L -Thyroxine promotes a proliferative airway smooth muscle phenotype in the presence of TGF- (cid:2) 1

L -Thyroxine promotes a proliferative airway smooth muscle phenotype in the presence of TGF- (cid:2) 1. Am J Physiol Lung Cell Mol Physiol 308: L301–L306, may reduce, whereas hyperthyroidism may aggravate, asthma symptoms. The mechanisms underlying this relationship are largely un-known. Since thyroid hormones have central roles in cell growth and differentiation, we hypothesized that airway remodeling, in particular increased airway smooth muscle (ASM) mass, may be involved. To address this hypothesis, we investigated the effects of triiodothyronine (T 3 ) and L -thyroxine (T 4 ) in the absence and presence of the proﬁ- brotic transforming growth factor (TGF)- (cid:2) 1 on human ASM cell phenotype switching. T 3 (1–100 nM) and T 4 (1–100 nM) did not affect basal ASM proliferation. However, when combined with TGF- (cid:2) 1 (2 ng/ml), T 4 synergistically increased the proliferative response, whereas only a minor effect was observed for T 3 . In line with a switch from a contractile to a proliferative ASM phenotype, T 4 reduced the TGF- (cid:2) 1-induced contractile protein expression by (cid:3) 50%. Cotreatment with T 3 reduced TGF- (cid:2) 1-induced contractile protein expression by (cid:3) 25%. The synergistic increase in proliferation was almost fully inhibited by the integrin (cid:4) v (cid:2) 3 antagonist tetrac (100 nM), whereas no signiﬁcant effects of the thyroid receptor antagonist 1–850 (3 (cid:5) M) were observed. Inhibition of MEK1/2, downstream of the integrin (cid:4) v (cid:2) 3 , also inhibited the T 4 - and TGF- (cid:2) 1-induced proliferative responses. Collectively, the results indicate that T 4 , and to a lesser extent T 3 , promotes a proliferative ASM phenotype in the presence of TGF- (cid:2) 1, which is predominantly mediated by the mem-brane-bound T 4 receptor (cid:4) v (cid:2) 3 . These results indicate that thyroid hormones may enhance ASM remodeling in asthma, which could be of relevance for hyperthyroid patients with this disease. VO 4 , 10 (cid:5) g/ml aprotinin, 10 (cid:5) g/ml leupeptin, and 10 (cid:5) g/ml pepstatin A, pH 8.0). Western blot analysis. Equal amounts of protein were separated on SDS-polyacrylamide gels and transferred onto nitrocellulose. To avoid nonspeciﬁc binding, membranes were blocked with blocking buffer (composition: 50 mM Tris·HCl, 150 mM NaCl, 0.1% Tween 20, and 5% nonfat dried milk powder) for 1 h at room temperature. Subsequently, the membranes were incubated with anti-sm- (cid:4) -actin and anti-calponin antibodies diluted in blocking buffer for 1 h at room temperature. After membranes were washed 3 times with 0.1% Tris-buffered saline-Tween 20 (0.1% TBS-T; composition: 50 mM Tris·HCl, 150 mM NaCl, and 0.1% Tween 20) for 10 min, membranes were incubated with HRP-labeled secondary antibodies for 1 h at room temperature, followed by an additional 3 washes with 0.1% TBS-T. With the use of enhanced chemiluminescence reagents, bands were recorded in the G:BOX iChemi gel documentation system equipped with GeneSnap image acquisition software (Syngene, Cambridge, UK). Band intensities were quantiﬁed by densitometry using GeneTools analysis software (Syngene). All bands were nor- malized to GAPDH.