MT1-MMP Controls Tumor-induced Angiogenesis through the Release of Semaphorin 4D

John R Basile,Kenn Holmbeck,Thomas H Bugge,J Silvio Gutkind

doi:10.1074/jbc.m609570200

Abstract

The semaphorins are a family of proteins originally identified as regulators of axon growth that recently have been implicated in blood vessel development. The plexins are high affinity receptors for the semaphorins and are responsible for initiation of signaling upon ligation. Emerging evidence indicates that many human cancers overexpress Semaphorin 4D, which promotes neovascularization upon stimulating its receptor, Plexin-B1, on endothelial cells. However, to exert its pro-angiogenic functions, Semaphorin 4D must be processed and released from its membrane bound form to act in a paracrine manner on endothelial cells. Here we show that Semaphorin 4D is a novel target for the membrane-tethered collagenase membrane type 1-matrix metalloproteinase. We demonstrate that this metalloproteinase, which is not expressed in normal or immortal but non-tumorigenic epithelial cell lines, was present in several head and neck squamous cell carcinoma cell lines and was required for processing and release of Semaphorin 4D into its soluble form from these cells, thereby inducing endothelial cell chemotaxis in vitro and blood vessel growth in vivo. These results suggest that the proteolytic cleavage of Semaphorin 4D may provide a novel molecular mechanism by which membrane type 1-matrix metalloproteinase controls tumor-induced angiogenesis.

Highlights

The semaphorins represent a large family of phylogenetically conserved molecules, both membrane-bound and secreted, originally identified by their ability to provide attractive and repulsive axon guidance cues during axon growth [1]
We show that Semaphorin 4D is a novel target for the membrane-tethered collagenase membrane type 1-matrix metalloproteinase. We demonstrate that this metalloproteinase, which is not expressed in normal or immortal but nontumorigenic epithelial cell lines, was present in several head and neck squamous cell carcinoma cell lines and was required for processing and release of Semaphorin 4D into its soluble form from these cells, thereby inducing endothelial cell chemotaxis in vitro and blood vessel growth in vivo
While both wild-type and knock-out mouse embryonic fibroblasts (MEFs) controls we identified an short hairpin RNA (shRNA) construct that caused a marked reducexhibited some chemotactic effects upon endothelial cells tion in MT1-matrix metalloproteinase (MMP) protein levels and MMP-2 (Fig. 3C, MT1-MMP: wt and KO, respectively), wells con- activation (Fig. 4C, lower panel, lane 4), which, when expressed taining wild-type MEFs transfected with Semaphorin 4D (Sema4D) (Fig. 3C, wt in HN12 cells, markedly reduced Sema4D release into the conand S4D) induced a robust endothelial cell migration, while ditioned medium (Fig. 4C, upper panel, lane 4)

Summary

The abbreviations used are

Sema4D, semaphorin 4D; HNSCC, head and neck squamous cell carcinoma(s); MMP, matrix metalloproteinase; MT1MMP, membrane type 1-MMP; MEF, mouse embryonic fibroblast; TIMP, tissue inhibitor of metalloproteinase; shRNA, short hairpin RNA. MT1-MMP was required for processing and release of Sema4D into its soluble form from these cells, thereby inducing endothelial cell chemotaxis in vitro and tumor-induced angiogenesis in vivo. These results suggest that the proteolytic cleavage of Sema4D may provide a novel molecular mechanism by which MT1-MMP controls tumor-induced angiogenesis

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION

Methods