Dysregulation of TGF-beta1 receptor activation leads to abnormal lung development and emphysema-like phenotype in core fucose-deficient mice

Abstract

The core fucosylation (alpha1,6-fucosylation) of glycoproteins is widely distributed in mammalian tissues, and is altered under pathological conditions. To investigate physiological functions of the core fucose, we generated alpha1,6-fucosyltransferase (Fut8)-null mice and found that disruption of Fut8 induces severe growth retardation and death during postnatal development. Histopathological analysis revealed that Fut8(-/-) mice showed emphysema-like changes in the lung, verified by a physiological compliance analysis. Biochemical studies indicated that lungs from Fut8(-/-) mice exhibit a marked overexpression of matrix metalloproteinases (MMPs), such as MMP-12 and MMP-13, highly associated with lung-destructive phenotypes, and a down-regulation of extracellular matrix (ECM) proteins such as elastin, as well as retarded alveolar epithelia cell differentiation. These changes should be consistent with a deficiency in TGF-beta1 signaling, a pleiotropic factor that controls ECM homeostasis by down-regulating MMP expression and inducing ECM protein components. In fact, Fut8(-/-) mice have a marked dysregulation of TGF-beta1 receptor activation and signaling, as assessed by TGF-beta1 binding assays and Smad2 phosphorylation analysis. We also show that these TGF-beta1 receptor defects found in Fut8(-/-) cells can be rescued by reintroducing Fut8 into Fut8(-/-) cells. Furthermore, exogenous TGF-beta1 potentially rescued emphysema-like phenotype and concomitantly reduced MMP expression in Fut8(-/-) lung. We propose that the lack of core fucosylation of TGF-beta1 receptors is crucial for a developmental and progressive/destructive emphysema, suggesting that perturbation of this function could underlie certain cases of human emphysema.

Fig. 1.

Semilethality and growth retardation in Fut8^-/- mice. (A) Survival ratio of Fut8^-/- (-/-, solid line), Fut8^+/- (+/-, broken line), and Fut8^+/+ (+/+, dotted line) mice after birth. (B) A 16-day-old Fut8^-/- pup (-/-) with a Fut8^+/+ littermate (+/+).

Fig. 2.

Emphysematous change and impaired ventilatory response in Fut8^-/- lung. (A) Representative histological sections (hematoxylin/eosin staining) of the lung of 18-day-old Fut8^+/+ (+/+), Fut8^+/- (+/-), and Fut8^-/- (-/-) pups show enlarged alveoli indicative of emphysema in Fut8^-/- mice. (B) Lung morphometry at different stages. The mean linear intercepts of pulmonary alveoli were calculated at postnatal 3-, 7-, 10-, 18-day-old and 8-week-old mice. The diameters of the pulmonary alveoli were shown as the mean ± SD from three independent experiments. Statistical analysis was performed by using Student's t test. *, P < 0.01; **, P < 0.001 (versus the matched age of Fut8^+/+ mice). P, postnatal. (C) Lung compliance test. Static pressure-volume curves were plotted by using 19- to 20-day-old Fut8^-/- (brown), Fut8^+/+ (pink), and Fut8^+/- (blue) pups. (D) Comparison of changes in respiratory minute volume in response to hypoxic or hypercapnic air in conscious 19- to 20-day-old littermate Fut8^+/+ (+/+), Fut8^+/- (+/-), and Fut8^-/- (-/-) mice.

Fig. 3.

Enhancement of some MMPs expression through down-regulation of TGF-β-receptor-mediated signaling in Fut8^-/- lung and embryonic fibroblasts. (A) RT-PCR analysis of emphysema-relating genes (see Table 1, which is published as supporting information on the PNAS web site). Total RNAs from 18-day-old Fut8^+/+ (+/+), Fut8^+/- (+/-), and Fut8^-/- (-/-) lungs were used as template. β-Actin RNA is shown as a loading control. (B) Effects of IL-1β and TGF-β1 on McolB expression. These fibroblasts were preincubated with or without TGF-β1(1ng/ml) for 3 h and then further incubated with or without IL-1β (2 ng/ml) for 24 h. Total RNA was isolated and used as template. (C) Effects of IL-1β and TGF-β on protein expression of MMP-12 secreted into culture media. These fibroblasts were incubated with or without IL-1β and TGF-β1 in the absence of FCS. After incubation for 24 h, the conditioned media were concentrated and subjected to electrophoresis for Western blot. (D) Binding of ¹²⁵I-TGF-β1 to its receptors on the cell surface. These cells from Fut8^+/+ (+/+) and Fut8^-/- (-/-) primary fibroblasts immortalized with SV40 large T, SW, and SK, respectively, or SK restored with Fut8 (SK+F8), were incubated with different amounts of radiolabeled TGF-β1 as indicated and 10 ng of cold TGF-β1 for 2 h on ice. Cell lysate radioactivity was measured. (E) ¹²⁵I-TGF-β1 was bound and cross-linked to its receptors on cell surface. The cultured cells were incubated with 250 pM ¹²⁵I-TGF-β1 for 2 h at 4°C, and then cross-linked with reagent BS³.(F) Analysis of fucosylation levels on TGF-β receptor II. TGF-β1 receptor II was immunoprecipitated from whole-cell lysates and then subjected to electrophoresis on 8% SDS/PAGE. After electroblotting, blots were probed by ALL lectin (Upper) and anti-TGF-β receptor II (Lower). (G) Effects of phosphorylated Smad2 levels on TGF-β1 stimulation. Serum-starved cells were treated with or without TGF-β1 at the indicated concentrations for 5 min and solubilized in lysis buffer as described in Materials and Methods. The cell lysates were detected by immunoblotting of anti-phospho-Smad2 antibody (Upper) and anti-Smad2 antibody (Lower). (H) The lung sections of 4-day-old mice were pretreated with hydroxygen blocking for 10 min at 37°C and then incubated with rabbit anti-human P-Smad2 antibody for 16 h at 4°C. The arrowheads indicate positive staining in Fut8^+/+ lung. (I) Therapeutic administration of recombinant TGF-β1 rescues emphysema-like changes in Fut8^-/- mice. The surviving postnatal 18-day-old Fut8^-/- mice were treated with or without recombinant TGF-β1 (50 or 100 ng/g of mouse body weight) for 20 times of injection every 2 days, and then the lung sections were subjected to hematoxylin/eosin staining. (J) Quantitative analyses of the pulmonary alveolar sizes were performed by mean linear intercept as described above. The diameters of the pulmonary alveoli were shown as the mean ± SD from three independent experiments. Statistical analysis was performed by using Student's t test. *, P < 0.01 (Fut8^-/- mice treated with TGF-β versus the matched age of mice without treatment).