The Role of HA in the Regulation of CD44 Membrane Dynamics

To determine whether cell-surface HA was necessary for CD44 co-localisation with EGFR and membrane dynamics and behaviour, Confocal Microscopy and FRAP analysis were used, following hyaluronidase treatment (Figure 3.12). Interestingly, CD44 co-localisation with EGFR was partially lost, following hyaluronidase treatment (Figure 3.12A-B). CD44 movement was restored in myofibroblasts incubated with hyaluronidase from both Streptomyces (S) and bovine testicular (BT) sources (Figure 3.12C-E). However, the MF indicated that hyaluronidase alone was not sufficient to restore mobility to the levels observed in fibroblasts. These data suggest that HAS2 production of the HA pericellular coat was partly responsible for the sequestration and anchoring of CD44 into lipid raft domains, where it co-localised with EGFR and enables the resulting differentiation signalling response.

Figure 3.1. Phenotypic Changes in Myofibroblast Differentiation. A. Cells were grown to 70% confluent

monolayers and growth arrested for 48 hours. Cells were then incubated in serum-free medium alone (fibroblasts) or in medium containing 10ng/ml TGF-β1 for 72 hours (myofibroblasts). The expression of α- SMA (green) was examined by immunocytochemistry, nuclei were visualised by Hoechst stain. Images shown are a representation of 5 independent experiments. Original magnification x400. The mRNA expression of B. α-SMA, C. EDA-FN, D. HAS2 and E. TSG-6, was analysed using QPCR. Results are shown as the mean ± s.e.m. of 3 individual experiments. **P=<0.01.

Figure 3.2. CD44 and EGFR Co-localisation in Myofibroblasts. A. Cells were grown to 70% confluent

monolayers and growth arrested for 48 hours. Cells were then incubated in serum-free medium alone (fibroblasts) or in medium containing 10ng/ml TGF-β1 for 72 hours (myofibroblasts). Confocal Laser Scanning Microscopy was used to examine the expression of EGFR (green; FITC) and CD44 (red; PE-R). Areas of co-localisation are shown in the merged images (yellow; enlarged images shown in blue boxes). Images shown are a representation of 5 independent experiments. Original magnification x630. B. Co- localisation of EGFR and CD44 was analysed by immunoprecipitation for EGFR, followed by immunoblotting for CD44. Image shown is representative of 3 individual experiments. C. Western blot analysis of total CD44 protein in fibroblasts and myofibroblasts. GAPDH was used as a loading control. Representative blot is shown. Densitometry graph shown is ± s.e.m. of 3 individual experiments. N/S = no

Figure 3.3. Both CD44 and EGFR are Required for Signal Transduction. A. Fibroblasts were transfected

with a scrambled siRNA sequence or siRNA targeting CD44, prior to treatments with or without AG1478 for 1 hour, before TGF-β1 for 72 hours. Phosphorylation of EGFR and ERK1/2 was analysed by Western blotting. Total EGFR and ERK1/2 proteins were used as loading controls. Image shown is representative of 3 independent experiments. B. Densitometric analysis of p-EGFR, normalised to total EGFR. Graph shows ± s.e.m. of 3 independent experiments. C. Densitometric analysis of p-ERK1/2, normalised to total ERK1/2. Graph shows ± s.e.m. of 3 independent experiments. D. QPCR was used to confirm CD44 mRNA knockdown by siCD44. Results shown are ± s.e.m. of 3 individual experiments. E. QPCR was used to analyse α-SMA mRNA, following siCD44 and 10μM AG1478 cellular treatments. Results shown are ± s.e.m. of 3 individual experiments. **P=<0.01.

Figure 3.4. The Membrane Dynamics of CD44 and EGFR. A. Sample time-lapse series of fluorescent

recovery, after photobleaching (FRAP) experiments. Original magnification x630. Fibroblasts or myofibroblasts were grown to 70% confluent monolayers on 22mm diameter glass coverslips, in 35mm 6- well tissue culture plates. Cells were growth arrested in serum-free medium for 48 hours. FRAP was performed at 37°C by photobleaching an approximately 10µm area of the cell membrane (indicated by white boxes on bright-field images). The recovery of fluorescence into this area (indicated by white arrows) was quantified and expressed as a fraction of the fluorescence intensity (FI) of a second region of membrane, outside of the photobleached area (FI Ratio). Complete quantified time-courses, average diffusion constants (D) and mobile fractions (MF) are shown for: B. EGFR in fibroblasts; C. CD44 in fibroblasts; D. EGFR in myofibroblasts; and E. CD44 in myofibroblasts. All results shown are representative of 5 independent experiments. Statistics shown are ± s.e.m. of 5 independent experiments.

Figure 3.5. Co-localisation is Lipid Raft Associated and Not Cytoskeletal. Cells were grown to 70% confluence, growth arrested for 48 hours and treated with 5µM

cytochalasin-B or 50µg/ml nystatin for 1 hour. Following 72 hours of incubation in serum-free media with or without 10ng/ml TGF-β1, cells were fixed and stained for A. α-SMA visualisation or B. EGFR (green) and CD44 (red) (areas of co-localisation in yellow). Original magnification x630. The expression of C. α-SMA mRNA and D. HAS2 mRNA, were assessed by QPCR. Results are shown as the mean ± s.e.m. of 3 individual experiments. E. Western blotting and densitometry analysis of total EGFR,

Figure 3.6. CD44 and EGFR Co-localisation with Lipid Raft Marker CTX-B. Fibroblasts or myofibroblasts were grown on 22mm diameter glass coverslips and

examined for A. EGFR (green; FITC) and CTX-B binding (red; TRITC); and B. CD44 (green; FITC) and CTX-B binding (red; TRITC), using Confocal Laser Scanning Microscopy (areas of co-localisation shown in yellow; areas of CTX-B binding with EGFR marked by white arrows). Images are representative of 5 individual experiments. Original magnification x630. C. Cells were treated with nystatin (50µg/ml) or MβCD (10mM) for 1 hour, before examination of EGFR or CD44 expression (green; FITC) and CTX-B binding (red; TRITC), (areas of EGFR or CD44 expression without CTX-B binding marked by white arrows). Images are representative of 3 individual experiments. Original magnification x630. D. Co-localisation of EGFR or CD44 with CTX-B was quantified (Mander’s Co-localisation Coefficient). Statistical analysis includes the average Pearson’s Correlation Coefficient (Rr) and Intensity Correlation Quotient (ICQ) for each experimental condition. Results shown are ± s.e.m. of 3 independent experiments. **P=<0.01.

Figure 3.7. Membrane Fraction Analysis in Fibroblasts and Myofibroblasts. Cells were grown to

confluence and growth arrested for 48 hours. Cells were incubated in A. serum-free medium alone (fibroblasts) or B. serum-free medium containing 10ng/ml TGF-β1 for 72 hours (myofibroblasts). Cellular membrane preparations were separated in a discontinuous OptiPrep gradient by ultracentrifugation. Fractions were analysed for the presence of EGFR and CD44. Cav-1 was used to detect fractions positive for lipid rafts, and EEA-1 for non-raft fractions. Lipid raft fractions were confirmed using CTX-HRP dot-blots for each fraction. Positive and negative control dot-blots were used to confirm the specificity of CTX-HRP. C. Quantification of the percentage of EGFR and CD44 found within lipid raft fractions 5-6 in fibroblasts and myofibroblasts. Results shown are ± s.e.m. of 3 independent experiments. D. Flow Cytometry analysis of cell surface expression of Cav-1 in fibroblasts and myofibroblasts. Unlabelled cells were used as intensity controls. Bar graph of relative intensity shown is ± s.e.m. of 3 individual experiments. N/S = no significance;

Figure 3.8. Lipid Raft Disruption Inhibits Myofibroblast Differentiation. Cells were grown to 70% confluence and growth arrested for 48 hours. Cells were incubated

with 50µg/ml nystatin for 1 hour, before incubation in serum-free medium alone or containing 10ng/ml TGF-β1 for 72 hours. A. α-SMA expression (green) was examined by immunocytochemistry. Nuclei visualised by Hoechst stain (blue). Naïve IgG was used for negative controls. Images are representative of 3 independent experiments. Original magnification: x400. B. Cells were examined for α-SMA mRNA using QPCR. Results are shown as the mean ± s.e.m. of 3 individual experiments. C. EGFR (green; FITC) and CD44 (red; PE-R) expression was examined by immunocytochemistry. Areas of co-localisation are shown in yellow. Nuclei visualised by Hoechst stain (blue). Naïve IgG was used for negative controls. Images are representative of 5 individual experiments. Original magnification: x400. D. The co-localisation of CD44 with EGFR was quantified (Mander’s Co-localisation Coefficient). Statistical analysis includes the average Pearson’s Correlation Coefficient (Rr) and Intensity Correlation Quotient (ICQ) for each experimental condition. Results shown are ± s.e.m. of 5 individual experiments. **P=<0.01.

Figure 3.9. Intracellular Signalling Through ERK1/2 and CaMKII. Confluent fibroblast monolayers were

growth arrested for 48 hours and were subsequently incubated with 10ng/ml TGF-β1, for up to 3 hours. The phosphorylation of A. ERK1/2 and B. CaMKII proteins was assessed by Western blot analysis at the indicated times. Western blot analysis for the appropriate total proteins was performed to ensure equal loading of protein samples. Following scanning densitometry, phosphorylated ERK1/2 and CaMKII expression was corrected for the expression of total ERK1/2 and CaMKII protein respectively; and is represented as ± s.e.m. of 3 separate experiments. Cells were pre-treated with 10µM MEK/ERK inhibitor PD98059(+) or left untreated(-) (C. and