Axin pathway activity regulates in vivo pY654-β-catenin accumulation and pulmonary fibrosis

Abstract

Epithelial to mesenchymal transition (EMT) and pulmonary fibrogenesis require epithelial integrin α3β1-mediated cross-talk between TGFβ1 and Wnt signaling pathways. One hallmark of this cross-talk is pY654-β-catenin accumulation, but whether pY654-β-catenin is a biomarker of fibrogenesis or functionally important is unknown. To clarify further the role of β-catenin in fibrosis, we explored pY654-β-catenin generation and function. α3β1 was required for TGFβ1-mediated activation of Src family kinases, and Src inhibition blocked both pY654 and EMT in primary alveolar epithelial cells (AECs). TGFβ1 stimulated β-catenin/Lef1-dependent promoter activity comparably in immortalized AECs stably expressing WT β-catenin as well as Y654E or Y654F β-catenin point mutants. But EMT was abrogated in the Tyr to Phe mutant. pY654-β-catenin was sensitive to the axin β-catenin turnover pathway as inhibition of tankyrase 1 led to high AEC axin levels, loss of pY654-β-catenin, and inhibition of EMT ex vivo. Mice given a tankyrase inhibitor (50 mg/kg orally) daily for 7 days beginning 10 days after intratracheal bleomycin had improved survival over controls. Treated mice developed raised axin levels in the lung that abrogated pY654-β-catenin and attenuated lung Snail1, Twist1, α-smooth muscle actin, and type I collagen accumulation. Total β-catenin levels were unaltered. These findings identify Src kinase(s) as a mediator of TGFβ1-induced pY654-β-catenin, provide evidence that pY654-β-catenin levels are a critical determinant of EMT and fibrogenesis, and suggest regulation of axin levels as a novel therapeutic approach to fibrotic disorders.

FIGURE 1.

Src activation mediates TGFβ1-induced pY654-β-catenin generation. A, pY654-β-catenin and p-Smad2 in immunoprecipitates and lysates were measured at various times after TGFβ1 stimulation. B, upper, pY654-β-catenin and its complex with p-Smad2 at 2 h were blocked by Src inhibitor PP2 (10 μm) but not inactive PP3. Lower, TGFβ1 activates Src family kinase(s) as revealed by p416-Src accumulation. PP2 or SU6656 (5 μm) blocked p416-Src and p14-caveolin1 at 2 h and attenuated Fn accumulation at 48 h. C, primary AECs were plated on Fn to allow latent TGFβ1 activation. Upper, AECs were cultured on Fn for 4 days. SB431542 and Src inhibitors inhibited TGFβ1-induced Col1, vimentin, and α-SMA as judged by immunoblotting and immunostaining. Lower, AECs were treated with TGFβ1 for 2 h ± PP2 or SU6656, and pY654β-catenin was detected by immunoprecipitation. D, WT or α3 integrin-null AECs plated on Fn were maintained in the presence of SB431542 (10 μm) for 2 days and then treated with TGFβ1 (4 ng/ml) plus dimethyl sulfoxide (control) or SU6656 (5 μm) for 4 h. Cell lysates were immunoblotted for Src kinase activation (pY416) and pY14-caveolin-1.

FIGURE 2.

pY654-β-catenin is required for EMT. A, AECs from WT or ^fl/fl β-catenin mice were treated with AdenoCre or AdenoGFP and seeded on Fn. After 4 days, cells were lysed and immunoblotted with the indicated antibodies. Absence of β-catenin correlated with decreased α-SMA expression. B, AECTs cultured on Matrigel form large cell aggregates (upper left). Cytospins of single cells were stained for β4 integrin or pro-surfactant protein C markers (upper right). Lower, AECTs seeded for 4 days on Fn in the presence (left) or absence (right) of SB431542 were stained for EMT markers E-cadherin (orange) and α-SMA (green). In the absence of inhibitor, AECTs undergo robust EMT. C, AECT-TOPflash expressing the indicated versions of exogenous β-catenin were treated with AdenoCre to remove endogenous β-catenin and seeded on Fn. After 4 days, starved cells were treated with LiCl (20 mm) and TGFβ1 (4 ng/ml) for 24 h. TOPflash luciferase activity is expressed as relative light units (RLU) and normalized to β-catenin expression. Data shown are representative of four independent experiments. Immunoblot of β-catenin (duplicate lanes) from one of the experiments is shown. D, left, AECTs expressing WT and mutant β-catenins were treated with AdenoCre and seeded on Fn. Cells were starved and treated with LiCl (20 mm) for 3 days. Twist1, α-SMA, and Col1 were induced in the presence of WT or Y654E-β-catenin, but not in the absence of β-catenin nor replacement by Y654F mutant. Right, Twist expression was normalized to total β-catenin expression, average of three independent experiments (p < 0.05).

FIGURE 3.

pY654-β-catenin does not significantly contribute to TGFβ1-induced TCF/β-catenin reporter activity. A, AECTs carrying the TOPflash reporter were seeded on Fn. Cells were lysed, and TOPflash activity was measured after treating with the indicated factors for 24 h: 20 mm LiCl, 4 ng/ml TGFβ1, 1 μm SIS3, 10 μm SB431542, 10 μm PP2 and PP3. B, A549 cells expressing TOPflash were treated with TGFβ1 ± LiCl or SU6656, and RLU was determined after 24 h. Relative activity normalized to β-gal expression. C, left, A549 cells were treated with control PBS or EGF (50 ng/ml) with or without Wnt3a for 2 h, and the pY654-β-catenin immunoprecipitates and the lysates were blotted for β-catenin. Right, A549 cells expressing TOPflash were treated with TGFβ1 (4 ng/ml) or EGF (50 ng/ml), and RLU was determined after 24 h. Relative activity was normalized to β-gal expression.

FIGURE 4.

Raised epithelial cell axin levels abrogate EMT. Primary AECs were plated on Fn and treated with either FT4001 (5 mm) or vehicle control. After 3 days: A, pY654-β-catenin and its complex with p-Smad2 detected by immunoprecipitation are decreased in the FT4001-treated cells, whereas total β-catenin and p-Smad2 levels are unaffected. B, immunoblots showed increased axin and decreased α-SMA proteins after FT4001 treatment (left). Quantification of α-SMA in 3 independent experiment confirms ∼75% loss of α-SMA with high axin levels (p < 0.05) (right). C, AECs were cultured on Fn without or with FT4001 (5 mm). After 3 days, immunostaining revealed suppression of Col1 (orange) and α-SMA (green) expression by FT4001.

FIGURE 5.

Tankyrase inhibition attenuates TGFβ1-stimulated EMT, bleomycin-induced injury, and fibrogenesis. A, mice injected intratracheally with bleomycin (2.5 units/kg). After 10 days, FT4001 50 mg/kg or vehicle alone was administered daily by gavage for 7 days. FT4001 significantly increased survival of bleomycin-treated mice by day 17 (p = 0.025). B, upper, Col1 (orange) and α-SMA (green) accumulation in bleomycin-injured lungs treated with FT4001 or vehicle. Images are composites of 20× fields tiled as described under “Experimental Procedures.” Middle and lower, representative H&E and Masson's trichrome staining of serial sections of lungs from vehicle- and FT4100-treated mice. Similar results were seen in lungs of three pairs of control and treated mice. C, pY654-β-catenin accumulation and EMT markers measured in protein extracts from snap-frozen lungs. pY654-β-catenin is strongly increased in bleomycin-treated mice and attenuated by FT4001. Axin and EMT markers (Col1, α-SMA, Snail1, Twist) are detected by immunoblotting. TGFβ1 signaling marker (p-Smad2) and total β-catenin are also shown. D, quantification (mean ± S.D. (error bars)) of EMT markers from three independent experiments (9 total mice given bleomycin and 15 given bleomycin + FT4001) analyzed by immunoblotting whole lung extracts. Data are expressed as relative -fold change over saline values (set at 1).