title: Bronchoalveolar Lavage Fluid Utilized Ex Vivo to Validate In Vivo Findings: Inhibition of Gap Junction Activity in Lung Tumor Promotion is Toll-Like Receptor 4-Dependent.
abstract: TLR4 protects against lung tumor promotion and pulmonary inflammation in mice. Connexin 43 (Cx43), a gap junction gene, was increased inTlr4wildtype compared toTlr4-mutant mice in response to promotion, which suggests gap junctional intercellular communication (GJIC) may be compromised. We hypothesized that the early tumor microenvironment, represented by Bronchoalveolar Lavage Fluid (BALF) from Butylated hydroxytoluene (BHT; promoter)-treated mice, would produce TLR4-dependent changes in pulmonary epithelium, including dysregulation of GJIC in theTlr4-mutant (BALBLps-d) compared to theTlr4-sufficient (BALB; wildtype) mice. BHT (4 weekly doses) was injected ip followed by BALF collection at 24 h. BALF total protein and total macrophages were significantly elevated in BHT-treated BALBLps-dover BALB mice, similar to previous findings. BALF was then utilized in anex vivomanner to treat C10 cells, a murine alveolar type II cell line, followed by the scrape-load dye transfer assay (GJIC), Cx43 immunostaining, and quantitative RT-PCR (Mcp-1, monocyte chemotactic protein 1). GJIC was markedly reduced in C10 cells treated with BHT-treated BALBLps-dBALF for 4 and 24 h compared to BALB and control BALF from the respective mice (p < 0.05).Mcp-1, a chemokine, was also significantly increased in the BHT-treated BALBLps-dBALF compared to the BALB mice, and Cx43 protein expression in the cell membrane altered. These novel findings suggest signaling from the BALF milieu is involved in GJIC dysregulation associated with promotion and links gap junctions to pulmonary TLR4 protection in a novelex vivomodel that could assist in future potential tumor promoter screening.
authors.affiliation: Department of Environmental and Occupational Health, Colorado School of Public Health, University of Colorado, Aurora, Colorado, USA.
authors.forename: Thomas
authors.initials: T
authors.lastname: Hill
authors.affiliation: Department of Environmental and Occupational Health, Colorado School of Public Health, University of Colorado, Aurora, Colorado, USA.
authors.forename: Ross S
authors.initials: RS
authors.lastname: Osgood
authors.affiliation: Department of Environmental and Occupational Health, Colorado School of Public Health, University of Colorado, Aurora, Colorado, USA.
authors.forename: Kalpana
authors.initials: K
authors.lastname: Velmurugan
authors.affiliation: Department of Environmental and Occupational Health, Colorado School of Public Health, University of Colorado, Aurora, Colorado, USA.
authors.forename: Carla-Maria
authors.initials: CM
authors.lastname: Alexander
authors.affiliation: Department of Pediatrics and Human Development, Michigan State University, Lansing, USA.
authors.forename: Brad L
authors.initials: BL
authors.lastname: Upham
authors.affiliation: Department of Environmental and Occupational Health, Colorado School of Public Health, University of Colorado, Aurora, Colorado, USA.
authors.forename: Alison K
authors.initials: AK
authors.lastname: Bauer
keywords: Bronchoalveolar Lavage Fluid (BALF); Butylated Hydroxytoluene (BHT); Connexin 43 (cx43); Gap Junction Intercellular Communication (GJIC); Gap junctions; Lung; Toll-like receptor 4 (TLR4); Tumor Promotion