Diverse functions of clusterin promote and protect against the development of pulmonary fibrosis

Abstract

Pulmonary fibrosis is a progressive scarring disorder of the lung with dismal prognosis and no curative therapy. Clusterin, an extracellular chaperone and regulator of cell functions, is reduced in bronchoalveolar lavage fluid of patients with pulmonary fibrosis. However, its distribution and role in normal and fibrotic human lung are incompletely characterized. Immunohistochemical localization of clusterin revealed strong staining associated with fibroblasts in control lung and morphologically normal areas of fibrotic lung but weak or undetectable staining in fibrotic regions and particularly fibroblastic foci. Clusterin also co-localized with elastin in vessel walls and additionally with amorphous elastin deposits in fibrotic lung. Analysis of primary lung fibroblast isolates in vitro confirmed the down-regulation of clusterin expression in fibrotic compared with control lung fibroblasts and further demonstrated that TGF-β₁ is capable of down-regulating fibroblast clusterin expression. shRNA-mediated down-regulation of clusterin did not affect TGF-β₁-induced fibroblast-myofibroblast differentiation but inhibited fibroblast proliferative responses and sensitized to apoptosis. Down-regulation of clusterin in fibrotic lung fibroblasts at least partly due to increased TGF-β₁ may therefore represent an appropriate but insufficient response to limit fibroproliferation. Reduced expression of clusterin in the lung may also limit its extracellular chaperoning activity contributing to dysregulated deposition of extracellular matrix proteins.

Figure 1

Localization of clusterin in normal human lung. Clusterin was detected immunohistochemically by staining formalin-fixed, paraffin embedded 3 μm sections of human control lung tissue. Representative images of clusterin (clu, A-C, brown/red, nuclei - blue) and elastic fibers ((D), grey/black) in tissue obtained from control lung (n = 3). Clusterin localizes to fibroblast-like cells (A), to small areas of bronchial epithelial cells (B) and to elastic fibers in blood vessels and alveolar walls (C,D) serial sections). Clusterin was not detectable in macrophages, alveolar epithelial cells (A) or endothelial cells (C). Different cell populations/structures are indicated by arrows: f - fibroblast-like cell, m - macrophage, e – alveolar epithelial cell, be - bronchial epithelial cell, en - endothelial cell, smc – smooth muscle cell, ef - elastic fibers. Scale bar represents 25 µm.

Figure 2

Localization of clusterin in IPF lung. Immunohistochemical staining for clusterin was performed on formalin-fixed, paraffin embedded 3 μm sections of IPF lung tissue (n = 3). Clusterin staining (clu, A, C-G, I brown/red, nuclei - blue) and staining for elastin (H, J, EvG, grey/black) in representative tissue sections. Clusterin is undetectable in αSMA positive myofibroblasts (A,B), forming and in cells overlying fibroblastic foci (A,C), compared to strong staining of fibroblast-like cells in morphologically normal non-fibrotic areas (D). Clusterin was observed sporadically in bronchial epithelial cells but more frequently than in controls (E). Similar to control lung, clusterin colocalized with elastin (G,H) and was undetectable in macrophages, smooth muscle and endothelial cells (F,G). Clusterin also colocalized with amorphous elastin aggregates in dense fibrotic regions (I,J). Different cell populations/structures are indicated by arrows; f - fibroblast-like cell, m - macrophage, he - hyperplastic epithelial cell, be - bronchial epithelial cell, en – endothelial cell, smc – smooth muscle cell, ef- elastic fibers. Scale bar represents 25 µm (A–J).

Figure 3

Clusterin gene expression and protein levels are decreased in fibrotic compared with control lung fibroblasts. Fibroblasts isolated from human control and fibrotic lung were grown in monolayer culture and clusterin mRNA and protein levels were detected via microarray, proteome profiler and immunofluoresecence analysis. (A) Microarray analysis of mRNA shows decreased clusterin gene expression in fibroblasts derived from fibrotic lungs (open circles; n = 5 IPF and 7 SSc) compared with controls (closed circles; n = 6). Proteome profiler array analysis (B, clusterin - CLU) and immunofluorescence staining of control and fibrotic lung fibroblasts (C,D) confirms low clusterin protein expression in fibrotic compared with control fibroblasts in vitro. (E) Semi-quantitative analysis of clusterin staining (C,D), clusterin signal (pixel intensity) normalized to cell numbers per visual field (n = 6). Data is representative of three individual experiments. *P < 0.05, ****P < 0.0001; Scale bar in D represents 10 µm.

Figure 4

TGF-β₁ associates with areas of decreased clusterin expression in fibrotic lung and down-regulates fibroblast clusterin mRNA and protein expression in vitro. Serial sections prepared from IPF lung (n = 3) were stained immunohistochemically to localize TGF-β₁ staining ((A), red/brown, nuclei blue) and clusterin ((B), red/brown, nuclei blue) in fibroblastic foci. (A,B) Representative images of immunohistochemical staining suggest that TGF-β₁ localizes to ECM, fibroblasts and macrophages, whilst staining for clusterin is weak or undetectable. (C) In vitro analysis of clusterin mRNA levels in TGF-β₁ stimulated (40 pM) human lung fibroblasts was performed via qRT-PCR and shows a time-dependent down-regulation of clusterin expression that was maximal at 24–48 h (n = 3) in response to TGF-β₁. Clusterin protein levels were also down-regulated in response to TGF-β₁ (40 pM) compared to control at 24 h and 48 h as demonstrated by western blotting at 24 h (D) and immunofluorescent staining at 48 h (E, red, nuclei - blue). (F) Semi-quantitative analysis of fluorescent signal of panel E: clusterin signal (pixel intensity) was normalized to cell numbers per visual field and compared to control (n = 6). Full-length western blots are presented in Supplementary Figure e4. Different cell populations/structures are indicated by arrows; f - fibroblast-like cell, m - macrophage, he - hyperplastic epithelial cell, ecm – extracellular matrix. *P < 0.05, **P < 0.01; scale bar 100 µm (A) and 10 µm (E).

Figure 5

Effect of clusterin deficiency on TGF-β₁-induced myofibroblast differentiation and collagen deposition. Lung fibroblasts were transduced with clusterin shRNA (shCLU, open bars) and mock shRNA vectors (grey bars) or remained untransfected (black bars). αSMA mRNA was assessed via qRT-PCR (A) and clusterin and αSMA protein levels detected by western blotting (B,C quantification). 48 h following TGF-β₁ stimulation (40 pM) αSMA mRNA and protein were increased. Basal and increased levels of αSMA mRNA and protein, however, did not vary between clusterin deficient, mock-transduced and control fibroblasts. Collagen mRNA (D) and deposition (E,F quantification) assessed by qRT-PCR and immunofluorescence staining were significantly increased in response to TGF-β₁. Although, basal and TGF-β₁ induced changes in collagen levels varied between clusterin deficient, mock and control fibroblasts, the overall fold-increase in collagen mRNA and deposition levels did not significantly change between clusterin deficient fibroblasts and control/mock fibroblasts. Data generated in A-F is representative of two individual experiments with fibroblasts derived from 2 donors. Full-length western blots are presented in Supplementary Figure e5. Scale bar in E represents 10 µm. **P < 0.01, ***P < 0.001 compared with untreated controls respectively.

Figure 6

Effect of clusterin deficiency on lung fibroblast proliferation.Lung fibroblasts were transduced with clusterin shRNA (shCLU, open bars) and mock shRNA vectors (grey bars) or remained untreated (black bars). Proliferation in shRNA-mediated clusterin deficient fibroblasts (A) or fibrotic lung fibroblasts (B) compared with controls was assessed in response to the indicated stimuli for 48 h or 72 h for FBS by counting DAPI-positive nuclei in a high-throughput immunofluorescence assay. Cell numbers were normalized to cell counts of controls (0.4% FBS in DMEM) and expressed as percent change in proliferation relative to control (n = 6). Data representatives of two individual experiments with fibroblasts derived from 1 donor per group. Significances compared with controls are marked with (#) symbol and significances between controls vs. shCLU or non-fibrotic vs. fibrotic lung fibroblasts are indicated with (*). ^*/#P < 0.05, ^**/##P < 0.01, ***/^###P < 0.001, ****/^####P < 0.0001.

Figure 7

Effect of clusterin deficiency on apoptosis. Lung fibroblasts were transduced with clusterin shRNA (shCLU, open circles) and mock shRNA vectors (grey circles) or remained untransfected (black circles). Lung fibroblasts were seeded and treated with FasL (3 nM – 6 nM) and/or exogenous clusterin (CLU, 125 nM) for 19 h or remained untreated. Apoptotic cells (Annexin V+ and Annexin V+/ DAPI+ cells) were assessed via FACS analysis post staining of apoptotic cells with annexin V – Alexa647 and DAPI (mean ± SEM, n = 5). (A) shRNA-induced clusterin deficiency sensitized fibroblasts to basal and FasL-induced apoptosis, and this could be overcome by addition of exogenous clusterin (B). (C) Basal apoptosis in representative control (9.37 ± 0.63%) and fibrotic (11.6 ± 0.69%) lung fibroblast isolates was not significantly different. Fibrotic lung fibroblasts were more resistant to FasL-induced apoptosis compared with controls (C) and exogenous clusterin tends to reduce basal and FasL-induced apoptotic levels further (D). Data representative of at least two individual experiments with fibroblasts derived from 1 donor per group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.