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.

Fig. 1.

Correlational cluster diagram of MT-SP1 and associated proteins. Transcriptional profiling of 19 genes was performed for nearly 2,000 samples from cell lines, normal tissues, and cancer tissues. Pearson's and Spearman's correlation coefficients were calculated for each gene paired with MT-SP1. Associated P values were also calculated and corrected by using the Bonferroni correction method (highly specific) and the false discovery rate (FDR) method (highly sensitive). Significantly correlated gene pairs (adjusted P < 0.05) are indicated by shading. Tissue categories without any significant correlations are not displayed. All correlations determined to be significant by using the Bonferroni method (darker shading) are significant by using the FDR method (lighter shading). Transcript levels of HAI-1, the proposed endogenous inhibitor of MT-SP1, are significantly correlated with transcript levels of MT-SP1 in the largest proportion of tissues.

Fig. 2.

MT-SP1 and RON, but not HAI-1, are expressed in peritoneal macrophages, and MT-SP1 cleaves MSP-1 at the activation site. (A) TaqMan was performed to determine MT-SP1, RON, and HAI-1 expression in primary mouse bone marrow-derived macrophages and peritoneal macrophages. MT-SP1 and RON are expressed at levels at least 10-fold greater in peritoneal macrophages than in bone marrow-derived macrophages. HAI-1 is not expressed in either cell type. (B) One hundred nanomolar MT-SP1 was incubated with 200 ng of pro-MSP-1 for 30 min at 37°C and then separated by SDS/PAGE. Approximately 50% of pro-MSP-1 is processed into the active α-β heterodimer in this time, as judged by silver stain. Decrease of the intensity of the pro-MSP-1 band is accompanied by a concomitant increase in the intensity of the band representing the β chain of MSP-1. (C) MT-SP1 activates pro-MSP-1 in a dose-dependent manner as is shown by disappearance of the full-length pro-MSP-1 by immunoblot (from left to right: 1 nM, 10 nM, and 100 nM).

Fig. 3.

MT-SP1-activated MSP-1 induces macrophage activation and morphology change. Primary mouse peritoneal macrophages were serum-starved and incubated in serum-free medium (SFM) in the absence or presence of 50 ng/ml MSP-1, 50 ng/ml pro-MSP-1, 40 nM HAI-1, and 400 nM anti-MT-SP1 antibody as indicated for 4 h. Upon treatment with MSP-1 or pro-MSP-1, the macrophages undergo a characteristic morphological change demonstrated by the development of an elongated shape and spiny protrusions. HAI-1 or anti-MT-SP1 antibody inhibition of endogenous MT-SP1 proteolytic activity abrogates the ability of pro-MSP-1 to induce this morphological change.

Fig. 4.

MT-SP1 activated MSP-1 inhibits LPS-induced nitric oxide production in macrophages. Mouse bone marrow macrophages were isolated and cultured in 24-well plates. Nitric oxide production was measured by the Griess reaction. The macrophages exhibit a robust nitric oxide production response to the positive control of 1 μg/ml LPS. (A) From left to right, the histogram represents cell cultured with (i) medium alone, (ii) 10 ng/ml MSP-1, (iii) 10 ng/ml pro-MSP-1, (iv) 10 ng/ml pro-MSP-1 plus 10 nM MT-SP1, (v) 1 μg/ml LPS, (vi) 1 μg/ml LPS plus 10 ng/ml MSP-1, (vii) 1 μg/ml LPS plus 10 ng/ml pro-MSP-1, and (viii) 10 ng/ml pro-MSP-1 plus 10 nM MT-SP1 plus 1 μg/ml LPS. The experiment was repeated twice in triplicate, and the data from these experiments were averaged and are reported ±SE. (B) The data are also presented as percent inhibition of NO production as a function of sample conditions.