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To assess the level of CTGF expression in Col1a2-CTGF fibroblasts compared to WT fibroblasts, mouse embryonic fibroblast (MEF) cells were isolated and cultured as described in Chapter 2. Phase contrast microscopy was used to examine the morphology of the cells and demonstrated that both the WT and Col1A2-CTGF MEFs assume an elongated, spindle shape morphology that is typical of fibroblastic cells (Fig. 3.5). When cells were plated at the same density, the Col1A2-CTGF cells reached confluence more rapidly than the WT cells, suggesting that fibroblast-specific CTGF overexpression may drive fibroblast proliferation (data not shown). As TGF-β is a transcriptional activator of CTGF (Leask and Abraham, 2006), it was interesting to find out whether exogenous TGF-β would have any effect on CTGF expression in Col1a2-CTGF fibroblasts. MEFs were quiesced for 24 hours in 0.5% BSA-supplemented serum-free medium prior to stimulation with TGF-β (4 ng / ml) for a further 24 hours. CTGF expression in the MEFs of WT and Col1a2-CTGF mice (n=3) was examined by lysing the cultured MEF cells to harvest the protein for Western blotting (Fig. 3.6 A). Using an anti-CTGF antibody, a single band of protein with a MW of 36 kDa detected in the Col1a2-CTGF cells was either relatively weak or absent in WT cells (p

= 0.0154). As a positive control for TGF-β treatment, a significant difference was found between the TGF-β-treated MEFs compared to those untreated (Fig. 3.6 B; p = 0.0149). Furthermore, the high basal level of CTGF in the Col1a2-CTGF MEFs was not maximal as expression could be further

Figure 3.5 Mouse embryonic fibroblast (MEF) cells isolated from Col1a2-CTGF transgenic mice show typical fibroblast morphology

By phase contrast microscopy cells appear spindle shaped and elongated (original magnification 100X). Inset shows higher magnification of image.

Mouse embryonic fibroblast (MEF) cells isolated from CTGF transgenic mice show typical fibroblast morphology

hase contrast microscopy cells appear spindle shaped and elongated (original magnification 100X). Inset shows higher magnification of image.

Mouse embryonic fibroblast (MEF) cells isolated from CTGF transgenic mice show typical fibroblast morphology

hase contrast microscopy cells appear spindle shaped and elongated (original magnification 100X). Inset shows higher magnification of image.

elevated by treatment with TGF-β (p = 0.0105). This suggested that TGF-β was able to induce expression of endogenous CTGF and perhaps also enhance the transgene expression controlled by the Col1a2 promoter, which is also responsive to TGF-β signalling.

Since CTGF is a secreted protein (Gressner and Gressner, 2008), the levels of secreted CTGF present in the conditioned media of MEFs were also assessed by Western blotting (Fig. 3.6 A). A protein of 36 kDa was detected in the medium of Col1A2-CTGF cells while there was no detectable CTGF in the conditioned medium from WT MEFs, similar to observations with the cell lysates. In contrast to the change in cellular CTGF levels, the change in secreted CTGF is more modest after TGF-β treatment in WT cells and does not match levels released by Col1a2-CTGF MEFs. A possible reason for this is that after being secreted, some of the CTGF may become anchored at the cell surface, perhaps via its C-terminal domains, rather than freely accumulating in the conditioned medium.

Although canonical TGF-β signalling has been shown to be dispensable in the induction of basal CTGF in Col1a2-CTFG MEFs (Sonnylal et al., 2010), when MEFs were incubated with the TGF-β neutralising antibody, 1D11 (p = 0.0024), or TGF-β receptor I inhibitor, SD208 (p = 0.0027) for 24 hours, both inhibitor pre-treatments attenuated expression of CTGF by the Col1a2-CTGF MEFs significantly compared to that in the absence of inhibitor treatment (Fig. 3.7). This suggested that there is some TGF-β component to CTGF expression involving TGF-β receptor I. CTGF induction may also be mediated by non-SMAD signalling such as through the pERK, p38 or JNK pathways, as reported previously (Ponticos et al., 2009; Sonnylal et al., 2010) .

Figure 3.6 CTGF expression is increased in Col1a2 embryonic fibroblasts (MEF) and enhanced by TGF

MEFs from WT and Col1A2

confluent and then maintained in serum

BSA for 24 hours prior to stimulation with TGF hours. (A) Western blotting of who

detect cell-associated and secreted CTGF protein, respectively. Whole cell lysate from cultured SSc skin fibroblasts was used as a positive control for CTGF detection. The two bands detected correspond to the 36 k

and its glycosylated form at 38 kDa. (B) Densitometric analysis of band intensities presented as relative density (arbitrary units). CTGF expression was normalised to the β

per group) ± SEM.

CTGF expression is increased in Col1a2 embryonic fibroblasts (MEF) and enhanced by TGF-β treatment

MEFs from WT and Col1A2-CTGF transgenic mice were cultured until confluent and then maintained in serum-free DMEM supplemented with 0.5%

BSA for 24 hours prior to stimulation with TGF-β (4 ng/ml) for a further 24 hours. (A) Western blotting of whole cell lysates and conditioned media to associated and secreted CTGF protein, respectively. Whole cell lysate from cultured SSc skin fibroblasts was used as a positive control for CTGF detection. The two bands detected correspond to the 36 k

and its glycosylated form at 38 kDa. (B) Densitometric analysis of band intensities presented as relative density (arbitrary units). CTGF expression was normalised to the β-tubulin loading control. Columns show mean (2 mice CTGF expression is increased in Col1a2-CTGF mouse

treatment

CTGF transgenic mice were cultured until free DMEM supplemented with 0.5%

ng/ml) for a further 24 le cell lysates and conditioned media to associated and secreted CTGF protein, respectively. Whole cell lysate from cultured SSc skin fibroblasts was used as a positive control for CTGF detection. The two bands detected correspond to the 36 kDa CTGF and its glycosylated form at 38 kDa. (B) Densitometric analysis of band intensities presented as relative density (arbitrary units). CTGF expression tubulin loading control. Columns show mean (2 mice

Figure 3.7 CTGF expression is attenuated by inhibitors TGF in Col1a2-CTGF mouse embryonic fibroblasts (MEFs)

MEFs from WT and Col1A2 confluent and then main

BSA for 24 hours pri 1D11 (10 µg/ml), or TGF

hours. (A) Western blotting of whole cell lysates. (B

of band intensities presented as relative density (arbitrary units). Optical density of bands corresponding to CTGF protein was normalised against optical density of the β

CTGF expression is attenuated by inhibitors TGF CTGF mouse embryonic fibroblasts (MEFs)

MEFs from WT and Col1A2-CTGF transgenic mice were cultured until confluent and then maintained in serum-free DMEM supplemented with 0.5%

BSA for 24 hours prior to incubation with the TGF-β-neutralising antibody, , or TGF-β receptor I inhibitor, SD208 (1 µM

hours. (A) Western blotting of whole cell lysates. (B) Densitometric analysis of band intensities presented as relative density (arbitrary units). Optical density of bands corresponding to CTGF protein was normalised against optical density of the β-tubulin loading control (n = 3, ** indicates p

CTGF expression is attenuated by inhibitors TGF-β signalling

CTGF transgenic mice were cultured until free DMEM supplemented with 0.5%

neutralising antibody, µM) for a further 24 ) Densitometric analysis of band intensities presented as relative density (arbitrary units). Optical density of bands corresponding to CTGF protein was normalised against

3, ** indicates p ≤ 0.01).

3.4.5 Attenuated lung fibrotic response to bleomycin in CTGF