Mechanisms of inflammation in spinal cord injury

An increase in protease activity is a hallmark event of the secondary injury cascade following contusion SCI. Elevated levels of protease activity result in the degradation of cytoskeletal components and myelin proteins essential for cellular function and survival. We have shown that a member of the cathepsin protease family is affected by SCI. The excessive release and activity of cathepsin B, a fairly ubiquitous lysosomal cysteine protease, has been implicated in several pathologies including tumor metastasis and progression, arthritis and Alzheimer's disease. Thus, our goal was to characterize any SCI-induced changes in cathepsin B expression. Following a T12 laminectomy and a moderate contusion (NYU device), the gene and protein profiles of cathepsin B in rats were examined using real-time PCR and immunoblots, respectively. Both the contusion injured animals and the time-matched sham controls exhibited elevated pro-enzyme protein levels (37 kDa form) at the lesion site, with significant differences between the two groups at 48 h, 72 h and 7 days post-SCI. Furthermore, there was a surge in the active species of the protein with significant differences at 72 h and 7 days post-SCI for the 30 kDa form and at 48 h. and 7 days for the 25 kDa form. Real-time PCR revealed increases in cathepsin B mRNA levels following contusion SCI as early as 6 h postinjury. These data indicate that SCI causes an up-regulation of cathepsin gene expression and protein levels, and suggest that this protease may be involved in the secondary injury cascade perhaps for as long as 1 week postinjury.     

Cathepsin B mRNA and protein expression following contusion spinal cord injury in rats

We provide the first data that cathepsin B (Cath B), a lysosomal cysteine protease, is up-regulated following contusion-spinal cord injury (SCI). Following T12 laminectomy and moderate contusion, Cath B mRNA and protein expression profiles were examined from 2 to 168 h post-injury in rats using real-time PCR and immunoblots, respectively. Contusion injury significantly increased [mRNA]Cath B in the injury site and adjacent segments over sham injury levels. While the largest [mRNA]Cath B induction (20-fold over naive) was seen in the injury site, the caudal segment routinely yielded [mRNA]Cath B levels greater than 10-fold over naive. Interestingly, sham injury animals also experienced mRNA induction at several time points at the injury site and in segments rostral and caudal to the injury site. Contusion injury also significantly elevated levels of Cath B proenzyme protein (37 kDa) over sham injury in the injury site (48, 72 and 168 h post-injury). Furthermore, significant protein increases of single and double chain Cath B (both active forms) occurred at the injury site at 72 and 168 h post-injury. Similar significant increases in Cath B protein levels were seen in areas adjacent to the injury site. The induction of Cath B mRNA and protein expression following contusion injury is previously undescribed and suggests that Cath B may potentially be involved in the secondary injury cascade, perhaps for as long as 1 week post-injury.     

A new nuclear protease with cathepsin L properties is present in HeLa and Caco‐2 cells

Recently many authors have reported that cathepsin L can be found in the nucleus of mammalian cells with important functions in cell‐cycle progression. In previous research, we have demonstrated that a cysteine protease (SpH‐protease) participates in male chromatin remodeling and in cell‐cycle progression in sea urchins embryos. The gene that encodes this protease was cloned. It presents a high identity sequence with cathepsin L family. The active form associated to chromatin has a molecular weight of 60 kDa, which is higher than the active form of cathepsin L described until now, which range between 25 and 35 kDa. Another difference is that the zymogen present in sea urchin has a molecular weight of 75 and 90 kDa whereas for human procathepsin L has a molecular weight of 38–42 kDa. Based on these results and using a polyclonal antibody available in our laboratory that recognizes the active form of the 60 kDa nuclear cysteine protease of sea urchin, ortholog to human cathepsin L, we investigated the presence of this enzyme in HeLa and Caco‐2 cells. We have identified a new nuclear protease, type cathepsin L, with a molecular size of 60 kDa, whose cathepsin activity increases after a partial purification by FPLC and degrade in vitro histone H1. This protease associates to the mitotic spindle during mitosis, remains in the nuclei in binuclear cells and also translocates to the cytoplasm in non‐proliferative cells. J. Cell. Biochem. 111: 1099–1106, 2010. © 2010 Wiley‐Liss, Inc.     

Molecular cloning and characterization of Cathepsin B from a scuticociliate, Uronema marinum

A cDNA encoding cathepsin B was cloned from the scuticociliate, Uronema marinum, which invades the olive flounder, Paralichthys olivaceus, leading to high mortalities in culturing fish. The full-length scuticociliate cathepsin B (ScCtB) gene contains an open reading frame of 1053 base pairs encoding 350 amino acids. A homology search revealed that ScCtB shares sequence identity with several piscine cathepsin Bs (48%-45%). The protein of ScCtB from U. marinum extracts was purified 12.8-fold by a one step purification process using a DEAE-Sephagel high performance liquid chromatography (HPLC) column. It had a molecular mass of 30 kDa, as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting, which was consistent with predicting molecular mass of mature protein (29.2 kDa) of ScCtB. The protease activity of the ScCtB enzyme was demonstrated by electrophoresis in a gelatin-acrylamide copolymerized gel. Its activity was quantified by cleaving a synthetic fluorogenic peptide substrate, Z-arginyl-arginyl-7-amido-4-methylcoumarin (Z-Arg-Arg-AMC). The optimum pH for the protease activity was 5.5. Typical of cysteine proteases, the enzyme was inhibited by trans-epoxysuccinyl-L-leucyl-amido(4-guanidino)butane (E-64) and leupeptin.     

A cathepsin B-like enzyme from mackerel white muscle is a precursor of cathepsin B

A cathepsin B-like enzyme from the white muscle of common mackerel Scomber japonicus was a cysteine protease that hydrolyzed Z-Arg-Arg-MCA, the substrate for cathepsin B. In a partial purified cathepsin B-like enzyme preparation at 4 degrees C left over time, a converted enzyme that hydrolyzes Z-Arg-Arg-MCA and Z-Phe-Arg-MCA appeared in the preparation. The converted enzyme was purified from the cathepsin B-like enzyme, characterized and was identified as mackerel cathepsin B. These results suggested that the mackerel cathepsin B-like enzyme was a precursor of cathepsin B. Mackerel cathepsin B formed in the purified cathepsin B-like enzyme preparation by adding of a small amount of the purified cathepsin B to the preparation. Therefore, mackerel cathepsin B-like enzyme was converted to the mature form of cathepsin B by autoactivation. The conversion of the cathepsin B-like enzyme (molecular mass 60 kDa) to cathepsin B (molecular mass 23 kDa) was detected by immunoblotting by using human anti-(cathepsin B) antibody. The intermediate forms of 40 kDa and 38 kDa were also detected during the conversion.     

Effects of cathepsins B and L inhibition on postischemic protein alterations in the brain

The effects of selective inhibition of cathepsins B and L on postischemic protein alterations in the brain were investigated in a rat model of middle cerebral artery occlusion (MCAO). Cathepsin B activity increased predominantly in the subcortical region of the ischemic hemisphere where the levels of collapsing mediator response protein 2, heat shock cognate 70 kDa protein, 60 kDa heat shock protein, protein disulfide isomerase A3 and albumin, were found to be significantly elevated. Postischemic treatment with Cbz-Phe-Ser(OBzl)-CHN₂, cysteine protease inhibitor 1 (CP-1), reduced infarct volume, neurological deficits and cathepsin B activity as well as the amount of heat shock proteins and albumin found in the brain. Our data strongly suggests that the decrease in heat shock protein levels and the significant reduction of serum albumin leakage into the brain following acute treatment with CP-1 is indicative of less secondary ischemic damage, which ultimately, is related to less cerebral tissue loss and improved neurological recovery of the animals.     

Expression and activation of ephexin is altered after spinal cord injury

Failure of axon regeneration after traumatic spinal cord injury (SCI) is attributable in part to the presence of inhibitory molecular interactions. Recent evidence demonstrates that activation of Eph signaling pathways leads to modulation of growth cone dynamics and repulsion through the activation of ephexin, a novel guanine nucleotide exchange factor (GEF). However, little is known about the expression and modulation of Eph molecular targets in the injured spinal cord. In this study, we determined the expression profile of ephexin after a moderate spinal cord contusion at thoracic level (T10) in young adult rats. Western-blot studies showed increased protein expression in injured rats at 4 and 7 days postinjury (DPI) when compared with control animals. The protein levels returned to normal at 14 DPI and remained steady until 28 DPI. However, immunoprecipitation studies of the phosphorylated ephexin demonstrated that this protein is activated by day 2 until 14 DPI. Expression of ephexin was noticeable in neurons, axons, microglia/macrophages, and reactive astrocytes, and co-localized with EphA3, A4, and A7. These results demonstrate the presence of ephexin in the adult spinal cord and its activation after SCI. Therefore, we show, for the first time, the spatiotemporal pattern of ephexin expression and activation after contusive SCI. Collectively, our data support our previous findings on the putative nonpermissive roles of Eph receptors after SCI and the possible involvement of ephexin in the intracellular cascade of events.     

Enzymatic and Molecular Biochemical Characterizations of Human Neutrophil Elastases and a Cathepsin G-like Enzyme

Human neutrophil elastase (HNE, EC 3. 4. 21. 37) is a causative factor of inflammatory diseases, including emphysema and rheumatoid arthritis. Enzymatic characterization is important for the development of new drugs involved in the regulation of this enzyme. In this study, we investigated the enzymatic and biochemical properties of five different elastolytic enzymes, with a molecular mass between 24 kDa and 72 kDa. Three elastases, molecular masses of 27, 29, 31 kDa, might be elastase isozymes that have the same NH₂-terminal amino acid sequences of Ile-Val-Gly-Gly-Arg-Arg-Ala. The 24-kDa enzyme, which showed the identical NH₂-terminal amino acid sequences to elastase, was a degraded fragment of native elastase. The elastolytic activity was conserved at the 6/7 domain of the NH₂-terminal region. The inhibitory characteristics of PMSF, DipF were the same as those of native elastases. The 72-kDa molecule, which showed elastolytic activity, might be a trimer formed between native elastases (31 kDa and 29 kDa) and a cathepsin G-like enzyme, which did not show elastolytic activity but enhanced the elastolytic activity of neutrophil elastase. Although this cathepsin G-like enzyme showed weak cathepsin G activity, it has distinguishable NH₂-terminal sequences of Ile-Val-Gly-Gly-Ser-Arg-Ala- from those of elastase or cathepsin G. The potentiation of elastolytic activity could be a result of the trimerization of native elastase with a cathepsin G-like enzyme, and was then weakly inhibited by serine protease inhibitors, such as PMSF, DipF. Therefore, we suggest the cathepsin G-like enzyme to be a novel enzyme, which has an important role in the development of inflammation.     

Synthesis and intracellular distribution of cathepsins E and D in differentiating murine Friend erythroleukemia cells.

The synthesis, accumulation, and cellular distribution of cathepsins E and D during the dimethyl sulfoxide (DMSO)-induced differentiation of Friend erythroleukemia cells were investigated. The cellular levels of cathepsins E and D rapidly increased within 1 day of DMSO induction and then sharply decreased over the next 7 days. Since the cells during 1 day of differentiation were morphologically the same as uninduced cells, the results suggest the importance of these enzymes in more cellular proteolysis for the following committed differentiation. While cathepsin D was present mostly in the sedimentable fraction of cells throughout the differentiation period, the distribution of cathepsin E varied to the stage of differentiation. The ratio of the soluble/sedimentable cathepsin E content was 1.1, 1.4, 0.9, and 0.7 in cells after 0, 1, 4, and 7 days of DMSO treatment, respectively. The maturation of reticulocytes to erythrocytes was accompanied by complete loss of the soluble cathepsin E and of all of the cellular cathepsin D. Immunoblotting analyses revealed that both uninduced and induced cells contained two forms of cathepsin E; a high molecular weight form (82 kDa) which was mainly associated with the sedimentable fraction and a low molecular weight form (74 kDa) which was found largely in the soluble fraction. The distribution of these two forms was not significantly changed throughout the differentiation period, but the 74-kDa protein was completely eliminated with maturation of reticulocytes to erythrocytes. Cathepsin D also appeared in two forms in both uninduced and induced cells; a minor (46 kDa) and a major (42 kDa) form which appear to have a precursor-product relationship.     

Activation of the P2X7 receptor induces migration of glial cells by inducing cathepsin B degradation of tissue inhibitor of metalloproteinase 1

The P2X₇ receptor is an ion‐gated channel, which is activated by high extracellular concentrations of adenosine triphosphate (ATP). Activation of P2X₇ receptors has been shown to induce neuroinflammatory changes associated with several neurological conditions. The matrix metalloproteinases (MMPs) are a family of endopeptidases that have several functions including degradation of the extracellular matrix, cell migration and modulation of bioactive molecules. The actions of MMPs are prevented by a family of protease inhibitors called tissue inhibitors of metalloproteinases (TIMPs). In this study, we show that ATP‐treated glial cultures from neonatal C57BL/6 mice release and increase MMP‐9 activity, which is coupled with a decrease in release of TIMP‐1 and an increase in activated cathepsin B within the extracellular space. This process occurs independently of NLRP3‐inflammasome formation. Treatment with a P2X₇ receptor antagonist prevents ATP‐induced MMP‐9 activity, inhibition of active cathepsin B release and allows for TIMP‐1 to be released from the cell. We have shown that cathepsin B degrades TIMP‐1, and inhibition of cathepsin B allows for release of TIMP‐1 and inhibits MMP‐9 activity. We also present data that indicate that ATP or cell damage induces glial cell migration, which is inhibited by P2X₇ antagonism, depletion of MMP‐9 or inhibition of cathepsin B.     

Cathepsin B-like cysteine proteases confer intestinal cysteine protease activity in Haemonchus contortus

Cathepsin B-like cysteine protease genes (cbls) constitute large multigene families in parasitic and nonparasitic nematodes. Although expressed in the intestine of some nematodes, the biological and biochemical functions of the CBL proteins remain unresolved. Di- and tetra-oligopeptides were used as fluorogenic substrates and irreversible/competitive inhibitors to establish CBL functions in the intestine of the parasitic nematode Haemonchus contortus. Cysteine protease activity was detected against diverse substrates including the cathepsin B/L substrate FR, the caspase 1 substrate YVAD, the cathepsin B substrate RR, but not the CED-3 (caspase 3) substrate DEVD. The pH at which maximum activity was detected varied according to substrate and ranged from pH 5.0 to 7.0. Individual CBLs were affinity isolated using FA and YVAD substrates. pH influenced CBL affinity isolation in a substrate-specific manner that paralleled pH effects on individual substrates. N-terminal sequencing identified two isolated CBLs as H. contortus GCP-7 (33 kDa) and AC-4 (37 kDa). N termini of each began at a position consistent with proregion cleavage and protease activation. Isolation of the GCP-7 band by each peptide was preferentially inhibited when competed with a diazomethane-conjugated inhibitor, Z-FA-CHN(2), demonstrating one functional difference among CBLs and among inhibitors. Substrate-based histological analysis placed CBLs on the intestinal microvilli. Data indicate that CBLs are responsible for cysteine protease activity described from H. contortus intestine. Results also support a role of CBLs in nutrient digestion.     

The influence of differential processing of procathepsin H on its aminopeptidase activity, secretion and subcellular localization in human cell lines

Cathepsin H is a unique member of the cysteine cathepsins that acts primarily as an aminopeptidase. Like other cysteine cathepsins, it is synthesized as an inactive precursor and activated by proteolytic removal of its propeptide. Here we demonstrate that, in human cells, the processing of the propeptide is an autocatalytic, multistep process proceeding from an inactive 41kDa pro-form, through a 30kDa intermediate form, to the 28kDa mature form. Tyr87P and Gly90P were identified as the two major endopeptidase cleavage sites, converting the 30kDa form into the mature 28kDa form. The level of processing differs significantly in different human cell lines. In monocyte-derived macrophages U937 and prostate cancer cells PC-3, the 28kDa form is predominant, whereas in osteoblasts HOS the processing from the 30kDa form to the 28kDa form is significantly lower. The aminopeptidase activity of the enzyme and its subcellular localization are independent of the product, however the 30kDa form was not secreted in HOS cells. The activity of the resulting cathepsin H in U937 cells was significantly lower than that in HOS cells, presumably due to the high levels of endogenous cysteine protease inhibitor cystatin F present specifically in this cell line. These results provide an insight into the dependence of human cathepsin H processing and regulation on cell type.     

Degradation of an Fc‐fusion recombinant protein by host cell proteases: Identification of a CHO cathepsin D protease

A host‐cell‐related proteolytic activity was identified in a recombinant Fc‐fusion protein production process. This report describes the strategy applied to characterize and isolate the enzyme responsible for this degradation by combining cell culture investigation and dedicated analytical tools. After isolation and sequencing of the clipped fragment generated in post‐capture material, enzymatic activity was traced in different culture conditions, allowing identification of viable CHO cells as the source of protease. Inhibitors and pH screenings showed that the enzyme belongs to an aspartic protease family and is preferably active at acidic pH. The protease was isolated by purification on a pepstatin A column and characterized as a protein related to cathepsin D. An additional metallo‐protease inhibited by EDTA was identified with an optimum activity at neutral pH. This study is an example of how quality and stability of therapeutic recombinant molecules are strongly influenced by cell culture parameters. Biotechnol. Bioeng. 2009; 104: 1132–1141. © 2009 Wiley Periodicals, Inc.     

Tumor necrosis factor induces the loss of sphingosine kinase-1 by a cathepsin B-dependent mechanism

Sphingosine kinase-1 (SK1) has emerged as a key component of cytokine responses, including roles in apoptosis, yet the specific mechanisms by which cytokines regulate SK1 in the apoptotic responses have not been studied. In this study, we show that prolonged treatment of MCF-7 cells with tumor necrosis factor (TNF) induces a dose- and time-dependent decrease in SK1 protein. Inhibition of the upstream caspase 8 by IETD significantly rescued TNF effects on SK1, yet the caspase 7 inhibitor DEVD failed to have any effect, suggesting that the decline in SK1 occurs downstream of the initiator caspase but upstream of the effector caspase. In addition to caspase activation, TNF caused disruption of lysosomes with relocation of the cysteine protease cathepsin B into the cytosol. Down-regulation of cathepsin B using small interfering RNA significantly restored SK1 levels following exposure to TNF, suggesting that SK1 loss was dependent on cathepsin B activity. The regulation of SK1 by the lysosomal protease was further supported by the colocalization of SK1 with the lysosome and cathepsin B in cells and the loss of the colocalization following exposure to TNF. The ability of cathepsin B to regulate SK1 was further corroborated by an in vitro approach where recombinant cathepsin B cleaved SK1 at multiple sites to produce several cleavage fragments. Therefore, these studies show that SK1 down-regulation by TNF is dependent on the "lysosomal pathway" of apoptosis and specifically on cathepsin B, which functions as an SK1 protease in cells.     

Mosquito cathepsin B-like protease involved in embryonic degradation of vitellin is produced as a latent extraovarian precursor

Here we report identification of a novel member of the thiol protease superfamily in the yellow fever mosquito, Aedes aegypti. It is synthesized and secreted as a latent proenzyme in a sex-, stage-, and tissue-specific manner by the fat body, an insect metabolic tissue, of female mosquitoes during vitellogenesis in response to blood feeding. The secreted, hemolymph form of the enzyme is a large molecule, likely a hexamer, consisting of 44-kDa subunits. The deduced amino acid sequence of this 44-kDa precursor shares high similarity with cathepsin B but not with other mammalian cathepsins. We have named this mosquito enzyme vitellogenic cathepsin B (VCB). VCB decreases to 42 kDa after internalization by oocytes. In mature yolk bodies, VCB is located in the matrix surrounding the crystalline yolk protein, vitellin. At the onset of embryogenesis, VCB is further processed to 33 kDa. The embryo extract containing the 33-kDa VCB is active toward benzoyloxycarbonyl-Arg-Arg-para-nitroanilide, a cathepsin B-specific substrate, and degrades vitellogenin, the vitellin precursor. Both of these enzymatic activities are prevented by trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64), a thiol protease inhibitor. Furthermore, addition of the anti-VCB antibody to the embryonic extract prevented cleavage of vitellogenin, strongly indicating that the activated VCB is involved in embryonic degradation of vitellin.     

Dl‐3‐n‐butylphthalide attenuates acute inflammatory activation in rats with spinal cord injury by inhibiting microglial TLR4/NF‐κB signalling

In this study, we examined the neuroprotective effects and anti‐inflammatory properties of Dl‐3‐n‐butylphthalide (NBP) in Sprague‐Dawley (SD) rats following traumatic spinal cord injury (SCI) as well as microglia activation and inflammatory response both in vivo and in vitro. Our results showed that NBP improved the locomotor recovery of SD rats after SCI an significantly diminished the lesion cavity area of the spinal cord, apoptotic activity in neurons, and the number of TUNEL‐positive cells at 7 days post‐injury. NBP inhibited activation of microglia, diminished the release of inflammatory mediators, and reduced the upregulation of microglial TLR4/NF‐κB expression at 1 day post‐injury. In a co‐culture system with BV‐2 cells and PC12 cells, NBP significantly reduced the cytotoxicity of BV‐2 cells following lipopolysaccharide (LPS) stimulation. In addition, NBP reduced the activation of BV‐2 cells, diminished the release of inflammatory mediators, and inhibited microglial TLR4/NF‐κB expression in BV‐2 cells. Our findings demonstrate that NBP may have neuroprotective and anti‐inflammatory properties in the treatment of SCI by inhibiting the activation of microglia via TLR4/NF‐κB signalling.     

Inhibition of cathepsin B reduces beta-amyloid production in regulated secretory vesicles of neuronal chromaffin cells: evidence for cathepsin B as a candidate beta-secretase of Alzheimer's disease

The regulated secretory pathway of neurons is the major source of extracellular A beta that accumulates in Alzheimer's disease (AD). Extracellular A beta secreted from that pathway is generated by beta-secretase processing of amyloid precursor protein (APP). Previously, cysteine protease activity was demonstrated as the major beta-secretase activity in regulated secretory vesicles of neuronal chromaffin cells. In this study, the representative cysteine protease activity in these secretory vesicles was purified and identified as cathepsin B by peptide sequencing. Immunoelectron microscopy demonstrated colocalization of cathepsin B with A beta in these vesicles. The selective cathepsin B inhibitor, CA074, blocked the conversion of endogenous APP to A beta in isolated regulated secretory vesicles. In chromaffin cells, CA074Me (a cell permeable form of CA074) reduced by about 50% the extracellular A beta released by the regulated secretory pathway, but CA074Me had no effect on A beta released by the constitutive pathway. Furthermore, CA074Me inhibited processing of APP into the COOH-terminal beta-secretase-like cleavage product. These results provide evidence for cathepsin B as a candidate beta-secretase in regulated secretory vesicles of neuronal chromaffin cells. These findings implicate cathepsin B as beta-secretase in the regulated secretory pathway of brain neurons, suggesting that inhibitors of cathepsin B may be considered as therapeutic agents to reduce A beta in AD.