scholarly journals The effects of serine proteinase inhibitors on bone resorption in vitro

2003 ◽  
Vol 178 (3) ◽  
pp. 437-447 ◽  
Author(s):  
A Tumber ◽  
S Papaioannou ◽  
J Breckon ◽  
MC Meikle ◽  
JJ Reynolds ◽  
...  
Keyword(s):  
Bone Resorption ◽  
Plasminogen Activator ◽  
Type I Collagen ◽  
Bone Matrix ◽  
Serine Proteinase ◽  
Tissue Type ◽  
Type I ◽  
Type Plasminogen Activator ◽  
Primary Mouse ◽  
Sites Of Action

The aims of this study were to identify the role and sites of action of serine proteinases (SPs) in bone resorption, a process which involves a cascade of events, the central step of which is the removal of bone matrix by osteoclasts (OCs). This resorbing activity, however, is also determined by recruitment of new OCs to future resorption sites and removal of the osteoid layer by osteoblasts (OBs), which enables OCs to gain access to the underlying mineralized bone. The resorption systems we have studied consisted of (i) neonatal calvarial explants, (ii) isolated OCs cultured on ivory slices, (iii) mouse OBs cultured on either radiolabelled type I collagen films or bone-like matrix, (iv) bone marrow cultures to assess OC formation and (v) 17-day-old fetal mouse metatarsal bone rudiments to assess OC migration and fusion. Two separate SP inhibitors, aprotinin and alpha(2)-antiplasmin dose-dependently inhibited (45)Ca release from neonatal calvarial explants: aprotinin (10(-6) M) was the most effective SP inhibitor, producing a maximum inhibitory effect of 55.9%.Neither of the SP inhibitors influenced either OC formation or OC resorptive activity. In contrast, each SP inhibitor dose-dependently inhibited OB-mediated degradation of both type I collagen fibrils and non-mineralized bone matrix. In 17-day-old metatarsal explants aprotinin produced a 55% reduction in the migration of OCs from the periosteum to the mineralized matrix after 3 days in culture but after 6 days in culture aprotinin was without effect on OC migration. Primary mouse osteoblasts expressed mRNA for urokinase type plasminogen activator (uPA), tIssue type plasminogen activator (tPA), the type I receptor for uPA, plasminogen activator inhibitor types I and II and the broad spectrum serine proteinase inhibitor, protease nexin I. In situ hybridization demonstrated expression of tPA and uPA in osteoclasts disaggregated from 6-day-old mouse long bones. We propose that the regulation of these various enzyme systems within bone tIssue determines the sites where bone resorption will be initiated.

Plasminogen Activator and Plasminogen Activator Inhibitor Associated with Granulomatous Inflammation: A Study with Murine Leprosy

1984 ◽  
Vol 52 (03) ◽  
pp. 243-249 ◽  
Author(s):  
S Izaki ◽  
T Hibino ◽  
Y Isozaki ◽  
P S Hsu ◽  
M Izaki ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Soluble Fraction ◽  
Proteinase Inhibitors ◽  
Granulomatous Inflammation ◽  
Serine Proteinase ◽  
Plasminogen Activator Inhibitor ◽  
Tissue Type ◽  
Weakly Alkaline ◽  
Heat Stable ◽  
Type Plasminogen Activator

SummaryPlasminogen activator that is associated with the development of hypersensitivity granulomas (gPA) was partially purified from a saline soluble fraction of murine lepromas elicited in “resistant” mice, C57BL/6N. The gPA was shown to consist of two subspecies (23,000 and 48,000 in molecular weight) with essentially identical enzymologic properties. The gPA was found to be a relatively heat stable weakly alkaline serine proteinase with trypsin-like characteristics in the specificity for synthetic substrates and proteinase inhibitors. It showed a high affinity for H- D-Ile-Pro-Arg-pNA (Km = 1.4 × 10-4 M) H-D-Val-Leu-Lys- pNA (Km = 5.2 × 10-4 M), and L-pyroGlu-Gly-Arg-pNA (Km = 9.3 × 10-4 M). The gPA did not demonstrate antigenic cross reaction with urokinase-type or tissue-type plasminogen activator.Two distinct enzymatic regulators of the gPA were also demonstrated in the saline soluble fraction of the hypersensitivity granulomas. The gPA and its regulation are assumed to be correlated with macrophage activation in the hypersensitivity granulomas

Carboxyterminal telopeptide of type I collagen, ICTP, as a marker of matrix degradation in neonatal mouse calvarial bones, in vitro

1992 ◽  
Vol 12 (5) ◽  
pp. 407-411 ◽  
Author(s):  
Östen Ljunggren ◽  
Sverker Ljunghall
Keyword(s):  
Parathyroid Hormone ◽  
Bone Resorption ◽  
Type I Collagen ◽  
Bone Matrix ◽  
Neonatal Mouse ◽  
Type I ◽  
Matrix Degradation ◽  
Dose Responses ◽  
Carboxyterminal Telopeptide

Bone resorption, in vitro, is often measured as the release of prelabelled45Ca from neonatal mouse calvarial bones, or from fetal rat long bones. In this report we describe a technique to measure the breakdown of bone-matrix, in vitro. We also describe a new way to dissect neonatal mouse calvarial bones, in order to obtain large amounts of bone samples. Twelve bone fragments were dissected out from each mouse calvaria and were thereafter cultured in CMRL 1066 culture medium in serum-free conditions in 0.5 cm2 multiwell culture dishes. Matrix degradation after treatment with parathyroid hormone was assessed by measuring the amount of carboxyterminal telopeptide of type I collagen (ICTP) by RIA. The data on matrix degradation was compared to the release of prelabelled45Ca from neonatal mouse calvarial bones. We found that the dose-responses for parathyroid hormone-induced release of prelabelled45Ca and ICTP were identical. In conclusion: RIA-analysis of the ICTP-release is an easy and accurate method to measure degradation of bone-matrix, in vitro. Furthermore, the new dissection technique, described in this report, makes it easy to obtain large amounts of bone samples and thus to perform extensive experiments, e.g. dose-responses for agents that enhance bone resorption.

Post-translational modification of the myxoma-virus anti-inflammatory serpin SERP-1 by a virally encoded sialyltransferase

2000 ◽  
Vol 347 (2) ◽  
pp. 375-382 ◽  
Author(s):  
Piers NASH ◽  
Michele BARRY ◽  
Bruce T. SEET ◽  
Kirstin VEUGELERS ◽  
Susy HOTA ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Eukaryotic Expression ◽  
Myxoma Virus ◽  
Tissue Type ◽  
Post Translational Modification ◽  
Type Plasminogen Activator ◽  
Anti Inflammatory ◽  
Virally Encoded ◽  
Sites Of Action

SERP-1 is a secreted serpin (serine-proteinase inhibitor) encoded by myxoma virus, a poxvirus pathogen of rabbits. SERP-1 is required for myxoma-virus virulence, and the purified protein has been shown to possess independent anti-inflammatory activity in animal models of restenosis and antigen-induced arthritis. As an inhibitor of serine proteinases, SERP-1 acts against tissue-type plasminogen activator, urokinase-type plasminogen activator, plasmin, thrombin and Factor Xa. In the present study, examination of SERP-1 glycosylation-site mutants showed that the N-linked glycosylation of Asn172 was essential for SERP-1 secretion, whereas mutation of Asn99 decreased secretion efficiency, indicating that N-linked glycosylation plays an essential role in the processing and trafficking of SERP-1. Furthermore, comparison of SERP-1 from wild-type myxoma virus and a virus containing a targeted disruption of the MST3N sialyltransferase locus demonstrated that SERP-1 is specifically modified by this myxoma-virus-encoded sialyltransferase, and is thus the first reported viral protein shown to by modified by a virally encoded glycosyltransferase. Sialylation of SERP-1 by the MST3N gene product creates a uniquely charged species of secreted SERP-1 that is distinct from SERP-1 produced from other eukaryotic expression systems, though this has no apparent effect upon the kinetics of in vitro proteinase inhibition. Rather, the role of viral sialylation of SERP-1 likely relates to masking antigenicity or targeting SERP-1 to specific sites of action in vivo.

scholarly journals Helodermatine, a kallikrein-like, hypotensive enzyme from the venom of Heloderma horridum horridum (Mexican beaded lizard).

1986 ◽  
Vol 164 (6) ◽  
pp. 1835-1845 ◽  
Author(s):  
A Alagon ◽  
L D Possani ◽  
J Smart ◽  
W D Schleuning
Keyword(s):  
Plasminogen Activator ◽  
Trypsin Inhibitor ◽  
Serine Proteinase ◽  
Tissue Type ◽  
Single Chain ◽  
Glandular Kallikrein ◽  
Arterial Blood ◽  
Type Plasminogen Activator ◽  
The Family ◽  
Pancreatic Trypsin Inhibitor

We have purified and characterized the major N-benzoyl-L-arginine ethyl ester hydrolase from the venom of Heloderma horridum horridum. The enzyme belongs to the serine proteinase family, and its activity vs. peptide amide substrates and human high-molecular-weight kininogen suggests a similarity to the family of kallikreins. This interpretation is corroborated by its reactivity with the natural inhibitors soybean trypsin inhibitor and Kunitz-type bovine pancreatic trypsin inhibitor (aprotinin). Injection of the enzyme (2-16 micrograms/kg) into anesthetized rabbits leads to a rapid dose-dependent transient decrease of the arterial blood pressure. Like glandular kallikrein it specifically converts single-chain tissue type plasminogen activator into its double chain form. In contrast to other kallikrein-like enzymes from snake venoms it shows no thrombin-like or plasminogen activator activity. The enzyme is a single-chain glycoprotein (Mr 63,000). The N-terminal sequence revealed significant homology to pig pancreatic kallikrein and to kallikrein like enzymes from Crotalus atrox and Crotalus adamanteus venom. This enzyme, which we name Helodermatine, is the first purified from Sauria with kallikrein-like properties.

Involvement of cyclic adenosine monophosphate-dependent protein kinase isozymes in tissue plasminogen activator secretion by rat Sertoli cells stimulated with follicle-stimulating hormone in vitro

1993 ◽  
Vol 128 (6) ◽  
pp. 555-562 ◽  
Author(s):  
V Laurent-Cadoret ◽  
F Guillou ◽  
Y Combarnous
Keyword(s):  
Protein Kinase ◽  
Plasminogen Activator ◽  
Sertoli Cells ◽  
Tissue Type ◽  
Type I ◽  
Dependent Protein Kinase ◽  
Tissue Type Plasminogen Activator ◽  
Camp Dependent Protein Kinase ◽  
Type Plasminogen Activator ◽  
Dependent Protein

This study was undertaken to investigate, in freshly isolated rat Sertoli cells, the physiological function of the type I and type II cyclic adenosine monophosphate (cAMP)-dependent protein kinase isozymes in tissue-type plasminogen activator secretion and the regulation of this cAMP process by follicle-stimulating hormone (FSH). Follicle-stimulating hormone-induced tissue-type plasminogen activator secretion depends upon intracellular cAMP levels. The changes in cAMP amounts required to activate maximally the tissue-type plasminogen activator secretion are extremely small, a cAMP threshold having to be reached for triggering the tissue-type plasminogen activator output. Intact Sertoli cells were incubated with combinations of cAMP analogs specific for each cAMP-dependent protein kinase type and complementary in their cAMP binding site on the cAMP-dependent protein kinase regulatory subunits: 8-aminohexylamino-cAMP = type 1, site 1; 8-thiomethyl-cAMP = type II, site 1 and N6-benzoyl-cAMP = types I/II, site 2. This allowed us to activate selectively each cAMP-dependent protein kinase type in a synergistic manner and then to evaluate their respective influence in the specific tissue-type plasminogen activator response. We establish that both of the cAMP-dependent protein kinase types are present and functional; the activity of the type I isozyme is preponderant (60%) in the cAMP-dependent tissue-type plasminogen activator secretion. Likewise, when these cAMP analogs were coupled with endogenously generated cAMP by FSH or forskolin, both of the cAMP-dependent protein kinase types were involved in the tissue-type plasminogen activator production. However, only tissue-type plasminogen activator secretion induced by FSH is mediated predominantly by the type I cAMP-dependent protein kinase, although the type II isozyme sustains an appreciable physiological role in the transmission pathway. We suggest some differences in the pattern of action between FSH and forskolin in Sertoli cells.

scholarly journals Fibrinolytic response to tumor necrosis factor in healthy subjects.

1991 ◽  
Vol 174 (3) ◽  
pp. 729-732 ◽  
Author(s):  
T van der Poll ◽  
M Levi ◽  
H R Büller ◽  
S J van Deventer ◽  
J P de Boer ◽  
...  
Keyword(s):  
Tumor Necrosis Factor ◽  
Plasminogen Activator ◽  
Tumor Necrosis ◽  
Tissue Type ◽  
Controlled Study ◽  
Type I ◽  
Plasmin Activity ◽  
Microvascular Thrombosis ◽  
Type Plasminogen Activator ◽  
Necrosis Factor

Tumor necrosis factor (TNF) may be involved in the disturbance of the procoagulant-fibrinolytic balance in septicemia, leading to microvascular thrombosis. To assess the dynamics of the fibrinolytic response to TNF in humans, we performed a crossover saline-controlled study in six healthy men, investigating the effects of a bolus intravenous injection of recombinant human TNF (50 micrograms/m2) on the stimulation and inhibition of plasminogen activation as well as on plasmin activity and inhibition. TNF induced a brief fourfold increase in the overall plasma plasminogen activator (PA) activity peaking after 1 h (p less than 0.0001), which was associated with rises in the antigenic levels of urokinase-type plasminogen activator (p less than 0.0001) and tissue-type plasminogen activator (p less than 0.0001). Plasminogen activator inhibitor type I antigen remained unchanged in the first hour, but showed a rapid eightfold increase thereafter (p less than 0.0001), which coincided with the decrease in PA activity. Generation of plasmin activity in the first hour was signified by an 11-fold rise in D-dimer levels (p less than 0.0001); inhibition of plasmin was reflected by a 36-fold rise in plasmin-alpha 2 antiplasmin complexes (p less than 0.0001), as well as by a transient 16% decrease in alpha 2-antiplasmin activity (p less than 0.01). In conclusion, TNF induced an early activation of the fibrinolytic system becoming maximal in 1 h, with a rapid inhibition thereafter. Earlier observations in the same subjects showed sustained coagulation activation for 6-12 h. The observed disbalance between the procoagulant and fibrinolytic mechanisms after TNF injection confirms the in vivo relevance of the effects of TNF on vascular endothelium in vitro and may explain the tendency towards microvascular thrombosis in septicemia.

scholarly journals Estrogen Reduces the Depth of Resorption Pits by Disturbing the Organic Bone Matrix Degradation Activity of Mature Osteoclasts

Endocrinology ◽  
2001 ◽  
Vol 142 (12) ◽  
pp. 5371-5378 ◽  
Author(s):  
Vilhelmiina Parikka ◽  
Petri Lehenkari ◽  
Mirja-Liisa Sassi ◽  
Jussi Halleen ◽  
Juha Risteli ◽  
...  
Keyword(s):  
Bone Resorption ◽  
Type I Collagen ◽  
Bone Matrix ◽  
Organic Matrix ◽  
Cysteine Proteases ◽  
Bone Collagen ◽  
Type I ◽  
Matrix Degradation ◽  
Carboxyl Terminal

Abstract Decreased E2 levels after menopause cause bone loss through increased penetrative resorption. The reversal effect of E2 substitution therapy is well documented in vivo, although the detailed mechanism of action is not fully understood. To study the effects of E2 on bone resorption, we developed a novel in vitro bone resorption assay in which degradation of inorganic and organic matrix could be measured separately. E2 treatment significantly decreased the depth of resorption pits, although the area resorbed was not changed. Electron microscopy further revealed that the resorption pits were filled with nondegraded collagen, suggesting that E2 disturbed the organic matrix degradation. Two major groups of proteinases, matrix metalloproteinases (MMPs) and cysteine proteinases, have been suggested to participate in organic matrix degradation by osteoclasts. We show here that MMP-9 released a cross-linked carboxyl-terminal telopeptide of type I collagen from bone collagen, and cathepsin K released another C-terminal fragment, the C-terminal cross-linked peptide of type I collagen. E2 significantly inhibited the release of the C-terminal cross-linked peptide of type I collagen into the culture medium without affecting the release of cross-linked carboxyl-terminal telopeptide of type I collagen in osteoclast cultures. These results suggest that organic matrix degradation is initiated by MMPs and continued by cysteine proteases; the latter event is regulated by E2.

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