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1.
Clin Pharmacol Ther ; 81(5): 756-60, 2007 May.
Article in English | MEDLINE | ID: mdl-17392723

ABSTRACT

Obesity is an epidemic with tremendous impact on both patients and health-care systems globally. This paper explores some of the questions related to the clinical development of new pharmacotherapies in the context of an evolving regulatory perspective. These include patient entry criteria, clinical database size, study designs, weight loss end points (including those for maintenance of weight loss and prevention of weight regain), clinically important patient-reported outcomes, comorbidity/risk factor end points, and challenges in establishing safety and efficacy in adolescent/pediatric patients, and approaches to the development of combination pharmacotherapies. Ultimately, patients, physicians, academia, industry, payers, and governments must continue to partner with regulators to help establish the appropriate balance between the known adverse consequences associated with inadequate treatment of the growing obesity epidemic and the concern for potential unknown risks that may be associated with the long-term use of new pharmacotherapies.


Subject(s)
Anti-Obesity Agents/therapeutic use , Legislation, Drug/trends , Obesity/drug therapy , Adolescent , Animals , Child, Preschool , Drug Therapy, Combination , European Union , Humans , Obesity/epidemiology , Risk Factors , United States , United States Food and Drug Administration , Weight Loss
2.
Arthritis Rheum ; 41(1): 110-21, 1998 Jan.
Article in English | MEDLINE | ID: mdl-9433876

ABSTRACT

OBJECTIVE: It has long been proposed that stromelysin is one of the major degradative matrix metalloproteinases responsible for the loss of cartilage in rheumatoid arthritis (RA) and osteoarthritis (OA). This hypothesis was tested by examining the arthritic paws of stromelysin 1 (SLN1)-deficient mice for loss of cartilage and for generation of neoepitopes that would be indicative of aggrecan cleavage. METHODS: The SLN1 gene was inactivated in murine embryonic stem cells, and knockout mice deficient in SLN1 activity were bred onto the B10.RIII background. The incidence and severity of collagen-induced arthritis (CIA) were compared in wild-type and knockout mice. Paws from mice with CIA were examined for loss of cartilage and for proteoglycan staining, as well as for the generation of the neoepitope FVDIPEN341. RESULTS: SLN1-deficient mice developed CIA, as did the wild-type N2 mice. Histologic analyses demonstrated no significant differences among the B10.RIII, wild-type, and knockout mice in loss of articular cartilage and proteoglycan staining. No decrease in the FVDIPEN341 epitope was observed in the SLN1-deficient mice. CONCLUSION: Disruption of the SLN1 gene neither prevents nor reduces the cartilage destruction associated with CIA. Moreover, SLN1 depletion does not prevent the cleavage of the aggrecan Asn341-Phe342 bond.


Subject(s)
Arthritis, Rheumatoid/genetics , Cartilage, Articular/pathology , Matrix Metalloproteinase 3/genetics , Osteoarthritis/genetics , Animals , Arthritis, Rheumatoid/chemically induced , Arthritis, Rheumatoid/pathology , Blotting, Northern , Cartilage, Articular/enzymology , Collagen , Epitopes/genetics , Epitopes/metabolism , Female , Gene Expression , Immunohistochemistry , Male , Matrix Metalloproteinase 3/deficiency , Metalloendopeptidases/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Osteoarthritis/chemically induced , Osteoarthritis/pathology , Phenotype , RNA, Messenger/analysis , Stem Cells
3.
J Clin Invest ; 100(1): 93-106, 1997 Jul 01.
Article in English | MEDLINE | ID: mdl-9202061

ABSTRACT

To examine the activity of matrix metalloproteinases (MMPs) and aggrecanase in control and diseased human articular cartilage, metabolic fragments of aggrecan were detected with monospecific antipeptide antibodies. The distribution and quantity of MMP-generated aggrecan G1 fragments terminating in VDIPEN341 were compared with the distribution of aggrecanase-generated G1 fragments terminating in NITEGE373. Both types of G1 fragments were isolated from osteoarthritic cartilage. The sizes were consistent with a single enzymatic cleavage in the interglobular domain region, with no further proteolytic processing of these fragments. Both neoepitopes were also detected by immunohistochemistry in articular cartilage from patients undergoing joint replacement for osteoarthritis (OA), rheumatoid arthritis (RA), and in cartilage from adults with no known joint disease. In control specimens, the staining intensity for both G1 fragments increased with age, with little staining in cartilage from 22-wk-old fetal samples. There was also an increase with age in the extracted amount of MMP-generated neoepitope in relation to both aggrecan and collagen content, confirming the immunohistochemical results. After the age of 20-30 yr this relationship remained at a steady state. The staining for the MMP-generated epitope was most marked in control cartilage exhibiting histological signs of damage, whereas intense staining for the aggrecanase-generated fragment was often noted in adult cartilage lacking overt histological damage. Intense staining for both neoepitopes appeared in the more severely fibrillated, superficial region of the tissue. Intense immunostaining for both VDIPEN- and NITEGE- neoepitopes was also detected in joint cartilage from patients with OA or RA. Cartilage in these specimens was significantly more degraded and high levels of staining for both epitopes was always seen in areas with extensive cartilage damage. The levels of extracted VDIPEN neoepitope relative to collagen or aggrecan in both OA and RA samples were similar to those seen in age-matched control specimens. Immunostaining for both types of aggrecan fragments was seen surrounding the cells but also further removed in the interterritorial matrix. In some regions of the tissue, both neoepitopes were found while in others only one was detected. Thus, generation and/or turnover of these specific catabolic aggrecan fragments is not necessarily coordinated. Our results are consistent with the presence in both normal and arthritic joint cartilage of proteolytic activity against aggrecan based on both classical MMPs and "aggrecanase."


Subject(s)
Arthritis, Rheumatoid/metabolism , Cartilage, Articular/metabolism , Endopeptidases/metabolism , Extracellular Matrix Proteins , Osteoarthritis/metabolism , Proteoglycans/metabolism , Adolescent , Adult , Aged , Aged, 80 and over , Aggrecans , Aging , Amino Acid Sequence , Arthritis, Rheumatoid/pathology , Arthritis, Rheumatoid/surgery , Cartilage, Articular/growth & development , Cartilage, Articular/pathology , Child , Child, Preschool , Chondroitin Sulfate Proteoglycans/metabolism , Epitopes/analysis , Female , Fetus , Gestational Age , Humans , Infant, Newborn , Knee Joint , Knee Prosthesis , Lectins, C-Type , Male , Middle Aged , Osteoarthritis/pathology , Osteoarthritis/surgery , Peptide Fragments/analysis , Reference Values
4.
J Biol Chem ; 271(45): 28212-9, 1996 Nov 08.
Article in English | MEDLINE | ID: mdl-8910438

ABSTRACT

Recombinant human inducible nitric-oxide synthase (rH-iNOS) was expressed in the baculovirus system and purified by a novel immunoaffinity column. rH-iNOS and its native counterpart from cytokine-stimulated primary hepatocytes exhibited similar molecular mass of 130 kDa on SDS-polyacrylamide gel electrophoresis, recognition by antipeptide antibodies, specific activities, and IC50 values for inhibitors. The active dimeric form exhibited a specific activity range of 114-260 nmol/min/mg at 37 degrees C and contained 1.15 +/- 0.04 mol of calmodulin/monomer. The enzyme exhibited a Soret lambdamax at 396 nm with a shoulder at 460 nm and contained 0. 28-0.64 mol of heme/monomer. Dithionite reduction under CO yielded an absorbance maximum at 446 nm, indicating a P450-type heme. Imidazole induced a type II difference spectrum, reversible by L-Arg. 2-Amino-5,6-dihydro-4H-1,3-thiazine (ADT) was competitive versus L-Arg (Ki = 22.6 +/- 1.9 nM), reversed the type II difference spectrum induced by imidazole (Kd = 17.7 nM), and altered the CO-ferrous absorbance of rH-iNOS. L-Arg did not perturb the CO-ferrous adduct directly, but it partially reversed the ADT-induced absorbance shift, indicating that both bind similarly to the protein but interact differently with the heme.


Subject(s)
Nitric Oxide Synthase/metabolism , Radiation-Protective Agents/pharmacology , Thiazines/pharmacology , Chromatography, High Pressure Liquid , Enzyme Induction , Humans , Kinetics , NADP/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
5.
Biochemistry ; 35(29): 9567-75, 1996 Jul 23.
Article in English | MEDLINE | ID: mdl-8755738

ABSTRACT

Nitric oxide synthase catalyzes the pyridine nucleotide-dependent oxidation of L-arginine to nitric oxide and L-citrulline. It is a specialized cytochrome P450 monooxygenase that is sensitive to inhibition by imidazole. Steady-state kinetic studies on recombinant human inducible nitric oxide synthase (rH-iNOS) demonstrate that imidazole and 1-phenylimidazole are competitive and reversible inhibitors versus L-arginine. Structure-activity relationship and pH dependence studies on the inhibition suggest that the neutral form of imidazole may be the preferred species and that the only modifications allowed without the loss of inhibition are at the N-1 position of imidazole. Optical spectrophotometric studies of rH-iNOS with imidazole and 1-phenylimidazole yielded type II difference spectra exhibiting Kd values of 63 +/- 2 and 28 +/- 3 microM, respectively. These values were in good agreement with the steady-state Ki of 95 +/- 10 and 38 +/- 4 microM, respectively, and confirms the site of binding is at the sixth axial ligand of the heme. Imidazole (2.2 mM) also perturbed the Kd of L-arginine from 3.03 +/- 0.45 to 209 +/- 10 microM. The observed increase in the Kd for L-arginine is consistent with imidazole being a competitive inhibitor versus L-arginine. The IC50 values of imidazole and 1-phenylimidazole were lower in the absence of exogenous BH4, and both inhibitors also competitively inhibited the BH4-dependent activation of the enzyme. These data taken together suggest that the L-arginine, dioxygen, and the BH4 binding sites are in close proximity in rH-iNOS. Furthermore, these studies demonstrate the usefulness of imidazole compounds as active site probes for recombinant human iNOS.


Subject(s)
Enzyme Inhibitors/pharmacology , Imidazoles/pharmacology , Nitric Oxide Synthase/chemistry , Arginine/analogs & derivatives , Arginine/metabolism , Binding Sites , Biopterins/analogs & derivatives , Biopterins/pharmacology , Enzyme Inhibitors/metabolism , Humans , Hydrogen-Ion Concentration , Imidazoles/metabolism , Isoenzymes/metabolism , Kinetics , Molecular Structure , Nitric Oxide Synthase/antagonists & inhibitors , Nitric Oxide Synthase/metabolism , Nitroarginine , Recombinant Proteins/chemistry , Spectrophotometry , Structure-Activity Relationship
6.
J Bone Miner Res ; 10(9): 1417-27, 1995 Sep.
Article in English | MEDLINE | ID: mdl-7502715

ABSTRACT

Periprosthetic osteolysis with or without aseptic loosening is a major clinical problem in total hip arthroplasty. While the macrophage response to prosthetic wear debris and its role in periprosthetic osteolysis has been extensively studied, information regarding other cell types (fibroblasts, osteoblasts) is limited. This study explored the response of fibroblasts to particulate wear debris. Fibroblasts isolated from interfacial membranes of patients with failed total hip replacements and normal synovial tissue, when challenged with small-sized ( < 3 microns) titanium (Ti) particles, responded with significantly enhanced expressions of collagenase, stromelysin and, to a much lesser extent, their tissue inhibitor of metalloproteinases (TIMP). These "regulated" expressions at both mRNA and protein levels were correlated with the size and composition of particles. De novo protein synthesis was required for the regulation of these mRNAs. A similar effect could be induced by the treatment of the cells with particle-free conditioned medium from Ti particle-stimulated fibroblasts. Furthermore, this conditioned medium significantly suppressed the mRNA levels of procollagen alpha 1 (I) and alpha 1 (III) in osteoblast-like MG-63 cells. It is concluded that fibroblasts stimulated with certain particle debris may play an important role in periprosthetic osteolysis by releasing bone-resorbing metalloproteinases and mediator(s) which resulted in suppressed collagen synthesis in osteoblasts.


Subject(s)
Hip Prosthesis/adverse effects , Osteolysis/pathology , Synovial Membrane/pathology , Titanium/adverse effects , Base Sequence , Bone Resorption/enzymology , Fibroblasts/drug effects , Fibroblasts/physiology , Humans , Metalloendopeptidases/metabolism , Molecular Sequence Data , Protein Biosynthesis , RNA, Messenger/metabolism , Reference Values , Stimulation, Chemical
7.
J Pharmacol Exp Ther ; 272(1): 452-9, 1995 Jan.
Article in English | MEDLINE | ID: mdl-7529313

ABSTRACT

The role of inducible nitric oxide synthase (iNOS) was examined in the hypotension and vascular hyporesponsiveness to norepinephrine (NE) invoked by lipopolysaccharide (LPS) in pentobarbital-anesthetized rats. Saline, dexamethasone (DEX), NG-monomethyl-L-arginine (LNMMA) or indomethacin (IND) were administered either pre-LPS (0.5 hr) or post-LPS (4.5 hr) treatment. Rats were then challenged with NE 10 min before LPS injection and 1, 4, and 5 hr after LPS. Administration of LPS produced a biphasic hypotension: an immediate hypotension, which partially recovered within 15 min and was unaffected by any of the pretreatments; and a secondary, more prolonged hypotension which was attenuated by DEX, LNMMA and IND. The NE-induced pressor effects were significantly attenuated 1, 4 and 5 hr post LPS. Pretreatment with LNMMA or DEX significantly attenuated the LPS-induced NE hyporesponsiveness 4 and 5 hr post LPS. LNMMA was the only post-LPS treatment able to reverse the NE hyporesponsiveness. The LPS-induced iNOS mRNA and protein expression was demonstrated in the liver, lung, spleen, heart, kidney and brain by Northern hybridization and Western blot analyses. Low levels of neuronal constitutive NOS mRNA and endothelial cell constitutive NOS mRNA were only detected in brain or myocardial tissue, respectively. Significant induction of iNOS mRNA and protein expression was also observed in the liver, lung and spleen of rats pretreated with DEX, LNMMA or IND. The continued expression of iNOS in the presence of a pharmacologically relevant dose of DEX suggests that DEX may not be an optimal pharmacological agent for defining the in vivo roles of iNOS.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Amino Acid Oxidoreductases/metabolism , Blood Pressure/drug effects , Lipopolysaccharides/pharmacology , Shock, Septic/metabolism , Vasodilation/drug effects , Amino Acid Oxidoreductases/genetics , Animals , Arginine/analogs & derivatives , Arginine/pharmacology , Dexamethasone/pharmacology , Female , Gene Expression/drug effects , Indomethacin/pharmacology , Nitric Oxide Synthase , Norepinephrine/pharmacology , RNA, Messenger/genetics , Rats , Rats, Sprague-Dawley , Tissue Distribution , omega-N-Methylarginine
8.
J Rheumatol ; 21(9): 1716-24, 1994 Sep.
Article in English | MEDLINE | ID: mdl-7799356

ABSTRACT

OBJECTIVE: To study the effects of the intraarticular injection of canine monocyte conditioned medium (cMCM) into dogs on proteoglycan fragment and stromelysin levels in the joint. METHODS: cMCM was injected intraarticularly into dogs, and the levels of proteoglycan fragments in synovial fluid (SF) as well as stromelysin levels in cartilage, synovium, and SF were assessed after 12 h. RESULTS: There was a 4-fold increase of proteoglycan fragment levels and a 6-fold increase in stromelysin levels in SF, and a 4.4-fold increase in stromelysin levels in cartilage extracts. Elevated mRNA levels were detected in both synovium and cartilage. By immunofluorescence staining, stromelysin was localized in chondrocytes throughout the cartilage and in synovial cells. CONCLUSION: Intraarticular injection of cMCM stimulated the expression of stromelysin mRNA and protein in cartilage and synovium and caused marked increases in stromelysin protein and proteoglycan fragment levels in SF.


Subject(s)
Arthritis/metabolism , Cartilage, Articular/chemistry , Metalloendopeptidases/analysis , Monocytes/physiology , Peptide Fragments/analysis , Proteoglycans/analysis , Synovial Fluid/chemistry , Synovial Membrane/chemistry , Animals , Cartilage, Articular/pathology , Culture Media, Conditioned , Dogs , Female , Fluorescent Antibody Technique , Immunohistochemistry , Injections, Intra-Articular , Matrix Metalloproteinase 3 , Metalloendopeptidases/biosynthesis , Metalloendopeptidases/genetics , RNA, Messenger/analysis , RNA, Messenger/genetics , Synovial Membrane/pathology
9.
J Biol Chem ; 269(9): 6765-72, 1994 Mar 04.
Article in English | MEDLINE | ID: mdl-7509810

ABSTRACT

Nitric oxide, a multifunctional effector molecule synthesized by nitric oxide synthase (NOS) from L-arginine, conveys signals for vasorelaxation, neurotransmission, and cytotoxicity. Three different NOS isoforms have been identified which fall into two distinct types, constitutive and inducible. The inducible NOS (iNOS) isoform is expressed in a variety of cell types and tissues in response to inflammatory agents and cytokines. The human iNOS (NOS2) gene was isolated on overlapping cosmid clones from a human genomic library using both the murine macrophage and the human hepatocyte iNOS cDNAs as probes. All isolated cosmids were part of a single genomic locus and no other genomic loci were identified or isolated. Analysis of this locus indicated that the human iNOS gene is approximately 37 kilobases in length and consists of 26 exons and 25 introns. Primer extension analysis of lipopolysaccharide and cytokine-stimulated human hepatocyte RNA mapped the transcriptional initiation site 30 base pairs downstream of a TATA sequence, and a 400-base pair 5'-flanking region was found to be structurally similar to the recently described murine iNOS promoter. Polymerase chain reaction analysis of a human/rodent genomic DNA somatic cell hybrid panel and fluorescent in situ hybridization indicated that the human iNOS gene is located on chromosome 17 at position 17cen-q11.2.


Subject(s)
Amino Acid Oxidoreductases/biosynthesis , Amino Acid Oxidoreductases/genetics , Chromosomes, Human, Pair 17 , Gene Expression Regulation, Enzymologic , Amino Acid Sequence , Animals , Base Sequence , Chromosome Mapping , Cloning, Molecular/methods , DNA/genetics , DNA/isolation & purification , DNA Primers , Enzyme Induction , Exons , Fibroblasts/enzymology , Genomic Library , Humans , Hybrid Cells , Introns , Liver/enzymology , Male , Molecular Sequence Data , Nitric Oxide Synthase , Polymerase Chain Reaction , Restriction Mapping , Rodentia , Sequence Homology, Nucleic Acid , Skin/enzymology , Transcription, Genetic
10.
Biochem Biophys Res Commun ; 196(3): 1330-4, 1993 Nov 15.
Article in English | MEDLINE | ID: mdl-7504476

ABSTRACT

To examine the potential contribution of endothelial cell cNOS (ec-cNOS) and inducible NOS (iNOS) in controlling vascular tone under normal versus inflammatory conditions, we performed Northern hybridizations to examine the differential expression of each NOS mRNA in human aortic endothelial cells (AOEC) and human aortic smooth muscle cells (AOSMC) cultured for 8 h in the presence or absence of cytokines (IL-1 beta, TNF-alpha, and IFN-gamma) and LPS. Cytokine/LPS treatment induced a 4.4 kb iNOS mRNA in the human AOSMC; in contrast, cytokine/LPS treatment down regulated the expression of ec-cNOS mRNA in the AOEC. No iNOS mRNA was detected in the AOEC under the conditions examined. These results suggest that under specific inflammatory conditions the generation of NO in vascular tissue switches from ec-cNOS in the endothelium to iNOS in the smooth muscle.


Subject(s)
Amino Acid Oxidoreductases/biosynthesis , Aorta, Thoracic/enzymology , Endothelium, Vascular/enzymology , Gene Expression Regulation, Enzymologic , Muscle, Smooth, Vascular/enzymology , RNA, Messenger/biosynthesis , Animals , Aorta, Thoracic/physiopathology , Blotting, Northern , Cells, Cultured , DNA Probes , Endothelium, Vascular/physiopathology , Enzyme Induction , Humans , Inflammation/enzymology , Isoenzymes/biosynthesis , Macrophages/enzymology , Mice , Muscle, Smooth, Vascular/physiopathology , Nitric Oxide Synthase , Poly A/biosynthesis , Poly A/isolation & purification , RNA, Messenger/isolation & purification , Reference Values , Restriction Mapping
11.
Arthritis Rheum ; 36(11): 1540-7, 1993 Nov.
Article in English | MEDLINE | ID: mdl-8240430

ABSTRACT

OBJECTIVE: To compare quantitatively the in vivo expression of collagenase messenger RNA (mRNA) and stromelysin mRNA in the joint tissues of human osteoarthritis (OA) and rheumatoid arthritis (RA) patients and in two animal models of acute inflammatory arthritis. METHODS: In vivo levels of metalloproteinase mRNA and protein were determined by quantitative Northern hybridization and by enzyme-linked immunosorbent assay, respectively. RESULTS: In synovium, mean levels of collagenase mRNA were similar to those of stromelysin mRNA; however, in cartilage, mean levels of collagenase mRNA were significantly lower. The ratios of collagenase mRNA to stromelysin mRNA levels in RA and OA cartilage reflected similar ratios of collagenase protein to stromelysin protein levels in synovial fluid. CONCLUSION: The regulation of collagenase mRNA expression in cartilage is distinct from that of stromelysin, suggesting distinct roles for these two metallo-proteinases in normal and abnormal physiologic functioning of cartilage.


Subject(s)
Cartilage, Articular/enzymology , Collagenases/genetics , RNA, Messenger/analysis , Synovial Membrane/enzymology , Adult , Aged , Animals , Arthritis, Rheumatoid/metabolism , Blotting, Northern , Cartilage, Articular/chemistry , Cells, Cultured , Culture Media, Conditioned/pharmacology , Disease Models, Animal , Dogs , Enzyme-Linked Immunosorbent Assay , Female , Humans , In Situ Hybridization , Interleukin-1/pharmacology , Male , Matrix Metalloproteinase 3 , Metalloendopeptidases/genetics , Middle Aged , Monocytes , Osteoarthritis/metabolism , Rabbits , Synovial Fluid/chemistry , Synovial Membrane/chemistry
12.
J Clin Invest ; 92(1): 179-85, 1993 Jul.
Article in English | MEDLINE | ID: mdl-8325982

ABSTRACT

We report here that a 92-kD gelatinolytic metalloproteinase is expressed as protein and mRNA in human osteoarthritic cartilage, but not in normal adult articular cartilage. Western immunoblotting demonstrated that the 92-kD gelatinolytic activity corresponded to 92-kD type IV collagenase/gelatinase (gelatinase B); mRNA for gelatinase B was identified by Northern blotting. Chondrocytes from normal cartilage also exhibited mRNA for 72-kD type IV collagenase/gelatinase (gelatinase A), tissue collagenase, and stromelysin-1, and these mRNAs were increased in osteoarthritic cartilage. Regional analysis of osteoarthritic cartilage samples from four individuals revealed that gelatinase B mRNA was expressed in grossly fibrillated areas; two of four nonfibrillated cartilage samples failed to exhibit the mRNA, but did have increased levels of mRNA for other neutral metalloproteinases. IL-1 alpha treatment of normal human cartilage explants or isolated chondrocytes induced increased levels of gelatinase B and increased mRNA for tissue collagenase and stromelysin-1. Under identical conditions, mRNA levels for gelatinase A were not increased indicating that regulation of this enzyme in human articular chondrocytes is distinct from that of other metalloproteinases. Our data showing expression of gelatinase B in fibrillated cartilage suggest that it is a marker of progressive articular cartilage degradation in osteoarthritis.


Subject(s)
Cartilage, Articular/enzymology , Collagenases/metabolism , Interleukin-1/pharmacology , Metalloendopeptidases/metabolism , Adult , Gene Expression/drug effects , Humans , Matrix Metalloproteinase 2 , Matrix Metalloproteinase 9 , Middle Aged , Molecular Weight , Organ Culture Techniques , RNA, Messenger/genetics
13.
Arthritis Rheum ; 35(10): 1227-33, 1992 Oct.
Article in English | MEDLINE | ID: mdl-1418008

ABSTRACT

OBJECTIVE: To examine the in vivo expression of the matrix metalloproteinase stromelysin in the synovium and articular cartilage of rabbits injected intraarticularly with recombinant human interleukin-1 beta (IL-1). METHODS: The direct isolation of messenger RNA (mRNA) from articular cartilage without the prior isolation of chondrocytes is described. The in vivo expression of stromelysin was examined at the mRNA level by Northern blot analysis, and at the protein level by in situ immunolocalization and by enzyme-linked immunosorbent assay. RESULTS: In the synovium of IL-1-injected joints, stromelysin mRNA levels were highest at 4 hours and declined to background levels within 24 hours. In the cartilage of IL-1-injected joints, stromelysin mRNA was elevated at 4 hours and continued to increase until 8 hours, before declining. Stromelysin mRNA expression preceded a similar increase in stromelysin protein levels in both synovium and cartilage. CONCLUSION: Intraarticular injection of IL-1 induced the endogenous expression of stromelysin mRNA and protein in both synovium and cartilage. The kinetics of stromelysin expression correlated well with the accumulation of stromelysin and proteoglycan in synovial fluids. Therefore, the de novo synthesis of stromelysin in cartilage may have contributed to the loss of proteoglycan from that tissue.


Subject(s)
Cartilage, Articular/enzymology , Interleukin-1/pharmacology , Metalloendopeptidases/metabolism , Synovial Membrane/enzymology , Animals , Female , Injections, Intra-Articular , Interleukin-1/administration & dosage , Kinetics , Matrix Metalloproteinase 3 , Metalloendopeptidases/genetics , Proteoglycans/metabolism , RNA, Messenger/metabolism , Rabbits , Recombinant Proteins/administration & dosage , Recombinant Proteins/pharmacology , Synovial Fluid/enzymology , Synovial Fluid/metabolism
14.
Matrix ; 12(3): 173-84, 1992 Jun.
Article in English | MEDLINE | ID: mdl-1406451

ABSTRACT

One of the best studied animal models of osteoarthritis is a dog model in which the anterior cruciate ligament of the hind limb stifle joint is transected (Pond-Nuki model). To determine whether stromelysin might play a role in this model, it was necessary to purify the enzyme for production of suitable probes. In the present study, dog synovial fibroblasts were stimulated to express a metalloproteinase that was demonstrated to be canine prostromelysin by Northern blot, protein purification and amino-terminal sequence analyses. Unlike rabbit synoviocytes, passaged dog synoviocytes did not express stromelysin mRNA in response to recombinant human IL-1, but expressed stromelysin mRNA only upon treatment with dog monocyte-conditioned medium (dMCM). The aminophenylmercuric acetate (APMA)-activatable metalloproteinase present in the culture supernatants of stimulated dog synoviocytes was purified using a combination of ion-exchange and dye matrix affinity chromatography. The purified canine metalloproteinase co-migrated on reducing SDS-PAGE with recombinant human prostromelysin-1 as a doublet with apparent molecular masses of 54 and 56 kDa. Similar to APMA-activated human prostromelysin-1, the APMA-activated canine metalloproteinase was inhibited by 1,10-phenanthroline or recombinant human tissue inhibitor of metalloproteinase (TIMP). The amino-terminal sequences of the canine pro- and APMA-activated enzymes were compared with those of human, rabbit and rat stromelysin. The striking homologies among the sequences demonstrated that the purified canine metalloproteinase was indeed canine prostromelysin. A rabbit anti-canine prostromelysin polyclonal antiserum was generated and used to localize the enzyme within cultured dog synoviocytes and articular cartilage stimulated with dMCM. The reagents developed in this study should be useful for examining the expression and distribution of prostromelysin in canine models of osteoarthritis.


Subject(s)
Cartilage, Articular/enzymology , Dogs/metabolism , Enzyme Precursors/biosynthesis , Metalloendopeptidases/biosynthesis , Phenylmercuric Acetate/analogs & derivatives , Synovial Membrane/enzymology , Amino Acid Sequence , Animals , Cells, Cultured , Collagenases/genetics , Culture Media/pharmacology , Cytokines/pharmacology , Disease Models, Animal , Dogs/genetics , Enzyme Induction/drug effects , Enzyme Precursors/genetics , Enzyme Precursors/isolation & purification , Fibroblasts/drug effects , Fibroblasts/enzymology , Glycoproteins/pharmacology , Humans , Leukocytes, Mononuclear/metabolism , Metalloendopeptidases/genetics , Metalloendopeptidases/isolation & purification , Molecular Sequence Data , Osteoarthritis/metabolism , Phenanthrolines/pharmacology , Phenylmercuric Acetate/pharmacology , Rabbits/genetics , Rats/genetics , Recombinant Proteins/pharmacology , Sequence Homology , Species Specificity , Stimulation, Chemical , Synovial Membrane/cytology , Tissue Inhibitor of Metalloproteinases
16.
Biochemistry ; 30(26): 6476-83, 1991 Jul 02.
Article in English | MEDLINE | ID: mdl-1647201

ABSTRACT

Stromelysin-1 is a member of a tissue metalloproteinase family whose members are all capable of degrading extracellular matrix components. A truncated form of human fibroblast prostromelysin 1 lacking the C-terminal, hemopexin-like domain has been expressed in Escherichia coli and purified to homogeneity. Treatment of this short form of prostromelysin with (aminophenyl)mercuric acetate resulted in activation and loss of the propeptide in a manner identical with the wild-type, full-length protein. Kinetic comparisons using Nle11-substance P as a substrate showed that the wild-type stromelysin and the truncated form of the enzyme had similar kcat and Km values. Likewise, both enzymes displayed similar Ki values for a hydroxamate-containing peptide inhibitor. Taken together, these results indicate that the C-terminal portion of stromelysin is not required for proper folding of the catalytic domain, maintenance of the enzyme in a latent form, activation with an organomercurial, cleavage of a peptide substrate, or interaction with an inhibitor. Moreover, the active short form of stromelysin displayed a reduction in the C-terminal heterogeneity, a characteristic degradation of the full-length stromelysin, and thereby provides a more suitable protein for future structural studies.


Subject(s)
Metalloendopeptidases/genetics , Amino Acid Sequence , Base Sequence , Binding Sites , Chromatography, Ion Exchange , Electrophoresis, Polyacrylamide Gel , Enzyme Precursors/isolation & purification , Enzyme Precursors/metabolism , Escherichia coli/genetics , Fibroblasts/enzymology , Humans , Hydroxamic Acids/pharmacology , Kinetics , Macromolecular Substances , Matrix Metalloproteinase 3 , Metalloendopeptidases/isolation & purification , Metalloendopeptidases/metabolism , Microbial Collagenase/genetics , Models, Structural , Molecular Sequence Data , Molecular Weight , Oligonucleotide Probes , Plasmids , Polymerase Chain Reaction/methods , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Restriction Mapping , Sequence Homology, Nucleic Acid
17.
J Immunol ; 145(12): 4154-66, 1990 Dec 15.
Article in English | MEDLINE | ID: mdl-2258613

ABSTRACT

Human IL-1 alpha, IL-1 beta, and TNF-alpha mRNA expression was examined in peripheral blood monocytes (PBM) from normal individuals and in primary synoviocytes isolated from patients with rheumatoid arthritis by Northern blot and in situ hybridization. Cells cultured in the presence or absence of LPS were analyzed using in vitro synthesized 35S-labeled sense or antisense RNA probes to determine the relative abundance and the cell type expressing each of the mRNA for these potent inflammatory mediators. The results indicated that 72% of the LPS-stimulated PBM expressed detectable levels of IL-1 alpha mRNA, 89% IL-1 beta mRNA, and 10% TNF-alpha transcripts. Thus, the majority of activated PBM produced both IL-1 alpha and IL-1 beta. Experiments combining immunofluorescence for IL-1 beta protein with in situ hybridization for TNF-alpha mRNA demonstrated that monocytes expressing TNF-alpha mRNA also produced IL-1 beta. Primary synoviocytes from four patients with RA were also examined for the mRNA expression of each cytokine. Northern blot analyses of total RNA isolated from 0 to 72 h after LPS- or mock-stimulation showed that IL-1 beta mRNA was the most abundantly expressed, followed by TNF-alpha. In situ hybridization revealed that IL-1 beta and TNF-alpha transcripts were detected exclusively in synovial tissue macrophages. IL-1 alpha mRNA was not detected in these cultures by either method.


Subject(s)
Arthritis, Rheumatoid/genetics , Interleukin-1/genetics , Macrophages/physiology , Monocytes/physiology , Synovial Membrane/physiopathology , Tumor Necrosis Factor-alpha/genetics , Blotting, Northern , Cells, Cultured , Gene Expression , Humans , In Vitro Techniques , Nucleic Acid Hybridization , RNA, Messenger/genetics , Synovial Membrane/pathology , Time Factors
18.
J Biol Chem ; 265(28): 17238-45, 1990 Oct 05.
Article in English | MEDLINE | ID: mdl-1698773

ABSTRACT

Primary and passaged human synovial fibroblasts isolated from rheumatoid pannus were treated with recombinant interleukin-1 (IL-1) alpha or beta, tumor necrosis factor-alpha (TNF), or phorbol myristate acetate (PMA) to determine the effects of these stimuli on the relative expression of stromelysin, collagenase, and tissue inhibitor of metalloproteinases (TIMP). The steady-state mRNA levels for these genes and glyceraldehyde-3-phosphate dehydrogenase were determined on Northern blots. Immunoblot analyses of the conditioned media using monoclonal antibodies generated against recombinant human stromelysin, collagenase, or TIMP showed that protein levels reflected the corresponding steady-state mRNA levels. The results revealed that 1) stromelysin and collagenase were not always coordinately expressed; 2) IL-1 was more potent than TNF or PMA in the induction of stromelysin expression; 3) neither IL-1 nor TNF significantly affected TIMP expression; 4) PMA induced both metalloproteinase and TIMP expression; and 5) the combination of IL-1 plus TNF had a synergistic effect on stromelysin expression. Dose response and time course experiments demonstrated that the synergistic effect of IL-1 plus TNF occurred at saturating concentrations of each cytokine and lasted for 7 days. In summary, the ability of IL-1 and TNF to preferentially induce stromelysin and collagenase expression, versus TIMP, may define a pivotal role for these cytokines in the pathogenesis of rheumatoid arthritis.


Subject(s)
Arthritis, Rheumatoid/metabolism , Glycoproteins/genetics , Interleukin-1/pharmacology , Metalloendopeptidases/genetics , Microbial Collagenase/genetics , Synovial Membrane/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Arthritis, Rheumatoid/enzymology , Arthritis, Rheumatoid/genetics , Cells, Cultured , Cloning, Molecular , DNA/genetics , DNA/isolation & purification , Drug Synergism , Fibroblasts/drug effects , Fibroblasts/enzymology , Fibroblasts/metabolism , Humans , Kinetics , Matrix Metalloproteinase 3 , Metalloendopeptidases/biosynthesis , Metalloendopeptidases/metabolism , Microbial Collagenase/biosynthesis , RNA/genetics , RNA/isolation & purification , RNA, Messenger/genetics , Synovial Membrane/enzymology , Tetradecanoylphorbol Acetate/pharmacology , Tissue Inhibitor of Metalloproteinases , Transcription, Genetic
19.
J Immunol ; 143(2): 718-26, 1989 Jul 15.
Article in English | MEDLINE | ID: mdl-2472451

ABSTRACT

FK506, a neutral macrolide with immunosuppressive properties, was shown to selectively and rapidly inhibit the accumulation of IL-2 mRNA, as well as the mRNAs of other early (E) phase T cell activation genes such as IL-3, IL-4, GM-CSF, TNF alpha, IFN-gamma, and c-myc in activated human peripheral blood T cells. The activity of FK506, when compared to Cyclosporin A, another immunosuppressant, was 10 to 100x more potent in its ability to inhibit IL-2 mRNA synthesis. FK506 inhibited IL-2 mRNA accumulation in Con A, Con A plus PMA, Ionomycin plus PMA, anti-CD3, and anti-CD3 plus PMA activated T cells. Transcripts from other T cell gene classes such as the immediate early (IE) phase gene, c-fos, the late phase (L) genes, transferrin receptor, IL-2R alpha-chain, and TNF-beta, and the constitutive class genes glyceraldehyde-3-phosphate dehydrogenase and class I MHC HLA-B7 were not affected by FK506. The macrolide Rapamycin, which is structurally related to FK506, had no inhibitory effect on IE, E, L, or constitutive class mRNAs, but it appeared to increase the levels of the E-phase transcripts that were inhibited in FK506 treated T cells. The effect of FK506 on inducible genes in non-T and non-lymphoid human cells was studied in LPS-induced monocytes and PMA or IL-1 activated synovial fibroblasts. FK506 did not affect expression of the mRNAs for IL-1 alpha or IL-1 beta in human monocytes, or of stromelysin, collagenase, or TIMP in synovial fibroblasts. Nuclear run-off transcription studies indicate that FK506 inhibits transcription of the IL-2 gene. These studies suggest that Cyclosporin A and FK506 may effect a common early event in the T cell activation pathway.


Subject(s)
Gene Expression Regulation/drug effects , Immunosuppressive Agents/pharmacology , Interleukin-2/genetics , Lymphocyte Activation/drug effects , Pyridines/pharmacology , T-Lymphocytes/metabolism , Base Sequence , Cell Differentiation/drug effects , Cell Division/drug effects , Growth Inhibitors/pharmacology , Humans , Interleukin-2/physiology , Molecular Sequence Data , RNA, Messenger/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/physiology , Tacrolimus , Transcription, Genetic/drug effects
20.
J Immunol ; 138(4): 1109-14, 1987 Feb 15.
Article in English | MEDLINE | ID: mdl-2949012

ABSTRACT

Although complementary DNA (cDNA) encoding human interleukin 1 beta (IL 1 beta) have been cloned in several laboratories, there are as yet no data demonstrating that recombinant IL 1 beta (rIL 1 beta) molecules expressed from such cDNA are faithful, fully active replicas of the native protein secreted by human monocytes. To this purpose, cDNA sequences corresponding to the exact NH2-terminus and amino acid sequence of mature, monocyte-derived human IL 1 beta were placed under control of the inducible trp-lac (TAC) fusion promoter and were expressed in E. coli strain JM105. rIL 1 beta was purified to homogeneity by high pressure liquid chromatography (HPLC). Yields of 10 to 20 mg of rIL 1 beta/liter/10(11) cells were obtained. Purified rIL 1 beta was then compared in a number of biochemical and biologic assays to purified native IL 1 beta. Native and rIL 1 beta co-migrated on SDS-polyacrylamide gels as 17.5 kd polypeptides and reacted with specific polyclonal antisera raised to three synthetic peptides of human IL 1 beta in immunoblot experiments. Amino acid sequence analysis of rIL 1 beta showed that the native amino terminus, an ALA residue, was faithfully maintained. Purified native and rIL 1 beta co-chromatographed on reverse-phase HPLC. Specific biologic activities of rIL 1 beta were indistinguishable from those of the native protein in murine thymocyte and human dermal fibroblast proliferation assays, with half-maximal stimulation occurring at concentrations of 25 pM in the murine thymocyte assay and 2 pM in the human dermal fibroblast assay. Similarly, native and rIL 1 beta competed equally well for high affinity IL 1 receptor binding sites, each exhibiting a Ki of 20 pM on MRC-5 human embryonic lung fibroblasts. These observations indicate that E. coli efficiently expresses large quantities of rIL 1 beta, which emulates exactly the properties of the native protein.


Subject(s)
Interleukin-1/biosynthesis , Recombinant Fusion Proteins/biosynthesis , Recombinant Proteins/biosynthesis , Amino Acid Sequence , Cloning, Molecular , DNA/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Fibroblasts/drug effects , Humans , Interleukin-1/genetics , Interleukin-1/pharmacology , Monocytes/analysis , Receptors, Immunologic/metabolism , Receptors, Interleukin-1 , Recombinant Fusion Proteins/pharmacology , Synovial Fluid/drug effects , T-Lymphocytes/drug effects
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