Pharmacological inhibitors to identify roles of cathepsin K in cell-based studies: a comparison of available tools

2009 ◽  
Vol 390 (9) ◽  
Author(s):  
Sylvie Desmarais ◽  
Frédéric Massé ◽  
M. David Percival
Keyword(s):  
Human Cell ◽  
Type I Collagen ◽  
Cathepsin K ◽  
The Other ◽  
Enzyme Assays ◽  
Type I ◽  
Treatment Of Osteoporosis ◽  
Cysteine Cathepsins ◽  
Rodent Cell ◽  
Enzyme Selectivity

Abstract Cathepsin K (Cat K) degrades bone type I collagen and is a target for the pharmacological treatment of osteoporosis. Further roles for Cat K have been recently described, some of which are supported by the use of purportedly selective Cat K inhibitors in human and rodent cell-based assays. Twelve commercial and non-commercial Cat K inhibitors were profiled against a panel of purified human, rat, and mouse cysteine cathepsins and in two cell-based enzyme occupancy assays for activity against Cat K, B, and L. Ten inhibitors, including the carbohydrazide Cat K inhibitor II (Boc-Phe-Leu-NHNH-CO-NHNH-Leu-Z), the non-covalent K4b, and the epoxide NC-2300, have either little Cat K selectivity, or appear poorly cell penetrant. The amino-acetonitrile-containing inhibitors L-873724 and odanacatib show greater than 100-fold human Cat K enzyme selectivity and have similar IC50 values against each cathepsin in cell-based and enzyme assays. The basic inhibitor balicatib has greater cellular potencies than expected on the basis of purified enzyme assays. The accumulation of [14C]-balicatib in fibroblasts is blocked by prior treatment of the cells with NH4Cl, consistent with balicatib having lysosomotropic properties. These results support the use of L-873724 and odanacatib as tools to identify novel roles for Cat K using human cell-based systems, but suggest using caution in the interpretation of studies employing the other compounds.

Zinc Inhibits Collagenolysis by Cathepsin K and Matrix Metalloproteinases in Demineralized Dentin Matrix

2017 ◽  
Vol 51 (6) ◽  
pp. 576-581 ◽  
Author(s):  
Pinar Altinci ◽  
Roda Seseogullari-Dirihan ◽  
Gulsen Can ◽  
David Pashley ◽  
Arzu Tezvergil-Mutluay
Keyword(s):  
Matrix Metalloproteinases ◽  
Mass Loss ◽  
Type I Collagen ◽  
Cathepsin K ◽  
Collagen Degradation ◽  
Type I ◽  
Level Control ◽  
Physiological Level ◽  
Cysteine Cathepsins ◽  
Demineralized Dentin

The enzymatic degradation of dentin organic matrix occurs via both the action of matrix metalloproteinases (MMPs) and cysteine cathepsins (CCs). Zinc can prevent collagen hydrolysis by MMPs. However, its effect on the activity of dentin-bound CCs is not known. The aim of this study was to investigate the effect of zinc on matrix-bound cathepsin K and MMP activity in dentin. Completely demineralized dentin beams were divided into test groups (n = 9) and incubated at 37°C in an incubation media (1 mL) containing ZnCl2 of 0.02 (physiological level, control), 0.2, 0.5, 1, 5, 10, 20, 30, or 40 mM. The dry mass changes of the beams were determined, and incubation media were analyzed for cathepsin K- and MMP-specific collagen degradation end products - CTX (C-terminal cross-linked telopeptide of type I collagen) and ICTP (cross-linked carboxy-terminal telopeptide of type I collagen) - at 1, 3, and 7 days of incubation. The mass loss of the beams decreased when the zinc level in the incubation media was ≥5 mM (p < 0.05). The release of liberated collagen degradation telopeptides decreased in accordance with the decrease in the mass loss rates of the beams. Cathepsin K-induced dentin collagen degradation can be strongly inhibited by zinc. Zinc levels of ≥5 mM can be considered as a reliable threshold for the stabilization of dentin matrices.

Type I Collagen Biosynthesis by Skin Fibroblasts from Patients with Idiopathic Juvenile Osteoporosis

1995 ◽  
Vol 89 (1) ◽  
pp. 69-73 ◽  
Author(s):  
Andrew E. Pocock ◽  
Martin J. O. Francis ◽  
Roger Smith
Keyword(s):  
Osteogenesis Imperfecta ◽  
Type I Collagen ◽  
The Other ◽  
Type I ◽  
Osteogenesis Imperfecta Type ◽  
Unrelated Control ◽  
Type Iii ◽  
Collagen Peptides ◽  
Idiopathic Juvenile Osteoporosis ◽  
Osteogenesis Imperfecta Type I

1. Skin fibroblast lines were cultured from nine patients who had the features of idiopathic juvenile osteoporosis, six relatives, five unrelated control subjects and three unrelated patients with osteogenesis imperfecta type I. Some patients with idiopathic juvenile osteoporosis were adults whose previous osteoporosis was in remission. Two patients with idiopathic juvenile osteoporosis were siblings and one patient with idiopathic juvenile osteoporosis had a daughter with severe osteogenesis imperfecta (type III). 2. The ratio of type III to type I collagen, synthesized by fibroblasts, was increased in two of the patients with osteogenesis imperfecta type I and in the daughter with osteogenesis imperfecta type III, but was normal in all the other patients with idiopathic juvenile osteoporosis and the other relatives. 3. Radiolabelled collagen was digested by cyanogen bromide and separated on SDS-PAGE. Unreduced collagen peptides migrated normally, except those from both the two siblings with idiopathic juvenile osteoporosis. In these two lines, abnormal migration suggested the presence of collagen I mutations. 4. The secretion of synthesized collagen by these two idiopathic juvenile osteoporosis lines and two others was reduced to only 43–45% as compared with a line from a 13-year-old control subject, which was defined as 100%. The three osteogenesis imperfecta type I lines secreted 18–37%, the other five idiopathic juvenile osteoporosis lines secreted 57–75%, the relatives (including the daughter with severe osteogenesis imperfecta) secreted 49–115% and the controls secreted 69–102%. 5. We conclude that qualitative abnormalities of type I collagen associated with a reduction in total secreted collagen synthesis may occur in a minority of patients with idiopathic juvenile osteoporosis; these patients could represent a subset of patients with this disorder.

Cells in regenerating deer antler cartilage provide a microenvironment that supports osteoclast differentiation

2001 ◽  
Vol 204 (3) ◽  
pp. 443-455
Author(s):  
C. Faucheux ◽  
S. Nesbitt ◽  
M. Horton ◽  
J. Price
Keyword(s):  
Type I Collagen ◽  
Mononuclear Cells ◽  
Osteoclast Differentiation ◽  
Cathepsin K ◽  
Collagen Matrix ◽  
Tartrate Resistant Acid Phosphatase ◽  
Type I ◽  
Deer Antler

Deer antlers are a rare example of mammalian epimorphic regeneration. Each year, the antlers re-grow by a modified endochondral ossification process that involves extensive remodelling of cartilage by osteoclasts. This study identified regenerating antler cartilage as a site of osteoclastogenesis in vivo. An in vitro model was then developed to study antler osteoclast differentiation. Cultured as a high-density micromass, cells from non-mineralised cartilage supported the differentiation of large numbers of osteoclast-like multinucleated cells (MNCs) in the absence of factors normally required for osteoclastogenesis. After 48 h of culture, tartrate-resistant acid phosphatase (TRAP)-positive mononuclear cells (osteoclast precursors) were visible, and by day 14 a large number of TRAP-positive MNCs had formed (783+/−200 per well, mean +/− s.e.m., N=4). Reverse transcriptase/polymerase chain reaction (RT-PCR) showed that receptor activator of NF κ B ligand (RANKL) and macrophage colony stimulating factor (M-CSF) mRNAs were expressed in micromass cultures. Antler MNCs have the phenotype of osteoclasts from mammalian bone; they expressed TRAP, vitronectin and calcitonin receptors and, when cultured on dentine, formed F-actin rings and large resorption pits. When cultured on glass, antler MNCs appeared to digest the matrix of the micromass and endocytose type I collagen. Matrix metalloproteinase-9 (MMP-9) may play a role in the resorption of this non-mineralised matrix since it is highly expressed in 100 % of MNCs. In contrast, cathepsin K, another enzyme expressed in osteoclasts from bone, is only highly expressed in resorbing MNCs cultured on dentine. This study identifies the deer antler as a valuable model that can be used to study the differentiation and function of osteoclasts in adult regenerating mineralised tissues.

scholarly journals Sequential, but not Concurrent, Incubation of Cathepsin K and L with Type I Collagen Results in Extended Proteolysis

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Akia N. Parks ◽  
Juhi Nahata ◽  
Naomi-Eliana Edouard ◽  
Johnna S. Temenoff ◽  
Manu O. Platt
Keyword(s):  
Type I Collagen ◽  
Cathepsin K ◽  
Type I

scholarly journals The Capacity of Periodontal Gel to Occupy the Spaces Inside the Periodontal Pockets Using Computational Fluid Dynamic

2019 ◽  
Vol 8 (1) ◽  
pp. 1
Author(s):  
Luca Levrini ◽  
Luigi Paracchini ◽  
Maria Giulia Nosotti
Keyword(s):  
Rheological Properties ◽  
Computational Fluid Dynamic ◽  
Type I Collagen ◽  
Saline Solution ◽  
Fluid Dynamic ◽  
The Other ◽  
Periodontal Pocket ◽  
Type I ◽  
Cat Scan ◽  
Different Types

The aim of the current work is to demonstrate the capacity of a new periodontal gel to occupy the spaces inside the periodontal pockets through Computational Fluid Dynamic (CFD). The test gel consists of two resorbable medical grade polymers (PEO, Poly Ethylen Oxide and HPMC, Hydroxy Propyl Metyl Cellulose), Type I Collagen, SAP (Vitamin C), and PBS (Saline Solution), while the control gel is 14% doxyclin controlled release gel, which is used for treating periodontal pockets with probing ≥5 mm after scaling and root plaining. The study examined the fluid dynamic analysis (Computational Fluid Dynamic—CFD) of two different gels, used in dentistry to treat periodontitis, in relation to both the geometry of the periodontal pocket and the function of two different types of needles that are used to distribute the preparation. The periodontal pocket was determined by reading DICOM images taken from the patient’s CAT scan. The results show that the H42® gel comes out uniformly compared to the other gel. Moreover, it is possible to observe how the rheological properties of the gel allow the fluid to spread evenly within the periodontal pocket in relation to the geometry of the needle. In particular, H42® gel exits in a constant way both from the first and the second exit. In fact, it was observed that by changing the geometry of the needle or the type of periodontal gel, the distribution of the gel inside the pocket was no longer homogeneous. Thus, having the correct rheological properties and correct needle geometries both speeds up the gel and optimizes the pressure distribution. Currently, the literature is still lacking, therefore further studies will be needed to confirm these results.

Mechanical Stress-Induced Orientation and Ultrastructural Change of Smooth Muscle Cells Cultured in Three-Dimensional Collagen Lattices

1994 ◽  
Vol 3 (6) ◽  
pp. 481-492 ◽  
Author(s):  
Keiichi Kanda ◽  
Takehisa Matsuda
Keyword(s):  
Smooth Muscle ◽  
Tensile Stress ◽  
Smooth Muscle Cells ◽  
Type I Collagen ◽  
Dynamic Stress ◽  
Three Dimensional ◽  
Muscle Cells ◽  
The Other ◽  
Type I ◽  
Oriented Parallel

The effect of tensile stress on the orientation and phenotype of arterial smooth muscle cells (SMCs) cultured in three-dimensional (3D) type I collagen gels was morphologically investigated. Ring-shaped hybrid tissues were prepared by thermal gelation of a cold mixed solution of type I collagen and SMCs derived from bovine aorta. The tissues were subjected to three different modes of tensile stress. They were floated (isotonic control), stretched isometrically (static stress) and periodically stretched and recoiled by 5% above and below the resting tissue length at 60 RPM frequency (dynamic stress). After incubation for up to four wk, the tissues were investigated under a light microscope (LM) and a transmission electron microscope (TEM). Hematoxylin and eosinstained LM samples revealed that, irrespective of static or dynamic stress loading, SMCs in stress-loaded tissues exhibited elongated bipolar spindle shape and were regularly oriented parallel to the direction of the strain, whereas those in isotonic control tissues were polygonal or spherical and had no preferential orientation. In Azan-stained samples, collagen fiber bundles in isotonic control tissues were somewhat retracted around the polygonal SMCs to form a random network. On the other hand, those in statically and dynamically stressed tissues were accumulated and prominently oriented parallel to the stretch direction. Ultrastructural investigation using a TEM showed that SMCs in control and statically stressed tissues were almost totally filled with synthetic organelles such as rough endoplasmic reticulums, free ribosomes, Golgi complexes and mitochondria, indicating that the cells remained in the synthetic phenotype. On the other hand, SMCs in dynamically stressed tissues had increased fractions of contractile apparatus, such as myofilaments, dense bodies and extracellular filamentous materials equivalent to basement membranes, that progressed with incubation time. These results indicate that periodic stretch, in concert with 3-D extracellular collagen matrices, play a significant role in the phenotypic modulation of SMCs from the synthetic to the contractile state, as well as cellular and biomolecular orientation.

scholarly journals Abnormal type I collagen metabolism by cultured fibroblasts in lethal perinatal osteogenesis imperfecta

1984 ◽  
Vol 217 (1) ◽  
pp. 103-115 ◽  
Author(s):  
J F Bateman ◽  
T Mascara ◽  
D Chan ◽  
W G Cole
Keyword(s):  
Osteogenesis Imperfecta ◽  
Cell Lines ◽  
Type I Collagen ◽  
The Other ◽  
Collagen Metabolism ◽  
Structural Defects ◽  
Collagen Molecule ◽  
Type I ◽  
Cultured Fibroblasts ◽  
Post Translational Modifications

Cultured skin fibroblasts from seven consecutive cases of lethal perinatal osteogenesis imperfecta (OI) expressed defects of type I collagen metabolism. The secretion of [14C]proline-labelled collagen by the OI cells was specifically reduced (51-79% of control), and collagen degradation was increased to twice that of control cells in five cases and increased by approx. 30% in the other two cases. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed that four of the OI cell lines produced two forms of type I collagen consisting of both normally and slowly migrating forms of the alpha 1(I)- and alpha 2(I)-chains. In the other three OI cell lines only the ‘slow’ alpha (I)′- and alpha 2(I)′-chains were detected. In both groups inhibition of the post-translational modifications of proline and lysine resulted in the production of a single species of type I collagen with normal electrophoretic migration. Proline hydroxylation was normal, but the hydroxylysine contents of alpha 1(I)′- and alpha 2(I)′-chains purified by h.p.l.c. were greater than in control alpha-chains. The glucosylgalactosylhydroxylysine content was increased approx. 3-fold while the galactosylhydroxylysine content was only slightly increased in the alpha 1(I)′-chains relative to control alpha 1(I)-chains. Peptide mapping of the CNBr-cleavage peptides provided evidence that the increased post-translational modifications were distributed throughout the alpha 1(I)′- and alpha 2(I)′-chains. It is postulated that the greater modification of these chains was due to structural defects of the alpha-chains leading to delayed helix formation. The abnormal charge heterogeneity observed in the alpha 1 CB8 peptide of one patient may reflect such a structural defect in the type I collagen molecule.

scholarly journals Novel roles of glycosaminoglycans in the degradation of type I collagen by cathepsin K

Glycobiology ◽  
2017 ◽  
Vol 27 (12) ◽  
pp. 1089-1098 ◽  
Author(s):  
Yota Tatara ◽  
Shinichiro Suto ◽  
Ken Itoh
Keyword(s):  
Type I Collagen ◽  
Cathepsin K ◽  
Type I

THE STRUCTURE OF PLATELET-REACTIVE SITES IN COLLAGENS

1987 ◽  
Author(s):  
M J Barnes ◽  
C M Fitzsimmons ◽  
L F Morton
Keyword(s):  
Platelet Aggregation ◽  
Type I Collagen ◽  
Quaternary Structure ◽  
The Other ◽  
Type I ◽  
Amino Acid Residues ◽  
Reactive Site ◽  
Parent Molecule ◽  
Type Iii ◽  
Helical Configuration

Collagen-induced platelet aggregation is an essential step in haemostasis and may be important in thrombosis, particularly that associated with the atherosclerotic plaque. We have located platelet-binding sites in collagen by fragmentation of the molecule with cyanogen bromide (CB) and measurement of the platelet aggregatory activity of the fragments following their renaturation to restore triple-helical configuration and polymerisation to introduce quaternary structure. We have found a high1y-reactive site in collagen type III located in the peptide α1(III)CB4 which was active at a concentration of less than 0.5μg/ml. The equivalent peptide from type I collagen α1(I)CB3 occurring in precisely the same location in the respective parent molecule (residues 403-551) and exhibiting a structure highly homologous to that of α1(III)CB4 was active only at concentrations higher than 200μg/ml. α1(I)CB7, the most active of the type I peptides, was able to cause platelet aggregation at around 5μg/ml whilst the equivalent type III peptide μ1(III)CB5 was inactive. Modification of specific amino acid residues indicated the importance of lysine in the activity of μ1(III)CB4 and of arginine in that of α1(I)CB7. Comparison of the structure of these peptides leads us to conclude that a reactive site comprises two basic residues, a specific distance apart, the conformation of one to the other dictated by collagen triple-helical configuration. One residue occurs in the sequence GlyPro(orHyp)LYS(orARG)GlyGlu, the other in GlyLYS(orARG)Pro(orHyp)GlyGlu. The lower reactivity of α1(I)CB7 relative to α1(III)CB4 can be attributed to the presence of two arginyl rather than lysyl residues and because the spacing of the two in CB4(G1y-LYS-Y-G1y-X-Y-G1y-X-LYS) represents a more favourable one than in CB7(Gly-X-ARG-Gly-X-Y-Gly-X-Y-Gly-ARG-Y).

scholarly journals Oral Administration of Quercetin or Its Derivatives Inhibit Bone Loss in Animal Model of Osteoporosis

2020 ◽  
Vol 2020 ◽  
pp. 1-21
Author(s):  
Yue-Yue Huang ◽  
Zi-Hao Wang ◽  
Li-Hui Deng ◽  
Hong Wang ◽  
Qun Zheng
Keyword(s):  
Animal Model ◽  
Oral Administration ◽  
Type I Collagen ◽  
Maximum Load ◽  
Type I ◽  
Bone Pathology ◽  
Treatment Of Osteoporosis ◽  
Beneficial Effects ◽  
Existing Problems ◽  
Item Checklist

Objectives. Quercetin (Q) and its derivatives are the major members of the naturally occurring flavonoid family, which possess beneficial effects on disease prevention including osteoporosis. The present study is aimed at further investigating the efficacy of the Q and its derivatives on bone pathology, bone-related parameters under imageology, bone maximum load, and serum bone metabolism indexes in animal model of osteoporosis. Potential mechanisms of Q and its derivatives in the treatment of osteoporosis as well as the existing problems regarding the modeling method and limitations of researches in this area were also summarized. Eight databases were searched from their inception dates to February 2020. Nineteen eligible studies containing 21 comparisons were identified ultimately. The risk of bias and data on outcome measures were analyzed by the CAMARADES 10-item checklist and Rev-Man 5.3 software separately. The results displayed the number of criteria met varied from 3/10 to 7/10 with an average of 5.05. The present study provided the preliminary preclinical evidence that oral administration of Q or its derivatives was capable of improving bone pathology, bone-related parameters under imageology and bone maximum load, increasing serum osteocalcin, alkaline phosphatase, and estradiol, and reducing serum c-terminal cross-linked telopeptide of type I collagen ( P < 0.05 ). No statistical difference was seen in survival rate, index of liver, or kidney function ( P > 0.05 ). Q and its derivatives partially reverse osteopenia probably via antioxidant, anti-inflammatory, promoting osteogenesis, inhibiting osteoclasts, and its estrogen-like effect. The findings reveal the possibility of developing Q or its derivatives as a drug or an ingredient in diet for clinical treatment of osteoporosis.

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