Coordinate expression and functional profiling identify an extracellular proteolytic signaling pathway

*Department of Pharmaceutical Chemistry, University of California, 600 16th Street, San Francisco, CA 94158;
^†The G. W. Hooper Foundation, University of California, 513 Parnassus Avenue, San Francisco, CA 94153; and
^§PDL Biopharma, Inc., 34801 Campus Drive, Fremont, CA 94555

Edited by James A. Wells, University of California, San Francisco, CA, and approved February 6, 2007 (received for review July 30, 2006)

Abstract

A multidisciplinary method combining transcriptional data, specificity profiling, and biological characterization of an enzyme may be used to predict novel substrates. By integrating protease substrate profiling with microarray gene coexpression data from nearly 2,000 human normal and cancerous tissue samples, three fundamental components of a protease-activated signaling pathway were identified. We find that MT-SP1 mediates extracellular signaling by regulating the local activation of the prometastatic growth factor MSP-1. We demonstrate MT-SP1 expression in peritoneal macrophages, and biochemical methods confirm the ability of MT-SP1 to cleave and activate pro-MSP-1 in vitro and in a cellular context. MT-SP1 induced the ability of MSP-1 to inhibit nitric oxide production in bone marrow macrophages. Addition of HAI-1 or an MT-SP1-specific antibody inhibitor blocked the proteolytic activation of MSP-1 at the cell surface of peritoneal macrophages. Taken together, our work indicates that MT-SP1 is sufficient for MSP-1 activation and that MT-SP1, MSP-1, and the previously shown MSP-1 tyrosine kinase receptor RON are required for peritoneal macrophage activation. This work shows that this triad of growth factor, growth factor activator protease, and growth factor receptor is a protease-activated signaling pathway. Individually, MT-SP1 and RON overexpression have been implicated in cancer progression and metastasis. Transcriptional coexpression of these genes suggests that this signaling pathway may be involved in several human cancers.

Footnotes

^¶To whom correspondence should be addressed at: University of California, Genentech Hall, 600 16th Street, San Francisco, CA 94158-2517. E-mail: craik{at}cgl.ucsf.edu
Author contributions: A.S.B. and C.S.C. designed research; A.S.B. and A.W. performed research; A.S.B., A.W., C.J.F., M.V., and K.W. contributed new reagents/analytic tools; A.S.B., A.W., and C.S.C. analyzed data; and A.S.B. and C.S.C. wrote the paper.
↵ ^‡Present address: Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0606514104/DC1.
Abbreviations:

MT-SP1,

membrane type serine protease 1;

MSP-1,

macrophage stimulating protein 1;

HAI-1,

hepatocyte growth factor activator inhibitor 1;

TTSP,

type two transmembrane serine protease;

PS-SCL,

positional scanning synthetic combinatorial library.
Freely available online through the PNAS open access option.