After incubation with Streptavidin/HRP (Invitrogen) for 45?min, nuclei were stained with DAPI (Dako)

After incubation with Streptavidin/HRP (Invitrogen) for 45?min, nuclei were stained with DAPI (Dako). of assays. Small HA oligosaccharides (sHA) efficiently inhibited these effects. Both siRNA-mediated reduction of CD44 expression and antibodies that block the conversation of CD44 with HA provided evidence that CXCL12-induced CXCR4 signaling depends on the binding of hHA to CD44. Consistently, CD44 and CXCR4 were found to actually interact in the presence of CXCL12, an interaction that could be inhibited by sHA. These findings provide novel insights into how microenvironmental components interact with cell surface receptors in multi-component complexes to regulate key aspects of tumor growth and progression. demonstrate a dependence of CXCL12/CXCR4 signaling on HA and CD44 in angiogenesis. Together these data reveal a novel mechanism through which HA in the microenvironment can influence tumor cell behavior and regulate angiogenesis. Results Opposing effects of hHA and sHA on CXCR4-induced signaling and cell motility Possible effects of HA on CXCL12-induced signaling were examined using the CXCR4-positive hepatoma cell collection HepG2iso31 and main human ECs (HUVECs). Both cell types express CXCR4 (Physique 1a) and the major HA receptor CD44. Hermes 3, an antibody that recognizes all CD44 isoforms, detected several bands in HepG2iso cells and HUVECs. The major band corresponds to CD44s, the smallest ubiquitously expressed CD44 isoform (examined in Orian-Rousseau32). The upper bands correspond to other CD44 isoforms expressed at lower levels. HepG2 cells that do not express any CD44 isoform served as a negative control (Physique 1a and Olaku monolayer wound closure assays. A wound of defined size was generated in confluent monolayers of HepG2iso cells. Migration of cells to fill the Paritaprevir (ABT-450) wound upon induction with CXCL12 alone strongly and significantly increased wound closure after 24?h (Physique 1d). This effect was potently augmented by preincubation with hHA in a statistically significant manner. Preincubation with hHA or sHA alone experienced no effect on wound closure compared with non-treated control cells. Consistent with the inhibitory effect of sHA on CXCL12-induced Erk phosphorylation, preincubation with sHA completely inhibited the augmented wound closure in response to CXCL12 (Physique 1d). Similar experiments were performed with Paritaprevir (ABT-450) HUVECs. Spontaneous wound closure was faster than that observed with HepG2iso cells. Nevertheless, a statistically significant increased wound closure in the presence of CXCL12 was observed after 24?h, which was further and significantly augmented by preincubation with hHA. In addition, sHA inhibited CXCL12-induced migration (Physique 1d). Proliferation assays revealed that CXCL12 did not induce proliferation of either HepG2iso cells or HUVECs to any significant extent during the time period of the wound closure assay (data not shown), ruling out that CXCL12 and hHA might induce monolayer wound closure by promoting cell proliferation. Opposing effects of hHA and sHA on CXCR4 activity in angiogenesis assays chemotaxis chamber made up of CXCL12 (200?ng/ml) or PBS as a control. Cell were transfected either with 5?nM CD44-specific siRNA (CD44 siRNA) or control siRNA (ctrl siRNA) as indicated. Lower panel: statistical analysis of the HepG2iso cell trajectories in three impartial experiments (meanS.D.): common Y, mean net distance (RU) traveled along the chemokine gradient (axis). Y/IXI ?1, percentages of HepG2iso cells traveling a longer distance in the direction of chemokine gradients (axis) than in the direction orthogonal to the gradients (axis) (*(TGFin HepG2iso cells in the presence or absence of the BU52 antibody (was examined using a mouse angiogenesis assay in which the ingrowth of vessels into a subcutaneous matrigel plug is evaluated. CXCL12 either alone or in combination with sHA or hHA was mixed with matrigel and then injected into the flanks of C57/BL6 mice (Physique 6a). Each animal was injected with a control matrigel plug on one flank and a plug made up of CXCL12 alone or in combination with hHA or sHA on the Rabbit polyclonal to JAKMIP1 other side. Vascularization of the matrigel plugs after 3 weeks in the mice was evaluated by quantifying blood vessels in sections of the plugs using CD31 immunostaining (Physique 6a). As expected, CXCL12 was able to significantly enhance vascularization of the matrigel plugs, an effect that was further significantly augmented in matrigel plugs made up of a mixture of hHA and CXCL12. In contrast, only few blood vessels were detected in the presence of sHA, comparable to the Paritaprevir (ABT-450) number observed in the absence of growth factor addition. The same experiment was performed in the presence of the KM81 antibody that prevents the binding of Paritaprevir (ABT-450) HA to CD44 (Physique 6b). This antibody dramatically and significantly blocked the formation of blood vessels.