks in vitro post culture with signal amplitudes up to 99 mV. Overall, our platform paves the way for longitudinal electrophysiological experiments on synaptic activity in human iPSC based disease models of neuronal networks, critical for understanding the mechanisms of neurological diseases and for developing drugs to treat them.

Aromatic-interaction-mediated inhibition of ß-amyloid assembly structures and cytotoxicity.

Abnormal aggregation of ß-amyloid (Aß) peptide plays an important role in the onset and progress of Alzheimer's disease (AD); hence, targeting Aß aggregation is considered as an effective therapeutic strategy. Here, we studied the aromatic-interaction-mediated inhibitory effect of oligomeric polypeptides (K8Y8, K4Y8, K8W8) on Aß42 fibrillization process. The polypeptides containing lysine as well as representative aromatic amino acids of tryptophan or tyrosine were found to greatly suppress the aggregation as evaluated by thioflavin T assay. Circular dichroism spectra showed that the ß-sheet formation of Aß42 peptides decreased with the polypeptide additives. Molecular docking studies revealed that the oligomeric polypeptides could preferentially bind to Aß42 through π-π stacking between aromatic amino acids and Phe19, together with hydrogen bonding. The cell viability assay confirmed that the toxicity of Aß42 to SH-SY5Y cells was markedly reduced in the presence of polypeptides. This study could be beneficial for developing peptide-based inhibitory agents for amyloidoses. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Chronic Hypoxia Increases Intracellular Ca(2+) Concentration via Enhanced Ca(2+) Entry Through Receptor-Operated Ca(2+) Channels in Pulmonary Venous Smooth Muscle Cells.

BACKGROUND: Hypoxic pulmonary hypertension (HPH) is characterized by pulmonary vascular remodeling. Intracellular Ca(2+)concentration ([Ca(2+)]i) is an essential signal for myocyte proliferation. Whether chronic hypoxia (CH) affects the basal [Ca(2+)]I and Ca(2+)entry through store- and/or receptor-operated calcium channels (SOCC, ROCC), and whether canonical transient receptor potential (TRPC) proteins are involved in CH-induced Ca(2+)influx and proliferation in pulmonary venous smooth muscle cells (PVSMCs) is examined. METHODS AND RESULTS: Rats were exposed to CH. PVSMCs were isolated from distal pulmonary veins. In freshly isolated PVSMCs, CH increased the basal [Ca(2+)]i; removal of Ca(2+)or application of SKF-96365 reversed the elevated [Ca(2+)]i, whereas nifedipine had no effect. Receptor-operated Ca(2+)entry (ROCE) was expressed in PVSMCs. In freshly isolated PVSMCs from CH rats, ROCE was enhanced, whereas store-operated Ca(2+)entry had no alteration. Furthermore, real-time polymerase chain reaction and western blotting showed that mRNA and protein expression level of TRPC6, but neither TRPC1 nor TRPC3, in pulmonary venous smooth muscle (PV) from CH rats and PVSMCs exposed to CH was greater than in normal PV and PVSMCs. The knockdown of TRPC6 in hypoxic PVSMCs with siRNA inhibited the enhanced ROCE and attenuated CH-induced PVSMCs proliferation. CONCLUSIONS: The enhanced Ca(2+)entry through ROCC, due to upregulated TRPC6, is a novel pathogenic mechanism contributing to the increased basal [Ca(2+)]iin PVSMCs and excessive PVSMC proliferation during the development of HPH.

Reinduced Wnt signaling limits regenerative potential of sensory axons in the spinal cord following conditioning lesion.

Conditioning lesion of the peripheral branch of dorsal column axons is a well-known paradigm enabling the central branch to regenerate after injury to the spinal cord. However, only a small number of regenerating axons enter grafted substrates, and they do not grow beyond the lesion. We found that conditioning lesion induces, in addition to growth-stimulating genes, related to receptor tyrosine kinase (Ryk), a potent repulsive receptor for Wnts. Wnts are expressed around the site of spinal cord injury, and we found that grafted bone marrow stromal cells secreting the Wnt inhibitors secreted frizzled-related protein 2 or Wnt inhibitory factor 1 enhanced regeneration of the central branch after peripheral conditioning lesion. Furthermore, we found that Wnt4-expressing grafts caused dramatic long-range retraction of the injured central branch of conditioned dorsal root ganglion neurons. Macrophages accumulate along the path of receding axons but not around Wnt4-expressing cells, suggesting that the retraction of dorsal column axons is not a secondary effect of increased macrophages attracted by Wnt4. Therefore, Wnt-Ryk signaling is an inhibitory force co-induced with growth-stimulating factors after conditioning lesion. Overcoming Wnt inhibition may further enhance therapies being designed on the basis of the conditioning-lesion paradigm.

Antagonistic functions of Dishevelleds regulate Frizzled3 endocytosis via filopodia tips in Wnt-mediated growth cone guidance.

How growth cones detect small concentration differences of guidance cues for correct steering remains a long-standing puzzle. Commissural axons engage planar cell polarity (PCP) signaling components to turn anteriorly in a Wnt gradient after midline crossing. We found here that Frizzled3, a Wnt receptor, undergoes endocytosis via filopodia tips. Wnt5a increases Frizzled3 endocytosis, which correlates with filopodia elongation. We discovered an unexpected antagonism between Dishevelleds, which may function as a signal amplification mechanism in filopodia where PCP signaling is activated: Dishevelled2 blocks Dishevelled1-induced Frizzled3 hyperphosphorylation and membrane accumulation. A key component of apical-basal polarity (A-BP) signaling, aPKC, also inhibits Dishevelled1-induced Frizzled3 hyperphosphorylation. Celsr3, another PCP component, is required in commissural neurons for anterior turning. Frizzled3 hyperphosphorylation is increased in Celsr3 mutant mice, where PCP signaling is impaired, suggesting Frizzled3 hyperphosphorylation does correlate with loss of PCP signaling in vivo. Furthermore, we found that the small GTPase, Arf6, which is required for Frizzled3 endocytosis, is essential for Wnt-promoted outgrowth, highlighting the importance of Frizzled3 recycling in PCP signaling in growth cone guidance. In a Wnt5a gradient, more Frizzled3 endocytosis and activation of atypical protein kinase C was observed on the side of growth cones facing higher Wnt5a concentration, suggesting that spatially controlled Frizzled3 endocytosis is part of the key mechanism for growth cone steering.

Lung function and incidence of chronic obstructive pulmonary disease after improved cooking fuels and kitchen ventilation: a 9-year prospective cohort study.

BACKGROUND: Biomass smoke is associated with the risk of chronic obstructive pulmonary disease (COPD), but few studies have elaborated approaches to reduce the risk of COPD from biomass burning. The purpose of this study was to determine whether improved cooking fuels and ventilation have effects on pulmonary function and the incidence of COPD. METHODS AND FINDINGS: A 9-y prospective cohort study was conducted among 996 eligible participants aged at least 40 y from November 1, 2002, through November 30, 2011, in 12 villages in southern China. Interventions were implemented starting in 2002 to improve kitchen ventilation (by providing support and instruction for improving biomass stoves or installing exhaust fans) and to promote the use of clean fuels (i.e., biogas) instead of biomass for cooking (by providing support and instruction for installing household biogas digesters); questionnaire interviews and spirometry tests were performed in 2005, 2008, and 2011. That the interventions improved air quality was confirmed via measurements of indoor air pollutants (i.e., SO2, CO, CO2, NO2, and particulate matter with an aerodynamic diameter of 10 µm or less) in a randomly selected subset of the participants' homes. Annual declines in lung function and COPD incidence were compared between those who took up one, both, or neither of the interventions. Use of clean fuels and improved ventilation were associated with a reduced decline in forced expiratory volume in 1 s (FEV1): decline in FEV1 was reduced by 12 ml/y (95% CI, 4 to 20 ml/y) and 13 ml/y (95% CI, 4 to 23 ml/y) in those who used clean fuels and improved ventilation, respectively, compared to those who took up neither intervention, after adjustment for confounders. The combined improvements of use of clean fuels and improved ventilation had the greatest favorable effects on the decline in FEV1, with a slowing of 16 ml/y (95% CI, 9 to 23 ml/y). The longer the duration of improved fuel use and ventilation, the greater the benefits in slowing the decline of FEV1 (p<0.05). The reduction in the risk of COPD was unequivocal after the fuel and ventilation improvements, with an odds ratio of 0.28 (95% CI, 0.11 to 0.73) for both improvements. CONCLUSIONS: Replacing biomass with biogas for cooking and improving kitchen ventilation are associated with a reduced decline in FEV1 and risk of COPD. TRIAL REGISTRATION: Chinese Clinical Trial Register ChiCTR-OCH-12002398.

Enhanced Biofilm Formation by Tetracycline in a Staphylococcus aureus Naturally Lacking ica Operon and atl.

Staphylococcus aureus is a major, widespread pathogen, and its biofilm-forming characteristics make it even more difficult to eliminate by biocides. Tetracycline (TCY) is a major broad-spectrum antibiotic, the residues of which can cause deleterious health impacts, and subinhibitory concentrations of TCY have the potential to increase biofilm formation in S. aureus. In this study, we showed how the biofilm formation of S. aureus 123786 is enhanced in the presence of TCY at specific subinhibitory concentrations. S. aureus 123786 used in this study was identified as Staphylococcal Cassette Chromosome mec III, sequence type239 and naturally lacking ica operon and atl gene. Two assays were performed to quantify the formation of S. aureus biofilm. In the crystal violet (CV) assay, the absorbance values of biofilm stained with CV at optical density (OD)540 nm increased after 8 and 16 hr of incubation when the concentration of TCY was 1/2 minimum inhibitory concentration (MIC), whereas at the concentration of 1/16 MIC, the absorbance values increased after 16 and 24 hr of incubation. In tetrazolium salt reduction assay, the absorbance value at OD490 nm of S. aureus 123786 biofilms mixed with 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium solution increased after 8 hr when the concentration of TCY was 1/4 MIC, which may be correlated with the higher proliferation and maturation of biofilm. In conclusion, the biofilm formation of S. aureus 123786 could be enhanced in the presence of TCY at specific subinhibitory concentrations.

Planar cell polarity proteins mediate ketamine-induced restoration of glutamatergic synapses in prefrontal cortical neurons in a mouse model for chronic stress.

Single administration of low-dose ketamine has both acute and sustained anti-depressant effects. Sustained effect is associated with restoration of glutamatergic synapses in medial prefrontal cortic (mFPC) neurons. Ketamine induced profound changes in a number of molecular pathways in a mouse model for chronic stress. Cell-cell communication analyses predicted that planar-cell-polarity (PCP) signaling was decreased after chronic administration of corticosterone but increased following ketamine administration in most of the excitatory neurons. Similar decrease of PCP signaling in excitatory neurons was predicted in dorsolateral prefrontal cortical (dl-PFC) neurons of patients with major depressive disorder (MDD). We showed that the basolateral amygdala (BLA)-projecting infralimbic prefrontal cortex (IL PFC) neurons regulate immobility time in the tail suspension test and food consumption. Conditionally knocking out Celsr2 and Celsr3 or Prickle2 in the BLA-projecting IL PFC neurons abolished ketamine-induced synapse restoration and behavioral remission. Therefore, PCP proteins in IL PFC-BLA neurons mediate synapse restoration induced by of low-dose ketamine.

Nicotine-induced epithelial-mesenchymal transition via Wnt/ß-catenin signaling in human airway epithelial cells.

Epithelial-mesenchymal transition (EMT) has been proposed to be a mechanism in airway remodeling, which is a characteristic of chronic obstructive pulmonary disease (COPD). Studies have shown that cigarette smoke and nicotine are factors that induce Wnt/ß-catenin activation, which is a pathway that has also been implicated in EMT. The main aim of this study was to test whether human bronchial epithelial cells are able to undergo EMT in vitro following nicotine stimulation via the Wnt3a/ß-catenin signaling pathway. We show that nicotine activates the Wnt3a signal pathway, which leads to the translocation of ß-catenin into the nucleus and activation of ß-catenin/Tcf-dependent transcription in the human bronchial epithelial cell (HBEC) line. This accumulation was accompanied by an increase in smooth muscle actin, vimentin, matrix metalloproteinases-9, and type I collagen expression as well as downregulation of E-cadherin, which are typical characteristics of EMT. We also noted that the release of TGF-ß(1) from these cells was stimulated by nicotine. Knockdown of Wnt3a with small interfering RNA (siRNA) prevented these effects, implying that ß-catenin activation in these responses is Wnt3a dependent. Furthermore, specific knockdown of TGF-ß(1) with TGF-ß(1) siRNA partially prevented nicotine-induced EMT, suggesting that TGF-ß(1) has a role in nicotine-mediated EMT in HBECs. These results suggest that HBECs are able to undergo EMT in vitro upon nicotine stimulation via the Wnt3a/ß-catenin signaling pathway.