To further examine whether T2 cells are able to undergo EMT-like changes, T2 cells were cultured with TGF-β (4 ng/ml) for 24 – 72 hours (h). Whole cell lysates were assessed for expression of EMT markers by Western blotting.
Although no significant changes were detected in levels of expression of the epithelial marker E-cadherin, TGF-β treatment was associated with significant increases in the expression of the mesenchymal cell markers CTGF at 24 hours (p = 0.0153), and α-SMA at 24 hours (p = 0.0003), 48 hours (p < 0.0001) and 72 hours (p = 0.0188). These results indicated that TGF-β treatment led to some EMT-like changes, although no significant changes were observed in the expression of the other EMT markers investigated, including the intermediate filament protein vimentin and transcription factor SOX-9 (Fig. 4.2).
As CTGF functions as a secreted protein, the levels of CTGF in the conditioned media of the T2 cells were also examined by Western blotting.
TGF-β treatment was associated with significantly higher expression of secreted CTGF at 24 hours (p = 0.0004), 48 hours (p = 0.0173) and 72 hours (p = 0.0353) (Fig. 4.3 A). As collagen type I is also considered a
Figure 4.2 TGF-β induced express T2 cells were cultured with TGF
lysates were assessed for expression of EMT markers by Western blotting (A) and quantified by densitometry (B). Target protein (E
SMA, vimentin, SOX
as a loading control. Histograms show mean ± SEM (arbitrary units, n=3 samples per group from 2 or more independent experiments). *
p < 0.0005, **** p <
β induced expression of mesenchymal proteins
T2 cells were cultured with TGF-β (4 ng/ml) for 24 – 72 hours (h). Whole cell lysates were assessed for expression of EMT markers by Western blotting (A) and quantified by densitometry (B). Target protein (E-cadherin, CTGF,
A, vimentin, SOX-9) expression was normalised against that of β
as a loading control. Histograms show mean ± SEM (arbitrary units, n=3 samples per group from 2 or more independent experiments). *
p < 0.0001, ns = no significance.
ion of mesenchymal proteins
72 hours (h). Whole cell lysates were assessed for expression of EMT markers by Western blotting cadherin, CTGF, α-9) expression was normalised against that of β-tubulin as a loading control. Histograms show mean ± SEM (arbitrary units, n=3 samples per group from 2 or more independent experiments). * p < 0.05, ***
Figure 4.3 TGF-β induced expression of secreted mesenchymal protein markers in T2 cells
T2 cells were cultured with TGF blot analysis of (A, left)
the conditioned media. Western blots were quantified by densitometry (right).
Expression of target proteins (CTGF, collagen type I) was normalised against β-tubulin in corresponding whole cell lysates
Histograms in A show mean (n=3 from 3
p < 0.05, *** p < 0.0005. Histogram in B is from a representative Western blotting image (n=1).
β induced expression of secreted mesenchymal protein markers in T2 cells
T2 cells were cultured with TGF-β (4 ng / ml) for 24 – 72 hours (h). Western blot analysis of (A, left) secreted CTGF and (B, left) collagen type I levels in the conditioned media. Western blots were quantified by densitometry (right).
Expression of target proteins (CTGF, collagen type I) was normalised against tubulin in corresponding whole cell lysates (as shown in Fig. 4.2).
Histograms in A show mean (n=3 from 3 independent experiments) ± SEM 0.0005. Histogram in B is from a representative Western blotting image (n=1).
β induced expression of secreted mesenchymal protein
72 hours (h). Western secreted CTGF and (B, left) collagen type I levels in the conditioned media. Western blots were quantified by densitometry (right).
Expression of target proteins (CTGF, collagen type I) was normalised against (as shown in Fig. 4.2).
independent experiments) ± SEM. * 0.0005. Histogram in B is from a representative Western
mesenchymal marker of EMT associated with tissue fibrosis (Kalluri, 2009), the levels of collagen type I in the conditioned media were also measured by Western blotting (Fig. 4.3 B). Fibronectin is another mesenchymal marker, but due to the variability experienced with Western blotting of this protein, (mainly because of its large molecular weight of 240 kDa, making it difficult to transfer from gel to membrane), fibronectin levels in the conditioned media were measured by enzyme linked immunosorbent assay (ELISA).
Significantly higher levels of fibronectin expression was observed in TGF-β-treated cells at 24 hours (p = 0.0004) and 48 hours (p = 0.0442) compared to untreated cells (Fig. 4.4). These results are consistent with previous studies reporting the involvement of TGF-β in the process of EMT in a human alveolar epithelial cell line, A549, characterised by expression of the same mesenchymal markers (Kasai et al., 2005). An important finding in the current study, which is supported by observations made by Kasai et al.
(Kasai et al., 2005), was the enhanced expression of CTGF identified as one of the features in a series of EMT-like changes induced by TGF-β treatment (Fig. 4.2).
4.2.3 TGF-β induced mRNA expression of CTGF and α-SMA in T2 cells without altering E-cadherin levels
To assess whether there were any changes in mRNA expression of EMT-related genes, T2 cells treated with TGF-β (4 ng/ml) subjected to qPCR analysis. Since results of the Western blot analysis indicated alterations in protein expression could already be detected at 24 hours, treatment of cells for qPCR analysis was also performed for 24 hours. Gene expression (copy number) was normalised against the geometric mean of the expression of three reference genes. As shown in Fig. 4.5, T2 cells treated with TGF-β expressed significantly higher levels of CTGF (p < 0.0001) and α-SMA (p = 0.0094). However, consistent with findings from the Western blot analysis showing that protein levels of E-cadherin were unchanged by TGF-β treatment (Fig. 4.2), no significant difference was found in expression of E-cadherin mRNA (p = 0.9545) between TGF-β-treated and control cells (Fig.
Figure 4.4 TGF-β induced fibronectin expressio T2 cells were treated with TGF
Fibronectin expression in the T2 cell conditioned medium was measured by ELISA. The level of fibronectin was normalised against amount of total protein as determi
lysates. Columns show the percentage of fibronectin compared to the control of no TGF-β treatment at 24 hours (first column).
3 samples per group) ± SEM. Unpaired t
β induced fibronectin expression in T2 cells T2 cells were treated with TGF-β (4 ng/ml) treatment for 24
Fibronectin expression in the T2 cell conditioned medium was measured by The level of fibronectin was normalised against amount of total protein as determined by BCA assay from the corresponding whole cell Columns show the percentage of fibronectin compared to the control
treatment at 24 hours (first column). Columns show mean (n 3 samples per group) ± SEM. Unpaired t-test, * p < 0.05.
n in T2 cells
β (4 ng/ml) treatment for 24 and 48 hours (h).
Fibronectin expression in the T2 cell conditioned medium was measured by The level of fibronectin was normalised against amount of total ned by BCA assay from the corresponding whole cell Columns show the percentage of fibronectin compared to the control Columns show mean (n =
Figure 4.5 TGF-β induced mRNA expression of CTGF and α cells without altering E
mRNA expression of E cultured with
TGF-Gene expression (copy number) was normalised against the geometric mean (G.M.) of the expression of three reference genes. Columns show mean (n 3 samples per group from 2 independent experiments) ± SEM. **
**** p < 0.0001.
β induced mRNA expression of CTGF and α cells without altering E-cadherin levels
mRNA expression of E-cadherin (A), CTGF (B) and α-SMA (C) in T2 cells -β (4 ng/ml) for 24 hours (h) were measured by qPCR.
Gene expression (copy number) was normalised against the geometric mean (G.M.) of the expression of three reference genes. Columns show mean (n 3 samples per group from 2 independent experiments) ± SEM. **
β induced mRNA expression of CTGF and α-SMA in T2
SMA (C) in T2 cells were measured by qPCR.
Gene expression (copy number) was normalised against the geometric mean (G.M.) of the expression of three reference genes. Columns show mean (n = 3 samples per group from 2 independent experiments) ± SEM. ** p < 0.01,
4.5 A). Increased expression of CTGF and α-SMA mRNA (Fig. 4.5 B and C) detected in TGF-β treated T2 cells compared to untreated cells suggest the increase in CTGF and α-SMA protein may be related to altered transcriptional regulation of these genes favouring de novo protein synthesis.
These alterations in mRNA expression levels suggest that TGF-β and CTGF may both play a role in EMT-like changes of alveolar epithelial cells.
4.2.4 TGF-β induces redistribution of E-cadherin and expression of