FIG. 3.

MT1-MMP upregulation by HA and thrombin is PI3K dependent. Phosphorylation of AKT (A) and p44/42 mitogen-activated protein kinase (B). U937 cells were serum-starved overnight and then stimulated with 50 μg/mL HA or 10 U/mL thrombin (Th) for 5 min. A representative Western blot from 2 independent experiments is shown. (C) MT1-MMP expression is PI3K dependent. Top panels: Western blot analysis of MT1-MMP expression in CB MNC, mPB, and CB CD34⁺ cells pretreated with 30 μM LY290042 (LY) or 50 μM PD98059 (PD) for 1 h and then stimulated with 50 μg/mL HA (left panel) or 10 U/mL thrombin (right panel) for 48 h. Equal amounts of protein (30 μg) were loaded as determined by the Bradford assay. The gel was stained with Coomassie blue to ensure equivalence of loading. Top panels: representative Western blot of 2 separate experiments is shown. Bottom panels: mean ± standard deviations from the densitometric analysis showing fold-increase relative to control. (D) ProMMP-2 activation is PI3K dependent. Zymographic analysis of the conditioned medium from co-cultures of HUVEC and CB CD34⁺ cells pretreated with 30 μM LY290042 (LY) or 50 μM PD98059 (PD) for 1 h and then stimulated with 50 μg/mL HA (left panel) or 10 U/mL thrombin (right panel) for 48 h. A representative zymogram from 2 independent experiments is shown. (E) CB CD34⁺ cells were pretreated with 30 μM LY290042 (LY) or 50 μM PD98059 (PD) for 1 h and then stimulated with 50 μg/mL HA for 2 h, plated on fibronectin-coated cover slips and immunostained with MT1-MMP and F-actin. (F) Trans-Matrigel chemoinvasion of CB CD34⁺ cells is PI3K dependent. CB CD34⁺ cells were pretreated with 30 μM LY290042 (LY) or 50 μM PD98059 (PD) and stimulated with 50 μg/mL HA or 10 U/mL thrombin for 2 h before trans-Matrigel chemoinvasion toward a low SDF-1 (20 ng/mL) gradient was performed. Data are pooled from triplicate chambers from 3 independent experiments, *P < 0.05. PI3K, phosphatidylinositol 3-kinase; MNC, mononuclear cells; mPB, mobilized peripheral blood.