Regulation of vascular endothelial growth factor production and angiogenesis by the cytoplasmic tail of tissue factor

Abstract

Tissue factor (TF), a transmembrane receptor for coagulation factor VII/VIIa, is aberrantly expressed in human cancers. We demonstrated a significant correlation between TF and vascular endothelial growth factor (VEGF) production in 13 human malignant melanoma cell lines (r(2) = 0.869, P < 0.0001). Two of these cell lines, RPMI-7951, a high TF and VEGF producer, and WM-115, a low TF and VEGF producer, were grown s.c. in severe combined immunodeficient mice. The high-producer cell line generated solid tumors characterized by intense vascularity, whereas the low producer generated relatively avascular tumors, as determined by immunohistologic staining of tumor vascular endothelial cells with anti-von Willebrand factor antibody. To investigate the structure-function relationship of TF and VEGF, a low-producer melanoma cell line (HT144) was transfected with a TF cDNA containing the full-length sequence, a cytoplasmic deletion mutant lacking the coding sequence for the distal three serine residues (potential substrates for protein kinase C), or an extracellular domain mutant, which has markedly diminished function for activation of factor X. Cells transfected with the full-length sequence produced increased levels of both TF and VEGF. Transfectants with the full-length sequence and the extracellular domain mutant produced approximately equal levels of VEGF mRNA. However, cells transfected with the cytoplasmic deletion mutant construct produced increased levels of TF, but little or no VEGF. Thus, the cytoplasmic tail of TF plays a role in the regulation of VEGF expression in some tumor cells.

Figure 1

Coexpression of TF and VEGF in human melanoma cell lines. Levels of TF and VEGF were measured by ELISA (10) in triplicate wells of triplicate cultures. Each point represents the mean of the results of three determinations. Linear regression analysis demonstrates a significant correlation between TF and VEGF production in these 13 melanoma cell lines (r² = 0.869, P < 0.0001). The TF was fully functional, as measured in a one-stage assay for procoagulant activity (PCA) (data not shown) (16), and the correlation between TF PCA and antigen expression was highly statistically significant (r² = 0.94, P < 0.0001).

Figure 2

Human melanoma cell lines grown as xenogeneic tumors in SCID mice. RPMI-7951 melanoma cells (a high TF and VEGF producer) or WM-115 melanoma cells (a low TF and VEGF producer) were inoculated s.c. (3 × 10⁶/mouse) on the same day. (A) The gross appearance of RPMI-7951 tumors was hemorrhagic, dark purplish in color, whereas the WM-115 tumors appeared pale and relatively avascular. The immunohistologic analysis of RPMI-7951 and WM-115 tumors from these SCID mice emphasized detection of VECs. (B) The RPMI-7951 tumor and (C) the WM-115 tumor were stained with rabbit anti-human von Willebrand factor, by using the standard immunoperoxidase method. The relative frequency of micro blood vessels is noted by arrow heads (magnifications: ×200 for B and C). The average number of blood vessels per a field for three RPMI-7951 tumors and five WM-115 tumors are 17.3 ± 4.0 (1 SD) and 3.2 ± 2.2, respectively.

Figure 3

Effects of transfection of TF cDNA, containing full-length sequence or the cytoplasmic tail deletion sequence into human melanoma cell line HT144. Filled bars represent the levels of TF and empty bars represent the levels of VEGF. The values are the mean ± 1 SD of triplicate assays from three separate experiments. Wild-type (WT) HT144 cells are low TF and VEGF producers. TF transfectants containing the full-length coding sequence (TFc5d) produced increased levels of both TF and VEGF. In contrast, TF transfectants containing the cytoplasmic tail deletion sequence (TFTSIIIB1) produced very little VEGF, although TF antigen production (and procoagulant activity, data not shown) was increased. Levels of VEGF in the nontransfected HT144 (WT) and HT144 transfected with the vector alone (Vector) were too low to illustrate on the graph, as was true for the vertical lines indicating SD for both TF and VEGF production by the WT cells and the cells containing vector, and for VEGF production by the TFTSIIIB-transfected cells.

Figure 4

Effects on VEGF transcription by transfection of TF cDNA, containing full-length sequence or the extracellular domain mutant sequence (Y¹⁵⁷ → A¹⁵⁷ and K¹⁵⁹ → A¹⁵⁹) into human melanoma cell line HT144. (a) A 2% agarose gel demonstrating RT-PCR products of VEGF-165 mRNA produced by transfectants with vector alone, TF sense, or TF^mut (157/159) plasmids. (b) Relative levels of VEGF mRNA production by transfectants with vector alone, TF sense, or TF^mut (157/159) plasmids. Relative levels of VEGF mRNA from each transfectant colony were determined by densitometric quantitation of RT-PCR products separated on 2% agarose gel as shown in a. Relative VEGF units were determined by dividing the intensity of densitometric values for VEGF by the intensity of densitometric values for GAPDH. Values of each bar graph represent mean ± 1 SD of at least seven separate transfectant colonies.