RESUMEN
Human peripheral blood mononuclear cells (lymphocyte-monocyte) in culture release a solube factor which can stimulate, up to 200-fold, production of prostaglandin E2 by isolated, adherent, rheumatoid synovial cells. Production of the factor by the mononuclear cells is enhanced by phytohemagglutinin. This factor is similar in apparent mol vt (10,000-20,000) to that which also stimulates collagenase production by the same cells.
Asunto(s)
Artritis Reumatoide/inmunología , Linfocitos/inmunología , Prostaglandinas E/biosíntesis , Membrana Sinovial/inmunología , Células Cultivadas , Humanos , Lectinas/farmacología , Activación de Linfocitos , Linfocitos/metabolismo , Monocitos/metabolismo , Estimulación QuímicaRESUMEN
1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) increases synthesis of heat shock proteins in monocytes and U937 cells and protects these cells from thermal injury. We examined whether 1,25-(OH)2D3 would also modulate the susceptibility of U937 cells to H2O2-induced oxidative stress. Cell viability was assessed by trypan blue exclusion and [3H]thymidine incorporation into DNA. Prior incubation for 24 h with 1,25-(OH)2D3 (25 pM or higher) unexpectedly increased H2O2 toxicity. Since cellular Ca2+ may be a mediator of cell injury we investigated effects of altering extracellular Ca2+ ([Ca2+]e) on 1,25-(OH)2D3-enhanced H2O2 toxicity as well as effects of 1,25-(OH)2D3 and H2O2 on cytosolic free Ca2+ concentration ([Ca2+]f). Basal [Ca2+]f in medium containing 1.5 mM Ca as determined by fura-2 fluorescence was higher in 1,25-(OH)2D3-pretreated cells than control cells (137 versus 112 nM, P less than 0.005). H2O2 induced a rapid increase in [Ca2+]f (to greater than 300 nM) in both 1,25-(OH)2D3-treated and control cells, which was prevented by a reduction in [Ca2+]e to less than basal [Ca2+]f. The 1,25(OH)2D3-induced increase in H2O2 toxicity was also prevented by preincubation with 1,25-(OH)2D3 in Ca2+-free medium or by exposing the cells to H2O2 in the presence of EGTA. Preexposure of cells to 45 degrees C for 20 min, 4 h earlier, partially prevented the toxic effects of H2O2 particularly in 1,25-(OH)2D3-treated cells, even in the presence of physiological levels of [Ca2+]e. Thus 1,25-(OH)2D3 potentiates H2O2-induced injury probably by increasing cellular Ca2+ stores. The 1,25-(OH)2D3-induced amplification of the heat shock response likely represents a mechanism for counteracting the Ca2+-associated enhanced susceptibility to oxidative injury due to 1,25-(OH)2D3.
Asunto(s)
Calcitriol/farmacología , Calcio/metabolismo , Peróxido de Hidrógeno/farmacología , Monocitos/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Proteínas de Choque Térmico/biosíntesis , Calor , Humanos , Monocitos/metabolismo , Monocitos/fisiología , Oxidación-ReducciónRESUMEN
We have shown in a variety of human wounds that collagenase-1 (MMP-1), a matrix metalloproteinase that cleaves fibrillar type I collagen, is invariably expressed by basal keratinocytes migrating across the dermal matrix. Furthermore, we have demonstrated that MMP-1 expression is induced in primary keratinocytes by contact with native type I collagen and not by basement membrane proteins or by other components of the dermal or provisional (wound) matrix. Based on these observations, we hypothesized that the catalytic activity of MMP-1 is necessary for keratinocyte migration on type I collagen. To test this idea, we assessed keratinocyte motility on type I collagen using colony dispersion and colloidal gold migration assays. In both assays, primary human keratinocytes migrated efficiently on collagen. The specificity of MMP-1 in promoting cell movement was demonstrated in four distinct experiments. One, keratinocyte migration was completely blocked by peptide hydroxymates, which are potent inhibitors of the catalytic activity of MMPs. Two, HaCaTs, a line of human keratinocytes that do not express MMP-1 in response to collagen, did not migrate on a type I collagen matrix but moved efficiently on denatured type I collagen (gelatin). EGF, which induces MMP-I production by HaCaT cells, resulted in the ability of these cells to migrate across a type I collagen matrix. Three, keratinocytes did not migrate on mutant type I collagen lacking the collagenase cleavage site, even though this substrate induced MMP-1 expression. Four, cell migration on collagen was completely blocked by recombinant tissue inhibitor of metalloproteinase-1 (TIMP-1) and by affinity-purified anti-MMP-1 antiserum. In addition, the collagen-mediated induction of collagenase-1 and migration of primary keratinocytes on collagen was blocked by antibodies against the alpha2 integrin subunit but not by antibodies against the alpha1 or alpha3 subunits. We propose that interaction of the alpha2beta1 integrin with dermal collagen mediates induction of collagenase-1 in keratinocytes at the onset of healing and that the activity of collagenase-1 is needed to initiate cell movement. Furthermore, we propose that cleavage of dermal collagen provides keratinocytes with a mechanism to maintain their directionality during reepithelialization.
Asunto(s)
Movimiento Celular/fisiología , Colágeno , Colagenasas/fisiología , Queratinocitos/enzimología , Animales , Línea Celular , Células Cultivadas , Colagenasas/biosíntesis , Colagenasas/genética , Inducción Enzimática , Factor de Crecimiento Epidérmico/farmacología , Gelatina , Humanos , Integrinas/metabolismo , Queratinocitos/citología , Queratinocitos/fisiología , Metaloproteinasa 1 de la Matriz , Ratones , Ratones SCIDRESUMEN
Plasminogen activators are highly selective proteases that activate the proenzyme plasminogen to the general protease, plasmin. We studied a porcine kidney cell line, originally isolated as a high producer of plasminogen activator, in which activities of cellular adenylate cyclase and cAMP-dependent protein kinase are increased in response to calcitonin. We found that salmon calcitonin, in the concentration range 0.03-300 nM, increased plasminogen activator production up to approximately 1,000-fold and concurrently inhibited cell multiplication; both of these effects were reversible. Human calcitonin was approximately 0.01 times as potent as salmon calcitonin, corresponding to potency differences observed in other biological systems. Plasminogen activator production was also stimulated by other agents that raise cellular cAMP levels such as cholera toxin, phosphodiesterase inhibitors, and vasopressin, but not to the same extent as by calcitonins. The rapidity and sensitivity of the plasminogen activator determination and other cellular responses may make it possible in the future to use this cell stain in a convenient bioassay for calcitonins and their analogues.
Asunto(s)
Calcitonina/farmacología , Túbulos Renales/enzimología , Activadores Plasminogénicos/metabolismo , Animales , División Celular/efectos de los fármacos , Línea Celular , AMP Cíclico/metabolismo , Porcinos , Factores de Tiempo , Vasopresinas/farmacologíaRESUMEN
Synovial tissue from patients with rheumatoid arthritis produces lysis of gels of reconstituted collagen fibrils in culture and releases soluble collagenase when cultured in collagen-free medium. Collagen molecules in solution at neutral pH at 20 degrees and 27 degrees C are cleaved by the synovial enzyme into (3/4) and (1/4) length fragments. In this respect the action of synovial enzyme is similar to that of amphibian collagenase and distinct from that of bacterial collagenase. At 37 degrees C reconstituted collagen fibrils and native fibers are attacked by the enzyme and further degraded to polypeptides of low molecular weight. These polypeptides are produced only after denaturation of the larger fragments, which occurs at temperatures near 37 degrees C.
Asunto(s)
Artritis Reumatoide/enzimología , Colagenasa Microbiana/metabolismo , Membrana Sinovial/enzimología , Isótopos de Carbono , Cromatografía en Gel , Colágeno , Técnicas de Cultivo , Electroforesis , Glicina , Calor , Humanos , Concentración de Iones de Hidrógeno , Péptidos , Desnaturalización Proteica , Soluciones , ViscosidadRESUMEN
Human peripheral blood lymphocytes incubated in culture for 1 to 3 days at 37 degree C, but not at 4 degree C, release a soluble factor which can stimulate, up to 400-fold, collagenase production by isolated, adherent, rheumatoid synovial cells. Production of lymphocyte factor is enhanced by phytohemagglutinin or concanavalin A. By gel filtration the factor has an apparent molecular weight of about 12,000.
Asunto(s)
Artritis Reumatoide/enzimología , Linfocitos/fisiología , Colagenasa Microbiana/biosíntesis , Membrana Sinovial/enzimología , Células Cultivadas , Inducción Enzimática , Calor , Humanos , Linfocinas/farmacologíaRESUMEN
Patients with Paget's disease of bone excrete, in the urine, polypeptides that have amino acid composition and other properties resembling those of fragments of collagen. The pattern of isotope incorporation in vivo suggests that these fragments are derived from collagen that has been synthesized and rapidly degraded, or that they are rapidly synthesized but not incorporated into tropocollagen molecules.
Asunto(s)
Colágeno/metabolismo , Osteítis Deformante/orina , Péptidos/orina , Aminoácidos/análisis , Isótopos de Carbono , Celulosa , Cromatografía , Cromatografía en Gel , Colágeno/análisis , Humanos , Hidroxiprolina/orina , Técnicas In Vitro , Péptidos/análisis , Prolina/metabolismoRESUMEN
Skin fibroblasts from two siblings with hydroxylysine-deficient collagen collagen (Ehlers-Danlos syndrome, type VI) contained normal levels of collagen prolyl hydroxylase activity but were markedly deficient in collagen lysyl hydroxylase activity. The deficiency was evident in all fractions of cell lysates, in low and high ionic strength buffers, and in detergent. Assays of mixtures of wild-type and mutant cell lysates indicated no activation of mutant enzyme by factors in wild-type cells or inhibition of normal enzyme by material in mutant cells. Wild type or mutant cells cultured with ascorbic acid (50 mug/ml of culture medium, added daily) contained approximately the same level of lysyl hydroxylase activity as cells cultured without ascorbate, but prolyl hydroxylase activity without ascorbate was depressed in both an average of 41%. The mutant lysyl hydroxylase was less stable at 37 degrees C than the wild type and did not form high molecular weight aggregates in low ionic strength buffers, as did the control enzyme. The activity of the mutant enzyme was maximally stimulated after dialysis against buffer solutions containing 10 mM dithiothreitol. When assayed in 100 muM dithiothreitol, the mutant enzyme exhibited a higher apparent Km for ascorbate (20 muM) than the wild type (4 muM). In 1.0 mM dithiothreitol the mutant enzyme's apparent Km for ascorbate was reduced to 5 muM. Wild type and mutant enzymes had the same apparent Km for alpha-keto-glutarate (20 muM). The properties of prolyl hydroxylase in wild type and mutant cells were identical: apparent Km's for ascorbate and alpha-ketoglutarate were 100 muM and 20 muM, respectively. If mutant enzyme protein with altered kinetic properties is the only enzyme functioning to hydroxylate lysyl residues in collagen, the variations in hydroxylysine content observed in collagen from different tissues in the subjects reported here could be in part due to differences in cofactor concentrations and in rate and sequence of events in collagen synthesis in different tissues.
Asunto(s)
Colágeno/metabolismo , Síndrome de Ehlers-Danlos/enzimología , Oxigenasas de Función Mixta/deficiencia , Ácido Ascórbico/farmacología , Cloromercuribenzoatos/farmacología , Ditiotreitol/farmacología , Síndrome de Ehlers-Danlos/genética , Fibroblastos/enzimología , Humanos , Hidroxilisina/deficiencia , Técnicas In Vitro , Cinética , Mercaptoetanol/farmacología , Mutación , Procolágeno-Prolina Dioxigenasa/metabolismoRESUMEN
The protein encoded by the protooncogene c-jun, included in the activator protein-1 (AP-1) complex, is probably the critical trans-acting factor controlling transcription of the procollagenase gene which is rate limiting for subsequent synthesis of procollagenase. Therefore, to elucidate possible mechanisms whereby IL-1 stimulates procollagenase synthesis, we measured levels of c-jun and procollagenase mRNA in human serum-starved dermal fibroblasts in response to human recombinant IL-1 beta (hrIL-1 beta). hrIL-1 beta or serum induced rapid increases in c-jun mRNA levels; mRNA levels declined rapidly after hrIL-1 beta and more slowly after exposure to serum. The increases in levels of c-jun mRNA preceded the increases in procollagenase mRNA. Whereas the increases in levels of procollagenase mRNA were blunted by cycloheximide, those of c-jun mRNA were enhanced. We interpret these results as follows: IL-1 or serum induce transcription of c-jun by mechanisms independent of new protein synthesis; c-JUN, the protein product of c-jun in the AP-1 complex, is an essential mediator of the effects of IL-1 or serum in the subsequent induction of expression of the procollagenase gene.
Asunto(s)
Fenómenos Fisiológicos Sanguíneos , Colagenasas , Proteínas de Unión al ADN/biosíntesis , Precursores Enzimáticos/biosíntesis , Fibroblastos/metabolismo , Interleucina-1/farmacología , Colagenasa Microbiana/biosíntesis , Proteínas Proto-Oncogénicas/biosíntesis , ARN Mensajero/biosíntesis , Factores de Transcripción/biosíntesis , Células Cultivadas , Medios de Cultivo , Fibroblastos/efectos de los fármacos , Fibroblastos/fisiología , Humanos , Proteínas Proto-Oncogénicas c-jun , ARN Mensajero/efectos de los fármacos , Proteínas Recombinantes/farmacologíaRESUMEN
Of the total urinary hydroxyproline in normal subjects and those with skeletal disorders, between 4 and 20% was nondialyzable. In some patients with Paget's disease of bone, hyperparathyroidism with osteitis fibrosa, hyperphosphatasia, and extensive fibrous dysplasia the total urinary hydroxyproline was sufficiently high to permit purification of this polypeptide hydroxyproline by gel filtration and ion exchange chromatography. The partially purified polypeptides had molecular weights between 4500 and 10,000 and amino acid compositions and physical properties resembling those of gelatin. The polypeptide fractions also contained neutral sugar and glucosamine. These fragments had been shown to be susceptible to cleavage by purified bacterial collagenase suggesting the presence of the sequence-Pro-X-Gly-Pro-Y-. After administration of proline-(14)C to patients with Paget's disease hydroxyproline-(14)C was excreted in the urine. The hydroxyproline-(14)C specific activity reached a peak in 2-4 hr and declined rapidly. The specific activity of the polypeptide (retentate) portion was severalfold greater than that of the raw urine and diffusate. When the labeled urines were subjected to gel filtration the hydroxyproline-(14)C fractions of highest molecular weight which were eluted first from the columns had the highest specific activities. Exposure of the hydroxyproline-(14)C-containing polypeptides to bacterial collagenase rendered them dialyzable. Four patients with hyperparathyroidism and osteitis fibrosa were studied before and after removal of a parathyroid adenoma, a period of transition from a predominance of bone collagen resorption to one of relatively increased bone collagen synthesis. The total urinary hydroxyproline fell rapidly after operation whereas the ratio of the polypeptide fraction to the total rose three- to fourfold. The results of these studies suggest that the urinary polypeptides represent fragments of collagen related to collagen synthesis. Changes in the ratio of these peptides to total hydroxyproline in the urine may serve as an index of new bone formation in patients with skeletal disorders.
Asunto(s)
Enfermedades Óseas/orina , Colágeno/biosíntesis , Hidroxiprolina/orina , Adenoma , Adolescente , Adulto , Anciano , Aminoácidos/análisis , Enfermedades Óseas/metabolismo , Isótopos de Carbono , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Femenino , Displasia Fibrosa Ósea/orina , Humanos , Hidroxiprolina/aislamiento & purificación , Hiperparatiroidismo/orina , Masculino , Colagenasa Microbiana/metabolismo , Persona de Mediana Edad , Peso Molecular , Osteítis Deformante/orina , Osteítis Fibrosa Quística/orina , Neoplasias de las Paratiroides , Péptidos/orinaRESUMEN
Fragments of synovium from patients with rheumatoid arthritis survive in defined tissue culture medium in the absence of added serum and, after 3-4 days, release into the medium enzyme capable of degrading undenatured collagen. Maximal activity is observed at pH 7-9 but the enzyme is inactive at pH 5. At temperatures of 20 degrees and 27 degrees C, collagen molecules in solution are cleaved into 3/4 and 1/4 length fragments with minimal loss of negative optical rotation, but with loss in specific viscosity of approximately 60%. Above 30 degrees C the fragments begin to denature and denaturation is complete at 37 degrees C. If the enzyme is not inhibited at this stage the large fragments are broken down further to polypeptides of low molecular weight. Reconstituted collagen fibrils and native fibers at 37 degrees C are cleaved to the low molecular weight fragments, although the fibrils are resistant to breakdown at lower temperatures (20 degrees -27 degrees C). It is proposed that the production of such an enzyme by inflamed and proliferating rheumatoid synovium may be responsible for some of the destruction of collagenous structures that accompanies rheumatoid arthritis.
Asunto(s)
Artritis Reumatoide/enzimología , Colagenasa Microbiana/metabolismo , Membrana Sinovial/enzimología , Adenosina Trifosfato/metabolismo , Animales , Isótopos de Carbono , Cromatografía en Gel , Colágeno/metabolismo , Técnicas de Cultivo , Electroforesis , Glicina/metabolismo , Cobayas , Histocitoquímica , Humanos , Concentración de Iones de Hidrógeno , Microscopía Electrónica , Peso Molecular , Proteínas/farmacología , Temperatura , Tripsina/farmacología , ViscosidadRESUMEN
An enzyme which degrades native collagen at neutral pH has been isolated from cultures of rheumatoid synovium in vitro, but little or no collagenolytic activity has been found in homogenates of fresh rheumatoid synovium. Similar to most other mammalian collagenases this synovial enzyme is readily inhibited by serum proteins. Proteins of synovial fluid are derived largely from serum and synovial fluid from noninflamed joints was found to inhibit synovial collagenase; the inhibitor was destroyed by trypsin, but not by hyaluronidase. Inhibitory activity was reduced in approximately one-half of the fluids from patients with rheumatoid arthritis. In a total of nine synovial fluids, collagenolytic activity was detectable. This activity was not present in constant amounts in synovial fluids aspirated at different times from the same patient and tended to vary inversely with the titer of inhibitory proteins. The collagenolytic activity in the synovial fluids from different patients was variably inhibited by serum proteins. Two distinct collagenases were detected in some rheumatoid synovial fluids and separated by gel filtration. One, labeled "B" enzyme, with an estimated molecular weight 20,000-25,000 resembled the collagenase obtained from synovial cultures. The other, labeled "A" enzyme degraded collagen fibrils as well as collagen in solution. Disc electrophoresis on acrylamide gels and electron microscopy of segment long spacing (SLS) aggregates of reaction products of the enzymes at 27 degrees C demonstrated that both "A" and "B" enzymes cleaved collagen molecules at a point three-quarters from the amino terminal end of the molecule. Thus collagen degradation in rheumatoid arthritis could result from the operation of these two collagenases.
Asunto(s)
Artritis Reumatoide/enzimología , Colagenasa Microbiana/análisis , Líquido Sinovial/enzimología , Proteínas Sanguíneas/farmacología , Cromatografía en Gel , Colágeno/metabolismo , Electroforesis Discontinua , Geles , Humanos , Colagenasa Microbiana/antagonistas & inhibidores , Microscopía Electrónica , Peso MolecularRESUMEN
In inflammatory diseases such as rheumatoid arthritis, functions of chondrocytes including synthesis of matrix proteins and proteinases are altered through interactions with cells of the infiltrating pannus. One of the major secreted products of mononuclear inflammatory cells is IL-1. In this study we found that recombinant human IL-1 beta suppressed synthesis of cartilage-specific type II collagen by cultured human costal chondrocytes associated with decreased steady state levels of alpha 1 (II) and alpha 1(IX) procollagen mRNAs. In contrast, IL-1 increased synthesis of types I and III collagens and levels of alpha 1(I), alpha 2(I), and alpha 1(III) procollagen mRNAs, as we described previously using human articular chondrocytes and synovial fibroblasts. This stimulatory effect of IL-1 was observed only when IL-1-stimulated PGE2 synthesis was blocked by the cyclooxygenase inhibitor indomethacin. The suppression of type II collagen mRNA levels by IL-1 alone was not due to IL-1-stimulated PGE2, since addition of indomethacin did not reverse, but actually potentiated, this inhibition. Continuous exposure of freshly isolated chondrocytes from day 2 of culture to approximately half-maximal concentrations of IL-1 (2.5 pM) completely suppressed levels of type II collagen mRNA and increased levels of types I and III collagen mRNAs, thereby reversing the ratio of alpha 1(II)/alpha 1(I) procollagen mRNAs from greater than 6.0 to less than 1.0 by day 7. IL-1, therefore, can modify, at a pretranslational level, the relative amounts of the different types of collagen synthesized in cartilage and thereby could be responsible for the inappropriate repair of cartilage matrix in inflammatory conditions.
Asunto(s)
Cartílago/citología , Colágeno/biosíntesis , Regulación de la Expresión Génica/efectos de los fármacos , Interleucina-1/farmacología , Cartílago/efectos de los fármacos , Cartílago/metabolismo , Colágeno/genética , Dinoprostona/biosíntesis , Electroforesis en Gel de Poliacrilamida , Humanos , Indometacina/farmacología , Procolágeno/genética , ARN Mensajero/metabolismoRESUMEN
Adherence to a substratum is a characteristic feature of monocyte-macrophages which may be required for several effector functions. Human peripheral blood monocytes selected by adherence were found to readhere preferentially at 1 h to fibronectin or to a biological matrix. There was then a progressive decrease in the number of adherent cells, and by 48 h only 8-20% of monocytes remained adherent. This loss of adherence occurred while monocytes remained viable by criteria such as exclusion of trypan blue or release of lactate dehydrogenase. 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) maintained the adherence of cultured monocytes to tissue culture plastic as well as to the biological matrix. This effect was concentration- and time-dependent, and suppressed by inhibitors of protein synthesis. Cellular proteins were labeled after incubation with [35S]methionine. Analysis by two-dimensional gel electrophoresis revealed increased labeling of several distinct proteins in 1,25-(OH)2D3-treated monocytes compared with control monocytes. The increased loss of adherence and decreased overall protein synthesis observed in monocytes incubated at 45 degrees C was partially prevented by preincubation of the cells with 1,25-(OH)2D3. We further evaluated the effects of thermal stress and 1,25-(OH)2D3 on protein synthesis by monocytes, and found that 1,25-(OH)2D3 increased the synthesis of heat shock proteins, protected normal protein synthesis, and increased the rate of recovery of normal protein synthesis after the thermal stress. These observations suggest that 1,25-(OH)2D3 influences monocytes by preserving the synthesis of proteins, including those critical for the maintenance of cell adherence.
Asunto(s)
Calcitriol/farmacología , Calor , Monocitos/citología , Adhesión Celular/efectos de los fármacos , Cicloheximida/farmacología , Electroforesis en Gel de Poliacrilamida , Proteínas de Choque Térmico/biosíntesis , Humanos , Técnicas In Vitro , Interferón gamma/farmacología , Peso Molecular , Monocitos/efectos de los fármacosRESUMEN
Interactions of cells with components of the extracellular matrix can modulate cellular functions. We measured binding of a major matrix protein to U937 cells, a human promonocytic line. Radioiodinated type I or type III human collagen was bound only to U937 cells differentiated to a more mature phenotype with 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). Binding was observed at 4 degrees C and was saturable; Scatchard analysis of the binding to 1,25-(OH)2D3-pretreated U937 cells indicated a single class of high-affinity binding sites. Preincubation of U937 cells with interferon gamma did not induce collagen binding. Collagen binding did not appear to be dependent on fibronectin binding. Surface proteins of U937 cells were 125I labeled and cell membrane proteins resolved by affinity chromatography on collagen-Sepharose. Major specifically labeled bands of 180, 155, and 125 kD were identified in membrane fractions from 1,25-(OH)2D3-pretreated U937 cells only. 1,25-(OH)2D3 appears to specifically regulate collagen binding to monocyte precursors.
Asunto(s)
Calcitriol/farmacología , Colágeno/metabolismo , Monocitos/metabolismo , Adhesión Celular/efectos de los fármacos , Diferenciación Celular , Fibronectinas/metabolismo , Humanos , Interferón gamma/farmacología , Cinética , Proteínas de la Membrana/análisis , Peso Molecular , Monocitos/efectos de los fármacos , Receptores de Calcitriol , Receptores de Esteroides/metabolismo , Factores de TiempoRESUMEN
Mice carrying a targeted mutation (r) in Col1a1, encoding a collagenase-resistant form of type I collagen, have altered skeletal remodeling. In hematoxylin and eosin-stained paraffin sections, we detect empty lacunae in osteocytes in calvariae from Col1a1(r/r) mice at age 2 weeks, increasing through age 10-12 months. Empty lacunae appear to result from osteocyte apoptosis, since staining of osteocytes/periosteal osteoblasts with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling is increased in Col1a1(r/r) relative to wild-type bones. Osteocyte perilacunar matrices stained with Ab that recognizes collagenase collagen alpha1(I) chain cleavage ends in wild-type but not Col1a1(r/r) calvariae. Increased calvarial periosteal and tibial/femoral endosteal bone deposition was found in Col1a1(r/r) mice from ages 3-12 months. Calcein labeling of calvarial surfaces was increased in Col1a1(r/r) relative to wild-type mice. Daily injections of synthetic parathyroid hormone for 30 days increased calcein-surface labeling in wild-type but caused no further increase in the already high calcein staining of Col1a1(r/r) bones. Thus, failure of collagenase cleavage of type I collagen in Col1a1(r/r) mice is associated with osteocyte/osteoblast death but increases bone deposition in a manner that mimics the parathyroid hormone-induced bone surface activation seen in wild-type mice.
Asunto(s)
Apoptosis , Remodelación Ósea/genética , Colágeno/metabolismo , Colagenasas/metabolismo , Osteoblastos/patología , Osteocitos/patología , Animales , Colágeno/genética , Fémur/patología , Ratones , Ratones Mutantes , Hormona Paratiroidea/farmacología , Tibia/patologíaRESUMEN
Parathyroid hormone (PTH) stimulates bone resorption by acting directly on osteoblasts/stromal cells and then indirectly to increase differentiation and function of osteoclasts. PTH acting on osteoblasts/stromal cells increases collagenase gene transcription and synthesis. To assess the role of collagenase in the bone resorptive actions of PTH, we used mice homozygous (r/r) for a targeted mutation (r) in Col1a1 that are resistant to collagenase cleavage of type I collagen. Human PTH(1-34) was injected subcutaneously over the hemicalvariae in wild-type (+/+) or r/r mice four times daily for three days. Osteoclast numbers, the size of the bone marrow spaces and periosteal proliferation were increased in calvariae from PTH-treated +/+ mice, whereas in r/r mice, PTH-induced bone resorption responses were minimal. The r/r mice were not resistant to other skeletal effects of PTH because abundant interstitial collagenase mRNA was detected in the calvarial periosteum of PTH-treated, but not vehicle-treated, r/r and +/+ mice. Calcemic responses, 0.5-10 hours after intraperitoneal injection of PTH, were blunted in r/r mice versus +/+ mice. Thus, collagenase cleavage of type I collagen is necessary for PTH induction of osteoclastic bone resorption.
Asunto(s)
Resorción Ósea/fisiopatología , Colagenasas/fisiología , Hormona Paratiroidea/farmacología , Fragmentos de Péptidos/farmacología , Animales , Resorción Ósea/inducido químicamente , Colagenasas/genética , Colagenasas/metabolismo , Femenino , Expresión Génica , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Mutagénesis , CráneoRESUMEN
The shape and function of adherent cells cultured from rheumatoid synovial membranes are influenced by immune cells, and their products. The synovial cells produce collagenase and prostaglandin E2 (PGE2), the levels of which are increased when the cells are incubated with the monokine, mononuclear cell factor/interleukin 1. The majority of adherent synovial cells are fibroblastlike in appearance and synthesize collagens and fibronectin; the synthesis of collagens and fibronectins are also increased by a monocyte factor. In the present study we found that the fibroblastlike cells expressed major histocompatibility complex class II (Ia-like) antigens after initial dispersion from the synovial membrane. Monocyte lineage antigens were detected on some round cells in early passage, but no T lymphocytes were identified in established cultures. There was loss of Ia expression on the fibroblastlike cells with age and passage in culture. The addition of the lymphokine, gamma interferon (recombinant), induced class II antigen (DR and DS/DQ) expression in early or late passage cells in a time- and dose-dependent manner and required protein synthesis. Furthermore, the adherent synovial fibroblastlike cells continued to be Ia-positive when examined as long as 10 d after the removal of gamma interferon. Ia expression was also induced by gamma interferon in normal skin fibroblasts. Synovial cells that could be induced to express Ia also bound a monoclonal antibody to type III collagen (a fibroblast marker). Gamma interferon, while inducing Ia expression, decreased the binding of type III collagen antibody on unstimulated as well as monokine-stimulated cells. Analysis of [3H]proline-labeled medium by SDS polyacrylamide gel electrophoresis showed that gamma interferon decreased the synthesis of type I and III collagens and fibronectin by adherent synovial cells in a dose-dependent manner. These findings suggest that Ia expression by synovial tissue cells is not cell-specific, but reflects one or several related events, such as the degree of T lymphocyte infiltration, the presence of factors that stimulate gamma interferon release, or an increased sensitivity of the cells to gamma interferon. Whereas the synthesis of class II antigens is enhanced by the lymphokine gamma interferon, and a monocyte factor(s) stimulates collagen, collagenase and PGE2 synthesis by the same cells, gamma interferon inhibits basal and monokine-induced collagen synthesis. Thus, lymphokines and monokines may influence the extent of fibrosis as contrasted to matrix destruction at various stages of the rheumatoid lesion by affecting the function of fibroblastlike synovial cells.
Asunto(s)
Colágeno/biosíntesis , Antígenos de Histocompatibilidad Clase II/biosíntesis , Interferón gamma/farmacología , Membrana Sinovial/citología , Células Cultivadas , Cicloheximida/farmacología , Dinoprostona , Electroforesis en Gel de Poliacrilamida , Fibronectinas/biosíntesis , Antígenos HLA-DR , Antígenos de Histocompatibilidad Clase II/análisis , Humanos , Interleucina-1/farmacología , Colagenasa Microbiana/biosíntesis , Monocinas , Prostaglandinas E/biosíntesis , Proteínas/farmacología , Membrana Sinovial/efectos de los fármacosRESUMEN
Cultured mononuclear cells from human peripheral blood produce a soluble factor (MCF) that stimulates collagenase and prostaglandin E2 (PGE2) release by cultured rheumatoid synovial cells up to several hundred fold. These target rheumatoid synovial cells lack conventional macrophage markers. To determine which mononuclear cells are the source of MCF, purified populations of monocyte-macrophages, thymus-derived (T) lymphocytes, and bone marrow-derived (B) lymphocytes were prepared. The monocyte-macrophages alone produced levels of MCF that were proportional to cell density but unaffected by phytohemagglutinin or pokeweed mitogen. No detectable collagenase activity was produced by the cultured monocyte-macrophages or lymphocytes. Purified T lymphocytes produced levels of MCF approximately or equal to 1--3% those of purified monocyte-macrophages in the presence or absence of the above lectins. Purified T lymphocytes modulated the production of MCF by the monocyte-macrophages, however, in a manner dependent upon relative cell densities and the presence of lectins. For example, at optimal ratios of T lymphocytes: monocyte-macrophages, MCF production was markedly stimulated by pokeweed mitogen. Thus, interactions of T lymphocytes and monocyte-macrophages could be important in determining levels of MCF, which regulate collagenase and PGE2 production by target synovial cells in inflammatory arthritis.
Asunto(s)
Artritis Reumatoide/metabolismo , Linfocinas/biosíntesis , Macrófagos/metabolismo , Colagenasa Microbiana/metabolismo , Monocitos/metabolismo , Prostaglandinas E/metabolismo , Membrana Sinovial/metabolismo , Linfocitos T/metabolismo , Artritis Reumatoide/patología , Células Cultivadas , Humanos , Técnicas In Vitro , Mitógenos de Phytolacca americana/farmacología , Membrana Sinovial/patologíaRESUMEN
Fibroblasts cultured from normal human dermis are heterogeneous with respect to growth kinetics, synthetic function, and morphologic features. There are many examples of clonal heterogeneity in apparently homogeneous connective tissue cell populations, and it has been suggested that selection of cell populations with particular phenotypic features is the basis for the development of pathologic connective tissue changes in inflammatory disorders. In these studies we report characterization of the pattern of matrix biosynthesis and responses to hormones in cells cloned from normal human dermis. The results indicate that cloned dermal fibroblasts are heterogeneous with respect to synthesis of collagens as well as their responses to prostaglandin E2 and parathyroid hormone. Selective expansion of clonal populations with unique patterns of matrix synthesis and cell surface receptors could provide the basis for abnormal connective tissue remodeling in certain pathologic states.