Disruption of canonical TGFβ-signaling in murine coronary progenitor cells by low level arsenic

Abstract

Exposure to arsenic results in several types of cancers as well as heart disease. A major contributor to ischemic heart pathologies is coronary artery disease, however the influences by environmental arsenic in this disease process are not known. Similarly, the impact of toxicants on blood vessel formation and function during development has not been studied. During embryogenesis, the epicardium undergoes proliferation, migration, and differentiation into several cardiac cell types including smooth muscle cells which contribute to the coronary vessels. The TGFβ family of ligands and receptors is essential for developmental cardiac epithelial to mesenchymal transition (EMT) and differentiation into coronary smooth muscle cells. In this in vitro study, 18hour exposure to 1.34μM arsenite disrupted developmental EMT programming in murine epicardial cells causing a deficit in cardiac mesenchyme. The expression of EMT genes including TGFβ2, TGFβ receptor-3, Snail, and Has-2 are decreased in a dose-dependent manner following exposure to arsenite. TGFβ2 cell signaling is abrogated as detected by decreases in phosphorylated Smad2/3 when cells are exposed to 1.34μM arsenite. There is also loss of nuclear accumulation pSmad due to arsenite exposure. These observations coincide with a decrease in vimentin positive mesenchymal cells invading three-dimensional collagen gels. However, arsenite does not block TGFβ2 mediated smooth muscle cell differentiation by epicardial cells. Overall these results show that arsenic exposure blocks developmental EMT gene programming in murine coronary progenitor cells by disrupting TGFβ2 signals and Smad activation, and that smooth muscle cell differentiation is refractory to this arsenic toxicity.

Keywords: Arsenic; Epicardial cells; Epithelial to mesenchymal transition; TGF-beta.

Figure 1. Arsenic exposure impacts viability of epicardial cells

Murine epicardial cells were incubated with the indicated concentrations of As(III) over 24 hours and 48 hours and subjected to MTS cell viability assay. Values are averages +/− S.D. of controls from triplicate samples at each dose representing three independent experiments. 24 hours cytotoxic IC₅₀=15.9 μM, and 48 hours cytotoxic IC₅₀=5.8μM. Asterisks (*P ≥ 0.05) and pound sign (#P ≥ 0.005) marks the first statistically significant observation for increase in cytotoxicity for 24 hour and 48 hour samples, respectively.

Figure 2. Arsenic decreases expression of key genes required for cardiac EMT

Murine epicardial cells were exposed to the indicated concentrations of As(III) for 18 hours. RNA was isolated from each treatment condition and RT-PCR analysis of cardiac EMT genes was performed. Heme oxygenase (Hmox) was used as a positive control for gene expression induction by arsenic. Clear bars, control; black bars, 1.34 μM; dark grey bars, 5 μM; light grey bars, 6.7 μM. *p < 0.05; # p < 0.005; + p < 0.0005 and & p < 0.00005. Statistical significance determined by one-tailed Student's T-test with equal variance. All samples performed in triplicate from a minimum of three independent experiments.

Figure 3. Arsenic blocks TGFβ2 stimulated Smad2/3 phosphorylation and nuclear localization

Epicardial cells were exposed to 1.34μM As(III) for 18 hours prior to stimulation with TGFβ2 for 20 minutes. A. Smad2/3 phosphorylation in whole cell lysates (top panels) and nuclear localization (bottom panels) was observed via immunoblotting. Actin used for loading control for total cell lysates and detection of LaminA for loading controls for nuclear lysates. B. Detection of Smad2/3 phosphorylation by immunostaining; control, top row;4ng/mlTGFβ2, second row;1.34μM As(III) + 4ng/mlTGFβ2, third row; and 1.34 μM As(III) alone, last row. Images at 1000× magnification, nuclei in blue (DAPI) and pSmad in red.

Figure 4. Arsenic blocks TGFβ2 mediated epicardial cell invasion and mesenchymal transformation

Epicardial cells were cultured on rat type I collagen gels and exposed to 1.34μM As(III) for 18 hours, followed by 4ng/ml TGFβ2 stimulation for 48 hours. Epicardial cells were subject to immunostaining for vimentin to visualize epithelial to mesenchymal transition. A. Control B. 4ng/ml TGFβ2 C. 1.34μM As(III) D. 1.34μM As(III) + 4ng/ml TGFβ2. Nuclei in blue (DAPI) and vimentin in red.

Figure 5. Arsenic does not block TGFβ2 mediated smooth muscle differentiation

SM22α-LacZmurine epicardial cells were exposed to 1.34μM As(III) for 18 hours and subsequently stimulated with 4ng/mL TGFβ2 for 24 hours. A. Chemiluminescence detection of β-galactosidase activity was used to assess SM22α promoter activity. Average values are shown +/− S.D. from triplicate samples performed in three independent experiments. B – E. X-GAL staining was performed to detect β-galactosidase expression under identical conditions to further assess extent of epicardial smooth muscle differentiation. B. Control C. 4ng/ml TGFβ2 D. 1.34μM As(III) E. 1.34μM As(III) + 4ng/ml TGFβ2. X-GAL positive cells are blue. Representative images from four independent experiments are shown at 400× magnification.