FIZZ1 stimulation of myofibroblast differentiation - PubMed (original) (raw)

FIZZ1 stimulation of myofibroblast differentiation

Tianju Liu et al. Am J Pathol. 2004 Apr.

Abstract

Bleomycin-induced pulmonary fibrosis is characterized by inflammation, emergence of myofibroblasts, and deposition of extracellular matrix. In an attempt to identify genes that may be involved in fibrosis, we used a 10,000 element (10 K) rat cDNA microarray to analyze the lung gene expression profiles in this model in the rat. Cluster analysis showed 628 genes were more than or equal to twofold up- or down-regulated, many of which were known to be involved in fibrosis. However, the most dramatic increase was observed with FIZZ1 (found in inflammatory zone; also known as RELM-alpha or resistin-like molecule-alpha), which was induced 17-fold to approximately 25-fold at the peak of expression. In situ hybridization analysis revealed FIZZ1 expression to localize primarily to alveolar and airway epithelium, which was confirmed in vitro by analysis of isolated type II alveolar epithelial cells. However FIZZ1 expression was not detected in isolated lung fibroblasts. Co-culture of FIZZ1-expressing type II cells with fibroblasts stimulated alpha-smooth muscle actin and type I collagen expression independent of transforming growth factor-beta. Transfection of a FIZZ1-expressing plasmid into fibroblasts or treatment with glutathione S-transferase-FIZZ1 fusion protein stimulated alpha-smooth muscle actin and collagen I production. These results suggest a novel role for FIZZ1 in myofibroblast differentiation in pulmonary fibrosis.

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Figures

Figure 1

Gene expression pattern (signature) of BLM-induced pulmonary fibrosis. Gene expression profiles of control and BLM-treated rat lung were analyzed by cDNA microarray analysis. Each experimental sample was hybridized against its matched control RNA obtained at the indicated time points (days). Expression data of 10,000 genes were analyzed by Cluster Analysis of Gene Expression Dynamics (CAGED) v 1.0 software. CAGED analysis revealed five distinct clusters, A to E. Each row represents a single gene and each column an average of the data from individual arrays of samples from three individual animals, at any given time point. Thirty-eight of the down-regulated genes are shown in cluster A. The up-regulated genes were separated into four groups: cluster B, consisting of 26 genes that were up-regulated early, clusters C and E, with 33 and 9 genes, respectively, that increased in the middle period of fibrosis. The late up-regulated genes displayed in cluster D were arrived at by applying a filter of any two time points showing fourfold difference on the 522 genes identified by CAGED analysis.

Figure 2

Kinetics of FIZZ1 induction from microarray data. The results from microarray analysis for FIZZ1 were expressed as fold increase over control lung tissue, and plotted as a function of time after BLM treatment. Means ± SE of triplicate animals are shown.

Figure 3

Kinetics of FIZZ1 mRNA expression by real-time RT-PCR. Lung tissue total RNAs were isolated after BLM or saline injection at the indicated time points, and subjected to Taqman real-time RT-PCR. GAPDH signals were used as internal controls. Results are shown as the threshold cycle (CT) at which an increase of reporter fluorescence (ΔRn) can first be detected. Amounts of FIZZ1 mRNA were normalized to GAPDH signals and expressed as 2−ΔΔCT. Means ± SE of triplicate samples are shown. All mean values in the BLM-treated groups were statistically different (P < 0.001) from their corresponding control (saline-treated) groups.

Figure 4

Localization of lung FIZZ1 expression by in situ hybridization. Lung tissue sections from a day 7 BLM (A, C, and D) or saline-treated (B) rat were analyzed by in situ hybridization for FIZZ1 mRNA expression. Digoxigenin-labeled sense (A) and anti-sense (B–D) oligonucleotides against FIZZ1 were used as probes. The positive signals (purple) were localized to airway (C, open arrows) and alveolar (D, solid arrows) epithelia in day 7 BLM-treated rat lung, but were essentially undetectable in control saline-treated lung (B). No signal was observed in day 7 BLM-treated lung section hybridized with the control sense probes (A). Original magnifications, ×400.

Figure 5

Effects of FIZZ1-expressing AECs on fibroblasts. FIZZ1-expressing AECs from control (Cont-AEC) or day 7 BLM-treated (BLM-AEC) rats were co-cultured with normal rat lung fibroblasts (Cont-RLF) or day 7 BLM-treated (BLM-RLF) as described in Materials and Methods. After 36 hours of incubation cell extracts were prepared for measurement of type I collagen by ELISA (right y axis in A), and of α-SMA by Western blotting (left y axis in A and ordinate in B). The Western blotting data were expressed as relative integration units (RIU) after measurement of net intensity of scanned bands from Western blots (in both A and B). TGF-β1 was added to fibroblasts as a positive control. Means ± SE of triplicate samples are shown. The stimulatory effects of BLM-AEC on both parameters were significantly different (P < 0.05) from their corresponding values for Cont-AEC and untreated fibroblasts. TGF-β1 effects were significantly higher than untreated cells. Cont-AEC did not have significant effects on fibroblasts. In B the effects of BLM-AEC co-culture on α-SMA expression in Cont-RLF were compared to those in BLM-RLF by Western blotting analysis. Both Cont-RLF and BLM-RLF expressed significantly higher α-SMA when co-cultured with BLM-AEC, and in each instance the expression in BLM-RLF was significantly higher than that in Cont-RLF. Results from triplicate samples were shown and expressed as in A.

Figure 6

Effects of forced FIZZ1 expression on fibroblasts. Normal rat lung fibroblasts that do not express FIZZ1 were transfected with either empty plasmid (empty vector) or FIZZ1-expressing plasmid (pEGFP-FIZZ1) and then analyzed for FIZZ1 (A), type I collagen (B), or α-SMA (C) expression as described in Materials and Methods. FIZZ1 mRNA was measured using real-time RT-PCR at the indicated time points after transfection, and expressed as described in the legend to Figure 3. The pEGFP-FIZZ1-induced increases in FIZZ1 mRNA levels were statistically significant (P < 0.05) at the 8-, 12-, and 24-hour time points (A). Type I collagen was measured at the indicated time points using ELISA, and the results were expressed as absorbance units at 405 nm. The increases induced by pEGFP-FIZZ1 transfection (relative to empty vector) were significant (P < 0.001) at both time points (B), whereas the effect on α-SMA expression was significant at P < 0.005 (C). Cell extract α-SMA content was quantitated by Western blotting 24 hours after transfection. Transfection with empty vector had no significant effects on fibroblasts (data not shown). Means ± SE of sextuplicate samples are shown.

Figure 7

Effects of FIZZ1 expression and GST-FIZZ1 fusion protein on fibroblasts. FIZZ1 expression plasmid (pEGFP-FIZZ1) was transfected into fibroblasts and the effects of sense and anti-sense FIZZ1 oligonucleotides were examined. FIZZ1 plasmid-transfected cells showed heightened α-SMA expression by Western blotting analysis, which was inhibited by anti-sense FIZZ1 oligonucleotides but not by sense oligonucleotides (a). When cell sonicates from FIZZ1 plasmid-transfected cells were added onto nontransfected fibroblasts, enhanced α-SMA expression was noted by Western blotting analysis relative to fibroblasts treated with sonicates from cells transfected with the empty vector at both 24 and 48 hours of incubation. (b). Finally, addition of GST-FIZZ1 fusion protein to fibroblasts stimulated α-SMA expression by Western blotting analysis, whereas GST had no significant effects. TGF-β1 was used as positive control (c). Representative results from three independent experiments are shown in each panel.

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FIZZ1 stimulation of myofibroblast differentiation - PubMed (original) (raw)