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First published online 18 October 2005
doi: 10.1242/jcs.02629

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Chemotaxis towards hyaluronan is dependent on CD44 expression and modulated by cell type variation in CD44-hyaluronan binding

George Tzircotis, Rick F. Thorne^* and Clare M. Isacke

Breakthrough Breast Cancer Research Centre, Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK

View larger version (23K): [in a new window]   Fig. 1. Directional migration of cells in a hyaluronan gradient. The directional migration of MDA-MB-468 and MDA-MB-231 cells was assayed in a Dunn chemotaxis chamber. The gradients employed were 0.25% to 10% FBS, 0 to 400 µg/ml umbilical cord hyaluronan, 0 to 400 µg/ml streptococcal hyaluronan or no gradient (0.25% FBS in both chambers). The data are presented as a circular histogram in which each 18° segment represents the total number of cells with an average angle of migration falling within that particular interval. The Rayleigh test for unimodal clustering is used to verify directional migration. If cells show statistically significant directional migration, the mean migration angle is displayed as a red arrow and the 95% confidence interval as a green sector. The number of cells analysed for each treatment is given.

View larger version (32K): [in a new window]   Fig. 2. MDA-MB-468 and MDA-MB-231 cells express CD44 and exhibit constitutive hyaluronan binding. (A) Cells cultured overnight were detached and stained for flow cytometry. For CD44 expression, cells were incubated either with anti-CD44 monoclonal antibody E1/2 followed by Alexa555 anti-mouse Ig (red graph) or with secondary antibody alone (black graph). For hyaluronan binding, cells were either incubated with FITC-conjugated hyaluronan (FITC-HA, green graph) or medium alone (black graph). (B) MDA-MB-468 cells cultured overnight on glass coverslips were incubated with FITC-hyaluronan for 1 hour at 37°C (green) and then fixed, permeabilised and stained with the anti-CD44 monoclonal antibody E1/2 followed by Alexa555 anti-mouse Ig (red). Nuclei were visualised with TO-PRO-3 (blue). Bar, 50 µm.

View larger version (34K): [in a new window]   Fig. 3. CD44 expression is required for sensing a hyaluronan gradient. (A,B) MDA-MB-468 cells were left untreated, mock transfected, transfected with control siRNA oligonucleotides or transfected with CD44 siRNA oligonucleotides. 72 hours later cells were detached from dishes and CD44 expression and hyaluronan binding assessed by flow cytometry or fluorescence microscopy as described in Fig. 2. In A, black lines represent binding of second antibody alone (CD44) or medium alone (FITC-HA) to untreated cells. (C) MDA-MB-468 cells transfected with CD44 or control siRNA oligonucleotides were assayed for their ability to migrate directionally in a gradient of FBS or umbilical cord hyaluronan as described in Fig. 1. Bar, 50 µm.

View larger version (65K): [in a new window]   Fig. 4. Effect of hyaluronan binding on the partitioning of CD44 into detergent-insoluble lipid rafts. MDA-MB-468 cells were incubated without or with 100 µg/ml umbilical cord hyaluronan before preparation of detergent-insoluble lipid rafts (RAFT) and soluble fractions (SOL) as described in the Materials and Methods. Protein equivalents (5 µg) were separated by electrophoresis and western blotted for CD44 (monoclonal antibody E1/2), DAF and -tubulin. Positions of molecular size markers are indicated in kDa.

View larger version (43K): [in a new window]   Fig. 5. Generation of a murine fibroblast line overexpressing human CD44s. MDA-MB-468 cells, MDA-MB-231 cells and NIH-3T3 cells transfected with pSR vector alone and NIH-3T3 cells expressing human CD44s were lysed and subjected to western blotting with anti-human CD44 monoclonal antibody E1/2, monoclonal antibody IM7, which recognises both human and murine CD44, or anti-murine CD44 monoclonal antibody KM201. Positions of molecular size markers are indicated in kDa.

View larger version (27K): [in a new window]   Fig. 6. NIH-3T3 fibroblasts do not migrate towards hyaluronan. (A) NIH-3T3 fibroblasts transfected with vector alone or human CD44s were assayed for their ability to migrate towards DCS (0.25-10.00% gradient) or umbilical cord hyaluronan (0-400 µg/ml gradient) as described in Fig. 1. (B) Cells were subjected to flow cytometry to monitor expression of murine CD44 using monoclonal antibody KM201 (blue), expression of human CD44 using monoclonal antibody E1/2 (red) and FITC-HA binding (green) as described in Fig. 2.

View larger version (30K): [in a new window]   Fig. 7. Hyaluronan binding of human CD44s expressing NIH-3T3 cells after hyaluronidase treatment. (A) NIH-3T3 cells transfected with vector alone or human CD44s were detached from culture dishes, resuspended in DMEM plus 10% DCS and treated with S. hyalurolyticus hyaluronidase. After washing twice in PBS, cells were incubated either with FITC-hyaluronan (FITC-HA, green) or CD44-Fc (red). As controls, cells were also incubated with Endo180-Fc (grey) or medium alone (black). Fc chimaeras were detected with PE-anti-human Fc. (B) NIH-3T3 cells transfected with vector alone or human CD44s were cultured on coverslips overnight and then treated with type IV-S hyaluronidase. After washing, cells were stained with FITC-hyaluronan (green) and anti-human CD44 monoclonal antibody E1/2 (red) as described in Fig. 2. Nuclei were visualised with TO-PRO-3 (blue). To illustrate the variation in FITC-hyaluronan binding following hyaluronidase treatment, two separate fields of view are shown (lower two rows). (C) MDA-MB-468 and MDA-MB-231 cells were analysed by flow cytometry for FITC-HA binding following treatment with either medium alone (green) or treatment with S. hyalurolyticus hyaluronidase (dotted green). Black lines represent binding of secondary antibody alone. Bar, 100 µm.

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