Post-ischemic protection of hepatocyte growth factor requires the type II transmembrane serine protease matriptase-A reciprocal regulation of the two for neuroprotection in stroke brain.

In a rat middle cerebral artery occlusion (MACo) model of ischemic stroke, intracerebroventricular administration of human recombinant hepatocyte growth factor (HGF) mitigated motor impairment and cortical infarction. Recombinant HGF reduced MCAo-induced TNFα and IL1ß expression, and alleviated perilesional reactivation of microglia and astrocyte. All of the aforementioned beneficial effects of HGF were antagonized by an inhibitor to the type II transmembrane serine protease matriptase (MTP). MCAo upregulated MTP mRNA and protein in the lesioned cortex. MTP protein, not the mRNA, was increased further by recombinant HGF but reduced when MTP inhibitor (MTPi) was added to the treatment. Changes of the endogenous active HGF by MCAo, HGF or MTPi paralleled with the changes of MTP protein under the same conditions whilst neither HGF mRNA nor the total endogenous HGF protein were altered. These data showed that the therapeutic effects of HGF in stroke brain is attributed to its proteolytic activation and that MTP is a main protease of the event. MCAo enhanced MTP mRNA and thus protein expression; the initial use of the recombinant active HGF stabilized MCAo-induced MTP protein and subsequent activation of endogenous latent HGF which in turn stabilized further MTP protein. A reciprocal regulation between MTP and HGF appears to be present where MTP promotes HGF activation and the active HGF prevents MTP protein turnover. This study, for the first time, shows that MTP can participate in neural protection in stroke brain through activation of HGF. The cycles of HGF-MTP regulation achieved preservation of the neurological activity.

Mitochondrial localization of St14-encoding transmembrane serine protease is involved in neural stem/progenitor cell bioenergetics through binding to F0F1-ATP synthase complex.

Knockdown of the suppression of tumorigenicity 14-encoding type II transmembrane serine protease matriptase (MTP) in neural stem/progenitor (NS/P) cells impairs cell mobility, response to chemo-attractants, and neurovascular niche interaction. In the present study, we showed by Western blot that a portion of MTP can be detected in the mitochondrial fraction of mouse NS/P cells by immunostaining that it is co-stained with the mitochondrial dye MitoTracker (Thermo Fisher Scientific, Waltham, MA, USA) inside the cells. Co-immunoprecipitation showed that MTP is bound to the ß subunit of mitochondrial F0F1-ATP synthase complex (ATP-ß). Cyto-immunofluorescence staining and an in situ proximity ligation assay further confirmed a physical interaction between MTP and ATP-ß. This interaction relied on the presence of both Cls/Clr urchin embryonic growth factor, bone morphogenic protein 1 and low-density lipoprotein receptor motifs of MTP. We found that NS/P cell mitochondrial membrane potential is impaired by MTP knockdown, and ATP synthesis and oxygen consumption rate are significantly reduced in MTP-knockdown NS/P cells. Among the oxidative phosphorylation functions, the greatest effect of MTP knockdown is the reduction by over 50% in the mitochondrial energy reserve capacity. This made MTP-knockdown NS/P cells unable to overcome hydrogen peroxide stress, which leads to cessation of cell growth. This work identifies 2 previously unknown functions for MTP: first as a binding protein in the mitochondrial F1F0-ATP synthase complex and second as a regulatory mechanism of mitochondrial bioenergetics. Mitochondrial MTP may serve a protective function for NS/P cells in response to stress.-Fang, J.-D., Tung, H.-H., Lee, S.-L. Mitochondrial localization of St14-encoding transmembrane serine protease is involved in neural stem/progenitor cell bioenergetics through binding to F0F1-ATP synthase complex.

Matriptase is required for the active form of hepatocyte growth factor induced Met, focal adhesion kinase and protein kinase B activation on neural stem/progenitor cell motility.

Hepatocyte growth factor (HGF) is a chemoattractant and inducer for neural stem/progenitor (NS/P) cell migration. Although the type II transmembrane serine protease, matriptase (MTP) is an activator of the latent HGF, MTP is indispensable on NS/P cell motility induced by the active form of HGF. This suggests that MTP's action on NS/P cell motility involves mechanisms other than proteolytic activation of HGF. In the present study, we investigate the role of MTP in HGF-stimulated signaling events. Using specific inhibitors of phosphatidylinositol-3-kinase (PI3K), protein kinase B (Akt) or focal adhesion kinase (FAK), we demonstrated that in NS/P cells HGF-activated c-Met induces PI3k-Akt signaling which then leads to FAK activation. This signaling pathway ultimately induces MMP2 expression and NS/P cell motility. Knocking down of MTP in NS/P cells with specific siRNA impaired HGF-stimulation of c-Met, Akt and FAK activation, blocked HGF-induced production of MMP2 and inhibited HGF-stimulated NS/P cell motility. MTP-knockdown NS/P cells cultured in the presence of recombinant protein of MTP protease domain or transfected with the full-length wild-type but not the protease-defected MTP restored HGF-responsive events in NS/P cells. In addition to functioning as HGF activator, our data revealed novel function of MTP on HGF-stimulated c-Met signaling activation.

Endogenous expression of matriptase in neural progenitor cells promotes cell migration and neuron differentiation.

Recent studies show that type II transmembrane serine proteases play important roles in diverse cellular activities and pathological processes. Their expression and functions in the central nervous system, however, are largely unexplored. In this study, we show that the expression of one such member, matriptase (MTP), was cell type-restricted and primarily expressed in neural progenitor (NP) cells and neurons. Blocking MTP expression or MTP activity prevented NP cell traverse of reconstituted basement membrane, whereas overexpression of MTP promoted it. The NP cell mobilization induced by either vascular endothelial growth factor or hepatocyte growth factor was also impaired by knocking down MTP expression. MTP acts upstream of matrix metalloproteinase 2 in promoting NP cell mobility. In embryonic stem cell differentiation to neural cells, MTP knockdown had no effect on entry of embryonic stem cells into the neural lineage. High MTP expression or activity, however, shifts the population dynamics from NP cells toward neurons to favor neuronal differentiation. This is the first report to demonstrate the direct involvement of type II transmembrane serine protease in NP cell function.