CTGF has been reported to act as a downstream mediator of TGF-β action in fibroblasts (Grotendorst, 1997) but the relationship of CTGF and TGF-β signalling in T2 cells remains largely unknown. In this study, TGF-β is shown to induce α-SMA expression and in order to investigate directly whether CTGF may be involved in this induction, T2 cells were treated with non- targeted control siRNA (siNTC) or CTGF-specific siRNA (siCTGF, 20 nM) prior to TGF-β (4 ng/ml) treatment for 24 hours (Fig. 4.7). Changes in CTGF and α-SMA mRNA expression were assessed by qPCR. Treatment with
Figure 4.6 T2 cells expressed mesenchymal cell markers after treatment with TGF-β
T2 cells were treated with TGF E-cadherin, CTGF,
immunofluorescence. Increased ex
was observed in the T2 cells treated with TGF controls. A decrease in E
membrane of the TGF
rabbit IgG (Ms IgG and Rb IgG) were used as controls for non
binding of primary antibodies. Representative images from 3 independent experiments. Magnification 400X.
T2 cells expressed mesenchymal cell markers after treatment
T2 cells were treated with TGF- β (4 ng/ml) for 48 hours. The expression of
cadherin, CTGF, α-SMA and vimentin were examined by
immunofluorescence. Increased expression of CTGF, α-SMA and vimentin was observed in the T2 cells treated with TGF- β compared to untreated controls. A decrease in E-cadherin expression was observed
membrane of the TGF-β-treated T2 cells compared to control. Mouse and it IgG (Ms IgG and Rb IgG) were used as controls for non
binding of primary antibodies. Representative images from 3 independent experiments. Magnification 400X.
T2 cells expressed mesenchymal cell markers after treatment
(4 ng/ml) for 48 hours. The expression of
SMA and vimentin were examined by
SMA and vimentin compared to untreated adherin expression was observed at the plasma treated T2 cells compared to control. Mouse and it IgG (Ms IgG and Rb IgG) were used as controls for non-specific binding of primary antibodies. Representative images from 3 independent
Figure 4.7 TGF-β induced mRNA e cells without altering E
mRNA expression of E cultured with TGF-
cells were also treated with either a con
a pool of siRNA targeting CTGF expression (siCTGF).
(copy number) was normalised against the geometric mean (G.M.) of the expression of three reference genes.
per group from 2 independent experiments) ± SEM. ** 0.0001.
β induced mRNA expression of CTGF and α hout 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 were measured by qPCR. cells were also treated with either a control non-target siRNA pool (siNTC) or a pool of siRNA targeting CTGF expression (siCTGF).
(copy number) was normalised against the geometric mean (G.M.) of the expression of three reference genes. Columns show mean (n
p from 2 independent experiments) ± SEM. ** p <
xpression of CTGF and α-SMA in T2
SMA (C) in T2 cells (4 ng/ml) for 24 hours were measured by qPCR. T2 target siRNA pool (siNTC) or a pool of siRNA targeting CTGF expression (siCTGF). Gene expression (copy number) was normalised against the geometric mean (G.M.) of the Columns show mean (n = 3 samples p < 0.01, **** p <
CTGF-specific siRNA (siCTGF) significantly reduced TGF-β-induced CTGF mRNA expression by 20 ± 0.1 % (p = 0.043) compared to control cells exposed to non-targeting control siRNA (siNTC). Concomitant with this knockdown in CTGF expression, a significant decrease of 43 ± 0.1 % (p = 0.0042) in TGF-β-induced α-SMA mRNA was observed in siCTGF-treated T2 cells compared to siNTC control. This shows that a partial knockdown of CTGF is able to reduce α-SMA expression induced by TGF-β treatment in T2 cells.
4.2.6 CTGF induces expression of mesenchymal cell proteins in T2 cells
As TGF-β treatment induced EMT-like changes in T2 cells, in a process which seemed to be at least partially dependent on CTGF, the question of whether CTGF alone may mediate EMT was investigated. In order to mimic the situation in which CTGF released by adjacent fibroblasts influences epithelial cells, as in the Col1a2-CTGF transgenic mice (Chapter 3), T2 cells were treated with recombinant CTGF. Initially, a range of recombinant CTGF concentrations (10 – 200 ng/ml) were tested with treatment times of 24 or 48 hours to determine the optimum dose and time of treatment.
Expression of the EMT markers, E-cadherin and α-SMA were assessed by Western blotting (Fig. 4.8). At 24 hours after the addition of exogenous CTGF to the T2 cell culture, a significant increase in α-SMA expression was already observed at the lowest concentration of CTGF at 10 ng/ml (p = 0.0039) compared to untreated cells. Peak α-SMA induction at 24 hours was achieved by treatment of the cells with 50 ng/ml of CTGF (p = 0.0037). Treatment with higher concentrations of CTGF did not induce significantly higher levels of α-SMA compared to CTGF at 50 ng/ml (Fig. 4.8 A). Elevated α-SMA expression was also observed after 48 hours of CTGF treatment, although in this case, the dose-dependent increase in α-SMA levels peaked at 100 ng/ml of CTGF (Fig. 4.8 B). Despite a rise in α-SMA expression, E-
Figure 4.8 CTGF induces expression of α cells
T2 cells were cultured with CTGF (10 hours (h). (Left) Western blot analysis of E and (right) densitometry.
expression of β-tubulin. Points show mean (n=3 samples per group) ± SEM. Unpaired t-test compared to control (0 ng/ml CTGF).*
CTGF induces expression of α-SMA and E
cultured with CTGF (10 – 200 ng/ml) for (A) 24 and (B) 48 hours (h). (Left) Western blot analysis of E-cadherin and α
and (right) densitometry. Target protein expression was normalised against tubulin. Points show mean (n=3 samples per group) ± SEM. test compared to control (0 ng/ml CTGF).* p < 0.05, ** p< 0.005.
SMA and E-cadherin in T2
ml) for (A) 24 and (B) 48 α-SMA expression normalised against tubulin. Points show mean (n=3 samples per group) ± SEM.
cadherin expression did not drop, as would be expected in EMT. Conversely, E-cadherin protein was significantly increased at the highest concentration of CTGF treatment at 24 hours and also at several concentrations at 48 hours (Fig. 4.8 A and B). The elevation of E-cadherin by CTGF may involve a mechanism other than EMT, which CTGF may be involved in (Tan et al., 2008).
The mesenchymal protein fibronectin was also induced by CTGF stimulation of T2 cells. Fibronectin levels in the conditioned media, measured by ELISA, were found to be significantly elevated after treatment of cells with 10 ng/ml (p = 0.0385) and 50 ng/ml (p = 0.0186) of CTGF for 48 hours (Fig. 4.9). However, induction of fibronectin by CTGF was lower compared to induction by TGF-β; 48 hours after treatment with TGF-β, fibronectin levels increased by 26.9 ± 3.12 % while CTGF treatment resulted in a more modest 14.0 ± 3.49 % increase compared to control.
Immunofluorescent staining demonstrated that addition of exogenous CTGF alone induced expression of α-SMA incorporated into stress fibres (Fig. 4.10) with no expression seen in untreated cells, confirming the results of the Western blotting analysis (Fig. 4.8). Detection of vimentin filaments were also increased in CTGF-treated T2 cells compared to control, reminiscent of the enhanced vimentin expression after TGF-β treatment of T2 cells (Fig 4.6). Although total E-cadherin levels as shown by Western blot were increased after CTGF treatment (Fig. 4.8), immunofluorescence showed a decrease in E-cadherin at the plasma membrane suggesting a disruption to E-cadherin function in maintaining cell-cell adhesion junctions.
4.2.7 CTGF induces a partial EMT phenotype in NMuMG cells
Like T2 cells, NMuMG cells are also epithelial cells, but are non-transformed mammary epithelial cells. In fact, NMuMG cells were the first cell type
Figure 4.9 CTGF induces expres
Levels of fibronectin in the conditioned media of T2 cell
(10 and 50 ng/ml) for 48 hours were assessed by ELISA. The level of fibronectin was normalised against amount of total protein as determined by BCA assay from the corresponding whole cell lysates. Points show mean normalised fibronect
SEM. * p < 0.05.
CTGF induces expression of fibronectin in T2 cells
Levels of fibronectin in the conditioned media of T2 cells treated with CTGF (10 and 50 ng/ml) for 48 hours were assessed by ELISA. The level of fibronectin was normalised against amount of total protein as determined by BCA assay from the corresponding whole cell lysates. Points show mean normalised fibronectin levels relative to the untreated negative control (n=3) ±
sion of fibronectin in T2 cells
s treated with CTGF (10 and 50 ng/ml) for 48 hours were assessed by ELISA. The level of fibronectin was normalised against amount of total protein as determined by BCA assay from the corresponding whole cell lysates. Points show mean in levels relative to the untreated negative control (n=3) ±
Figure 4.10 Localisation of CTGF cells
T2 cells were treated with CTGF (100 ng/ml) for 48 h
E-cadherin, α-SMA and vimentin were examined by immunofluorescence (green staining). Increased expression of
in the T2 cells treated with CTGF compared to untreated controls. A decrease in E-cadher
especially around the plasma membrane.
blue. Representative images from 3 independent experiments. Magnification 400X.
Localisation of CTGF-induced EMT protein markers in T2
T2 cells were treated with CTGF (100 ng/ml) for 48 hours. The expression of SMA and vimentin were examined by immunofluorescence . Increased expression ofα -SMA and vimentin was observed in the T2 cells treated with CTGF compared to untreated controls. A cadherin was detected in treated compared to control T2 cells, especially around the plasma membrane. DAPI-stained nuclei are shown in Representative images from 3 independent experiments. Magnification 400X.
EMT protein markers in T2
ours. The expression of SMA and vimentin were examined by immunofluorescence SMA and vimentin was observed in the T2 cells treated with CTGF compared to untreated controls. A in was detected in treated compared to control T2 cells, stained nuclei are shown in Representative images from 3 independent experiments.
reported to undergo EMT after TGF-β treatment (Maeda et al., 2005). Since NMuMG cells provide a classic in vitro system for studying EMT, to verify whether CTGF was able to induce EMT in vitro, NMuMG cells treated with CTGF (200 ng/ml) for 24 – 72 hours. Expression of E-cadherin, and α-SMA protein were assessed by Western blotting (Fig. 4.11 A) and quantified by densitometry (Fig. 4.11 B and C). Treatment with the prototypical inducer of EMT, TGF-β (4 ng/ml), was associated with a time-dependent decrease in E- cadherin levels, as well as a time-dependent accumulation of α-SMA. The change in E-cadherin expression was -0.5529 ± 0.1362 arbitrary units (p = 0.0154) at 24 hours, with expression diminishing to a change of -0.9758 ± 0.1593 arbitrary units (p = 0.0036) at 48 hours, and -0.5516 ± 0.1300 arbitrary units (p = 0.0132) at 72 hours compared to controls at each time- point. No significant change was observed with α-SMA expression until 48 hours of TGF-β treatment compared with control (0.5177 ± 0.1487 arbitrary units (p = 0.0253), which increased further at 72 hours (1.155 ± 0.3299 arbitrary units ( 0.0249).
In contrast to treatment with T2 cells, CTGF induced a significant decrease in E-cadherin expression by NMuMG cells, albeit a more modest change compared to the effects of TGF-β. At 24 hours, the difference in E-cadherin expression between control and CTGF-treated NMuMG cells was -0.7450 ± 0.1818 arbitrary units (p = 0.0149). Similarly at 48 hours, the difference was -0.8058 ± 0.2023 arbitrary units (p = 0.0163). However, no significant differences were observed in α-SMA protein levels in CTGF-treated NMuMG cells compared to control, in contrast to that observed in CTGF-treated T2 cells, which resulted in the induction of α-SMA.