Journal: Environmental Health Perspectives

Article Title: Mechanisms and Consequences of Variable TRPA1 Expression by Airway Epithelial Cells: Effects of TRPV1 Genotype and Environmental Agonists on Cellular Responses to Pollutants in Vitro and Asthma

doi: 10.1289/EHP11076

Figure Lengend Snippet: (A) Volcano plot of RNA sequencing data from NHBE cells with either the TRPV1 I585I/I or I585I/V genotypes. Summary data can be found in Excel Table S1. (B) TRPA1 , (C) TRPV1 , (D) NLRP2 , and (E) IGFBP2 mRNA expression in NHBE cells having either the TRPV1 I585I/I or I585I/V genotype ( n = 6 or 7 donors/genotype). Data (Rq) are the mean ± SD , relative to β 2 M mRNA and the average for target gene expression in NHBEs with the I585I/I genotype. * p ≤ 0.05 and ** p < 0.01 using a one-tailed unpaired Student’s t -test. Summary data can be found in Excel Table S2 (B,C) Excel Table S3 (D,E). Note: IGFBP2, insulin like growth factor binding protein 2; NHBE, normal human bronchial epithelial (cells); NLRP2, nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing 2; Rq, relative quantification; SD, standard deviation; TRPA1, transient receptor potential cation channel subfamily A member 1; TRPV1, transient receptor potential cation channel subfamily V member 1; β 2 M , β 2 -microglobulin .

Article Snippet: NHBEs immortalized with cyclin-dependent kinase 4 (CDK4) and telomerase reverse transcriptase (hTERT), or HBEC3-KT cells, were from ATCC (CRL-4051) and were grown in airway epithelial cell basal medium supplemented with bronchial epithelial cell growth kit, 30 μ g / mL Geneticin, and 250 ng / mL puromycin (ATCC).

Techniques: RNA Sequencing, Expressing, Targeted Gene Expression, One-tailed Test, Binding Assay, Quantitative Proteomics, Standard Deviation

Journal: Environmental Health Perspectives

Article Title: Mechanisms and Consequences of Variable TRPA1 Expression by Airway Epithelial Cells: Effects of TRPV1 Genotype and Environmental Agonists on Cellular Responses to Pollutants in Vitro and Asthma

doi: 10.1289/EHP11076

Figure Lengend Snippet: (A) TRPA1 and (B) TRPV1 mRNA expression in HBEC3-KT cells following 24-h treatment with nonivamide ( 10 μ M ) or LJO-328 ( 25 μ M ; white bars), and co-treatment with pine WSPM in media ( 20 μ g / cm 2 ; gray hashed bars). Data (Rq) are the mean ± SD for target gene mRNA expression relative to β 2 M mRNA and cells treated with media containing 0.2% DMSO only ( n = 3 – 6 ). Control and WSPM co-treated groups were analyzed independently comparing all treatment groups using one-way ANOVA and Tukey’s multiple comparisons test. * p ≤ 0.05 , *** p < 0.001 , and **** p < 0.0001 . #### Indicates significant difference ( p < 0.0001 ) from all WSPM co-treated groups using two-way ANOVA and a Bonferroni multiple comparisons test comparing the corresponding ± WSPM groups. Summary data can be found in Excel Table S4. Note: ANOVA, analysis of variance; DMSO, dimethyl sulfoxide; HBEC3-KT, telomerase reverse transcriptase and CDK4–immortalized normal human bronchial epithelial (cells); LJO-328, N -(4- tert -butylbenzyl)- N -(1-[3-fluoro-4-(methylsulfonylamino)phenyl]ethyl)thiourea; nonivamide, 2-mercaptoethanol, n -vanillylnonanamide; Rq, relative quantity; SD, standard deviation; TRPA1, transient receptor potential cation channel subfamily A member 1; TRPV1, transient receptor potential cation channel subfamily V member 1; WSPM, wood smoke particulate matter; β 2 M , β 2 -microglobulin .

Article Snippet: NHBEs immortalized with cyclin-dependent kinase 4 (CDK4) and telomerase reverse transcriptase (hTERT), or HBEC3-KT cells, were from ATCC (CRL-4051) and were grown in airway epithelial cell basal medium supplemented with bronchial epithelial cell growth kit, 30 μ g / mL Geneticin, and 250 ng / mL puromycin (ATCC).

Techniques: Expressing, Control, Reverse Transcription, Standard Deviation

Journal: Environmental Health Perspectives

Article Title: Mechanisms and Consequences of Variable TRPA1 Expression by Airway Epithelial Cells: Effects of TRPV1 Genotype and Environmental Agonists on Cellular Responses to Pollutants in Vitro and Asthma

doi: 10.1289/EHP11076

Figure Lengend Snippet: Temporal changes in (A) TRPA1 and (B) TRPV1 mRNA expression in HBEC3-KT cells following treatment with media containing 0.2% DMSO (open circles, solid lines) or pine WSPM in media ( 20 μ g / cm 2 ; closed circles, dashed lines). Data (Rq) are the mean ± SD for target gene mRNA expression relative to β 2 M mRNA and the control ( n = 3 ). * p ≤ 0.05 and **** p < 0.0001 using repeated measures two-way ANOVA comparing the control and WSPM-treated groups at each time and correction using Bonferroni’s multiple comparisons test. Summary data can be found in Excel Table S6. Note: ANOVA, analysis of variance; DMSO, dimethyl sulfoxide; HBEC3-KT, telomerase reverse transcriptase and CDK4–immortalized normal human bronchial epithelial (cells); Rq, relative quantification; SD, standard deviation; TRPA1, transient receptor potential cation channel subfamily A member 1; TRPV1, transient receptor potential cation channel subfamily V member 1; WSPM, wood smoke particulate matter; β 2 M , β 2 -microglobulin .

Article Snippet: NHBEs immortalized with cyclin-dependent kinase 4 (CDK4) and telomerase reverse transcriptase (hTERT), or HBEC3-KT cells, were from ATCC (CRL-4051) and were grown in airway epithelial cell basal medium supplemented with bronchial epithelial cell growth kit, 30 μ g / mL Geneticin, and 250 ng / mL puromycin (ATCC).

Techniques: Expressing, Control, Reverse Transcription, Quantitative Proteomics, Standard Deviation

Journal: Environmental Health Perspectives

Article Title: Mechanisms and Consequences of Variable TRPA1 Expression by Airway Epithelial Cells: Effects of TRPV1 Genotype and Environmental Agonists on Cellular Responses to Pollutants in Vitro and Asthma

doi: 10.1289/EHP11076

Figure Lengend Snippet: TRPA1 (white bars) and TRPV1 (gray hashed bars) mRNA expression in HBEC3-KTs transfected with scramble, GAPDH , and TRPV1 siRNA ( 500 pmol / mL ) 24 h posttransfection. Data (Rq) are the mean ± SD for target gene mRNA expression relative to β 2 M mRNA and the average for cells transfected with scramble siRNA ( n = 3 ). Data for TRPA1 and TRPV1 were analyzed independently comparing all three siRNAs using one-way ANOVA and Tukey’s multiple comparisons test. *** p < 0.001 and **** p < 0.0001 . Summary data can be found in Excel Table S7. Note: ANOVA, analysis of variance; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HBEC3-KT, telomerase reverse transcriptase and CDK4–immortalized normal human bronchial epithelial (cells); Rq, relative quantification; SD, standard deviation; siRNA, small interfering RNA; TRP, transient receptor potential; TRPA1, transient receptor potential cation channel subfamily A member 1; TRPV1, transient receptor potential cation channel subfamily V member 1; β 2 M , β 2 -microglobulin .

Article Snippet: NHBEs immortalized with cyclin-dependent kinase 4 (CDK4) and telomerase reverse transcriptase (hTERT), or HBEC3-KT cells, were from ATCC (CRL-4051) and were grown in airway epithelial cell basal medium supplemented with bronchial epithelial cell growth kit, 30 μ g / mL Geneticin, and 250 ng / mL puromycin (ATCC).

Techniques: Expressing, Transfection, Reverse Transcription, Quantitative Proteomics, Standard Deviation, Small Interfering RNA

Journal: Environmental Health Perspectives

Article Title: Mechanisms and Consequences of Variable TRPA1 Expression by Airway Epithelial Cells: Effects of TRPV1 Genotype and Environmental Agonists on Cellular Responses to Pollutants in Vitro and Asthma

doi: 10.1289/EHP11076

Figure Lengend Snippet: (A) TRPA1 mRNA expression in HBEC3-KT cells following 24-h treatment with media containing 0.2% DMSO or the NF- κ B inhibitor BMS-345541 (BMS; 10 μ M ). **** p < 0.0001 using a one-tailed unpaired Student’s t -test. (B) TRPA1 , (C) TRPV1 , and (D) IL8 mRNA expression in HBEC3-KT cells following 4- and 24-h treatment with TNF α ( 50 ng / mL ) or 0.01% BSA in media ( n = 3 ). Data (Rq) are the mean ± SD for target gene mRNA expression relative to β 2 M mRNA and the control. * p ≤ 0.05 , ** p < 0.01 , and **** p < 0.0001 using two-way ANOVA and Tukey’s multiple comparisons test comparing all treatments groups. (E) Calcium flux in HBEC3-KT cells treated for 24 h with either media containing 0.01% BSA and 0.2% DMSO, TNF α ( 50 ng / mL ), BMS-345541 ( 10 μ M ), or TNF α and BMS subsequently stimulated by the addition of AITC ( 150 μ M ). Data were normalized to ionomycin ( n = 5 ). Raw images are shown in Figure S6. **** p < 0.0001 using one-way ANOVA and Tukey’s multiple comparison test comparing all groups. Summary data can be found in Excel Table S8. Note: AITC, allyl isothiocyanate; ANOVA, analysis of variance; BSA, bovine serum albumin; DMSO, dimethyl sulfoxide; HBEC3-KT, telomerase reverse transcriptase and CDK4–immortalized normal human bronchial epithelial (cells); IL8, interleukin-8; NF- κ B , nuclear factor kappa light chain enhancer of activated B cells; Rq, relative quantification; SD, standard deviation; TNF α , tumor necrosis factor-alpha; TRPA1, transient receptor potential cation channel subfamily A member 1; TRPV1, transient receptor potential cation channel subfamily V member 1; β 2 M , β 2 -microglobulin .

Article Snippet: NHBEs immortalized with cyclin-dependent kinase 4 (CDK4) and telomerase reverse transcriptase (hTERT), or HBEC3-KT cells, were from ATCC (CRL-4051) and were grown in airway epithelial cell basal medium supplemented with bronchial epithelial cell growth kit, 30 μ g / mL Geneticin, and 250 ng / mL puromycin (ATCC).

Techniques: Expressing, One-tailed Test, Control, Comparison, Reverse Transcription, Quantitative Proteomics, Standard Deviation

Journal: Environmental Health Perspectives

Article Title: Mechanisms and Consequences of Variable TRPA1 Expression by Airway Epithelial Cells: Effects of TRPV1 Genotype and Environmental Agonists on Cellular Responses to Pollutants in Vitro and Asthma

doi: 10.1289/EHP11076

Figure Lengend Snippet: (A) Quantification of nuclear pNF- κ B /p65 in HBEC3-KTs treated for 24 h with media, TNF α ( 50 ng / mL ), nonivamide ( 10 μ M ), or LJO-328 ( 25 μ M ). pNF- κ B /p65 intensity was normalized to p84, and NF- κ B (total) was normalized to β -actin prior to calculating the pNF- κ B /NF- κ B ratio. Data represents the mean ± SD ( n = 3 ). ** p < 0.01 using one-way ANOVA and Tukey’s multiple comparisons test comparing all groups. (B) Representative western blot image where, from left to right, are the molecular weight standard (MW), control (1), TNF α (2), nonivamide (3), and LJO-328 treatments (4). Raw western blot data are shown in Figure S7. Summary data can be found in Excel Table S9. Note: ANOVA, analysis of variance; HBEC3-KT, telomerase reverse transcriptase and CDK4–immortalized normal human bronchial epithelial (cells); LJO-328, N -(4- tert -butylbenzyl)- N -(1-[3-fluoro-4-(methylsulfonylamino)phenyl]ethyl)thiourea; nonivamide, 2-mercaptoethanol, n -vanillylnonanamide; NF- κ B , nuclear factor kappa light chain enhancer of activated B cells; p84, rabbit nuclear matrix protein; pNF- κ B , phospho-nuclear factor kappa light chain enhancer of activated B cells; SD, standard deviation; TNF α , tumor necrosis factor-alpha.

Article Snippet: NHBEs immortalized with cyclin-dependent kinase 4 (CDK4) and telomerase reverse transcriptase (hTERT), or HBEC3-KT cells, were from ATCC (CRL-4051) and were grown in airway epithelial cell basal medium supplemented with bronchial epithelial cell growth kit, 30 μ g / mL Geneticin, and 250 ng / mL puromycin (ATCC).

Techniques: Western Blot, Molecular Weight, Control, Reverse Transcription, Standard Deviation

Journal: Environmental Health Perspectives

Article Title: Mechanisms and Consequences of Variable TRPA1 Expression by Airway Epithelial Cells: Effects of TRPV1 Genotype and Environmental Agonists on Cellular Responses to Pollutants in Vitro and Asthma

doi: 10.1289/EHP11076

Figure Lengend Snippet: (A) TRPA1 , (B) TRPV1 , and (C) IL8 mRNA expression in HBEC3-KT cells following 4- or 12-h treatment with media containing 0.2% DMSO or the PKC activator and the TRPV1 sensitizer PMA. Data (Rq) are the mean ± SD for target gene mRNA expression relative to β 2 M mRNA and the control ( n = 3 ). *** p < 0.001 and **** p < 0.0001 using two-way ANOVA and Dunnett’s multiple comparison test comparing to the 4 and 12 h gene-specific control. (D) Effects of 12-h treatment with the PKC inhibitor Go6983 ( 10 μ M ) and p38 MAPK inhibitor PD169316 ( 10 μ M ) on TRPA1 mRNA expression in HBEC3-KT and SAECs compared with cells treated with media containing 0.2% DMSO. Data (Rq) are the mean ± SD for target gene mRNA expression relative to β 2 M mRNA and the control. **** p < 0.0001 using multiple t -tests comparing treatment vs. the respective cell type–specific control. (E) TRPV1 and IL8 mRNA expression in HBEC3-KT cells following 12-h treatment with media containing 0.2% DMSO or Go6983 ( 10 μ M ). * p ≤ 0.05 and **** p < 0.0001 using two-way ANOVA and Bonferroni’s multiple comparisons test to compare the gene-specific control and treatment group. (F) TRPV1-mediated calcium flux in HEK-293 cells stably overexpressing human TRPV1 with and without 12-h treatment with media containing 0.2% DMSO or the PKC inhibitor Go6983 ( 10 μ M ). Data are the mean ± SD for change in fluorescence relative to media-treated cells normalized to the maximum response (100%) and fit using the log[agonist] vs. normalized response-variable slope equation ( n = 3 ). ** p < 0.01 and **** p < 0.0001 using two-way ANOVA and a Bonferroni test. Raw data are graphed in Figure S8. Summary data can be found in Excel Table S10 (A–C) and Excel Table S11 (D–F). Note: ANOVA, analysis of variance; DMSO, dimethyl sulfoxide; HBEC3-KT, telomerase reverse transcriptase and CDK4–immortalized normal human bronchial epithelial (cells); IL8, interleukin-8; p38 MAPK, p38 mitogen-activated protein kinase; PD169316, a p38 MAPK inhibitor; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; Rq, relative quantification; SAECs, small airway epithelial cells; SD, standard deviation; TRPA1, transient receptor potential cation channel subfamily A member 1; TRPV1, transient receptor potential cation channel subfamily V member 1; β 2 M , β 2 -microglobulin .

Article Snippet: NHBEs immortalized with cyclin-dependent kinase 4 (CDK4) and telomerase reverse transcriptase (hTERT), or HBEC3-KT cells, were from ATCC (CRL-4051) and were grown in airway epithelial cell basal medium supplemented with bronchial epithelial cell growth kit, 30 μ g / mL Geneticin, and 250 ng / mL puromycin (ATCC).

Techniques: Expressing, Control, Comparison, Stable Transfection, Fluorescence, Reverse Transcription, Quantitative Proteomics, Standard Deviation

Journal: Environmental Health Perspectives

Article Title: Mechanisms and Consequences of Variable TRPA1 Expression by Airway Epithelial Cells: Effects of TRPV1 Genotype and Environmental Agonists on Cellular Responses to Pollutants in Vitro and Asthma

doi: 10.1289/EHP11076

Figure Lengend Snippet: (A) NLRP2 mRNA expression in HBEC3-KT cells following 24-h treatment with media containing 0.2% DMSO or the NF- κ B inhibitor BMS-345541 ( 10 μ M ). Data (Rq) are the mean ± SD for target gene mRNA expression relative to β 2 M mRNA and the control ( n = 3 ). **** p < 0.0001 using a one-tailed, unpaired Student’s t -test. (B) Representative western blot image for NLRP2 in HBEC3-KT cells transfected with 100 pmol / mL NLRP2 siRNA, where, from left to right, are the molecular weight standards (MW), scramble siRNA (1), GAPDH siRNA (2), and NLRP2 siRNA 1 and 2 transfected cell lysates (3 and 4). (C) Quantification of NLRP2 protein in siRNA-transfected HBEC3-KTs. Raw data are provided in Figure S9. Data are the mean ± SD of the ratio of NLRP2 to β -actin band density ( n = 3 ). * p ≤ 0.05 and ** p < 0.01 using one-way ANOVA and Tukey’s multiple comparison test comparing all groups. (D–G) NLRP2 , TRPA1 , TRPV1 , and IL8 mRNA expression in HBEC3-KTs 24 h after NLRP2 siRNA-2 ( 100 pmol ) transfection, and (H) IL8 mRNA expression following TRPV1 siRNA ( 500 pmol ) transfection compared with the respective control siRNA and GAPDH siRNA groups. Data (Rq) are the mean ± SD for target gene mRNA expression relative to β 2 M mRNA and the scramble control ( n = 3 ). ** p < 0.01 , *** p < 0.001 , and **** p < 0.0001 using one-way ANOVA and Tukey’s multiple comparison test comparing all groups. Summary data can be found in Excel Table S12. Note: ANOVA, analysis of variance; BMS, BMS-345541; DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HBEC3-KT, telomerase reverse transcriptase and CDK4–immortalized normal human bronchial epithelial (cells); IL8, interleukin-8; NF- κ B , nuclear factor kappa light chain enhancer of activated B cells; NLRP2, nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing 2; Rq, relative quantification; SD, standard deviation; siRNA, small interfering RNA; TRPA1, transient receptor potential cation channel subfamily A member 1; TRPV1, transient receptor potential cation channel subfamily V member 1; β 2 M , β 2 -microglobulin .

Article Snippet: NHBEs immortalized with cyclin-dependent kinase 4 (CDK4) and telomerase reverse transcriptase (hTERT), or HBEC3-KT cells, were from ATCC (CRL-4051) and were grown in airway epithelial cell basal medium supplemented with bronchial epithelial cell growth kit, 30 μ g / mL Geneticin, and 250 ng / mL puromycin (ATCC).

Techniques: Expressing, Control, One-tailed Test, Western Blot, Transfection, Molecular Weight, Comparison, Reverse Transcription, Binding Assay, Quantitative Proteomics, Standard Deviation, Small Interfering RNA

Journal: Environmental Health Perspectives

Article Title: Mechanisms and Consequences of Variable TRPA1 Expression by Airway Epithelial Cells: Effects of TRPV1 Genotype and Environmental Agonists on Cellular Responses to Pollutants in Vitro and Asthma

doi: 10.1289/EHP11076

Figure Lengend Snippet: (A–C) DDIT3 and (D–F) IL8 mRNA expression in NHBE cells having either the TRPV1 I585I/I or I585I/V genotype 24 h after treatment with media containing either 0.2% DMSO, DEP (CFA 10 μ g / cm 2 ), or CFA ( 180 μ g / cm 2 ) treatment ( n ≥ 4 donors/genotype). (A,B,D,E) Data (Rq) are the mean ± SD for target gene mRNA expression normalized to β 2 M mRNA, analyzed using a paired one-tailed t -test. Fold change in (C) DDIT3 and (F) IL8 mRNA expression in DEP- and CFA-treated cells normalized to media-treated controls for each donor. * p ≤ 0.05 using multiple t -tests to compare genotype effects for each particle. Summary data can be found in Excel Table S13. Note: CFA, coal fly ash; DDIT3, DNA damage-inducible transcript-3; DEP, diesel exhaust particles; DMSO, dimethyl sulfoxide; IL8, interleukin-8; NHBE, normal human bronchial epithelial (cells); Rq, relative quantification; SD, standard deviation; TRPV1, transient receptor potential cation channel subfamily V member 1; β 2 M , β 2 -microglobulin .

Article Snippet: NHBEs immortalized with cyclin-dependent kinase 4 (CDK4) and telomerase reverse transcriptase (hTERT), or HBEC3-KT cells, were from ATCC (CRL-4051) and were grown in airway epithelial cell basal medium supplemented with bronchial epithelial cell growth kit, 30 μ g / mL Geneticin, and 250 ng / mL puromycin (ATCC).

Techniques: Expressing, One-tailed Test, Quantitative Proteomics, Standard Deviation

Journal: Environmental Health Perspectives

Article Title: Mechanisms and Consequences of Variable TRPA1 Expression by Airway Epithelial Cells: Effects of TRPV1 Genotype and Environmental Agonists on Cellular Responses to Pollutants in Vitro and Asthma

doi: 10.1289/EHP11076

Figure Lengend Snippet: Schematic summarizing the authors hypothesis for how TRPV1, NF- κ B , PKC, and p38 MAPK may regulate TRPA1, TRPV1, and NLRP2 expression in AECs. The summary is based on the cumulative results of this and other referenced studies. (A) TRPV1 I585I/I and (B) TRPV1 I585I/V. Note: AECs, airway epithelial cells; Ca 2 + , calcium ions; Go6983, a PKC inhibitor; NF- κ B , nuclear factor kappa light chain enhancer of activated B cells; NLRP2, nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing 2; MAPK, p38 mitogen-activated protein kinase; PD169316, a p38 MAPK inhibitor; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; TRPA1, transient receptor potential cation channel subfamily A member 1; TRPV1, transient receptor potential cation channel subfamily V member 1.

Article Snippet: NHBEs immortalized with cyclin-dependent kinase 4 (CDK4) and telomerase reverse transcriptase (hTERT), or HBEC3-KT cells, were from ATCC (CRL-4051) and were grown in airway epithelial cell basal medium supplemented with bronchial epithelial cell growth kit, 30 μ g / mL Geneticin, and 250 ng / mL puromycin (ATCC).

Techniques: Expressing, Binding Assay