Cathepsin C aggravates neuroinflammation via promoting production of CCL2 and CXCL2 in glial cells and neurons in a cryogenic brain lesion.

OBJECTIVE: Chemokines regulate infiltration of immune cells to brain in inflammation. Cathepsin C (CatC), a lysosomal protease, has been found to participate in neuroinflammation. However, how CatC affects chemokines expression in neuroinflammation triggered by traumatic brain injury (TBI) remains unclear. Here, we investigated the effects of CatC on chemokines and neuroinflammation in TBI. METHODS: The present study used CatC knockdown (KD) and overexpression (OE) mice to generate cryogenic brain lesion model and determined effects of CatC on expression of chemokines CCL2, CCL5 and CXCL2 and infiltration of immune cells in acute and chronic phases of the lesion. Further, cellular sources of various chemokines were demonstrated in vitro. Values were compared with wild type (WT) mice. RESULTS: The results found that 6 h after lesion, CatC expression,IL-1ß and TNF-α mRNA and protein expression were strongly induced in the lesions; CCL2 and CXCL2 mRNA and protein expression were increased in CatC OE mice, while decreased in CatC KD mice. On the 3rd day after lesion, macrophages and neutrophils were mainly infiltrated to the lesions. Simultaneously, Iba-1+ cells in CatC OE mice were increased, while MPO + cells in CatC KD mice were decreased. In contrast, on the 28th day after lesion, a few lymphocytes were infiltrated surrounding new blood vessels. CatC OE mice showed larger volumes of scar areas, higher expression of CCL2,CXCL2,IL-1ß,TNF-α,IL-6 and iNOS, as well as stronger GFAP+ and Iba-1+ signals, while CatC KD mice had reversed effects. No significant differences of CCL5 expression were found in various genotype mice. Further, in vitro study demonstrated CatC-induced expression of CCL2 were mainly derived from microglia and neurons, while CXCL2 derived from microglia and astrocytes. CONCLUSION: Our data indicate that CatC aggravates neuroinflammation via promoting production of CCL2 and CXCL2 in glial cells and neurons in a cryogenic brain lesion, providing potential cellular and molecular targets for future intervention of TBI and other neuroinflammatory diseases.

Up-regulation of microglial cathepsin C expression and activity in lipopolysaccharide-induced neuroinflammation.

BACKGROUND: Cathepsin C (Cat C) functions as a central coordinator for activation of many serine proteases in inflammatory cells. It has been recognized that Cat C is responsible for neutrophil recruitment and production of chemokines and cytokines in many inflammatory diseases. However, Cat C expression and its functional role in the brain under normal conditions or in neuroinflammatory processes remain unclear. Our previous study showed that Cat C promoted the progress of brain demyelination in cuprizone-treated mice. The present study further investigated the Cat C expression and activity in lipopolysaccharide (LPS)-induced neuroinflammation in vivo and in vitro. METHODS: C57BL/6 J mice were intraperitoneally injected with either 0.9% saline or lipopolysaccharide (LPS, 5 mg/kg). Immunohistochemistry (IHC) and in situ hybridization (ISH) were used to analyze microglial activation, TNF-α, IL-1ß, IL-6, iNOS mRNAs expressions and cellular localization of Cat C in the brain. Nitrite assay was used to examine microglial activation in vitro; RT-PCR and ELISA were used to determine the expression and release of Cat C. Cat C activity was analyzed by cellular Cat C assay kit. Data were evaluated for statistical significance with paired t test. RESULTS: Cat C was predominantly expressed in hippocampal CA2 neurons in C57BL/6 J mice under normal conditions. Six hours after LPS injection, Cat C expression was detected in cerebral cortical neurons; whereas, twenty-four hours later, Cat C expression was captured in activated microglial cells throughout the entire brain. The duration of induced Cat C expression in neurons and in microglial cells was ten days and three days, respectively. In vitro, LPS, IL-1ß and IL-6 treatments increased microglial Cat C expression in a dose-dependent manner and upregulated Cat C secretion and its activity. CONCLUSIONS: Taken together, these data indicate that LPS and proinflammatory cytokines IL-1ß, IL-6 induce the expression, release and upregulate enzymatic activity of Cat C in microglial cells. Further investigation is required to determine the functional role of Cat C in the progression of neuroinflammation, which may have implications for therapeutics for the prevention of neuroinflammation-involved neurological disorders in the future.

High level expression, purification and activation of human dipeptidyl peptidase I from mammalian cells.

Dipeptidyl peptidase I (DPPI) plays a crucial role in maturation of many regulatory peptides and has been suggested as a pharmaceutical target in several inflammatory diseases. It is also a useful processing enzyme for the generation of authentic protein products by catalyzing the removal of N-terminal fusion peptides. We used a robust transient transfection system in human embryonic kidney 293 cells to exploit expression and activation of DPPI from chicken, rat and man for the development of an industrial production process. The expression of human and rat DPPI was significantly higher in the human HEK293 cell line than that obtained with avian DPPI. A CHO K1SV stable cell line was selected as the optimal stable host system for production of human DPPI yielding expression levels higher than 1.5 g/L. The secreted pro-DPPI underwent auto-maturation during defined buffer conditions during the purification steps. Active human DPPI was purified with a three-step purification strategy employing: Butyl Sepharose 4 Fast Flow, Sephadex G-25 Medium and Q Sepharose Fast Flow chromatography. The final yield of active enzyme was approximately 1 g/L cell culture. The enzyme exhibited exopeptidase activity against both a dipeptide-p-nitroanilide substrate and N-terminally extended MEAE-hGH (Met-Glu-Ala-Glu-human growth hormone). In conclusion, an efficient production process for recombinant human DPPI has been developed including a highly efficient and stable CHO cell system and an efficient purification procedure, which is simple and easy to scale for industrial purposes. The present data facilitates not only industrial applications of DPPI as a processing enzyme, but also provides active enzyme useful in the identification of small molecule inhibitors.

Expression and purification of active recombinant cathepsin C (dipeptidyl aminopeptidase I) of kuruma prawn Marsupenaeus japonicus in insect cells.

Cathepsin C (CTSC) is a lysosomal cysteine protease belonging to the papain superfamily. Our previous study showed that CTSC precursor (zymogen) is localized exclusively in cortical rods (CRs) of mature oocyte in the kuruma prawn Marsupenaeus japonicus, suggesting that CTSC might have roles on regulating release and/or formation of a jelly layer. In this study, enzymically active CTSC of the kuruma prawn was prepared by recombinant expression in the High Five insect cell line. The recombinant enzyme with a polyhistidine tag at its C-terminus was considered to be initially secreted into the culture medium as an inactive form of zymogen, because Western blot with anti-CTSC antibody detected a 51 kDa protein corresponding to CTSC precursor. After purification by affinity chromatography on nickel-iminodiacetic acid resin, the enzyme displayed three forms of 51, 31, and 30 kDa polypeptides. All of the forms can be recognized by antiserum raised against C-terminal polyhistidine tag, indicating that the 31 and 30 kDa forms were generated from 51 kDa polypeptide by removal of a portion of the N-terminus of propeptide. Following activation at pH 5.5 and 37 degrees C for 40 hours under native conditions, the recombinant CTSC (rCTSC) exhibited increased activity against the synthetic substrate Gly-Phe-beta-naphthylamide and optimal pH at around 5. The purified rCTSC will be useful for further characterization of its exact physiological role on CRs release and/or formation of a jelly layer in kuruma prawn.

P. gingivalis regulates the expression of Cathepsin B and Cystatin C.

Porphyromonas gingivalis is a major etiological agent of periodontitis that could affect the expression of Cathepsins B and C by disrupting the balance between these enzymes and their inhibitor, Cystatin C. We tested this hypothesis by infecting human oral epithelial cells with P. gingivalis or activating solely by its lipopolysaccharide. The mRNA level, the enzymatic activity, and the protein expression of Cathepsin B were increased (three-fold) in a dose-dependent manner, while those of Cystatin C decreased (five-fold). No changes were observed for Cathepsin C. Although activation by lipopolysaccharides led to a delayed imbalance (2 days) between Cathepsin B and Cystatin C, this imbalance took place very rapidly during the infection (< 6 hrs), indicating that the whole bacterium contains components that initiate rapid changes in the transcription rates of Cathepsin B and Cystatin C and selectively modify the molecular pathways that lead to this imbalance.

Production of active bovine cathepsin C (dipeptidyl aminopeptidase I) in the methylotrophic yeast Candida boidinii.

The heterologous production of active bovine cathepsin C (CTC; dipeptidyl aminopeptidase I) was investigated. Attempts to express CTC in Escherichia coli were hampered by formation of inclusion bodies that were partially degraded. To overcome this impediment, secretion of recombinant CTC was attempted in the methylotrophic yeast Candida boidinii. A DNA fragment encoding bovine procathepsin C was synthesized based on preferred codon usage in C. boidinii and placed downstream of the C. boidinii proteinase A signal sequence resulting in secretion of active CTC into the culture medium. The gene was expressed under the control of the methanol-inducible formate dehydrogenase gene promoter. Production levels were significantly improved by using a protease-deficient strain, changing medium composition, and by lowering the temperature of induction. When the recombinant C. boidinii was grown for 90 h in a jar-fermenter, active CTC was secreted with a yield of up to approximately 12 mg/l.

Importance of the propeptide in the biosynthetic maturation of rat cathepsin C.

Cathepsin C is a cysteine dipeptidyl-aminopeptidase. Active cathepsin C is found in lysosomes as a 200-kDa multimeric enzyme. Subunits constituting this assembly all arise from the proteolytic cleavage of a single precursor giving rise to three peptides: the propeptide, the alpha- and the beta-chains. Some features of the propeptide such as its length, its high level of glycosylation and its retention in the active lysosomal form of the enzyme suggest an important contribution of the proregion in the transport, maturation and expression of cathepsin C. In order to assess some aspects of this contribution, we transiently expressed mutant molecules of rat cathepsin C either lacking three of the four glycosylation sites, partially deleted in the proregion, or mutated at tryptophan 39 also located in the proregion, and studied their biosynthesis. Our results show that at least one of the three glycosylation sites in the propeptide must be glycosylated in order to obtain targeting and maturation of cathepsin C. We also show that a deletion of 14 amino acids and mutation W39S in the propeptide totally abolishes the biosynthetic processing of the enzyme. These results demonstrate that in addition to its role as a chaperone or in maintaining the latency of the enzymatic activity, the propeptide is required for proper transport and expression of newly synthesized cathepsin C.

Acute doxorubicin (adriamycin) dosage does not reduce cardiac protein synthesis in vivo, but decreases diaminopeptidase I and proline endopeptidase activities.

Anthracycline antibiotics are effective anticancer agents but their use is limited due to unwanted adverse side effects. The toxic effects of doxorubicin (adriamycin) include the development of defined cardiac lesions leading to cardiomyopathy in some patients. This has been reported to be due to reductions in cardiac protein synthesis. However, virtually all of these previous studies have failed to consider the specific radioactivity of the precursor pool in their measurements or have carried out their studies in vitro. To further resolve the above we measured fractional rates of cardiac protein synthesis using the "flooding dose" method in rats treated with adriamycin (5 mg/kg body wt). Controls were identically treated and injected with saline. At 2.5 or 24 h after adriamycin injection, rates of protein synthesis were measured with a flooding dose of l-[4-(3)H]phenylalanine. Measurements included free (S(i)) and protein-bound (S(b)) phenylalanine-specific radioactivities, the protein synthetic capacity (RNA/protein ratio; C(s)), the fractional rates of protein synthesis calculated from the ratio S(b)/S(i), and the protein synthetic efficiency calculated from the ratio k(s)/C(s). Complementary analyses included assays of lysosomal (cathepsins B, D, H, and L and diaminopeptidases I and II) and cytoplasmic proteases (alanyl aminopeptidase, arginyl aminopeptidase, leucyl aminopeptidase, diaminopeptidase IV, tripeptidyl aminopeptidase, and proline endopeptidase). These enzymes constitute the most active proteases in this tissue and represent an index of protein degradation capacity in cardiac muscle. The results showed that in 2.5-h dosed rats, adriamycin had no effect on S(i), S(b), C(s), k(s), or k(RNA) (P > 0.05, not significant (NS) in all instances). In 2.5-h dosed rats, levels of diaminopeptidase I activity were reduced (P < 0.05), whereas the activities of other proteases were not significantly altered (NS in all instances). In 24-h dosed rats, adriamycin reduced cardiac S(b) (P < 0.001), which would normally be interpreted as a reduction in protein synthesis. However, S(i) was also decreased in 24-h adriamycin-injected rats (P < 0.025%). C(s) was not changed (NS). Consequently, the calculated k(s) and k(RNA) values were not significantly affected in 24-h adriamycin-dosed rats (NS). There were also significant reductions in proline endopeptidase activities in rats exposed for 24 h to adriamycin. The activities of other proteases were not significantly affected at this time point (NS in all instances). In conclusion, adriamycin reduces amino acid labeling of cardiac proteins, an effect that is a consequence of altered free phenylalanine-specific radioactivities. There was some evidence of limited altered intracellular proteolysis.