Proteases of the nematode Caenorhabditis elegans

Crude homogenates of the soil nematode Caenorhabditis elegans exhibit strong proteolytic activity at acid pH. Several kinds of enzyme account for much of this activity: cathepsin D, a carboxyl protease which is inhibited by pepstatin and optimally active toward hemoglobin at pH 3; at least two isoelectrically distinct thiol proteases (cathepsins Ce1 and Ce2) which are inhibited by leupeptin and optimally active toward Z-Phe-Arg-7-amino-4-methylcoumarin amide at pH 5; and a thiol-independent leupeptin-insensitive protease (cathepsin Ce3) with optimal activity toward casein at pH 5.5. Cathepsin D is quantitatively most significant for digestion of macromolecular substrates in vitro, since proteolysis is inhibited greater than 95% by pepstatin. Cathepsin D and the leupeptin-sensitive proteases act synergistically, but the relative contribution of the leupeptin-sensitive proteases depends upon the protein substrate.     

A His-Leu-Leu sequence near the carboxyl terminus of the cytoplasmic domain of the cation-dependent mannose 6-phosphate receptor is necessary for the lysosomal enzyme sorting function.

The determinants on the cytoplasmic tail of the cation-dependent mannose 6-phosphate receptor (CD-MPR) required for lysosomal enzyme sorting have been analyzed. Mouse L cells deficient in the mannose 6-phosphate/insulin-like growth factor-II receptor were transfected with normal bovine CD-MPR cDNA or cDNAs containing mutations in the 67-amino acid cytoplasmic tail and assayed for their ability to target the lysosomal enzyme cathepsin D to lysosomes. Cells expressing the wild-type bovine CD-MPR sorted 67 +/- 2% of newly synthesized cathepsin D compared with the base-line value of 47 +/- 1%. The presence of mannose 6-phosphate in the medium did not affect the efficiency of cathepsin D sorting, indicating that the routing of the ligand-receptor complex is completely intracellular. Mutant receptors with the carboxyl-terminal His-Leu-Leu-Pro-Met67 residues deleted or replaced with alanines sorted cathepsin D below the base-line value. A mutant receptor with the outermost Pro-Met residues replaced with alanines sorted cathepsin D better than the wild-type receptor, indicating that the essential residues for sorting are the His-Leu-Leu sequence. Disruption of a putative casein kinase II phosphorylation site at Ser57 had no detectable effect on sorting. The mutant receptor with the five-amino acid deletion was able to bind to a phosphopentamannose affinity column, proving that its ligand binding site was grossly intact. Resialylation experiments showed that this mutant receptor recycled from the cell surface to the Golgi at a rate similar to the normal CD-MPR, indicating that the defect in sorting is at the level of the Golgi.     

Effects of differentiation-inducing agents on synthesis, maturation and secretion of cathepsin D in U937 and HL-60 cells

Treatment of human monocyte U937 and promyelocyte HL-60 cultures with agents known to induce differentiation (12-O-tetra-decanoylphorbol 13-acetate, calcitriol and dimethylsulfoxide) accelerates the maturation of cathepsin D and enhances the incorporation of [35S]methionine into cathepsin D. The most pronounced effects are obtained with calcitriol, which at a concentration of 10(-7) M increases the incorporation of [35S]methionine into cathepsin D from 0.08% to 0.4% of the detergent-soluble radioactivity. In addition, this treatment enhances the secretion of cathepsin D from about 8% to greater than or equal to 16% of the newly synthesized enzyme. In the presence of 10mM NH4Cl approximately half of the produced cathepsin D is secreted in both control and calcitriol-treated cells. It appears that in U937 cells two mechanisms are involved in sorting of cathepsin D. One of these is sensitive to NH4Cl and its efficiency is selectively decreased in cells pretreated with calcitriol.     

Molecular cloning, characterization and expression analysis of cathepsin D gene from turbot Scophthalmus maximus

Cathepsin D is a lysosomal endoproteolytic aspartic proteinase which also has been found in endosomes of macrophage. It is thought to play key roles in the developmental and physiological process of animals. The EST sequence of turbot (Scophthalmus maximus L.) cathepsin D was obtained from a subtractive cDNA library. In the present study, 5'-RACE and 3'-RACE were carried out to obtain the complete cDNA sequence of turbot cathepsin D, which contained a 91 bp 5'-UTR, a 1191 bp open reading frame encoding 396 amino acids, and a 329 bp 3'-UTR. The deduced amino acid sequence of the cathepsin D consisted of a signal peptide of 18 aa, a leader peptide extending 43 aa, and a mature peptide of 335 aa. BLAST analysis revealed that turbot cathepsin D shared high similarity with other known cathepsin D, and it showed significant homology with that of Barramundi (Lates calcarifer B., 89% aa similarity). Quantitative real-time PCR (q PCR) demonstrated that the highest expression level of the turbot cathepsin D was in liver. After turbot were challenged with Vibrio harveyi, the lowest expression levels of cathepsin D in liver, spleen and head kidney were detected at 8 h. This result was different from the expression of MHCII of which the expression lever was increased upon challenge. The expression levels of cathepsin D in liver and head kidney increased gradually after 8 h and exceeded the background level after 24 h. In spleen, the expression level was reinforced after 8 h and kept at level that was higher than the original level after 12 h. The results suggested that cathepsin D might process antigens for presentation to the immune system and have synergetic effect with apoptosis pathway until 12 h after injection.     

Identification of a Putative Structural Gene for Cathepsin D in Caenorhabditis Elegans

Mutants of Caenorhabditis elegans having about 10% of wild-type activity of the aspartyl protease cathepsin D have been isolated by screening. Mutant homozygotes have normal growth rates and no obvious morphological or developmental abnormalities. The mutant gene (cad-1) has been mapped to the right extremity of linkage group II. Heterozygous animals (cad-1/+) show intermediate enzyme levels and animals heterozygous for chromosomal deficiencies of the right extremity of linkage group II have 50% of wild-type activity. Cathepsin D purified from a mutant strain has a lower activity per unit mass of pure enzyme. These data suggest that cad-1 is a structural gene for cathepsin D.     

Transformation of leukotriene D4 catalyzed by lysosomal cathepsin H of rat liver

Among several intracellular protease tested, cathepsin H transformed leukotriene D4 to E4 with a release of glycine in a stoichiometric quantity. Under the optimal conditions the rate of leukotriene D4 transformation by cathepsin H was about 3% of the hydrolysis rate of alpha-N-benzoyl-DL-arginine-2-naphthylamide which is commonly utilized as a very efficient substrate to test the peptidase activity of the enzyme. Leukotriene C4 was not transformed to leukotriene D4 by cathepsin H. Neither cathepsin B nor C was active with leukotrienes C4 and D4.     

Changes Induced in Astrocyte Cathepsin D by Cytokines and Leupeptin

Cathepsin D is widely, but unevenly, distributed among cells and is capable of degrading a number of neural peptides and proteins. The present study was undertaken to examine the level of cathepsin D in astrocytes that might be relevant to its induction in inflammatory demyelination. Primary astrocytes were cultured from neonatal rat cerebrums according to the method of McCarthy and de Vellis. Based on staining for cell markers, cultures were greater than 95% astrocytes and less than 3% microglia. Under serum-free conditions, leupeptin induced a 1.4- to 2.0-fold increase, maximal by 48 hours, in cathepsin D protein quantified by a radioimmunoassay. Cathepsin D enzymatic activity, inhibitable by pepstatin, also increased. Northern blot analysis demonstrated that leupeptin also increased cathepsin D mRNA expression. Kinetic analysis indicated that maximal cathepsin D mRNA levels are detected 24 h after stimulation with leupeptin. Exposure of astrocytes under the same conditions to rat recombinant interferon-gamma, human recombinant tumor necrosis factor-alpha, human recombinant interleukin-1 beta, lipopolysaccharide, calcium ionophore, or a combination of these reagents did not increase the level of cathepsin D above controls. These results indicate that astrocytic cathepsin D mRNA and protein can be induced by selected materials. Furthermore, the effects attributed to leupeptin as a proteinase inhibitor may be modified by its ability to increase cathepsin D activity.     

Cathepsin D of rat spleen. Affinity purification and properties of two types of cathepsin D.

Two types of cathepsin D were purified from rat spleen by a rapid procedure involving an acid precipitation of tissue extract, affinity chromatography with pepstatin--Sepharose 4B and concanavalin-A--Sepharose 4B, and chromatography on Sephadex G-100 and DEAE-Sephacel. The purified major enzyme (85% of the cathepsin D activity after DEAE-Sephacel chromatography), termed cathepsin D-I, represented about a 1000-fold purification over the homogenate and about a 20% recovery. The purified minor enzyme (15%), termed cathepsin D-II, represented about a 900-fold purification and about a 3% recovery. Both enzymes showed four (pI: 4.2, 4.9, 6.1 and 6.5) and three (pI: 4.6, 5.6 and 5.8) multiple forms after isoelectric focusing, respectively. The purified enzymes appeared homogeneous on electrophoresis in polyacrylamide gel and had a molecular weight of about 44000. In sodium dodecylsulfate/polyacrylamide gel electrophoresis both enzymes showed a single protein band corresponding to a molecular weight of 44000. The enzymes had similar amino acid compositions except for serine, proline and methionine. Cathepsin D-I contained 6.6% carbohydrate, consisting of mannose, glucose, galactose, fucose and glucosamine in a ratio of 8:2:1:1:5 with a trace of sialic acid. The properties of purified enzymes were also compared.     

Increased cathepsin D-like activity in cortex, tubules, and glomeruli isolated from rats with experimental nephrotic syndrome.

We have examined the activity and distribution of cathepsin D (EC 3.4.23.5), a major renal lysosomal endoproteinase, in the various anatomical and functional areas of normal rat kidney. Cathepsin D-like activities (delta A280/h per mg of protein) in normal rat tissues were: cortex, 0.78 +/- 0.05, n = 37; medulla, 0.62 +/- 0.03, n = 12; papilla, 0.63 +/- 0.04, n = 12; tubules, 0.74 +/- 0.04, n = 28; glomeruli, 0.59 +/- 0.03, n = 28; and liver, 0.41 +/- 0.02, n = 28. Enzyme activity was maximal at pH 3.0-3.5 and inhibited more than 90% by pepstatin (6.7 micrograms/ml), suggesting that the enzyme is cathepsin D. In subsequent experiments we measured cathepsin D-like activity in cortex, tubules and glomeruli isolated from rats with puromycin aminonucleoside (PAN)-induced nephrotic syndrome. Treated animals (15 mg of PAN/100g body wt., intraperitoneally) developed proteinuria beginning 4 days after injection and exceeding 900 mg/24h on day 9. In two separate experiments involving 52 animals we observed a significant increase in cathepsin D-like activity in cortex (+82.7%), tubules (+109.6%) and glomeruli (+54.7%) isolated from PAN-treated rats killed during marked proteinuria (day 9, mean total urinary protein excretion: 937 +/- 94 mg/24h). This increase was observed whether the activity was expressed per mg of DNA or per mg of protein. Increased cathepsin D-like activity was first observed in cortex and tubules coincident with the onset of proteinurea (day 4, mean total urinary protein excretion: 112 +/- 23 mg/24h). In contrast with the significant elevation of renal cathepsin D-like activity, the activity (nmol/h per mg of protein) of alpha-L-fucosidase (EC 3.2.1.51), a non-proteolytic enzyme, was markedly decreased in the identical samples used for the measurement of cathepsin D-like activity: cortex (-46.4%); tubules (-46.1%); and glomeruli (-38.5%). In addition to changes in renal enzyme activities, PAN-treated rats excreted large amounts of cathepsin D-like activity in their urine (beginning on day 3) compared with nearly undetectable cathepsin D-like activity in the urine from control rats. The significant increases in glomerular and tubular cathepsin D activity may reflect an important role for this enzyme in the pathophysiology associated with PAN-induced nephrotic syndrome.     

Prolactin crinophagy is induced in the estrogen-stimulated male rat pituitary

Summary The phenomenon of crinophagy in rat pituitary mammotrophs, or lysosomal uptake of prolactin secretory granules, was confirmed by means of double-label immunogold electron microscopy, and shown to be induced in estrogen-stimulated male rats. Rabbit antibodies to rat cathepsin D were used to label lysosomes, and to rat prolactin to label secretory granules. The pituitaries were fixed in 4% formaldehyde and 1% glutaraldehyde, embedded in Lowicryl K4M, and thin sections were exposed successively to primary antibodies, biotin-labelled second antibodies, and streptavidin-gold, with an amplification procedure for cathepsin D. Cathepsin D and prolactin were detected separately on opposite sides of the sections, using 5-nm and 15-nm gold particles. Lysosomal uptake of prolactin secretory granules was not observed in untreated control rats. It was detected in about 26% of lysosome-containing mammotroph cell sections in estrogen-stimulated rats and at 7 h after estrogen withdrawal, but fell to 14% at 24 h and to 2% at 72 h after estrogen withdrawal.     

Prolactin crinophagy is induced in the estrogen-stimulated male rat pituitary

The phenomenon of crinophagy in rat pituitary mammotrophs, or lysosomal uptake of prolactin secretory granules, was confirmed by means of double-label immunogold electron microscopy, and shown to be induced in estrogen-stimulated male rats. Rabbit antibodies to rat cathepsin D were used to label lysosomes, and to rat prolactin to label secretory granules. The pituitaries were fixed in 4% formaldehyde and 1% glutaraldehyde, embedded in Lowicryl K4M, and thin sections were exposed successively to primary antibodies, biotin-labelled second antibodies, and streptavidin-gold, with an amplification procedure for cathepsin D. Cathepsin D and prolactin were detected separately on opposite sides of the sections, using 5-nm and 15-nm gold particles. Lysosomal uptake of prolactin secretory granules was not observed in untreated control rats. It was detected in about 26% of lysosome-containing mammotroph cell sections in estrogen-stimulated rats and at 7 h after estrogen withdrawal, but fell to 14% at 24 h and to 2% at 72 h after estrogen withdrawal.     

Expression of proteolytic enzymes during Dictyostelium discoideum spore germination

The specific activity of cathepsin B-like, cathepsin D-like, and leucine aminopeptidase enzymes was measured in dormant, aging, and germinating spores of wild-type and mutant Dictyostelium discoideum.The activity of leucine aminopeptidase was relatively constant during spore aging and spore germination. The level of cathepsin D-like activity was highest in young dormant spores but decreased during germination or aging.The level of cathepsin B-like activity remained constant in wild-type spores which were aged for 13 days. The dormant spores of spontaneous germination mutants initially contained low levels of cathepsin B-like activity which increased during aging. Thus, there was no correlation between the level of endogenous cathepsin B activity and the ability to be autoactivated or heat-activated. The level of cathepsin B-like activity does not have a role in the generation of energy for the swelling stage of germination. Finally, the combined level of endogenous and exogenous cathepsin B activity increased more than 20-fold during the emergence of myxamoebae suggesting that the enzyme(s) may play a role at this development stage of germination.     

Olfactomedin 4 Inhibits Cathepsin C-Mediated Protease Activities, Thereby Modulating Neutrophil Killing of Staphylococcus aureus and Escherichia coli in Mice

Neutrophils kill bacteria generally through oxidative and nonoxidative mechanisms. Whereas much research has focused on the enzymes essential for neutrophil killing, little is known about the regulatory molecules responsible for such killing. In this study, we investigated the role of olfactomedin 4 (OLFM4), an olfactomedin-related glycoprotein, in neutrophil bactericidal capability and host innate immunity. Neutrophils from OLFM4(-/-) mice have increased intracellular killing of Staphylococcus aureus and Escherichia coli in vitro. The OLFM4(-/-) mice have enhanced in vivo bacterial clearance and are more resistant to sepsis when challenged with S. aureus or E. coli by i.p. injection. OLFM4 was found to interact with cathepsin C, a cysteine protease that plays an important role in bacterial killing and immune regulation. We demonstrated that OLFM4 inhibited cathepsin C activity in vitro and in vivo. The cathepsin C activity in neutrophils from OLFM4(-/-) mice was significantly higher than that in neutrophils from wild-type littermate mice. The activities of three serine proteases (neutrophil elastase, cathepsin G, and proteinase 3), which require cathepsin C activity for processing and maturity, were also significantly higher in OLFM4(-/-) neutrophils. The bacterial killing and clearance capabilities observed in OLFM4(-/-) mice that were enhanced relative to wild-type mice were significantly compromised by the additional loss of cathepsin C in mice with OLFM4 and cathepsin C double deficiency. These results indicate that OLFM4 is an important negative regulator of neutrophil bactericidal activity by restricting cathepsin C activity and its downstream granule-associated serine proteases.     

Cathepsin B inactivation attenuates hepatocyte apoptosis and liver damage in steatotic livers after cold ischemia-warm reperfusion injury

Hepatic steatosis predisposes the liver to cold ischemia-warm reperfusion (CI/WR) injury by unclear mechanisms. Because hepatic steatosis has recently been associated with a lysosomal pathway of apoptosis, our aim was to determine whether this cell-death pathway contributes to CI/WR injury of steatotic livers. Wild-type and cathepsin B-knockout (Ctsb(-/-)) mice were fed the methionine/choline-deficient (MCD) diet for 2 wk to induce hepatic steatosis. Mouse livers were stored in the University of Wisconsin solution for 24 h at 4 degrees C and reperfused for 1 h at 37 degrees C in vitro. Immunofluorescence analysis of the lysosomal enzymes cathepsin B and D showed a punctated intracellular pattern consistent with lysosomal localization in wild-type mice fed a standard diet after CI/WR injury. In contrast, cathepsin B and D fluorescence became diffuse in livers from wild-type mice fed MCD diet after CI/WR, indicating that lysosomal permeabilization had occurred. Hepatocyte apoptosis was rare in both normal and steatotic livers in the absence of CI/WR injury but increased in wild-type mice fed an MCD diet and subjected to CI/WR injury. In contrast, hepatocyte apoptosis and liver damage were reduced in Ctsb(-/-) and cathepsin B inhibitor-treated mice fed the MCD diet following CI/WR injury. In conclusion, these findings support a prominent role for the lysosomal pathway of apoptosis in steatotic livers following CI/WR injury.     

Characterization of hemoglobin-hydrolyzing acidic proteinases in human and rat neutrophils

The nature and levels of hemoglobin (Hb)-hydrolyzing acidic proteinases including cathepsin D and cathepsin E, which were most active at pH 3.5-4.0, were enzymatically and immunochemically compared between human and rat neutrophils. By subcellular fractionation and immunoprecipitation with discriminative antibodies specific for each enzyme, cathepsin D was shown to be present in the granular content fraction of both human and rat neutrophils and to account for about 35% of the total Hb-hydrolyzing activity. Cathepsin E was observed mainly in the cytoplasmic fraction of rat neutrophils from peripheral blood and peritoneal exudates and accounted for about 65% of the total activity, but it was not detected in human blood neutrophils. Immunoelectron microscopy on rat neutrophils revealed that cathepsin D was exclusively confined to lysosomes, whereas cathepsin E was localized mainly in the cytoplasmic matrix and often in the perinuclear spaces and the rough endoplasmic reticulum. The non-cathepsin D activity in human neutrophils, which represented about 65% of the total activity, appeared to be due to a serine proteinase, since it was inhibited by diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride and was not inhibited by agents specific for aspartic-, cysteine-, or metallo proteinases. The enzyme(s) responsible for this activity was largely associated with the granular membranes, and a half of it could be described as an integral membrane protein on the basis of phase separation with Triton X-114 at 35 degrees C. The levels of these Hb-hydrolases in gingival crevicular fluid from human chronic inflammatory periodontitis patients were examined in order to clarify their participation in the periodontal tissue breakdown.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and properties of cathepsin D from porcine spleen.

Cathepsin D was purified from porcine spleen to near homogeneity as determined by gel electrophoresis. The isolation scheme involved an acid precipitation of tissue extract, DEAE-cellulose and Sephadex G-200 chromatography, and isoelectric focusing. The end product represented about a 1000-fold purification and about a 10% recovery. The purified enzyme was the major isoenzyme, which represented 60% of cathepsin D present in porcine spleen. Two minor isoenzymes of cathepsin D were present in small amounts. The purified enzyme resembled porcine pepsin in molecular weight (35,000), amino acid composition, and inactivation by specific pepsin inactivators. The pH activity curve of the purified enzyme showed two optima near pH 3 and 4. The relative activities at these optimal pH values were affected by salt concentration. Experimental evidence indicated that the two-optima phenomenon is a property of a single enzyme species.     

Evidence that Dot-dependent and -independent factors isolate the Legionella pneumophila phagosome from the endocytic network in mouse macrophages

Legionella pneumophila survives within macrophages by evading phagosome–lysosome fusion. To determine whether L. pneumophila resides in an intermediate endosomal compartment or is isolated from the endosomal pathway and to investigate what bacterial factors contribute to establishment of its vacuole, we applied a series of fluorescence microscopy assays. The majority of vacuoles, aged 2.5 min to 4 h containing post-exponential phase (PE) L. pneumophila , appeared to be separate from the endosomal pathway, as judged by the absence of transferrin receptor, LAMP-1, cathepsin D and each of four fluorescent probes used to label the endocytic pathway either before or after infection. In contrast, more than 70% of phagosomes that contained Escherichia coli , polystyrene beads, or exponential phase (E) L. pneumophila matured to phagolysosomes, as judged by co-localization with LAMP-1, cathepsin D and fluorescent endosomal probes. Surprisingly, neither bacterial viability nor the putative Dot/Icm transport complex was absolutely required for vacuole isolation; although phagosomes containing either formalin-killed PE wild-type or live PE dotA or dotB mutant L. pneumophila rapidly accumulated LAMP-1, less than 20% acquired lysosomal cathepsin D or fluorescent endosomal probes. Therefore, a Dot-dependent factor(s) isolates the L. pneumophila phagosome from a LAMP-1-containing compartment, and a formalin-resistant Dot-independent activity inhibits vacuolar accumulation of endocytosed material and delivery to the degradative lysosomes.     

Cloning of cDNA for mosquito lysosomal aspartic protease. Sequence analysis of an insect lysosomal enzyme similar to cathepsins D and E.

A cDNA coding for the lysosomal aspartic protease from the mosquito (mLAP) was cloned and sequenced. The mLAP cDNA is 1420 base pairs long with an open reading frame of 387 amino acids. The deduced amino acid sequence contains a signal pre-propeptide sequence of 18 amino acids followed by 369 amino acids with a 35-amino acid putative pro-enzyme domain in the NH2-terminal. The amino acid sequence of mLAP is 92 and 81% similar to human cathepsin D and cathepsin E, respectively. Typical cleavage sites for cathepsin D processing into light and heavy chains are lacking in mLAP. A single glycosylation site occurs in the mLAP sequence at a position corresponding to the first glycosylation site of cathepsins D. The mLAP sequence shares putative phosphorylation determinants, which in cathepsins D are linked to the formation of mannose 6-phosphate. In the mosquito fat body, lysosomal enzymes specifically degrade organelles involved in the biosynthesis and secretion of vitellogenin. The mLAP mRNA accumulates to its highest level 24 h after initiation of vitellogenin synthesis and 12 h before the peak of mLAP protein accumulation and its enzymatic activity. Translational regulation of mLAP mRNA may occur. The 5'-untranslated region of mLAP mRNA is similar to elements conferring negative translational control by steroids.     

Low temperature blocks transport and sorting of cathepsin D in fibroblasts

The transport of newly synthesized cathepsin D in fibroblasts at 16-28 degrees C was compared to that at 37 degrees C. At 37 degrees C newly synthesized cathepsin D passes the trans Golgi within 30-60 min, becomes segregated from the secretory route into prelysosomal organelles within 1-2 h and processed to mature forms in dense lysosomes within 1.5-3 h after synthesis. The small fraction of cathepsin D that escapes transport into lysosomes is secreted within less than 2 h. At 16-28 degrees C the transport of cathepsin D to lysosomes is inhibited in a temperature-dependent manner. At 16-28 degrees C cathepsin D precursors are slowly transported to the trans Golgi. The cathepsin D precursors accumulate at a site that is in continuity with the secretory pathway and located within or distal of the trans Golgi and proximal to the site where cathepsin D precursors leave the secretory pathway as complexes with mannose 6-phosphate receptors. The arrest at this site is not complete. The receptor-dependent segregation of the cathepsin D precursors released from the block is impaired at less than or equal to 26 degrees C. The inhibition of segregation results in an increased, albeit retarded secretion of cathepsin D. The fraction of cathepsin D precursors that is segregated from the secretory pathway encounters a further low temperature block in prelysosomal organelles. There cathepsin D precursors are proteolytically processed to an intermediate form, which accumulates transiently.(ABSTRACT TRUNCATED AT 250 WORDS)     

Overexpression of cathepsin K in mice decreases collagen deposition and lung resistance in response to bleomycin-induced pulmonary fibrosis

Background

Lung fibrosis is a devastating pulmonary disorder characterized by alveolar epithelial injury, extracellular matrix deposition and scar tissue formation. Due to its potent collagenolytic activity, cathepsin K, a lysosomal cysteine protease is an interesting target molecule with therapeutic potential to attenuate bleomycin-induced pulmonary fibrosis in mice. We here tested the hypothesis that over-expression of cathepsin K in the lungs of mice is protective in bleomycin-induced pulmonary fibrosis.

Methods

Wild-type and cathepsin K overexpressing (cathepsin K transgenic; cath K tg) mice were challenged intratracheally with bleomycin and sacrificed at 1, 2, 3 and 4 weeks post-treatment followed by determination of lung fibrosis by estimating lung collagen content, lung histopathology, leukocytic infiltrates and lung function. In addition, changes in cathepsin K protein levels in the lung were determined by immunohistochemistry, real time RT-PCR and western blotting.

Results

Cathepsin K protein levels were strongly increased in alveolar macrophages and lung parenchymal tissue of mock-treated cathepsin K transgenic (cath K tg) mice relative to wild-type mice and further increased particularly in cath K tg but also wild-type mice in response to bleomycin. Moreover, cath K tg mice responded with a lower collagen deposition in their lungs, which was accompanied by a significantly lower lung resistance (R_L) compared to bleomycin-treated wild-type mice. In addition, cath K tg mice responded with a lower degree of lung fibrosis than wild-type mice, a process that was found to be independent of inflammatory leukocyte mobilization in response to bleomycin challenge.

Conclusion

Over-expression of cathepsin K reduced lung collagen deposition and improved lung function parameters in the lungs of transgenic mice, thereby providing at least partial protection against bleomycin-induced lung fibrosis.