Molecular Cloning and Functional Characterization of Mouse α3(IV)NC1

Abstract

Non-collagenous α3 chain of type IV collagen or α3(IV)NC1, a 28 kDa C-terminal domain of collagen type IV is a specific inhibitor of endothelial cell translation and angiogenesis. In the present study we have cloned and expressed mouse α3(IV)NC1 in baculovirus system. The recombinant protein was expressed in soluble form and tested for several of its biological functions. We identified that this recombinant mouse α3(IV)NC1 specifically inhibited proliferation, translation and tube formation of endothelial cells. Also, we show that α3(IV)NC1 treatment results in apoptosis specifically in proliferating endothelial cells. In addition we report for the first time that mouse α3(IV)NC1 inhibits migration and p38 MAPK phosphorylation in addition to inhibition of FAK/Akt/mTOR/4E-BP1 signaling. In mice α3(IV)NC1 treatment reduced tumor growth and CD-31 positive endothelial vasculature in tumors. Collectively, our data demonstrate the expression of biologically active form of mouse α3(IV)NC1 in Sf-9 cells and provide important mechanistic insights on α3(IV)NC1 antiangiogenic actions in endothelial cells.

Figure 1

Cloning, expression and purification of mouse α3(IV)NC1. Panel A. Dot-blot analysis of the plaques containing recombinant virus expressing α3(IV)NC1: Plaques (a and b) lanes 1, 2 and 3 were found to be positive. The wells c and d (1 and 2) serve as negative controls containing non-recombinant AcNPV virus infected cell extracts, and well e (1, 2 and 3) corresponds to un-infected Sf9 cell extracts serves as negative control. α3(IV)NC1 cDNA was used as positive control in lane 3 (c and d). Panel B. Time course expression of α3(IV)NC1 in Sf9 insect cells at 12, 24, 48 and 72 hr of post infection: Lane 1 shows uninfected cell extracts at 0 hr (control). Each lane contains about 30 μg of crude cell extract and the figure is a coomassie-stained gel. Western immunoblot analysis of the time course expression of α3(IV)NC1 using α3(IV)NC1 antibody are shown in lower Panel. Panel C. Purification of α3(IV)NC1 using affinity matrix: Lane 1 shows 10 μg of flow through, lanes 2 and 3 shows 1 μg of affinity purified α3(IV)NC1 before and after dialysis.

Figure 2

Functional characterization of mouse α3(IV)NC1. Panel A. The graph summarizes results from three independent protein synthesis experiments on the relative uptake of radioactive [³⁵S] methionine at 48 hrs of α3(IV)NC1 treatment. *Indicates P < 0.03 (1 μM α3(IV)NC1 treatment compared to FCS. **Indicates P < 0.01 (rapamycin treatment compared to FCS). Panel B. The graph summarizes results from three independent proliferation experiments on the relative uptake of [³H]-thymidine incorporation. *Indicates P < 0.01 (1 μM α3(IV)NC1 treatment compared to FCS. **Indicates P < 0.005 (1 μM α1(IV)NC1 treatment compared to FCS). Panel C. Cell Viability assay: The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide assay (MTT) was used to evaluate HUVECs or MLEC cells viability after treatment with α3(IV)NC1. α3(IV)NC1 decreased the cell viability in a dose-dependent manner. *Indicates P < 0.005 (with and without 1 μM α3(IV)NC1 treatment. Panel D. Control and α3(IV)NC1 treated cells were lysed, and caspase-3 activity was detected. DEVD and TNF-α were used as positive controls. *Indicates P < 0.005 (TNF-α alone or with TNF-α with DEVD treatment. In panel A-C −FCS and +FCS represents cells grown in 0.1% and 10% FCS medium.

Figure 3

Cell signaling experiments with mouse α3(IV)NC1. Panel A. Migration of HUVECs or MLEC cells with and without mouse α3(IV)NC1. Panel B. Tube formation of endothelial cells on Matrigel matrix with and without α3(IV)NC1. Migration and tube formation of endothelial were viewed using a light microscope at 100x magnification. Panel C-E. Serum-starved HUVEC or MLEC cells were plated on fibronectin coated culture plate supplemented with 1 μ α3(IV)NC1 for the indicated times and cytosolic extracts were analyzed by western blotting. Immunoblots of phosphorylated FAK or Akt or p38 (top blot) and total FAK or Akt of p38 (lower blot) were shown. Panel F. mTOR kinase Assay: Autoradiograph of the autophosphorylated mTOR (top blot) and phosphorylated 4E-BP1 (lower) isolated from (HA)-mTOR transfected HUVECs shown. (P) and FN represents phosphorylated protein and fibronectin.

Figure 4

Inhibition of tumor growth and tumor angiogenesis in 129/Sv mice. Panel A. We injected mouse α3(IV)NC1 protein to SCC-PSA1 tumor bearing mice daily for 15 days. Data are representative of three such independent experiments. The results are shown as the mean ± SEM and p < 0.001 compared to mice with and without α3(IV)NC1 injection. Panel B. Frozen sections (4-μm) from tumor tissue were stained with anti-CD31 antibody and the number of CD31 positive blood vessels were counted in 6 fields. The blood vessel quantification results were shown as the mean ± SEM. *Indicates p < 0.001; compared to mice with and without mouse α3(IV)NC1 treatment. Scale bar corresponds to 50 μM. Arrows indicated CD31 positive endothelial vasculature.