Coordinate expression and functional profiling identify an extracellular proteolytic signaling pathway

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.cancer ͉ macrophage activation ͉ protease substrate specificity ͉ proteomics D espite the successful physiological and biochemical characterization of many proteases, the vast majority of the Ͼ2% of the human genome that encodes proteases has yet to be functionally classified. Although many approaches demonstrate the sufficiency of a protease to cleave a given substrate, very few are able to address the physiological relevance of such in vitro findings. Cell-surface proteolysis is suggested to play a major role in cancer progression and metastasis through the processing of macromolecules important for regulating the extracellular environment. The cell-surface localization, high activity, and exquisite specificity of type II transmembrane serine proteases (TTSPs) suggest a role in outside-in signaling and interaction with the microenvironment. We elected to apply a multifaceted approach to identify physiologically relevant substrates of one prominent member of this family, membrane type serine protease 1 (MT-SP1/matriptase).Members of the TTSP family, such as hepsin and MT-SP1, are highly expressed in many cancers, including those of the prostate, breast, colon, and ovary (1-9). Both overexpression and inhibition studies have supported the role of MT-SP1 in tumorigenesis and tumor growth. Targeted overexpression of MT-SP1 in squamous epithelia in mice results in skin-limited nodules of squamous cell carcinoma that become metastatic in the presence of the chemical carcinogen DMBA (10). ...

Proteolysis-induced N-terminal Ectodomain Shedding of the Integral Membrane Glycoprotein CUB Domain-containing Protein 1 (CDCP1) Is Accompanied by Tyrosine Phosphorylation of Its C-terminal Domain and Recruitment of Src and PKCδ

“…The existence of interactions between CDCP1 and matriptase is also supported by a recent report showing that these proteins co-immunoprecipitate from SW480 and SW620 colon cancer cell lysates using an antibody against the tetraspanin CD9 (35). In addition, it has been reported that the mRNA expression patterns of this protease:substrate pair are significantly correlated in many tissue types (38). It has also been shown in separate reports that both matriptase (39) and CDCP1 (12) are expressed during prostate cancer progression.…”

Membrane-Bound Serine Protease Inhibitor HAI-1 Is Required for Maintenance of Intestinal Epithelial Integrity

“…10,36 Indeed, Hgfac knockout mice also showed enhanced susceptibility to DSS-induced colitis, possibly due to insufficient activation of HGF after acute mucosal injury. 37 Interestingly, a recent gene-centric mapping analysis suggests that the MST1 gene encoding MSP, which is another cytokine activated by HGFAC and matriptase, 38,39 may be involved in IBD pathogenesis. 40 Therefore, altered pericellular activation of MSP may also be involved in the susceptibility of HAI-1/SPINT1-deficient mice to DSS colitis.…”

Epithelial Integrity Is Maintained by a Matriptase-Dependent Proteolytic Pathway

“…[3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] The surface of metazoan organs is lined by specialized epithelia that are endowed with multiple general and organ-specific functions. These include the maintenance of ion gradients, macromolecular transport, absorption, secretion of lipids, mucins, hormones and growth factors, and exclusion of pathogens.…”