Brain, Vol. 125, No. 6, 1283-1296,
June 2002
© 2002 Guarantors of Brain
Activity of a newly identified serine protease in CNS demyelination
Departments of 1 Neurology and 2 Immunology, Mayo Medical and Graduate Schools, Rochester, Minnesota, 3 Institute of Molecular Biophysics and Department of Chemistry, Florida State University, Tallahassee, Florida and 4 Department of Neurology, University of California at San Francisco, San Francisco, California, USA
Correspondence to: Isobel A. Scarisbrick, Department of Neurology, 428 Guggenheim Building, Mayo Clinic Rochester, 200 First Street, SW, Rochester, MN 55905, USA E-mail: scarisbrick.isobel{at}mayo.edu
Received December 12, 2001. Revised January 12, 2002. Accepted January 24, 2002.
 |
Summary |
|---|
 Top Summary IntroductionMethodsResultsDiscussionReferences |
|---|
We have identified a novel serine protease, myelencephalon-specific protease (MSP), which is preferentially expressed in the adult CNS, and therein, is abundant in both neurones and oligodendroglia. To determine the potential activity of MSP in CNS demyelination, we examined its expression in multiple sclerosis lesions and in two animal models of multiple sclerosis: Theiler’s murine encephalomyelitis virus (TMEV) and myelin oligodendrocyte glycoprotein (MOG)-induced experimental allergic encephalomyelitis (EAE) in marmosets. High levels of MSP were present within infiltrating mononuclear cells, including macrophages and T cells, which characteristically fill sites of demyelination, both in multiple sclerosis lesions and in animal models of this disease. The functional consequence of excess MSP on oligodendroglia was determined in vitro by evaluating the effects of recombinant MSP (r-MSP) on oligodendrocyte survival and process number. Application of excess r-MSP resulted in a dramatic loss of processes from differentiated oligodendrocytes, and a parallel decrease in process outgrowth from immature cells. Transfection of oligodendrocyte progenitors with an MSP–green fluorescent protein construct produced similar changes in oligodendrocyte process number. Importantly, r-MSP did not affect oligodendrocyte survival or differentiation towards the sulphatide-positive lineage. We further demonstrate that myelin basic protein, and to a lesser extent myelin oligodendrocyte glycoprotein, can serve as MSP substrates. These studies support the hypothesis that excess MSP, as is present in inflammatory CNS lesions, promotes demyelination.
Keywords: demyelination; glia; inflammation; multiple sclerosis; proteolytic enzyme
Abbreviations: DIG = digoxigenin; EK = enterokinase; GFAP = glial fibrillary acidic protein; GFP = green fluorescent protein; MMP = matrix metalloproteinases; MSP = myelencephalon-specific protease; MSP-IR = MSP immunoreactivity; PLP = proteolipid protein; r-MSP = recombinant MSP; SNK = Student–Newman–Keuls; TMEV = Theiler’s murine encephalomyelitis virus
|
Introduction |
|---|
|
TopSummaryIntroductionMethodsResultsDiscussionReferences |
|---|
Serine proteases are best characterized for their roles in the blood coagulation and fibrinolytic systems, but there is considerable evidence concerning the central functions of proteolytic cascades mediated by these enzymes in the nervous system (Gingrich and Traynelis, 2000
). Established or proposed roles of serine proteases and their endogenous serpin inhibitors include: cell migration; neurite outgrowth and pathfinding; synaptic remodelling; cell excitability; and both glial and neuronal cell survival (Moonen et al., 1982; Monard, 1988; Seeds et al., 1990; Liu et al., 1994a, b; Houenou et al., 1995; Tsirka et al., 1995, 1997; Davies et al., 2001). These events are mediated, in part, by the ability of serine proteases to cleave, thereby activating growth factor precursor proteins, to degrade components of the extracellular matrix, and to bind to cell surface receptors, activating intracellular signalling cascades. The enzymatic activity of serine proteases is tightly regulated, afforded in part by a series of specific endogenous serpin inhibitors. Imbalances between proteases and their inhibitors, due to injury or disease, have been shown to result in CNS pathogenesis, including neuronal degeneration (Tsirka et al., 1995, 1997).
Myelencephalon-specific protease (MSP), is a serine protease that has been cloned in our laboratory, which is preferentially expressed in the CNS and predicted to have trypsin-like activity and a broad range of substrate specificity (Scarisbrick et al., 1997). The human homologue of MSP has also been identified and has been termed protease M (Anisowicz et al., 1996), neurosin (Yamashiro et al., 1997), zyme (Little et al., 1997) and, most recently, kallikrein 6 (hK6) (Yousef and Diamandis, 2001). Whereas only three kallikrein genes were originally thought to exist in humans, 14 members have now been identified, aligned on chromosome 19q (Yousef and Diamandis, 2001). While several of these newly identified genes have been reported to be expressed in brain, much still needs to be learned regarding their normal physiological roles, and their potential contributions to CNS pathogenesis. For example, changes in both neurosin (hK8) and MSP (hK6) levels, in brain samples, CSF or sera, have been observed in progressive neurodegenerative disorders such as Alzheimer’s and Parkinson’s disease (Diamandis et al., 2000a; Okui et al., 2001; Shimizu-Okabe et al., 2001). hK6 has been shown to cleave amyloid precursor protein in vitro (Little et al., 1997) and to be elevated in the sera of ovarian cancer patients (Diamandis et al., 2000b).
We are particularly interested in the potential activity of MSP (hK6) in CNS function and dysfunction, since unlike most other serine proteases identified to date, MSP is robustly expressed in the CNS, but exhibits a more limited distribution in non-neural tissues (Scarisbrick et al., 1997; Yamashiro et al., 1997). We previously showed that MSP is abundantly expressed by neurones and in a subpopulation of white matter glia in the human and rodent CNS (Scarisbrick et al., 1997, 2001). Remarkably, within normal white matter, MSP expression is almost exclusively associated with oligodendroglia (Scarisbrick et al., 1997, 2000; Yamanaka et al., 1999). Moreover, in response to glutamate receptor-mediated excitotoxic injury in the adult rat spinal cord, a common mediator of CNS injury, we have demonstrated that MSP mRNA is upregulated in both neural and glial elements (Scarisbrick et al., 1997).
Given the abundant expression of MSP in oligodendroglia of the adult CNS, and regulation by injury, in this study we set out to determine its potential involvement in CNS demyelinating disease. To accomplish this we examined MSP expression in human multiple sclerosis lesions, and in acute and subacute lesions of experimental allergic encephalomyelitis (EAE) in the common marmoset, Callithrix jacchus, and in the spinal cord of mice chronically infected with the Daniel’s strain of Theiler’s murine encephalomyelitis virus (TMEV). The pathological significance of changes observed has been evaluated by determining the effects of excess of recombinant MSP (r-MSP) on oligodendrocyte differentiation and myelin degradation in vitro. Collectively, these studies indicate that MSP is a multifunctional serine protease, which may participate in multiple effector pathways governing demyelination of the CNS.
|
Methods |
|---|
|
TopSummaryIntroductionMethodsResultsDiscussionReferences |
|---|
Human multiple sclerosis lesions
This study was performed on paraffin-embedded and formalin-fixed archival material from autopsies with clinically (Poser et al., 1983
) and pathologically confirmed multiple sclerosis. Paraffin-embedded 5-µm sections were stained with routine neuropathological stains including haematoxylin–eosin (H and E), Luxol fast blue/periodic acid Schiff (LFB/PAS), and Bielschowsky silver impregnation axonal stain, as well as immunocytochemistry for the following markers: anti-proteolipid protein (MCA839, Serotec, Raleigh, NC, USA), anti-myelin oligodendrocyte glycoprotein (a gift from Dr Piddlesden, University of Cardiff, Cardiff, UK), and anti-MSP (see below). All cases underwent detailed neuropathological examination and were screened for white matter demyelinating lesions. Demyelinating activity was classified according to recently established criteria (Lassmann et al., 1998). Active demyelinating lesions were diffusely infiltrated by macrophages containing myelin proteins as markers of recent and ongoing myelin phagocytosis. Inactive demyelinated lesions were completely demyelinated without signs of remyelination.
TMEV model of multiple sclerosis
Four- to 8-week-old female SJL/J (H-2s) mice (Jackson Laboratories, Bar Harbor, Mass., USA) were intracerebrally injected with 2 x 106 p.f.u. of the Daniel’s strain of TMEV, in a 10 µl volume. Care and handling of mice was in accord with the guidelines of both the NIH and Mayo Clinic Animal Care and Use Committee. At 30, 45, 90, 120 and 180 days post-infection, mice were anaesthetized with pentobarbital (150 mg/kg) and perfused with 4% paraformaldehyde. Spinal cords were blocked transversely at 1 mm, cryoprotected in 25% sucrose, frozen on dry ice and sectioned transversely at 20 µm. Alternatively, blocks were embedded in paraffin and cut at 5 µm. Unfixed spinal cords were obtained at the same time points, snap frozen and stored at –70°C until analysis.
MOG-induced EAE
Marmosets were obtained from Clea, Japan, or the New England Regional Primate Research Center, and housed in the primate colony at the University of California, San Francisco, according to all guidelines of the Institutional Animal Care and Use Committee. EAE was induced by immunization with 100 µg of recombinant rat 
MOG (extracellular domain, containing amino acids 1–125), emulsified in complete Freund’s adjuvant, followed by intravenous injection of 1010 killed Bordetella pertussis organisms on the day of immunization and 48 h later (Genain and Hauser, 1997). Clinical signs of EAE developed between 19 and 23 days after immunization. Animals were euthanized with worsening signs during the acute phase (40–42 days after immunization), under deep barbiturate anaesthesia, by intracardiac perfusion with 4% paraformaldehyde. Slabs of spinal cord were processed for paraffin and 5-µm thick sections were stained with H and E, or processed to localize MSP-immunoreactivity (IR).
Histochemistry
Immunostaining of MSP in mouse, marmoset and human tissue sections was accomplished using purified biotin-conjugated or unconjugated mouse monoclonal MSP antibodies (Scarisbrick et al., 2000) or rabbit polyclonal antibodies (Blaber et al., 2002), each of which yielded identical staining patterns in the tissues examined. Cell-specific markers used in double labelling studies were: monoclonal anti-glial fibrillary acidic protein (anti-GFAP); Cy3 conjugate (Sigma, St Louis, Mo., USA); rat anti-mouse F480 IgG (Serotec, Raleigh, NC, USA); biotinylated isolectin B4 (Sigma); rat anti-mouse CD4; or rat biotinylated anti-mouse CD8b.2 (PharMingen, San Diego, Calif., USA). Bound antibodies were detected using mouse adsorbed, fluorochrome-conjugated, species appropriate secondary antibodies (Jackson ImmunoResearch, West Grove, Pa., USA), or with the avidin–biotin immunoperoxidase technique (Vector Laboratories, Burlingame, Calif., USA). In all cases, control for the specificity of immunostaining included staining as above with the omission of primary antibody.
In situ hybridization
Examination of MSP and proteolipid protein (PLP) mRNA expression in TMEV-infected mouse spinal cord was accomplished using digoxigenin (DIG)-labelled cRNA probes (Scarisbrick et al., 1997, 1999). The MSP-specific probe was prepared by transcription from the MSP cDNA construct pM514, containing 435 base pairs (bp) of rat MSP (nucleotides 220–655), and the PLP probe from construct pGPLP-1, containing 250 bp of mouse PLP (nucleotides 34–285). Hybridization was performed as described previously (Scarisbrick et al., 1999), and in some cases hybridized slides were further processed to localize MSP-IR, GFAP-IR or isolectin B4-IR, as detailed above.
Recombinant MSP
Rat r-MSP was expressed in the baculovirus system, as described in detail elsewhere (Blaber et al., 2002). Briefly, the zymogen form of r-MSP with a 44 amino acid synthetic pro-sequence including an enterokinase (EK) recognition sequence and 6x histidine tag, was expressed in baculovirus expression system, purified in a single step utilizing the His-tag fusion and nickel affinity resin and shown to be 98% pure by Coomassie Blue-stained SDS–PAGE. The homogeneity of purified MSP was confirmed using N-terminal sequencing, mass spectrometry and size exclusion high-performance liquid chromatography. After activation by EK (Roche Diagnostics Corp., Indianapolis, Ind., USA), mature r-MSP was further purified by G-50 superfine (Pharmacia Corp., Kalamazoo, Mich., USA) size exclusion chromatography, to eliminate enterokinase and the cleaved propeptide.
Degradation of myelin basic protein and MOG by MSP
Rat myelin basic protein (MBP) was isolated from adult rat brain (Deibler et al., 1975) and was incubated with r-MSP in 40 mM phosphate, 150 mM NaCl (pH 7.4) at 100 : 1 mass ratio. The final concentration of r-MSP in the reaction mixture was 35.3 µM. The reaction mixture was incubated at 37°C, time points were taken at 0, 1, 4 and 16 h post-incubation, snap frozen on dry ice and kept at –80°C. The digestion pattern of rat MBP was analysed by loading 10 µl of sample (equivalent to 5 µg of rat MBP) per lane on 16.5% Tricine SDS–PAGE under reducing conditions.
Recombinant rat myelin oligodendrocyte glycoprotein (MOG), as described above, was incubated with r-MSP in the same conditions as rat MBP, except that the final concentration of MOG was 31.5 µM. The digested sample was resolved on 16.5% Tricine SDS–PAGE for analysis in the same manner.
Oligodendrocyte cell culture systems
Two oligodendrocyte culture systems were used; purified oligodendrocyte progenitors and the bipotential CG4 oligodendrocyte cell line (Louis et al., 1992). Mixed primary glial cell cultures were prepared from the telencephala of PN-1 Sprague–Dawley rats, and OL progenitors obtained from these, by overnight shaking and differential adhesion as described in detail in McCarthy and De Vellis (1980). Purified OL progenitors were plated onto poly-L-ornithine coated glass coverslips, at a density of 20 x 103/cm2 and grown in Dulbecco’s minimal essential media (DMEM) containing: 4.5 mg/ml glucose, 2 mM glutamine, N2 supplement (Gibco-BRL, Grand Island, NY, USA), 5 µg/ml insulin, 30 nM T3, 10 ng/ml biotin, 50 U/ml penicillin–streptomycin, 0.1 mg/ml sodium pyruvate (Sigma), and 10 ng/ml each of PDGF-AA (platelet derived growth factor AA) and bFGF (basic fibroblast growth factor) (R & D Systems, Minneapolis, Minn., USA). Undifferentiated CG4 cells were grown in Ham’s DMEM F12 containing the same supplements.
To examine the effects of excess exogenous r-MSP, telencephalon-derived or CG4 O2A progenitor cells were differentiated toward the oligodendrocyte lineage by replacement of mitogenic factors, PDGF and FGF, with 0.05% bovine serum albumin (BSA). The effect of r-MSP on differentiated oligodendrocytes was examined by exposing cells differentiated for 72 h, to 1 or 10 µg/ml (40 or 400 nM) of r-MSP for an additional 72 h, with media changes containing fresh r-MSP every 24 h. To evaluate the effect of r-MSP on oligodendrocyte differentiation, progenitors were plated in differentiation media as above, but media were supplemented with 1 or 10 µg/ml of r-MSP after a 30 min culture period, allowing for cellular attachment. As above, cells were then allowed to differentiate for a further 72 h before analysis. To distinguish between cell surface or substrate effects, in a third paradigm, CG4 O2A cells were treated in one of three ways: (i) cells were plated and media changed to differentiation media containing 1 or 10 µg/ml of r-MSP 30 min after plating; (ii) cells were resuspended in, and incubated for 30 min with, 1 or 10 µg/ml of r-MSP, spun down and resuspended in protease-free differentiation media before plating; or (iii) prior to plating, the polyornithine coated coverslip was incubated for 1 h at 37°C with 1 or 10 µg/ml of r-MSP. Cells were differentiated for a further 24 h prior to analysis. Control wells were supplemented with an equal volume of vehicle (40 mM NaOAc, 100 mM NaCl, pH 4.5), alone. To visualize oligodendrocyte processes, coverslips were briefly rinsed in HEPES-buffered saline solution (HBSS), and stained live in HBSS containing 1% BSA, for the presence of cell surface sulphatide, using the monoclonal antibody (mAb) O4 (Sommer and Schachner, 1981), and fluorescein (FITC)-conjugated secondary antibodies (Jackson ImmunoResearch). Labelled cells were fixed in 2% paraformaldehyde and coverslipped with 90% glycerol (pH 8.0), containing 10 µg/ml of the nuclear stain bisbenzamide (Sigma).
In each cell culture paradigm, process outgrowth and cell number were evaluated from six (165 mm2) fields per coverslip, which were imaged digitally (40x objective) using an Olympus AX70 microscope, fitted with a SPOT colour digital camera (Diagnostic Instruments, Inc., Sterling Heights, Mich., USA). The number of oligodendrocyte O4-IR processes that crossed horizontal lines of a 0.25 inch grid superimposed on each image were counted for each field. Additionally, in each field counts were also made of the total number of O4-positive cells and all cells stained with bisbenzamide. On average, 120 cells were counted per culture condition in each experiment. The mean and standard error of counts from triplicate wells were calculated and analysed by one-way analysis of variance and the Student–Newman–Keuls (SNK) post hoc test. All experiments were performed in triplicate and repeated at least twice using independent cell culture preparations.
Overexpression of MSP in CG4 cells
To prepare the green fluorescent protein (GFP)–rat MSP construct, the full-length MSP clone, without stop codon, was amplified by polymerase chain reaction from vector SB12–42B, and subcloned in-frame with the cycle 3 GFP protein of pcDNA3.1/CT-GFP-TOPO® (Invitrogen, Carlsbad, Calif., USA). For cellular transfection, vectors containing the MSP–GFP construct, or GFP alone, were digested with BglII, ethanol precipitated and resuspended in sterile water. Proliferating CG4 cells grown on polyornithine-coated 60 mm dishes at a density of 2 x 105 per 35 mm well were transfected with 2 µg of DNA, using FuGENE 6 reagent (Roche Diagnostics). Cells successfully transfected were identified by expression of Cycle 3 GFP when viewed with a 40x objective on an inverted Olympus IX70 microscope, fitted with a FITC filter set. GFP-positive cells were imaged digitally, using both fluorescence and phase microscopy, at 24, 48 and 96 h post-transfection, and the number of processes evaluated by counting those that crossed a superimposed 0.25 inch grid. At each time point the number of processes associated with cells transfected with MSP–GFP or GFP alone were compared using the Mann–Whitney rank sum test.
|
Results |
|---|
|
TopSummaryIntroductionMethodsResultsDiscussionReferences |
|---|
Expression of MSP in human multiple sclerosis lesions
To determine the potential involvement of MSP in the development of multiple sclerosis lesions, we examined the appearance of MSP-IR in actively demyelinating CNS plaques. Multiple sclerosis lesions demonstrated prominent MSP-IR within inflammatory cells in areas with on-going demyelination, and within the central demyelinated core of plaques in association with reactive astrocytes (Fig. 1A–D). In three actively demyelinating lesions from a chronic autopsy case of multiple sclerosis, MSP-IR was abundant along the plaque border between the actively demyelinating plaque edge and the surrounding periplaque white matter. Alignment of sequential sections stained for LFB/PAS or MSP revealed that the intense rim of MSP-IR was located on the lesion side, bordering normal white matter (Fig. 1B and D). This pattern of MSP-IR created the appearance of ‘rings’ around actively demyelinating lesions. Within these rings, MSP-IR was associated with inflammatory cells and reactive astrocytes (Fig. 1D).
|
Regulated expression of MSP in animal models of multiple sclerosis
To profile the activity of MSP in CNS demyelination, we have examined its expression in two previously characterized animal models of multiple sclerosis. Callithrix jacchus is an outbred new-world primate that is susceptible to immunization with myelin antigens, producing a form of EAE with close clinical and neuropathological similarities to human multiple sclerosis (Genain and Hauser, 1997
). TMEV is a mouse model of virus-induced immune-mediated demyelination characterized by CNS mononuclear cell infiltration and widespread spinal cord demyelination (Lindsley and Rodriguez, 1989; Miller and Gerety, 1990). Consistent with previous experience with MOG-induced EAE in C. jacchus, all animals examined herein displayed multifocal, perivascular inflammatory infiltrates of mononuclear cells and macrophages, accompanied by prominent concentric demyelination throughout the CNS (Genain and Hauser, 1997).
We previously demonstrated in rat and human that MSP in normal white matter is largely confined to oligodendroglia (Fig. 1E), with significant levels of expression also in CNS grey matter; this pattern was conserved in both the marmoset and mouse spinal cord (Fig. 2A–D) (Scarisbrick et al., 2000, 2001). Parallel to observations in human multiple sclerosis lesions, sites of demyelination in spinal cord and brain of the marmoset were infiltrated with MSP-IR inflammatory cells (Fig. 2A and B). Similarly, in areas of demyelination in TMEV-infected mice, examined from 30 to 180 days post-infection, a striking increase in the number of cells associated with MSP-IR and MSP mRNA was observed (Figs 2C and D, and 3A–I). To determine the identity of MSP producing cells within inflammatory lesions, immunohistochemical double labelling techniques were used to examine TMEV-induced lesions (Fig. 3A–F). These experiments demonstrated MSP-IR associated with F480- and isolectin B4-positive macrophages, in addition to CD4 and CD8 immunoreactive T cells. To confirm MSP production by phagocytic macrophages, MSP mRNA was co-localized within isolectin B4-positive cells (Fig. 3G–I). While observed infrequently in the normal cord (Scarisbrick et al., 2000), the presence of MSP-IR in GFAP-positive astrocytes was also seen (data not shown). In active lesions, it was difficult to delineate MSP-IR oligodendroglia, due to the high levels of MSP expression by inflammatory cells. However, even within chronic TMEV lesions, PLP mRNA-producing oligodendroglia were present (Fig. 2E). Together, these findings underscore the potential pathogenic importance of elevated MSP expression by inflammatory cells in CNS immune-mediated demyelination.
|
|
Degradation of myelin-specific proteins by MSP
The potential involvement of MSP in myelin turnover was assessed by determining the ability of r-MSP to degrade the myelin-associated proteins, rat MBP and rat myelin oligodendrocyte glycoprotein (
MOG, amino acids 1–125). Although the aqueous digestion conditions used in this in vitro analysis were not identical to the physiological conditions in which these proteins are naturally located, these studies did demonstrate that both rat MBP and MOG are subject to degradation upon incubation with r-MSP at physiological pH. The degree of digestion of each protein by r-MSP, however, was different (Fig. 4A and B). While rat MBP was readily fragmented, degradation of MOG was much slower. The digestion of rat MBP was so extensive that no intact rat MBP remained within 1 h of incubation with r-MSP, and no fragments with more than 
6 kDa molecular weight could be resolved on 16.5% Tricine SDS–PAGE after overnight digestion (Fig. 4A, lanes 3 and 5). There was a distinct pattern of four lower molecular weight rat MBP fragments clearly seen 1 h post r-MSP digestion (Fig. 4A, lane 3). N-terminal sequencing of these fragments revealed that cleavage occurred after an arginine residue in each case (Blaber et al., 2002). It should be noted that MOG showed a rapid molecular weight reduction from 15.5 to 14.7 kDa immediately after addition of r-MSP (Fig. 4B, lane 2). N-terminal sequencing showed this to be the result of digestion after an arginine residue within an N-terminal leader sequence originating from the cloning vector. The internal sites of MOG were more resistant to digestion by r-MSP; however, slow hydrolysis was apparent.
|
Effect of r-MSP on oligodendrocyte process outgrowth
To determine the significance of elevated MSP at sites of active demyelination to oligodendrocyte survival and function, oligodendroglia cultured from the post-natal Day 1 (PN-1) rat brain were exposed, at different stages of differentiation, to excess r-MSP in vitro, over a 72 h culture period. Exposure of differentiated oligodendrocytes to 1 or 10 µg/ml (40 or 400 nM) of active r-MSP resulted in a 2-fold decrease in the number of sulphatide (O4)-positive processes per cell, compared with control wells exposed to vehicle alone (P
0.005; SNK post hoc test) (Fig. 5A). A parallel 2-fold decrease in process outgrowth was observed when oligodendrocyte progenitors were differentiated in the presence of r-MSP (P < 0.005; SNK, data not shown). Notably, neither of these treatments had a significant effect on the total number of cells stained by the nuclear stain or bisbenzamide, or the percentage of those immunoreactive for O4 (Fig. 5B and C). The effects of excess MSP in these experiments was identical in the case of both telencephalon-derived O2A cells and for CG4 oligodendrocytes.
|
The effect of r-MSP on process stability and outgrowth may have been mediated by activity of the enzyme at the cellular surface, on the polyornithine-coated substratum or both. To distinguish between these possibilities, CG4 oligodendrocyte precursors were exposed to 1 or 10 µg/ml of r-MSP shortly after plating as above, were resuspended and pre-incubated in media containing r-MSP before plating, or were plated in r-MSP free media onto polyornithine-coated coverslips that had been pre-treated with r-MSP. Assessment of O4-positive processes, O4-positive cells and total cell number after a 24 h period revealed that exposure of cells to excess r-MSP shortly after plating, or before plating, decreased process outgrowth by
2-fold (SNK P < 0.05) (Fig. 6A). In contrast, pre-treatment of the substratum had no significant effect on the number of O4-positive processes. Again, none of the treatment protocols affected total cell number or the percentage of O4-positive cells (Fig. 6B and C). We conclude from these experiments that the primary activity of excess r-MSP on immature oligodendrocytes was to affect their ability to extend processes, a result already apparent by 24 h and which was likely mediated primarily on the cellular side, not on the substratum.
|
Effects of MSP overexpression on oligodendrocyte differentiation
To determine whether excess endogenous MSP had similar effects to excess protease applied exogenously, the full-length clone for MSP was inserted into a vector containing a C-terminal GFP fusion protein. CG4 oligodendrocyte precursors were transfected with the MSP–GFP construct, or vector containing GFP alone, and the number of processes in cells expressing GFP assayed at 12, 48 or 96 h post-transfection (Fig. 7). While neither construct affected oligodendrocyte survival over the period examined, MSP-overexpressing cells had significantly fewer processes from the 48 h time point onwards (P < 0.001, 48 h; P = 0.005, 96 h; Mann–Whitney rank sum test). Thus, overexpression of MSP within the cell had the same effect as applying excess protease exogenously, i.e. a decrease in oligodendrocyte processes.
|
|
Discussion |
|---|
|
TopSummaryIntroductionMethodsResultsDiscussionReferences |
|---|
This study shows for the first time the potential involvement of the newly identified serine protease MSP in the pathogenesis of demyelinating disease, and the biological activity of r-MSP in an oligodendrocyte cell culture system. We demonstrate that MSP, now known as hK6 in humans (Yousef and Diamandis, 2001
), is expressed at high levels in inflammatory cells at sites of active demyelination in human multiple sclerosis lesions, and in both autoimmune-mediated disease in the common marmoset and TMEV-induced demyelination in the mouse. The localization of MSP within both macrophages and T cell subsets at sites of demyelination and its demonstrated ability to degrade myelin-specific proteins, coupled with the profound negative effect of an excess on oligodendrocyte process outgrowth and integrity, support the hypothesis that MSP may be a key effector molecule contributing to inflammatory cell-mediated demyelination.
Multiple sclerosis is an inflammatory demyelinating disease of the CNS and represents the most common cause of neurological disorder in young adults in North America and Europe. While considerable progress has been made in understanding genetic susceptibility and pathogenesis of this disease (Noseworthy et al., 2000), there is still no known uniformly effective treatment strategy. The enzymatic digestion of both barriers to the CNS and myelin, by serine proteases, is known to contribute to the development and progression of multiple sclerosis (Cuzner et al., 1978; Alvord et al., 1979; Cammer et al., 1986; Gijbels et al., 1993; Proost et al., 1993; Norga et al., 1995). The identification and characterization of key enzymatic players, therefore, may suggest new therapeutic targets to reduce lesion load and promote remyelination, even in cases of chronic disease.
Role of inflammatory cell MSP
The functional consequence of robust MSP expression by inflammatory cells at sites of inflammation and demyelination in multiple sclerosis may be several-fold. The signal peptide within MSP (Scarisbrick et al., 1997), the demonstration of high levels in human CSF and serum (Diamandis et al., 2000b, c; Okui et al., 2001) and what is known about other serine proteases all suggest MSP is secreted by cells, exerting its activity, at least in part, in the extracellular space. A direct outcome of inflammatory cell MSP production therefore may be its secretion to facilitate transendothelial migration of inflammatory cells into, and within, the CNS. This possibility is corroborated by the demonstration herein of dense MSP expression by all inflammatory cell subsets examined, including macrophages, as well as CD4 and CD8 T cells, not only within perivascular cuffs, but also within the parenchyma of the CNS. A role for secreted MSP in facilitating cell migration is further supported by our previous work, demonstrating that MSP cleaves components of the extracellular matrix, including laminin, fibronectin and collagen, each a component of the blood–brain barrier basal lamina (Blaber et al., 2002). It remains to be determined whether MSP is upregulated in activated inflammatory cells or constitutively expressed therein. Our own work (Scarisbrick et al., 1997) and that of others (Yamashiro et al., 1997; Yousef et al., 1999; Petraki et al., 2001) has demonstrated at least low to moderate levels of MSP mRNA expression in the normal human spleen, thymus and lymph nodes.
The potential role of MSP in inflammatory cell migration is supported by what has been shown for other neutral proteases, including the matrix metalloproteinases (MMPs), such as MMP-9, and tissue plasminogen activator (tPA). Each, like MSP, has been shown to be present in subsets of inflammatory cells in animal models and in human multiple sclerosis lesions (Gijbels et al., 1993; Chandler et al., 1995; Cuzner et al., 1996; Maeda and Sobel, 1996; Gveric et al., 2001; Lindberg et al., 2001). MMP-9 (type IV collagenase) has further been shown to affect the transmigration of lymphocytes in vitro (Leppert et al., 1996; Stuve et al., 1996) and in vivo (Rosenberg et al., 1995). An important consideration in attempting to target proteolysis to abrogate CNS inflammation, as suggested for the MMPs (Kieseier et al., 1999), is the distribution of each enzyme outside the brain. Indeed, inhibition of MMP activity can result in widespread deposition of extracellular matrix components. Although MSP is also expressed in human peripheral tissues (Petraki et al., 2001), it exhibits a more limited distribution relative to both MMPs and tPA (Scarisbrick et al., 1997), and therefore may represent an important alternative enzymatic target to modulate CNS inflammation.
Effect of MSP on oligodendroglia
In addition to inflammatory cell transmigration, MSP may participate in more indirect ‘bystander’ activities at sites of demyelination. Our data suggest that a potential consequence of elevated MSP at sites of inflammation may be a ‘dying back’ of oligodendroglial processes (Rodriguez, 1989). It has previously been established that infiltrating immune cells can cause both oligodendrocyte and myelin damage in multiple sclerosis, and in animal models of this disease (Huitinga et al., 1990; Tran et al., 1998). For example, immune cells and CNS resident cells can release toxic molecules such as tumour necrosis factor- (Selmaj and Raine, 1988; Renno et al., 1995), interleukin-1 (Bauer et al., 1993), reactive oxygen species (Ruuls et al., 1995), nitric oxide (Okuda et al., 1995) and MMPs (Chandler et al., 1995), each of which has been implicated as a cytopathic mediator of demyelination. We demonstrate that application of exogenous r-MSP, causes a retraction of oligodendroglial process from mature cells, and inhibits process extension from immature cells in vitro. Moreover, the effect of excess MSP on process extension was shown to be mediated either by overexpression internally, using an MSP–GFP construct, by exogenous application over the period of culture or by pre-incubation with oligodendrocytes prior to culture, but not by the pre-treatment of the substratum alone. These studies thereby demonstrate that MSP may exert its activity, at least in part, at the cellular surface, perhaps by modifying cell surface integrin receptors, thereby affecting the ability of oligodendrocytes to maintain or extend processes.
While the presence of excess, unregulated levels of MSP appears to compromise the integrity of oligodendroglial processes, it should be noted that MSP is densely produced by oligodendrocytes of the normal adult rodent and human CNS, and is normally localized to the growth tips of oligodendrocytes in cell culture (Scarisbrick et al., 2000, 2001). In this regard, it is also important to emphasize that while excess MSP had a profound effect on oligodendrocyte process number, it did not affect oligodendrocyte survival or differentiation towards the sulphatide-positive lineage. These data support the hypothesis that regulated expression of MSP promotes normal oligodendrocyte function, but that an excess, as is present in inflammatory demyelinating lesions, may have a detrimental effect, both on the processes of existing oligodendroglia and any precursors known to be in the area poised to effect repair (Chang et al., 2000).
MSP-mediated myelin breakdown
Evidence is also presented that myelin degradation may be an additional consequence of excess MSP at sites of CNS inflammation. We demonstrate that MBP, and to a lesser extent MOG, both potentially important auto-antigens in multiple sclerosis (Linington et al., 1993; Genain et al., 1994), are cleaved by MSP in vitro. Taken with the demonstration of MSP within macrophages and T cell subsets, secreted MSP is clearly in a position to participate in myelin breakdown at sites of CNS inflammation. In addition, since both MSP protein and mRNA are present within macrophages, it is likely that MSP participates in the breakdown of myelin debris taken up by these cells. It remains for future work to determine whether cleavage of myelin proteins by MSP results in the generation of encephalitogenic epitopes, known to be presented to TH1-positive T cells by macrophages, and thought to drive epitope spreading in demyelinating disease (Miller et al., 1997; Katz-Levy et al., 2000). It will be important to identify and characterize the activators and endogenous inhibitor(s) of MSP, as well as regulators of this enzyme at the transcriptional level, in order to better understand the full range of MSP activity in the orchestration of the immuno-inflammatory response and the pathogenesis of CNS demyelination.
|
Acknowledgements |
|---|
The authors wish to thank Dr C. Linington for generously providing the rat MOG (1–125) plasmid. This research was supported by grant PP0725 from the National Multiple Sclerosis Society to I.A.S. and PP0757 to M.B., and by grants R01NS32129 and P01-NS38468 from the National Institutes of Health.
|
References |
|---|
|
TopSummaryIntroductionMethodsResultsDiscussionReferences |
|---|
Alvord EC, Hruby S, Sires LR. Degradation of myelin basic protein by cerebrospinal fluid: preservation of antigenic determinants under physiological conditions. Ann Neurol 1979; 6: 474–82.[Web of Science][Medline]
Anisowicz A, Sotiropoulou G, Stenman G, Mok SC, Sager R. A novel protease homolog differentially expressed in breast and ovarian cancer. Mol Med 1996; 2: 624–36.[Web of Science][Medline]
Bauer J, Berkenbosch F, Van Dam AM, Dijkstra CD. Demonstration of interleukin-1 beta in Lewis rat brain during experimental allergic encephalomyelitis by immunocytochemistry at the light and ultrastructural level. J Neuroimmunol 1993; 48: 13–21.[Web of Science][Medline]
Blaber SI, Scarisbrick IA, Bernett MJ, Dhanarajan P, Seavy MA, Jin Y, et al. Enzymatic properties of rat myelencephalon-specific protease. Biochemistry 2002; 41: 1165–73.[Medline]
Cammer W, Brosnan CF, Basile C, Bloom BR, Norton WT. Complement potentiates the degradation of myelin proteins by plasmin: implications for a mechanism of inflammatory demyelination. Brain Res 1986; 364: 91–101.[Web of Science][Medline]
Chandler S, Coates R, Gearing A, Lury J, Wells G, Bone E. Matrix metalloproteinases degrade myelin basic protein. Neurosci Lett 1995; 201: 223–6.[Web of Science][Medline]
Chang A, Nishiyama A, Peterson J, Prineas J, Trapp BD. NG2-positive oligodendrocyte progenitor cells in adult human brain and multiple sclerosis lesions. J Neurosci 2000; 20: 6404–12.
Cuzner ML, Davison AN, Rudge P. Proteolytic enzyme activity of blood leukocytes and cerebrospinal fluid in multiple sclerosis. Ann Neurol 1978; 4: 337–44.[Web of Science][Medline]
Cuzner ML, Gveric D, Strand C, Loughlin AJ, Paemen L, Opdenakker G, et al. The expression of tissue-type plasminogen activator, matrix metalloproteases and endogenous inhibitors in the central nervous system in multiple sclerosis: comparison of stages in lesion evolution. J Neuropathol Exp Neurol 1996; 55: 1194–204.[Web of Science][Medline]
Davies B, Kearns IR, Ure J, Davies CH, Lathe R. Loss of hippocampal serine protease BSP1/neuropsin predisposes to global seizure activity. J Neurosci 2001; 21: 6993–7000.
Deibler GE, Martenson RE, Kramer AJ, Kies MW. The contribution of phosphorylation and loss of COOH-terminal arginine to the microheterogeneity of myelin basic protein. J Biol Chem 1975; 250: 7931–8.
Diamandis EP, Yousef GM, Petraki C, Soosaipilla AR. Human kallikrein 6 as a biomarker of Alzheimer’s disease. Clin Biochem 2000a; 33: 663–7.[Web of Science][Medline]
Diamandis EP, Yousef GM, Soosaipillai AR, Bunting P. Human kallikrein 6 (zyme/protease M/neurosin): a new serum biomarker of ovarian carcinoma. Clin Biochem 2000b; 33: 579–83.[Web of Science][Medline]
Diamandis EP, Yousef GM, Soosaipillai AR, Grass L, Porter A, Little S, et al. Immunofluorometric assay of human kallikrein 6 (zyme/protease M/neurosin) and preliminary clinical applications. Clin Biochem 2000c; 33: 369–75.[Web of Science][Medline]
Genain CP, Hauser SL. Creation of a model for multiple sclerosis in Callithrix jacchus marmosets. [Review]. J Mol Med 1997; 75: 187–97.[Web of Science][Medline]
Genain CP, Lee-Parritz D, Nguyen MH, Massacesi L, Joshi N, Ferrante R, et al. In healthy primates, circulating autoreactive T cells mediate autoimmune disease. J Clin Invest 1994; 94: 1339–45.
Gijbels K, Proost P, Masure S, Carton H, Billiau A, Opdenakker G. Gelatinase B is present in the cerebrospinal fluid during experimental autoimmune encephalomyelitis and cleaves myelin basic protein. J Neurosci Res 1993; 36: 432–40.[Web of Science][Medline]
Gingrich MB, Traynelis SF. Serine proteases and brain damage – is there a link? [Review]. Trends Neurosci 2000; 23: 399–407.[Web of Science][Medline]
Gveric D, Hanemaaijer R, Newcombe J, van Lent NA, Sier CF, Cuzner ML. Plasminogen activators in multiple sclerosis lesions: implications for the inflammatory response and axonal damage. Brain 2001; 124: 1978–88.
Houenou LJ, Turner PL, Li L, Oppenheim RW, Festoff BW. A serine protease inhibitor, protease nexin I, rescues motoneurons from naturally occurring and axotomy-induced cell death. Proc Natl Acad Sci USA 1995; 92: 895–9.
Huitinga I, van Rooijen N, de Groot CJ, Uitdehaag BM, Dijkstra CD. Suppression of experimental allergic encephalomyelitis in Lewis rats after elimination of macrophages. J Exp Med 1990; 172: 1025–33.
Katz-Levy Y, Neville KL, Padilla J, Rahbe S, Begolka WS, Girvin AM, et al. Temporal development of autoreactive Th1 responses and endogenous antigen presentation of self myelin epitopes by central nervous system-resident APCs in Theiler’s virus-infected mice. J Immunol 2000; 165: 5304–14.
Kieseier BC, Seifert T, Giovannoni G, Hartung H-P. Matrix metalloproteinases in inflammatory demyelination: targets for treatment. [Review]. Neurology 1999; 53: 20–5.
Lassmann H, Raine CS, Antel J, Prineas JW. Immunopathology of multiple sclerosis: report on an international meeting held at the Institute of Neurology of the University of Vienna. J Neuroimmunol 1998; 86: 213–7.[Web of Science][Medline]
Leppert D, Waubant E, Burk MR, Oksenberg JR, Hauser SL. Interferon beta-1b inhibits gelatinase secretion and in vitro migration of human T cells: a possible mechanism for treatment efficacy in multiple sclerosis. Ann Neurol 1996; 40: 846–52.[Web of Science][Medline]
Lindberg RL, De Groot CJ, Montagne L, Freitag P, van der Valk P, Kappos L, et al. The expression profile of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) in lesions and normal appearing white matter of multiple sclerosis. Brain 2001; 124: 1743–53.
Lindsley MD, Rodriguez M. Characterization of the inflammatory response in the central nervous system of mice susceptible or resistant to demyelination by Theiler’s virus. J Immunol 1989; 142: 2677–82.[Abstract]
Linington C, Berger T, Perry L, Weerth S, Hinze-Selch D, Zhang Y, et al. T cells specific for the myelin oligodendrocyte glycoprotein mediate an unusual autoimmune inflammatory response in the central nervous system. Eur J Immunol 1993; 23: 1364–73.[Web of Science][Medline]
Little SP, Dixon EP, Norris F, Buckley W, Becker GW, Johnson M, et al. Zyme, a novel and potentially amyloidogenic enzyme cDNA isolated from Alzheimer’s disease brain. J Biol Chem 1997; 272: 25135–42.
Liu Y, Fields RD, Festoff BW, Nelson PG. Proteolytic action of thrombin is required for electrical activity-dependent synapse reduction. Proc Natl Acad Sci USA 1994a; 91: 10300–4.
Liu Y, Fields RD, Fitzgerald S, Festoff BW, Nelson PG. Proteolytic activity, synapse elimination, and the Hebb synapse. [Review]. J Neurobiol 1994b; 25: 325–35.[Web of Science][Medline]
Louis JC, Muir D, Varon S. Autocrine inhibition of mitotic activity in cultured oligodendrocyte-type-2 astrocyte (O-2A) precursor cells. Glia 1992; 6: 30–8.[Web of Science][Medline]
Maeda A, Sobel RA. Matrix metalloproteinases in the normal human central nervous system, microglia nodules, and multiple sclerosis lesions. J Neuropathol Exp Neurol 1996; 55: 300–9.[Web of Science][Medline]
McCarthy KD, de Vellis J. Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J Cell Biol 1980; 85: 890–902.
Miller SD, Gerety SJ. Immunologic aspects of Theiler’s murine encephalomyelitis virus (TMEV)-induced demyelinating disease. Semin Virol 1990; 1: 263.
Miller SD, Vanderlugt CL, Begolka WS, Pao W, Yauch RL, Neville KL, et al. Persistent infection with Theiler’s virus leads to CNS autoimmunity via epitope spreading. Nat Med 1997; 3: 1133–6.[Web of Science][Medline]
Monard D. Cell-derived proteases and protease inhibitors as regulators of neurite outgrowth. [Review]. Trends Neurosci 1988; 11: 541–4.[Web of Science][Medline]
Moonen G, Grau-Wagemans M-P, Selak I. Plasminogen activator-plasmin system and neuronal migration. Nature 1982; 298: 753–5.[Medline]
Norga K, Paemen L, Masure S, Dillen C, Heremans H, Billiau A, et al. Prevention of acute autoimmune encephalomyelitis and abrogation of relapses in murine models of multiple sclerosis by the protease inhibitor D-penicillamine. Inflamm Res 1995; 44: 529–34.[Web of Science][Medline]
Noseworthy JH, Lucchinetti C, Rodriguez M, Weinshenker BG. Multiple sclerosis. [Review]. New Engl J Med 2000; 343: 938–52.
Okuda Y, Nakatsuji Y, Fujimura H, Esumi H, Ogura T, Yanagihara T, et al. Expression of the inducible isoform of nitric oxide synthase in the central nervous system of mice correlates with the severity of actively induced experimental allergic encephalomyelitis. J Neuroimmunol 1995; 62: 103–12.[Web of Science][Medline]
Okui A, Kominami K, Uemura H, Mitsui S, Yamaguchi N. Characterization of brain-related serine protease, neurosin (human kallikrein 6), in human cerebrospinal fluid. Neuroreport 2001; 12: 1345–50.[Web of Science][Medline]
Petraki CD, Karavana VN, Skoufogiannis PT, Little SP, Howarth DJ, Yousef GM, et al. The spectrum of human kallikrein 6 (zyme/protease M/neurosin) expression in human tissues as assessed by immunohistochemistry. J Histochem Cytochem 2001; 49: 1431–41.
Poser CM, Paty DW, Scheinberg L, McDonald WI, Davis FA, Ebers GC, et al. New diagnostic criteria for multiple sclerosis: guidelines for research protocols. Ann Neurol 1983; 13: 227–31.[Web of Science][Medline]
Proost P, Van Damme J, Opdenakker G. Leukocyte gelatinase B cleavage releases encephalitogens from human myelin basic protein. Biochem Biophys Res Commun 1993; 192: 1175–81.[Web of Science][Medline]
Renno T, Krakowski M, Piccirillo C, Lin JY, Owens T. TNF-alpha expression by resident microglia and infiltrating leukocytes in the central nervous system of mice with experimental allergic encephalomyelitis. Regulation by Th1 cytokines. J Immunol 1995; 154: 944–53.[Abstract]
Rodriguez M. Multiple sclerosis: basic concepts and hypothesis. [Review]. Mayo Clin Proc 1989; 64: 570–6.[Web of Science][Medline]
Rosenberg GA, Estrada EY, Dencoff JE, Stetler-Stevenson WG. Tumor necrosis factor-alpha-induced gelatinase beta causes delayed opening of the blood–brain barrier: an expanded therapeutic window. Brain Res 1995; 703: 151–5.[Web of Science][Medline]
Ruuls SR, Bauer J, Sontrop K, Huitinga I, ’t Hart BA, Dijkstra CD. Reactive oxygen species are invovled in the pathogenesis of experimental allergic encephalomyelitis in Lewis rats. J Neuroimmunol 1995; 56: 207–17.[Web of Science][Medline]
Scarisbrick IA, Towner MD, Isackson PJ. Nervous system-specific expression of a novel serine protease: regulation in the adult rat spinal cord by excitotoxic injury. J Neurosci 1997; 17: 8156–68.
Scarisbrick IA, Isackson PJ, Windebank AJ. Differential expression of brain-derived neurotrophic factor, neurotrophin-3 and neurotrophin-4/5 in the adult rat spinal cord: regulation by the glutamate receptor agonist kainic acid. J Neurosci 1999; 19: 7757–69.
Scarisbrick IA, Asakura K, Blaber S, Blaber M, Isackson PJ, Bieto T, et al. Preferential expression of myelencephalon-specific protease by oligodendrocytes of the adult rat spinal cord white matter. Glia 2000; 30: 219–30.[Web of Science][Medline]
Scarisbrick IA, Isackson PJ, Ciric B, Windebank AJ, Rodriguez M. MSP, a trypsin-like serine protease, is abundantly expressed in the human nervous system. J Comp Neurol 2001; 431: 347–61.[Web of Science][Medline]
Seeds NW, Haffke S, Christensen K, Schoonmaker J. Cerebellar granule cell migration involves proteolysis. Adv Exp Med Biol 1990; 265: 169–78.[Medline]
Selmaj KW, Raine CS. Tumor necrosis factor mediates myelin and oligodendrocyte damage in vitro. Ann Neurol 1988; 23: 339–46.[Web of Science][Medline]
Shimizu-Okabe C, Yousef GM, Diamandis EP, Yoshida S, Shiosaka S, Fahnestock M. Expression of the kallikrein gene family in normal and Alzheimer’s disease brain. Neuroreport 2001; 12: 2747–51.[Web of Science][Medline]
Sommer I, Schachner M. Monoclonal antibodies (O1 to O4) to oligodendrocyte cell surfaces: an immunocytological study in the central nervous system. Dev Biol 1981; 83: 311–27.[Web of Science][Medline]
Stuve O, Dooley NP, Uhm JH, Antel JP, Francis GS, Williams G, et al. Interferon beta-1b decreases the migration of T lymphocytes in vitro: effects on matrix metalloproteinase-9. Ann Neurol 1996; 40: 853–63.[Web of Science][Medline]
Tran EH, Hoekstra K, van Rooijen N, Dijkstra CD, Owens T. Immune invasion of the central nervous system parenchyma and experimental allergic encephalomyelitis, but not leukocyte extravasation from blood, are prevented in macrophage-depleted mice. J Immunol 1998; 161: 3767–75.
Tsirka SE, Gualandris A, Amaral DG, Strickland S. Excitotoxin-induced neuronal degeneration and seizure are mediated by tissue plasminogen activator. Nature 1995; 377: 340–4.[Medline]
Tsirka SE, Rogove AD, Bugge TH, Degen JL, Strickland S. An extracellular proteolytic cascade promotes neuronal degeneration in the mouse hippocampus. J Neurosci 1997; 17: 543–52.
Yamanaka H, He X, Matsumoto K, Shiosaka S, Yoshida S. Protease M/neurosin mRNA is expressed in mature oligodendroyctes. Brain Res Mol Brain Res 1999; 71: 217–4.[Medline]
Yamashiro K, Tsuruoka N, Kodama S, Tsujimoto M, Yamamura Y, Tanaka T, et al. Molecular cloning of a novel trypsin-like serine protease (neurosin) preferentially expressed in brain. Biochim Biophys Acta 1997; 1350: 11–4.[Medline]
Yousef GM, Diamandis EP. The new human tissue kallikrein gene family: structure, function, and association to disease. [Review]. Endocr Rev 2001; 22: 184–204.
Yousef GM, Luo LY, Scherer SW, Sotiropoulou G, Diamandis EP. Molecular characterization of zyme/protease M/neurosin (PRSS9), a hormonally regulated kallikrein-like serine protease. Genomics 1999; 62: 251–9.[Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  G. Pampalakis, E. Prosnikli, T. Agalioti, A. Vlahou, V. Zoumpourlis, and G. Sotiropoulou A Tumor-Protective Role for Human Kallikrein-Related Peptidase 6 in Breast Cancer Mediated by Inhibition of Epithelial-to-Mesenchymal Transition Cancer Res., May 1, 2009; 69(9): 3779 - 3787. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. P. Christophi, C. A. Hudson, M. Panos, R. C. Gruber, and P. T. Massa Modulation of Macrophage Infiltration and Inflammatory Activity by the Phosphatase SHP-1 in Virus-Induced Demyelinating Disease J. Virol., January 15, 2009; 83(2): 522 - 539. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Yoon, G. Laxmikanthan, J. Lee, S. I. Blaber, A. Rodriguez, J. M. Kogot, I. A. Scarisbrick, and M. Blaber Activation Profiles and Regulatory Cascades of the Human Kallikrein-related Peptidases J. Biol. Chem., November 2, 2007; 282(44): 31852 - 31864. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Oikonomopoulou, K. K. Hansen, M. Saifeddine, I. Tea, M. Blaber, S. I. Blaber, I. Scarisbrick, P. Andrade-Gordon, G. S. Cottrell, N. W. Bunnett, et al. Proteinase-activated Receptors, Targets for Kallikrein Signaling J. Biol. Chem., October 27, 2006; 281(43): 32095 - 32112. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. F. Angelo, A. R. Lima, F. M. Alves, S. I. Blaber, I. A. Scarisbrick, M. Blaber, L. Juliano, and M. A. Juliano Substrate Specificity of Human Kallikrein 6: SALT AND GLYCOSAMINOGLYCAN ACTIVATION EFFECTS J. Biol. Chem., February 10, 2006; 281(6): 3116 - 3126. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. A. Borgono, I. P. Michael, and E. P. Diamandis Human Tissue Kallikreins: Physiologic Roles and Applications in Cancer Mol. Cancer Res., May 1, 2004; 2(5): 257 - 280. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||