Figure 4.
Overexpression of Cu,Zn-SOD modulates Jmjd3 expression and activity. (A) BMDMs were obtained from WT and Cu,Zn-SOD transgenic (Cu,Zn-SODTg) mice. Total RNA was isolated, and jmjd3 mRNA was measured by quantitative RT-PCR and is expressed in arbitrary units normalized to β-actin mRNA (n = 3); *P < 0.05. (B) Nuclear fractions from WT and Cu,Zn-SODTg macrophages were isolated, and Jmdj3/ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX) enzyme activity was measured (n = 4); *P < 0.05. (C) BMDMs were obtained from WT, Cu,Zn-SODTg (Tg), and STAT6−/− mice. Nuclear fractions were isolated and immunoblot analysis was performed for methylated histone H3 (H3K27 me3). Figure is representative of five experiments. (D) Macrophages were infected with a replication-deficient adenovirus vector expressing either an empty vector (cytomegalovirus [CMV]) or Cu,Zn-SOD vector (Cu,Zn-SOD) for 24 hours. After 24 hours, cells were transfected with either STAT6WT or STAT6C528S vectors for 24 hours. Total RNA from macrophages was isolated and jmjd3 gene expression was measured by quantitative RT-PCR. Results show arbitrary units normalized to β-actin mRNA. *P < 0.05 versus STAT6WT and **P < 0.05 versus all other groups; n = 4. (E) Macrophages were transfected with empty, STAT6WT, or STAT6C528S vectors. After 24 hours, cells were treated with PEG or PEG-SOD for 1 hour followed by chrysotile exposure for 3 hours. Lysates were obtained under nonreducing conditions. Samples were separated on a polyacrylamide gel in the presence or absence of dithiothreitol (DTT). Representative of three replicates. (F) Macrophages were transfected with empty, STAT6WT, or STAT6C528S in combination with scrambled or Cu,Zn-SOD siRNA. After 72 hours, cells were exposed to chrysotile for 3 hours. Lysates were obtained under nonreducing conditions. Samples were separated on a polyacrylamide gel in the presence or absence of DTT. Representative of three replicates. (G) Nuclear fractions from alveolar macrophages from normal subjects and patients with asbestosis were isolated and Jmjd3/UTX enzyme activity was measured (n = 3); *P < 0.05. (H) Total RNA was isolated from alveolar macrophages from normal subjects and patients with asbestosis. Arginase 1 gene expression was measured by quantitative RT-PCR (n = 6); *P < 0.05. (I) Macrophages were transfected with scrambled or Jmjd3 siRNA. After 72 hours, cells were exposed to chrysotile (10 μg/cm2) for 4 hours. Total RNA was isolated, and tnf-α and fizz1 gene expression was measured by quantitative RT-PCR (n = 3); *P < 0.05 versus scrambled (−), **P < 0.05 versus scrambled (+) in both tnf-α and fizz1. Inset, jmjd3 gene expression was measured (n = 3). Ns, not significant; Ox, oxidized; Red, reduced; Scr, scrambled.