Cr(VI)-stimulated STAT3 tyrosine phosphorylation and nuclear translocation in human airway epithelial cells requires Lck

Abstract

Chronic inhalation of low amounts of Cr(VI) promotes pulmonary diseases and cancers through poorly defined mechanisms. SFKs (Src family kinases) in pulmonary airway cells may mediate Cr(VI) signalling for lung injury, although the downstream effectors of Cr(VI)-stimulated SFKs and how they relate to pathogenic gene induction are unknown. Therefore SFK-dependent activation of transcription factors by non-cytotoxic exposure of human bronchial epithelial cells to Cr(VI) was determined. Protein-DNA binding arrays demonstrated that exposing BEAS 2B cells to 5 microM Cr(VI) for 4 and 24 h resulted in increased protein binding to 25 and 43 cis-elements respectively, while binding to 12 and 16 cis-elements decreased. Of note, Cr(VI) increased protein binding to several STAT (signal transducer and activator of transcription) cis-elements. Cr(VI) stimulated acute tyrosine phosphorylation and nuclear translocation of STAT1 over a 4 h period and a prolonged activation of STAT3 that reached a peak between 48 and 72 h. This prolonged activation was observed for both STAT3alpha and STAT3beta. Immunofluorescent confocal microscopy confirmed that Cr(VI) increased nuclear localization of phosphorylated STAT3 for more than 72 h in both primary and BEAS 2B human airway cells. Cr(VI) induced transactivation of both a STAT3-driven luciferase reporter construct and the endogenous inflammatory gene IL-6 (interleukin-6). Inhibition with siRNA (small interfering RNA) targeting the SFK Lck, but not dominant-negative JAK (Janus kinase), prevented Cr(VI)-stimulated phosphorylation of both STAT3 isoforms and induction of IL-6. The results suggest that Cr(VI) activates epithelial cell Lck to signal for prolonged STAT3 activation and transactivation of IL-6, an important immunomodulator of lung disease progression.

Figure 1. Low doses of Cr(VI) do not affect cell viability

BEAS 2B cells were exposed to various doses of Cr(VI) ranging from 1 to 20 μM for 72 h. Immediately following this exposure, the cells were incubated with 1 μM calcein AM and washed and retention of dye was measured by fluorescence. The results shown are from two independent experiments performed in triplicate. **P<0.01, ***P<0.001.

Figure 2. Cr(VI) affects a variety of transcription factors

Triplicate flasks of BEAS 2B cells were exposed to 5 μM Cr(VI) for 4 or 24 h, after which nuclear proteins were isolated and quantified. Each treatment group was pooled and samples were analysed for protein binding to DNA cis-elements by the protein/DNA arrays I and II. Densitometric analysis of the blot was performed and results are presented as fold change over control.

Figure 3. Cr(VI) induced the phosphorylation of STAT3

Following exposure of BEAS 2B cells to 5 μM Cr(VI) for 1, 4 or 24 h, cytosolic and nuclear protein fractions were isolated and quantified. Then, 15 μg of nuclear protein and 30 μg of cytosolic protein were used for Western blot analysis with antibodies against total and phosphorylated STAT1, STAT3 and STAT5 proteins. The duplicate samples were from separate cell cultures and are representative of duplicates from three independent experiments.

Figure 4. Subchronic exposure to Cr(VI) increases STAT3 phosphorylation

(A) Total cell lysates were isolated from BEAS 2B cells exposed either to 5 μM Cr(VI) for 48 or 72 h or to 0.01 μg/ml of IL-6 for 30 min prior to the end of the experiment. Antibodies to pY-STAT3 and STAT3 were used in the Western blotting. (B) Cytosolic and nuclear protein fractions from BEAS 2B cells exposed for 72 h to 5 μM Cr(VI). Western-blot analysis was performed on 15 μg of nuclear protein or 30 μg of cytosolic protein. (C) The purity of the nuclear and cytosolic fractions was demonstrated by probing for the presence or absence of the nuclear protein, lamin A/C, or cytosolic β-tubulin. The experiments were performed in duplicate cell cultures and are representative of three independent experiments.

Figure 5. Cr(VI) induces phosphorylation of STAT3 in the nucleus

Immunofluorescence confocal microscopy was used to confirm that Cr(VI) increases nuclear levels of phosphorylated STAT3. BEAS 2B cells grown on coverslips or pHBE cells grown on transwell membranes in air/liquid interface cultures were exposed to 5 μM Cr(VI) (72 h) or to 0.01 μg/ml of IL-6 (30 min). Following fixation and permeabilization, cells were immunostained with antibodies against either pY-STAT3 or total STAT3 (green), as described in the Materials and methods section. (B, D) These Figures show (A) and (C) merged with images of the nuclei stained with TO-PRO3 (blue). These results were from three independent experiments performed in duplicate.

Figure 6. Cr(VI) induces STAT3-dependent transactivation and IL-6 mRNA levels

(A) BEAS 2B cells were transfected with pSTAT3-luc. After 24 h, cells were treated with 5μM Cr(VI) for either 24 or 48 h. Luciferase activity in cell lysates was then measured and results are reported as means±S.D. in relative light units (RLU) for three separate experiments. **Significance at P<0.01. (B) BEAS 2B grown under normal culture conditions and pHBE cells cultured in transwells under an air/liquid interface were incubated in the presence or absence of 5 μM Cr(VI) for 72 h. Total RNA was then collected and assayed for IL-6 or β-actin mRNA levels using quantitative real-time RT–PCR. The results are reported as means±S.D. of fold change from respective controls to account for differences in basal levels in IL-6 mRNA between the two cell types and culture conditions. Statistical differences are designated by *P<0.05 and **P<0.01 (n=3 for BEAS 2B cells and n=4 for pHBE cells).

Figure 7. Lck is required for full Cr(VI)-stimulated STAT3 phosphorylation

(A) BEAS 2B cells at 70–80% confluence were transiently transfected with plasmids containing either pRK5 or dominant-negative (dn) JAK2. After 24 h, cells were exposed to Cr(VI) (5 μM) for 72 h or to 0.01 μg/ml of IL-6 for 30 min prior to the end of the experiment. Western-blot analysis was performed on 30 μg of total cell lysate. (B) BEAS 2B cells were transiently transfected with various amounts of siRNA specific to human Lck. Total cell lysates were obtained after 96 h and Lck and β-actin protein was measured by Western-blot analysis. (C) BEAS 2B cells were transiently transfected with 200 nM siRNA specific to human Lck or non-specific, random siRNA (NS). After 72 h, cells were exposed to Cr(VI) (5 μM) for an additional 72 h. IL-6 (0.01 μg/ml) was added for 30 min prior to the end of the experiment. Nuclear proteins were extracted at the end of the experiments and probed for pY-STAT3 and β-actin by Western-blot analysis. The results are representative of three separate experiments.