Phenotypic control of gap junctional communication by cultured alveolar epithelial cells

Abstract

We examined phenotype-specific changes in gap junction protein [connexin (Cx)] expression and function by cultured rat alveolar type II cells. Type II cells cultured on extracellular matrix in medium containing keratinocyte growth factor (KGF) and 2% fetal bovine serum (FBS; KGF/2) retained expression of surfactant protein C and the 180-kDa lamellar body membrane protein (lbm180). These markers were lost when cells were cultured in medium containing 10% FBS (MEM/10). With RT-PCR, cells cultured in MEM/10 showed transient increases in Cx43 and Cx46 mRNA expression, whereas Cx32 and Cx26 decreased and Cx30.3 and Cx37 were unchanged. Transient changes in Cx32, Cx43, and Cx46 protein expression were confirmed by immunoblot. In contrast, cells cultured in KGF/2 retained expression of Cx32 and showed increased expression of Cx30.3 and Cx46 mRNAs, compared with that in day 0 cells. With immunofluorescence microscopy, Cx32 and Cx43 were at the plasma membrane of cells grown in KGF/2, whereas Cx46 was exclusively intracellular. Type II cells cultured in MEM/10 showed ∼3- to 4-fold more intercellular transfer of microinjected lucifer yellow through gap junctions than cells grown in 2% FBS. Thus type II cells dynamically alter gap junctional communication, and distinct alveolar epithelial cell phenotypes express different connexins.

Extracellular Matrix Influences Alveolar Epithelial Claudin Expression and Barrier Function

“…The microenvironment can also promote alveolar cells to undergo an epithelial-to-mesenchyme transition, which contributes to pulmonary fibrosis (23,24). Consistent with this observed plasticity, alveolar epithelial cells cultured under different conditions show differences in expression and function of gap junction proteins (25)(26)(27). However, it remains to be determined how the cell microenvironment influences tight junction protein expression.…”

“…SpragueDawley rat type II alveolar epithelial cells were isolated from lungs lavaged and perfused with elastase using the method of Dobbs and colleagues (37), with modifications (26). For routine preparations, cells were biopanned with IgG-coated culture dishes to remove alveolar macrophages and other Fc receptor-expressing cells.…”

“…Changes in cell phenotype also were assessed using surfactant protein C (SP-C) and connexin32 as type II cell markers and receptor for advanced gylcosylation end products and caveolin-1 as type I cell markers (26,41) (Figure 6). Under the culture conditions tested, neither SP-C or connexin32 was detectable by immunoblot, although it was present in control purified type II cells.…”

Alveolar epithelial disintegrity in pulmonary fibrosis

“…They consist of an array of transmembrane channels composed of connexins (Cx) that connect to similar structures in the adjacent cells. Differential expression of various connexins in the lung, especially Cx43 and Cx46, has an important role in the regulation of normal lung homeostasis and remodeling in response to epithelial cell injury (1,31). Fibroblasts from patients with IPF demonstrate significant reduction in Cx43 mRNA with alteration to GJ intercellular communication compared with fibroblasts from normal subjects (53).…”

Interaction with surrounding normal epithelial cells influences signalling pathways and behaviour of Src-transformed cells

“…Where indicated, the following inhibitors were used: 4-AP (2 mM, SigmaAldrich), (S)-(-)-Blebbistatin (30 M, Toronto Research Chemicals, ON, Canada), U0126 (10 M, Promega), PF573228 (20 M, TOCRIS Bioscience, Ellisville, MO), and aGA (50 M, Sigma-Aldrich). It should be noted that aGA has been reported to efficiently block gap junctions in MDCK cells (Abraham et al, 1999;De Blasio et al, 2004). LY294002, ML-7, and Y27632 were from Calbiochem (Darmstadt, Germany) and were all used at 10 M.…”