mRNA Expression Of Cathepsin Research Articles

Sulindac (K-80003) with nab-paclitaxel and gemcitabine overcomes drug-resistant pancreatic cancer

The Nab-paclitaxel combined with gemcitabine (AG) regimen is the main chemotherapy regimen for pancreatic cancer, but drug resistance often occurs. Currently, the ability to promote sensitization in drug-resistant cases is an important clinical issue, and the strategy of repurposing conventional drugs is a promising strategy. This study aimed to identify a classic drug that targets chemotherapy resistance’s core signaling pathways and combine it with the AG regimen to enhance chemosensitivity. We also aimed to find reliable predictive biomarkers of drug combination sensitivity. Using RNA sequencing, we found that abnormal PI3K/Akt pathway activation plays a central role in mediating resistance to the AG regimen. Subsequently, through internal and external verification of randomly selected AG-resistant patient-derived organoid (PDO) and PDO xenograft models, we discovered for the first time that the classic anti-inflammatory drug sulindac K-80003, an inhibitor of the PI3K/Akt pathway that we focused on, promoted sensitization in half (14/28) of AG-resistant pancreatic ductal adenocarcinoma cases. Through RNA-sequencing, multiplex immunofluorescent staining, and immunohistochemistry experiments, we identified cFAM124A as a novel biomarker through which sulindac K-80003 promotes AG sensitization. Its role as a sensitization marker is explained via the following mechanism: cFAM124A enhances both the mRNA expression of cathepsin L and the activity of the cathepsin L enzyme. This dual effect stimulates the cleavage of RXRα, leading to large amounts of truncated RXRα, which serves as a direct target of K-80003. Consequently, this process results in the pathological activation of the PI3K/Akt pathway. In summary, our study provides a new treatment strategy and novel biological target for patients with drug-resistant pancreatic cancer.

Osteogenesis, Osteoclastogenesis and their Crosstalk in Lipopolysaccharide-induced Periodontitis in Mice.

To determine the crosstalk of osteogenesis and osteoclastogenesis of alveolar bone in lipopolysaccharide (LPS)-induced periodontitis in mice. A representative periodontitis model was established by treating mice with LPS, and osteoblasts and osteoclasts were cultured. Osteoblasts and osteoclasts were cocultured to determine the effects of LPS on the crosstalk of osteogenesis and osteoclastogenesis. Quantitative polymerase chain reaction (qPCR) was performed to determine the expression of osteoclastogenesis makers underlying the potential mechanisms. The morphological and pathological changes in alveolar bone were observed in LPSinduced mice and LPS dose-dependently suppressed osteogenesis. The mRNA expression of cathepsin K, as a marker of osteoclasts, was accordingly downregulated in the coculture. The mRNA expression of osteoprotegerin was increased, while that of receptor activator of nuclear factor-κB ligand (RANKL) was decreased with an increased concentration of LPS. Moreover, the mRNA expression of toll-like receptor 4 (TLR4) was upregulated by LPS, whereas TLR4 knockout partially recovered osteoclast differentiation in the upper layer of the coculture. LPS dose-dependently suppressed osteogenesis but had a bidirectional effect on osteoclastogenesis. The combined effects of LPS on osteogenesis, osteoclastogenesis and their crosstalk via TLR4 account for alveolar bone loss in periodontitis.

Oxidized Black Phosphorus Nanosheets as an Inorganic Antiresorptive Agent

Black phosphorus (BP) is a newly discovered two-dimensional material that has promising applications from bioelectronics to biomedicine. However, facile oxidation of BP leads to changes in surface ...

Pila globosa snail extract inhibits osteoclast differentiation via downregulation of nuclear factor κB and nuclear factor of activated T-Cells c1 signaling pathways

Background: Osteoporosis, a skeletal disease, that leads to increased fracture risk, features an enhanced osteoclast formation and bone resorption. Identification of agents that modulate aberrant osteoclast formation and function is important for the treatment of osteoporosis. Objective: The current study describes for the first time that Pila globosa snail extract inhibits osteoclastogenesis in vitro and thus suppresses bone loss in ovariectomy-induced rat model. Materials and Methods: Ovariectomized (Ovx) rats were treated with P. globosa snail extract and compared with sham, Ovx, and Ovx treated with zoledronate groups. Serum levels of C-terminal crosslinking telopeptides of type-1 collagen (CTX-1), TRAP5b, and antioxidant markers were determined. mRNA expressions of cathepsin K (CTSK), TRAP, calcitonin receptor (CTR), and matrix metalloproteinase 9 (MMP-9) were also assessed. Immunoblots of nuclear factor of activated T-cells c1 (NFATc1), c-Fos, TNF receptor-associated factor 6, c-Jun, and nuclear factor κB (NFκB) proteins were analyzed. Results and Discussion: P. globosa snail extract induced a decrease in the activation of NFκB, c-Fos, and NFATc1, which resulted in the downregulation of target genes, CTSK, TRAP, CTR, and MMP-9. P. globosa snail extract decreased the serum markers of bone resorption, C-terminal telopeptides of type-1 collagen (CTX-1), and tartrate-resistant acid phosphatase 5b (TRAP5b), reflecting the reduced number and activity of osteoclasts. Moreover, the results also suggested that the protective effect of P. globosa snail extract against osteoporosis is associated with the reduction of oxidative stress as evidenced by decreased malondialdehyde and increased serum antioxidant markers, superoxide dismutase, catalase, and glutathione. Conclusion: The upshot of the study suggests that the P. globosa snail extract represents a potential treatment option against osteolytic bone disease.

Sequential alteration of microglia and astrocytes in the rat thalamus following spinal nerve ligation

BackgroundSpinal reactive astrocytes and microglia are known to participate to the initiation and maintenance of neuropathic pain. However, whether reactive astrocytes and microglia in thalamic nuclei that process sensory-discriminative aspects of pain play a role in pain behavior remains poorly investigated. Therefore, the present study evaluated whether the presence of reactive glia (hypertrophy, increased number and upregulation of glial markers) in the ventral posterolateral thalamic nucleus (VPL) correlates with pain symptoms, 14 and 28 days after unilateral L5/L6 spinal nerve ligation (SNL) in rats.MethodsMechanical allodynia and hyperalgesia (von Frey filament stimulation) as well as ambulatory pain (dynamic weight bearing apparatus) were assessed. Levels of nine glial transcripts were determined by quantitative real-time PCR on laser microdissected thalamic nuclei, and levels of proteins were assessed by Western blot. We also studied by immunohistofluorescence the expression of glial markers that label processes (GFAP for astrocytes and iba-1 for microglia) and cell body (S100β for astrocytes and iba-1 for microglia) and quantified the immunostained surface and the number of astrocytes and microglia (conventional counts and optical dissector method of stereological counting).ResultsDifferential, time-dependent responses were observed concerning microglia and astrocytes. Specifically, at day 14, iba-1 immunostained area and number of iba-1 immunopositive cells were decreased in the VPL of SNL as compared to naïve rats. By contrast, at day 28, GFAP-immunostained area was increased in the VPL of SNL as compared to naïve rats while number of GFAP/S100β immunopositive cells remained unchanged. Using quantitative real-time PCR of laser microdissected VPL, we found a sequential increase in mRNA expression of cathepsin S (day 14), fractalkine (day 28), and fractalkine receptor (day 14), three well-known markers of microglial reactivity. Using Western blot, we confirmed an increase in protein expression of fractalkine receptor at day 14.ConclusionsOur results demonstrate a sequential alteration of microglia and astrocytes in the thalamus of animals with lesioned peripheral nerves. Furthermore, our data report unprecedented concomitant molecular signs of microglial activation and morphological signs of microglial decline in the thalamus of these animals.

Meta-Analysis of Large-Scale Toxicogenomic Data Finds Neuronal Regeneration Related Protein and Cathepsin D to Be Novel Biomarkers of Drug-Induced Toxicity.

Undesirable toxicity is one of the main reasons for withdrawing drugs from the market or eliminating them as candidates in clinical trials. Although numerous studies have attempted to identify biomarkers capable of predicting pharmacotoxicity, few have attempted to discover robust biomarkers that are coherent across various species and experimental settings. To identify such biomarkers, we conducted meta-analyses of massive gene expression profiles for 6,567 in vivo rat samples and 453 compounds. After applying rigorous feature reduction procedures, our analyses identified 18 genes to be related with toxicity upon comparisons of untreated versus treated and innocuous versus toxic specimens of kidney, liver and heart tissue. We then independently validated these genes in human cell lines. In doing so, we found several of these genes to be coherently regulated in both in vivo rat specimens and in human cell lines. Specifically, mRNA expression of neuronal regeneration-related protein was robustly down-regulated in both liver and kidney cells, while mRNA expression of cathepsin D was commonly up-regulated in liver cells after exposure to toxic concentrations of chemical compounds. Use of these novel toxicity biomarkers may enhance the efficiency of screening for safe lead compounds in early-phase drug development prior to animal testing.

Characterization of cathepsin B gene from orange-spotted grouper, Epinephelus coioides involved in SGIV infection

The lysosomal cysteine protease cathepsin B of papain family is a key regulator and signaling molecule that involves in various biological processes, such as the regulation of apoptosis and activation of virus. In the present study, cathepsin B gene (Ec-CB) was cloned and characterized from orange-spotted grouper, Epinephelus coioides. The full-length Ec-CB cDNA was composed of 1918 bp and encoded a polypeptide of 330 amino acids with higher identities to cathepsin B of teleosts and mammalians. Ec-CB possessed typical cathepsin B structural features including an N-terminal signal peptide, the propeptide region and the cysteine protease domain which were conserved in other cathepsin B sequences. Phylogenetic analysis revealed that Ec-CB was most closely related to Lutjanus argentimaculatus. RT-PCR analysis showed that Ec-CB transcript was expressed in all the examined tissues which abundant in spleen, kidney and gill. After challenged with Singapore grouper iridovirus (SGIV) stimulation, the mRNA expression of cathepsin B in E. coioides was up-regulated at 24 h post-infection. Subcellular localization analysis revealed that Ec-CB was distributed predominantly in the cytoplasm. When the fish cells (GS or FHM) were treated with the cathepsin B specific inhibitor CA-074Me, the occurrence of CPE induced by SGIV was delayed, and the viral gene transcription was significantly inhibited. Additionally, SGIV-induced typical apoptosis was also inhibited by CA-074Me in FHM cells. Taken together, our results demonstrated that the Ec-CB might play a functional role in SGIV infection.

Cetylpyridinium Chloride Inhibits Receptor Activator of Nuclear Factor-κB Ligand-Induced Osteoclast Formation

Osteoclasts are responsible for bone erosion in diseases as diverse as osteoporosis, periodontitis, and rheumatoid arthritis. Antiseptic products have received recent attention as potential therapeutic and preventive drugs in human disease. The purpose of this study was to investigate the effect of the antiseptic cetylpyridinium chloride (CPC) on osteoclast formation using mouse bone marrow-derived macrophages (BMMs). CPC inhibited receptor activator of nuclear factor (NF)-κB ligand (RANKL)-induced osteoclast formation in a dose-dependent manner without causing cytotoxicity. The mRNA expression of cathepsin K, calcitonin receptor (CTR), and Prdm1 in osteoclasts was reduced by CPC. In experiments to elucidate its mechanism of action, CPC was found to suppress RANKL-induced expression of c-Fos and nuclear factor of activated T cells (NFATc1), transcription factors that are essential for osteoclast differentiation. CPC also inhibited RANKL-induced activation of extracellular signal-regulated kinase (ERK) and NF-κB and expression of cyclooxygenase (COX)-2. These results collectively suggest that CPC inhibits osteoclast differentiation by suppressing the activation of ERK and NF-κB and reducing the expression of COX-2, c-Fos, and NFATc1. CPC may therefore be a useful drug in the prevention of bone loss.

Cathepsin L in the orange-spotted grouper, Epinephelus coioides: molecular cloning and gene expression after a Vibrio anguillarum challenge

The orange-spotted grouper, Epinephelus coioides, is an important fish maricultured in many Asian countries. In the present study, the full-length cDNA of cathepsin L, an immunity related gene of fishes, was isolated from E. coioides using rapid amplification of cDNA ends (RACE). It is 1,443 bp in length, including an open reading frame (ORF) of 1,011 bp. The open reading frame encoded a preproprotein of 336 amino acids (aa), which consisted of a signal peptide of 16 aa, a proregion peptide of 98 aa and a mature peptide of 222 aa. The preproprotein contained an oxyanion hole (Gln), a catalytic triad formed by Cys, His and Asn, and the conserved ERWNIN, GNFD and GCNGG motifs, all characteristic of cathepsin L. Homology analysis revealed that the deduced amino acid sequence of E. coioides cathepsin L shared 80.1-94.8 % identity with those of reported fishes. Tissue-dependent mRNA expression analysis showed that the cathepsin L transcript was expressed in all the examined tissues of the healthy E. coioides, being highest in the liver and moderate in the heart, gonad and intestine. After Vibrio anguillarum stimulation, the mRNA expression of cathepsin L in E. coioides was significantly increased in the skin, fin, gills, liver, blood, spleen, head kidney and intestine, with the highest observed in the spleen (10.6-fold) at 12 h post-injection and the next in blood (7.5-fold) at 8 h post-injection. These results provided initial information for further studies on the physiological and immunological roles of the cathepsin L gene in the orange-spotted grouper.

AVL-292, a Bruton's Tyrosine Kinase Inhibitor Impacts Bone Resorption by Abrogating Osteoclast Sealing Zone Formation in Multiple Myeloma

Abstract 1822The activation of Bruton's tyrosine kinase (Btk), a member of the Tec family of tyrosine kinases regulates B-cell development and activation, and plays a key role in antibody production. Interestingly, Btk and the other Tec kinase family, Tec, regulate OC differentiation via Receptor Activator of Nuclear Factor κ B (RANK) signaling. Moreover, patients with X-linked agammaglobulinemia (XLA) who harbor Btk null mutations have impaired OC function.Here we show that, a potent and specific inhibitor of Btk, AVL-292, inhibits OC function in MM patients. AVL-292 is a highly selective, covalent Btk inhibitor that potently silences Btk enzymatic activity (IC50 < 0.5nM) with high selectivity towards Btk with lack of significant inhibition against other kinases involved in BCR signaling (Syk, Lyn). We examined the mechanism of action of AVL-292 in the context of OCs function. OC derived from MM patient monocytes were assayed with or without AVL-292. Interestingly, OC function was inhibited in the presence AVL-292 as demonstrated by pit formation assay. However, mRNA expression of Cathepsin K and TRAP, markers of OC differentiation, were increased in the presence of AVL-292. These data suggest AVL-292 inhibits OC function without affecting the OC differentiation.It has been shown that BTK and Tec regulation of OC differentiation is related to calcium (Ca2+) signaling by increasing Ca2+ flux through direct phosphorylation of phospholipase C2 (PLCγ2). To delineate the mechanism of action of the BTK inhibitor against OC function, the RANK signaling proteins were detected by western blotting and Ca2+ concentration was measured by flourescence. AVL-292 inhibited phosphorylation of the BTK substrate, PLCγ2 in OCs. This was associated with an inhibition of intracellular Ca2+ release by AVL-292 which otherwise increased with RANKL stimulation in OCs.Given the effect of AVL-292 on RANK signaling we next evaluated its effect on Proline-rich tyrosine Kinase 2 (Pyk2) and c-Src. Pyk2 plays a role in OC activation and localizes to the sealing zone in OC. RANK signaling activates c-Src, which phosphorylates Pyk2. Moreover c-Src controls OC bone resorption by regulating actin organization via cortactin. Interestingly, AVL-292 inhibited c-Src and Pyk2 phosphorylation. Furthermore, AVL-292 inhibited cortactin protein and mRNA expression, and upregulated c-Cbl protein (E3 ubiquitin ligase for c-Src) expression in OCs derived from MM patient monocytes.These data demonstrate that the novel BTK inhibitor AVL-292 inhibits OC function through inhibition of OC sealing zone formation. These results suggest a potential novel mechanism of Btk inhibition with AVL-292 on osteoclast function and therefore bone resorption. Disclosures:Evans:Celgene: Employment. Singh:Celgene: Employment. Westlin:Celgene: Employment. Raje:Onyx: Consultancy; Celgene: Consultancy; Millenium: Consultancy; Acetylon: Research Funding; Amgen: Research Funding; Eli-Lilly: Research Funding.

Molecular cloning and mRNA expression of cathepsin C in white shrimp, Litopenaeus vannamei

The cDNA encoding cathepsin C from the haemocytes of Litopenaeus vannamei (designated LVcathepsinC) was cloned by rapid amplification of cDNA ends (RACE) techniques. The full length of LVcathepsinC cDNA was 2026 bp, with an open reading frame of 1356 bp, which encoded a polypepetide of 451 amino acid residues with a theoretical molecular weight of 50.76 kDa and an estimated isoelectric point of 6.01. LVcathepsinC showed high similarity to other organisms on performing blast analysis. Fluorescent quantitative real-time reverse transcriptase-polymerase chain reaction was used to examine the expression of the LVcathepsinC gene in haemocytes of L. vannamei after the challenge of bacteria Vibrio alginolyticus. There was a clear time-dependent expression pattern of LVcathepsinC after the bacterial challenge, and the mRNA expression reached a maximum level at 4 h post challenge, and then returned to the control level after 8 h. The up-regulated mRNA expression of LVcathepsinC in L. vannamei after bacterial challenge indicates that the LVcathepsinC gene is inducible and may be involved in immune response.

Mineral Trioxide Aggregate Inhibits Osteoclastic Bone Resorption

Mineral trioxide aggregate (MTA), a commonly used endodontic repair material, is useful for both basic and clinical research, and the effect of MTA on osteoblast differentiation has been well-defined. However, the effects of MTA on osteoclastic bone resorption are not fully understood. Hence, the aim of this study is to examine the effect of MTA solution in the regulation of osteoclast bone-resorbing activity using osteoclasts formed in co-cultures of primary osteoblasts and bone marrow cells. MTA solution dose-dependently reduced the total area of pits formed by osteoclasts. The reduction of resorption induced by 20% MTA treatment was due to inhibition of osteoclastic bone-resorbing activity and had no effect on osteoclast number. A 20% MTA solution disrupted actin ring formation, a marker of osteoclastic bone resorption, by reducing phosphorylation and kinase activity of c-Src, and mRNA expressions of cathepsin K and mmp-9. A high concentration of MTA solution (50%) induced apoptosis of osteoclasts by increasing the expression of Bim, a member of the BH3-only (Bcl-2 homology) family of pro-apoptotic proteins. Taken together, our results suggest that MTA is a useful retrofilling material for several clinical situations because it both stimulates osteoblast differentiation and inhibits bone resorption.

Expression of Cathepsins in Human Skin Photoaging

Cathepsins are involved in regulatory mechanisms in human skin, but their role in photoaged skin remains unknown. This study investigates the role of cathepsin B, D, K, and G in skin photoaging in vivo and in vitro. Cathepsin-induced changes in skin as a result of chronic UV irradiation were detected by immunohistochemistry methods. Protein cathepsin expressions in UVA-induced premature senescence in fibroblasts in vitro were detected by Western blot technique. Cathepsin mRNA expression in photoaged skin and fibroblasts was detected by real-time reverse transcription-polymerase chain reaction. Immunohistochemistry and Western blot show lower protein expression of cathepsin B, D, and K in photoaged skin and fibroblasts, while cathepsin G was higher. The mRNA expression of cathepsin B, D, and K of the photoaged skin in vivo decreased to 20 ± 0.5, 25 ± 1.6 and 22 ± 0.8%, while cathepsin G mRNA increased to 2.24 ± 0.09 times that of control. In photoaged fibroblasts, cathepsin B, D, and K mRNA was downregulated to 64 ± 2.9, 24 ± 2.1 and 9 ± 0.5% while cathepsin G mRNA was upregulated to 1.42 ± 0.06 times that of control fibroblasts. These experiments suggest that cathepsin B, D, K, and G may act as biomarkers in photoaged human skin.

Expressions of cathepsin B in photoaging skin and its significance.

Objective To investigate the expression of cathepsin B in photoaging skin and its signifi-cance.Methods Skin specimens were obtained from the right forearm(sun-exposed sites)and buttock (unexposed sites)of 6 healthy volunteers with informed consent and subjected to immunohistochemistry for the detection of cathepsin B expression.Primary human fibroblasts derived from the prepuce of children aged 3 to 6 years were cultured in vitro;after 10 to 15 passages,cells were divided into four groups to be treated with methoxsalen of 50 mg/L for 24 hours followed by ultraviolet A(UVA)exposure(premature senescence group),phosphate buffered saline(PBS)only(control group),UVA exposure only(UVA group),methoxsalen only(methoxsalen group).Then,the protein and mRNA expressions of cathepsin B were detected by Western blot and RT-PCR,respectively,in these fibroblasts 1,2,3 weeks after the treatment.Results Cathepsin B was observed in both exposed and unexposed sites of all volunteers,and the average absorbence of cathepsin B was significantly lower in exposed sites than in unexposed sites(0.2130±0.7997 vs 0.4520±0.5921,t=5.37,P<0.05).Decreased protein expression of cathepsin B was also noted in the premature senescence group compared with the other three groups.Moreover,the gray ratio between cathepsin B protein and glyceraldehyde phosphate dehydrogenase(GAPDH)in premature senescence group reduced from 28.099±O.054 before treatment to 25.1 03±0.102 in week 1 and 17.693±0.099 in week 3 after UVA exposure.RT-PCR revealed that the mRNA expression level of cathepsin B in fibroblasts of premature senescence group decreased by 36 percent compared with that in the control group.Conclusions The expression of cathepsin B decreases in photoaged skin as well as in UVA-exposed fibroblasts in a time-dependent manner,which may be associated with the self-repair of photoaged skin. Key words: Skin aging; Cathepsin B; Fibroblasts

Molecular cloning and mRNA expression of cathepsin C gene in black tiger shrimp ( Penaeus monodon)

Cathepsin C (dipeptidyl-peptidase I, DPPI) is a lysosomal cysteine proteinase belonging to the papain superfamily, which is capable of removing dipeptides sequentially from the amino terminus of peptide and protein substrates. In the present study, the cDNA of a cathepsin C was cloned from black tiger shrimp Penaeus monodon (designated PmcathepsinC) by homology cloning and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of PmcathepsinC consisted of 2051 nucleotides with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, and an open reading frame (ORF) of 1350 bp encoding a polypeptide of 449 amino acid residues with a predicted molecular weight of 50.0 kDa and theoretical isoelectric point of 5.65. The high identity of PmcathepsinC with Cathepsin C in other organisms indicated that PmcathepsinC should be a new member of the Cathepsin C family. By fluorescent quantitative real-time PCR, mRNA transcript of PmcathepsinC was detectable in all the examined tissues with higher level in ovary and heart. The temporal expression of PmcathepsinC mRNA in the hepatopancreas was up-regulated by lipopolysaccharide (LPS) stimulation and reached the maximum level at 4 h post-stimulation, and then dropped back to the original level gradually. These results indicated that PmcathepsinC was a constitutive and inducible acute-phase protein that perhaps involved in the immune defense of P. monodon.

CDNA cloning, mRNA expression and recombinant expression of a cathepsin L-like cysteine protease from Neobenedenia melleni (Monogenea: Capsalidae)

The monogenean Neobenedenia melleni is a worldwide virulent pathogen to many species of sea-caged fishes. Its outbreaks have caused fearful economic losses in fish mariculture. Cysteine proteases play vital biological roles in various important physiological and pathological processes. In the present paper the full-length cDNA of cathepsin L-like cysteine protease was isolated from the N. melleni using rapid amplification of cDNA ends (RACE). It is 1070 bp long and contains an open reading frame (ORF) of 1008 bp. The prepro-cathepsin L consists of a signal peptide of 17 amino acids (aa), a pro-region peptide of 100 aa and a mature peptide of 218 aa. The consensus motifs flanking conserved catalytic active sites (C 25, H 159 and N 175, papain numbering), which are present in all papain-like cysteine proteases, were identified in the sequence. The cathepsin L gene comprises three introns and four exons. mRNA expression of cathepsin L was determined using semi-quantitative RT-PCR analysis. The cathepsin L mRNA was expressed in oncomiracidia, larvae and adult, but not in newly shed eggs and encysted oncomiracidia. The coding region of cathepsin L cDNA for the mature cathepsin L protein was subcloned into an expression plasmid pET-22b(+) and fused with a 6-His-Tag. Moreover, we have successfully developed an expression system in Escherichia coli to overproduce the recombinant cathepsin L of N. melleni. The present study is the first report of the cloning and description of cDNA encoding a cysteine protease from a monogenean, and provides essential information for further studies at the protein level, such as the function, regulation mechanism and possibility for use as a target molecule for an anti-monogenean drug.

Cathepsin L Activity Is Increased in Alveolar Macrophages and Bronchoalveolar Lavage Fluid of Smokers

Elastinolytic enzymes derived from alveolar macrophages (AM) are considered to play an important role in the development of emphysema associated with cigarette smoking. In this study, the enzyme activity and mRNA expression of cathepsin L were quantitated in AM and bronchoalveolar lavage (BAL) fluid obtained from current smokers and compared with those from nonsmokers. Activity was measured with the synthetic substrate Z-Phe-Arg-MCA combined with a novel cathepsin B inhibitor, CA-074. We found that the specific activity of cathepsin L was significantly elevated in BAL cells from smokers (7.1 +/- 0.7 mumol/mg protein/h, mean +/- SEM) compared with cells from nonsmokers (2.9 +/- 0.3) (p < 0.01). The expression of cathepsin L mRNA in BAL cells as determined by dot-blot analysis was also higher in BAL cells from smokers, which was comparable to the increase in the enzyme activity. About 5 to 6% of the specific activity of cathepsin L in BAL cell lysates was detected in unconcentrated BAL fluid; specific activity was also significantly higher in samples from smokers (0.38 +/- 0.04 mumol/mg protein/h) than from nonsmokers (0.14 +/- 0.02). In addition, procathepsin L (42 kD) and the mature form of cathepsin L (33 kD) were demonstrated in BAL fluid by immunoblot analyses. These data suggest that cigarette smoking induces mRNA expression and the synthesis of cathepsin L in AM and the release of procathepsin from AM into extracellular milieu. Furthermore, increased activity levels of cathepsin L in extracellular compartments may contribute to the proteolysis of elastin in the process of lung destruction associated with cigarette smoking.