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J Sodek

Publications and source records attributed to J Sodek.

At least 91 records · Page 5Linked to original sources
PubMed1991

Expression of bone matrix proteins associated with mineralized tissue formation by adult rat bone marrow cells in vitro: inductive effects of dexamethasone on the osteoblastic phenotype.

The nature and tissue distribution of non-collagenous bone proteins synthesized by adult rat bone marrow cells, induced to differentiate in the presence of dexamethasone (DEX) and beta-glycerophosphate (beta-GP), was studied in vitro to determine the potential role of these proteins in bone formation. Northern hybridization analysis revealed a strong induction of bone sialoprotein (BSP) and osteocalcin in DEX-treated cultures, whereas the constitutive expression of secreted phosphoprotein I (SPP-1), type I collagen, SPARC, and alkaline phosphatase was stimulated 6-, 5-, 3-, and 2.5-told, respectively. Metabolic labeling of proteins showed that the sialoproteins (SPP-1 and BSP) were mostly secreted into the culture medium in the non-mineralizing (-beta-GP) cultures, but were the predominant non-collagenous proteins associated with the hydroxyapatite of the bone nodules in mineralizing cultures (+ beta-GP). Extraction of the tissue matrix with 4 M GuHCl and digestion of the demineralized tissue matrix with bacterial collagenase revealed that some BSP was also associated non-covalently and covalently with the collagenous matrix. SPP-1 was present in two distinct, 44 kDa and 55 kDa, forms in the conditioned medium of all cultures and was preferentially associated with the hydroxyapatite in the mineralizing cultures. In comparison, SPARC was abundant in culture media but could not be detected in de-mineralizing extracts of the mineralized tissue. Radiolabeling with [35SO4] demonstrated that both SPP-1 and BSP synthesized by bone cells are sulfated, and that a 35 kDa protein and some proteoglycan were covalently associated with the collagenous matrix in +DEX cultures. Labeling with [32PO4] was essentially confined to the sialoproteins; the 44 kDa SPP-1 incorporating significantly more [32PO4] than the 55 kDa SPP-1 and the BSP. These studies demonstrate that BSP and osteocalcin are only expressed in differentiated osteoblasts and that most of the major non-collagenous bone proteins associate with the bone mineral. However, some novel proteins together with some of the BSP are associated with the collagenous matrix where they can influence hydroxyapatite formation.

Alkaline Phosphatase↗
PubMed1991

Immunohistochemical localization of bone sialoprotein in foetal porcine bone tissues: comparisons with secreted phosphoprotein 1 (SPP-1, osteopontin) and SPARC (osteonectin).

Bone sialoprotein (BSP) is a prominent component of bone tissues that is expressed by differentiated osteoblastic cells. Affinity-purified antibodies to BSP were prepared and used in combination with biotin-conjugated peroxidase-labeled second antibodies to demonstrate the distribution of this protein in sections of demineralized foetal porcine tibia and calvarial bone. Staining for BSP was observed in the matrix of mineralized bone and also in the mineralized cartilage and associated cells of the epiphysis, but was not observed in the hypertrophic zone nor in any of the soft tissues including the periosteum. In comparison, SPP-1 (osteopontin) and SPARC (osteonectin), which are also major proteins in porcine bone, were observed in the cartilage as well as in the mineralized bone matrix. In addition, SPARC was also present in soft connective tissues. Although SPP-1 distribution was more restricted than SPARC, hypertrophic chondrocytes, periosteal cells and some stromal cells in the bone marrow spaces were stained in addition to osteoblastic cells. The variations in the distribution and cellular expression of BSP, SPARC and SPP-1 in bone and mineralizing cartilage indicate these proteins perform different functions in the formation and remodelling of mineralized connective tissues.

Animals↗
PubMed1991

Differential regulation of the 55 and 44 kDa forms of secreted phosphoprotein 1 (SPP-1, osteopontin) in normal and transformed rat bone cells by osteotropic hormones, growth factors and a tumor promoter.

Confluent cultures of rat bone cells synthesize several forms of secreted phosphoprotein 1 (SPP-1, osteopontin), the major phosphorylated forms of which migrate at 55 and 44 kDa on 15% cross-linked SDS-PAGE gels and correspond to the transformation-associated proteins pp 69 and pp 62. A clonal rat calvarial cell line (RCA 11), which expressed the highest level of SPP-1, produced only the 55 kDa form of the phosphorylated protein, whereas normal rat calvarial cells enriched in osteoblastic cells (RC IV cells) produced mostly the 55 kDa form, with small amounts of the 44 kDa form. In contrast, a 44 kDa form was the major [32PO4]-labelled SPP-1 synthesized by a rat osteocarcoma cell line (ROS 17/2.8 cells) with lesser amounts of the 55 kDa SPP-1. When [35S]methionine was used to measure protein synthesis, only the 55 kDa SPP-1 could be clearly detected in confluent cultures of each cell population, indicating that the 55 kDa SPP-1 is the prominent form of SPP-1 synthesized by each cell population. Following treatment of the normal rat bone cells for 24 h with osteotropic hormones (vit D3, PTH and RA), growth factors (PDGF, EGF, TGF-beta), a tumor promoter (TPA) and a plant lectin (Con A), the 55 kDa [35S]methionine labelled SPP-1 was increased 1.7-8.3-fold. Similar, but generally lower responses were observed in the clonal RCA 11 cell line, whereas the ROS 17/2.8 cells were more refractory, showing only a strong response to vit D3. In general, vit D3 produced the strongest stimulation in all populations with TGF-beta producing a good response in the non-transformed cells and RA in the RC IV cells. In contrast, PTH was inhibitory in both RCA 11 and ROS 17/2.8 cells. In most, but not all, cases the alteration in SPP-1 synthesis reflected similar changes in SPP-1 mRNA and in the intensity of the [32PO4]-labelled 55 kDa SPP-1. Collectively, these studies demonstrate that bone cells produce several forms of SPP-1 which are differentially regulated in normal and transformed cells through both transcriptional and posttranscriptional mechanisms.

Animals↗
PubMed1991

Biosynthesis of bone proteins by fetal porcine calvariae in vitro. Rapid association of sulfated sialoproteins (secreted phosphoprotein-1 and bone sialoprotein) and chondroitin sulfate proteoglycan (CS-PGIII) with bone mineral.

To study the biosynthesis of bone proteins, fragments of fetal porcine calvariae were cultured in the presence of 50 micrograms/ml ascorbate and 10 mM beta-glycerophosphate and individual cultures labeled for either 4 h or 48 h with [35S]-methionine, Na2[35SO4], Na3[32PO4] or [14C]-glycine plus [14C]-proline. The radiolabeled proteins in tissue extracts were obtained by sequential extraction with 4 M GuHCl (G1-extract), 0.5 M EDTA (E-extract), and again with 4 M GuHCl (G2-extract) and analyzed together with the radiolabeled proteins secreted into the medium. SPP-1 (secreted phosphoprotein 1, osteopontin) was the major non-collagenous protein deposited into the bone matrix, with lesser amounts of BSP (bone sialoprotein), osteocalcin and chondroitin sulfate proteoglycans (CS-PG II and CS-PG III). SPP-1 was also the major phosphorylated protein and was recovered, together with several fragmented forms, almost entirely in the demineralizing extracts. Moreover, approximately one-half of the [35SO4] incorporated into E-extract proteins was present in SPP-1, the remainder being incorporated into PGs with smaller amounts associated with BSP. Over 65% of the [35SO4] in the proteoglycans of the demineralizing extracts was recovered in the small CS-PG III with less than 35% in CS-PG II, the bone homologue of DS-PG II (decorin). In contrast, CS-PG II was the predominant small proteoglycan in culture media and in guanidine extracts. Some sulfated BSP was also observed in guanidine extracts and small amounts appeared to bind to collagen. Radiolabeled SPARC (osteonectin), a prominent protein of fetal porcine bone, was not detected in the mineralized bone tissues but was prominent in the culture medium. These results demonstrate that following secretion, the major proteins expressed by osteoblastic cells are initially incorporated into different tissue compartments, with most of the sulfated sialoproteins and CS-PG III associating rapidly with the hydroxyapatite crystals. The initial distribution of these proteins is of importance in the evaluation of their role in bone formation and mineralization.

Animals↗
PubMed1991

Localization of bone sialoprotein (BSP) expression to sites of mineralized tissue formation in fetal rat tissues by in situ hybridization.

Bone sialoprotein (BSP) is a major protein in the mineralized matrix of bone and dentine. To study the relationship between the expression of BSP and the formation of mineralized connective tissues, a cDNA probe to rat BSP was prepared for in situ hybridization analysis of developing fetal rat bones and teeth. When used for Northern hybridization analysis of rat bone marrow cells induced to differentiate into osteogenic cells by dexamethasone, the BSP cDNA revealed a specific induction of 1.6- and 2.0-kb mRNA species of BSP. In tissue sections a strong hybridization signal associated with osteoblasts was observed in areas of endochondral bone formation in the long bone metaphysis and condylar cartilage, and in the intramembranous bone of the calvaria and mandible. Hybridization reflecting a lower degree of expression was evident in cells of the transitional zone of mineralizing cartilage and in odontoblasts forming incisor dentine. Expression of BSP was also demonstrated in the hypertrophic cartilage cells in the long bone and condylar process. In contrast, expression of BSP could not be detected in the reserve or proliferative chondrocytes, fibroblasts and muscle cells. These studies demonstrate that the expression of BSP in bones and teeth is essentially restricted to cells directly involved in the formation of mineralizing connective tissue matrices, indicating that BSP has a specific role in biological mineralization and that it is a useful marker of bone formation.

Animals↗
PubMed1991

Collagenase activity in recurrent periodontitis: relationship to disease progression and doxycycline therapy.

Previous reports have suggested that active progression of periodontitis may be correlated with increased collagenolytic activity, and that improved clinical conditions after tetracycline treatment may be explained by inhibition of host collagenase. Eighty-two patients with a recent history of periodontal abscesses and/or loss of gingival attachment level (GAL) despite active periodontal therapy were enrolled in a double-blind, randomized, placebo-controlled trial. Clinical measurements, sampling of gingival crevicular fluid (GCF) and subgingival scaling were performed every 2 months. If any site exhibited greater than 2 mm loss of GAL or a periodontal abscess, patients were administered either 100 mg doxycycline per day for 3 weeks or placebo. During 12 months of monitoring, 55 patients exhibited recurrent active disease and were then randomly assigned to either the doxycycline (n = 30) or placebo (n = 25) groups. Analysis of active collagenase and latent collagenase in GCF samples were determined by functional assays and quantitated after SDS-PAGE and fluorography. Collagenase activities were assayed at sites exhibiting active destruction (study site), at sites with pocket depth comparable to the study site but without active destruction, and at healthy sites. Clinical measurements of GAL and collagenase activity were made at intervals between 1 wk and 7 months after completion of the drug regime. Within 7 months, 15 out of 19 patients on placebo exhibited recurrent disease compared to 13 out of 29 patients on doxycycline. Collagenase activity exhibited large variations among patients and was analyzed as presence or absence of active collagenase with a logistic model.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗
PubMed1991

Evidence for polymorphonuclear leukocyte collagenase and 92-kilodalton gelatinase in gingival crevicular fluid.

Analysis of inflammatory exudate collected from sites of experimental periodontitis in cynomolgus monkeys has revealed the presence of collagenase and a 92-kDa gelatinase that comigrated after electrophoresis with the 92-kDa gelatinase released from polymorphonuclear leukocytes. Since neutralizing antibodies to fibroblast collagenase had no effect on the collagenase activity and bacterial collagenases could not be detected, polymorphonuclear leukocytes appear to be the major source of collagenolytic proteinases in inflammatory fluid from gingiva.

Animals↗
PubMed1991

Mechanism of collagen phagocytosis by human gingival fibroblasts: importance of collagen structure in cell recognition and internalization.

Phagocytosis of extracellular collagen by fibroblasts appears to be the principal pathway of collagen degradation in the physiological turnover of connective tissues. To study the mechanism of collagen phagocytosis, subconfluent gingival fibroblasts were serum-starved and incubated for up to 16 h with collagen-coated fluorescent latex beads. Internalization of beads was measured either by flow cytometry or by image analysis. Phagocytosis was blocked by inactivation of protein kinase C with staurosporin, and was also decreased significantly (32%) when cells were pre-incubated for 6h with cycloheximide. Phagocytosis of collagen-coated beads appeared to be receptor-mediated, since internalization was inhibited threefold by the cell-attachment blocking peptide (GRGDSP). The process of internalization was influenced by the type of collagen and its molecular structure. Thus, internalization was decreased in the order: type I greater than V greater than III collagen, and internalization of type I collagen was reduced significantly by digestion with either bacterial (45%) or vertebrate (38%) collagenase. However, collagen denaturation, which facilitates binding to fibronectin, did not effect internalization. Although concanavalin A stimulated both phagocytosis (71%) and collagenase synthesis, PMA and IL-1, which also increase collagenase expression, did not affect phagocytosis, indicating that phagocytosis of collagen-coated beads does not require collagenase. Moreover, analysis of tissue inhibitor of metalloproteinase expression revealed no difference between phagocytic and non-phagocytic cells. Collectively, these results demonstrate that collagen phagocytosis is regulated through protein kinase C and is also dependent upon cellular recognition and collagen structure, but not on the expression of collagenase.

Alkaloids↗
PubMed1990

Concanavalin A produces a matrix-degradative phenotype in human fibroblasts. Induction and endogenous activation of collagenase, 72-kDa gelatinase, and Pump-1 is accompanied by the suppression of the tissue inhibitor of matrix metalloproteinases.

The lectin concanavalin A (ConA) causes fibroblasts to acquire an arborized morphology and to express elevated levels of collagenase. The temporal and mechanistic aspects of ConA regulation of matrix metalloproteinases (MMPs) and the tissue inhibitor of matrix metalloproteinases (TIMP) were characterized in early passage human fibroblasts. Collagenase (MMP-1), measured by functional assays in the absence of TIMP and also as immunoprecipitated [35S]methionine-labeled protein, was increased 10-20-fold following ConA (20 micrograms/ml, 2 x 10(-7) M) treatment for 24-72 h, with active collagenase comprising approximately 20% of the total collagenase activity. By comparison, MMP-2 (72-kDa gelatinase; molecular mass, 72 kDa, +dithiothreitol; 66 kDa, -dithiothreitol), analyzed by enzymography and following affinity purification, was increased less than 2-fold by ConA and was present entirely as an activated, 61-kDa (+dithiothreitol; 59 kDa, -dithiothreitol) form. Northern hybridization analyses revealed that ConA elevated the steady-state mRNA levels for MMPs; collagenase mRNA increased approximately 16-fold, MMP-2 increased 2-fold, and Pump-1, a recently described MMP gene, was induced. Concomitantly, a 10-fold reduction in TIMP protein and mRNA levels by ConA occurred. In comparison, 12-O-tetradecanoylphorbol-13-acetate (50 ng/ml, 8 x 10(-8) M), which also stimulates collagenase expression strongly (greater than 30-fold), elevated TIMP protein and mRNA levels (2- and 3-fold, respectively) and did not affect MMP-2 expression. The changes in MMP and TIMP mRNA levels induced by ConA were blocked by the protein synthesis inhibitor cycloheximide, and the half-lives of collagenase and MMP-2 mRNAs (53 and 46 h, respectively) were unaffected, indicating that ConA exerts its effects transcriptionally, through pathways requiring de novo protein synthesis. Increased transcription of the mmp genes was confirmed by nuclear run-on analyses; mmp-1 transcription was increased by greater than 25-fold, mmp-2 by approximately 3-fold, and Pump-1 by approximately 7-fold. In contrast, Timp gene transcription was reduced by approximately 80%, revealing reciprocal regulation of MMPs and TIMP during the induction of a resorptive cell phenotype. Decreased amounts of collagen and fibronectin, but not of SPARC (secreted protein, acidic and rich in cysteine) in the conditioned medium was the result of MMP activity since steady-state mRNA levels and transcription of the respective matrix protein genes were unaffected by ConA.

Blotting, Northern↗
PubMed1990

Characterization of fetal porcine bone sialoproteins, secreted phosphoprotein I (SPPI, osteopontin), bone sialoprotein, and a 23-kDa glycoprotein. Demonstration that the 23-kDa glycoprotein is derived from the carboxyl terminus of SPPI.

Demineralizing extracts of porcine bone contain two large 66-80-kDa sialoproteins and smaller 20- and 23-kDa glycoproteins with similar chemical properties. Each protein was characterized following extraction from fetal calvariae and purification under dissociative conditions using Sepharose CL-6B, followed by fast protein liquid chromatography fractionation on hydroxyapatite and Mono Q resins. Unlike the large sialoproteins, the 20- and 23-kDa glycoproteins did not contain sialic acid. Nevertheless, affinity-purified antibodies raised against the 23-kDa protein recognized both the 20-kDa protein and a 67-kDa sialoprotein on immunoblots. These antibodies also immunoprecipitated a 60-kDa [35S]methionine-labeled protein produced by cell-free synthesis of calvarial bone mRNA, indicating that the smaller proteins were derived from the 67-kDa protein. The two sialoproteins were shown by primary sequence analysis to be secreted phosphoprotein I (SPPI, osteopontin, bone sialoprotein I) and bone sialoprotein (BSP, bone sialoprotein II). The SPPI was also characterized by its susceptibility to thrombin which produced a 23-kDa fragment, similar to the glycoprotein isolated, and a 30-kDa fragment. Amino-terminal sequence analysis of the 23- and 20-kDa proteins revealed that these proteins were derived from the carboxyl-terminal half of the SPPI molecule, the proteins showing 58% identity with human and rat, and 50% identity with mouse, SPPI sequences. Both the 23- and 20-kDa proteins appeared to be generated by the activity of an endogenous trypsin-like protease that cleaves at Arg-Ser (residues 155-156) and Lys-Ala (residues 182-183) bonds. Radiolabeling studies performed in vitro showed that the 23-kDa fragment was detectable in mineralized tissue within 4 h. The fragment was phosphorylated but, unlike SPPI, was not sulfated. The rapid generation of the 23-kDa glycoprotein and its presence in different bone tissues at different developmental stages indicate that the fragmentation of SPPI is important in bone formation and remodeling.

Amino Acid Sequence↗
PubMed1990

Immunohistochemical localization of SPARC (osteonectin) and denatured collagen and their relationship to remodelling in rat dental tissues.

To study this relationship, specific antibodies were used to determine the distribution of these proteins in mature rat dental tissues. Staining for SPARC with affinity-purified polyclonal antibodies was prominent throughout molar and incisor ligaments, endosteal tissue, dental pulp and muscle. More moderate staining was observed in other soft tissues including the lamina propria of gingiva, whereas the staining of demineralized bone was weak and in dentine was barely detectable. A monoclonal antibody (MBP 322), raised against a denatured form of a small collagenous bone protein, reacted strongly with osteoblastic cells but more moderately with alveolar bone. A strong reaction, indicative of unfolded collagen, was also evident throughout the dental pulp and molar ligament, whereas in the incisor ligament staining was largely restricted to the tooth-related half. Moderate staining with this antibody was also observed in other soft tissues and in dentine. The monoclonal antibody also stained the nuclei of certain cells; notably, whereas most of the fibroblasts in the tooth-related half of the incisor ligament were stained strongly, only occasional nuclei of fibroblasts in the molar ligament and in the bone-related half of the incisor ligament showed immunoreactivity. The differential staining of nuclei provides evidence for phenotypic differences between fibroblast populations within these tissues. The prominence of SPARC in the ligament tissues is consistent with their embryonic characteristics, whereas unfolded collagen recognized by the MBP 322 antibody may indicate sites of rapid collagen remodelling.

Animals↗
PubMed1990

Identification of polymorphonuclear leukocyte collagenase and gelatinase activities in mouthrinse samples: correlation with periodontal disease activity in adult and juvenile periodontitis.

In previous studies, elevations in the levels of active and latent collagenase in gingival crevicular fluid (GCF) have been correlated positively with periodontal disease activity. To provide a simple diagnostic approach for testing collagenolytic activity, the feasibility of using a 3.0 ml water mouthrinse to collect GCF simultaneously from all sites in the mouth was assessed. Patients with adult periodontitis (AP, n = 23) and local juvenile periodontitis (LJP, n = 7) were sampled before periodontal therapy and some (12 AP, 4 LJP) were also assessed longitudinally after scaling and root planing, administration of antibiotics, and following periodontal surgery. Healthy patients (n = 19) were used as controls. The levels of active collagenase, procollagenase, and collagenase inhibitor activity were determined by functional assays and quantitated after SDS-PAGE and fluorography. Gelatinase and progelatinase were assayed by enzymography on gelatin-substrate gels. Active collagenase levels were found to be significantly higher (14- to 20-fold) in AP and LJP patients compared to controls, whereas matrix metalloproteinase activity was not detected in mouthrinses from edentulous patients. Collagenase inhibitor levels were generally low in all groups of subjects tested. Following clinical treatment the levels of active collagenase and gelatinase were reduced; the reduction was significant for active collagenase after tetracycline treatment and scaling in LJP patients. Of the clinical indices recorded (gingival index, plaque index, and pocket depth) there were no significant correlations with enzyme activity but similar trends were observed between the changes in active collagenase and gingival index. In patients with untreated periodontal disease, collagenase occurred predominantly in the active form. N-ethylmaleimide (NEM) and p-aminophenylmercuric acetate (AMPA) were equally effective as activators of the latent collagenase, indicating that the collagenase was derived from PMNs, which were also the source of gelatinase. The results of these studies indicate that measurement of active collagenase and gelatinase in mouthrinse samples is potentially useful in the diagnosis and assessment of periodontal disease activity.

Adolescent↗
PubMed1989

Sulphation of secreted phosphoprotein I (SPPI, osteopontin) is associated with mineralized tissue formation.

Secreted phosphoprotein I (SPPI) is a prominent structural protein in mineralized connective tissues. Rat bone cells in culture produce several forms of SPPI that differ in post-translational modifications such as phosphorylation and sulphation. To determine the significance of protein sulphation in bone formation, the synthesis of SPPI was studied in vitro using rat bone marrow cells (RBMC) which form bone-like tissue when grown in the presence of dexamethasone (Dex) and beta-glycerophosphate (beta-GP). In the presence of 10(-7) M Dex SPPI expression was stimulated 4-5-fold. Radiolabelling multilayered RBMCs for 48 h with [35S]-methionine, Na2[35SO4], or Na3[32PO4] revealed that two major phosphorylated forms of SPPI were secreted into the culture medium: a highly phosphorylated form migrating at 44 kDa on 15% SDS-PAGE and a less phosphorylated 55 kDa form. In the mineralized tissue formed in the presence of Dex and beta-GP, both forms of SPPI, in addition to proteoglycans and a 67 kDa protein, incorporated significant amounts of [35SO4]. Sulphation of SPPI was not observed in the absence of mineral formation, indicating that the sulphation of SPPI is closely associated with mineralization and that it can be used as a sensitive and specific marker for the osteoblastic phenotype.

Animals↗
PubMed1989

Multiple forms of SppI (secreted phosphoprotein, osteopontin) synthesized by normal and transformed rat bone cell populations: regulation by TGF-beta.

Metabolic labeling has revealed that rat bone cell populations in culture synthesize several forms of the secreted phosphoprotein, SppI. Most cell populations produced two major [32PO4]-labeled forms that behaved anomolously on SDS-PAGE migrating at 60 kDa and 56 kDa on 10% gels and 55 kDa and 44 kDa on 15% gels. Minor forms of intermediate sizes were also resolved. In normal bone cells the 60 kDa form was predominant and was the only form produced by the clonal bone cell line, RCA 11, whereas the 56 kDa a form predominated in the transformed bone cell line, ROS 17/2.8. In all populations [35S]-methionine-labeling revealed SppIs at approximately 60 kDa but no 56 kDa form. Each form of SppI was specifically cleaved by thrombin which generated fragments of approximately 28 kDa. Transforming growth factor beta 1 increased SppI mRNA levels 3 to 6-fold within 24 h in the normal bone cells, but no increase occurred in the ROS 17/2.8 cells. The elevated expression of SppI was reflected in a selective increase in the synthesis of the [32PO4]-and [35S]-methionine-labeled 60 kDa SppIs.

Animals↗
PubMed1989

Independent regulation of collagenase, 72-kDa progelatinase, and metalloendoproteinase inhibitor expression in human fibroblasts by transforming growth factor-beta.

The effects of transforming growth factor-beta (TGF-beta) on fibroblast collagenolytic activity were investigated to determine if modulation of matrix metalloendoproteinase activity could augment the stimulation of connective tissue formation by TGF-beta. Quiescent human fibroblast cultures were incubated in the continuous presence of 1.0 ng/ml (40 pM) TGF-beta in culture medium supplemented with 0.2% (v/v) serum and containing [35S]methionine. Aliquots of conditioned cell culture media, harvested daily for 4 days, were processed individually to separate procollagenase and a 72-kDa progelatinase from metalloendoproteinase inhibitor (TIMP) and plasminogen activator inhibitor (PAI-1) using tandem minicolumns of heparin- and gelatin-Sepharose. The fractionated 54-kDa procollagenase was quantitated, after p-amino-phenylmercuric acetate activation, by functional assays using soluble [14C] glycine-labeled collagen as substrate. In cultures treated with TGF-beta, procollagenase expression was progressively decreased (approximately 50% on day 1, approximately 75% on day 2) to undetectable levels on days 3 and 4. This decrease occurred despite a 1.6-fold increase in the synthesis of total secreted protein. Contrasting the effect on procollagenase, TGF-beta increased the synthesis of a 72-kDa progelatinase (characterized as a matrix neutral metalloproteinase and likely to be MMP-2) up to 1.8-fold, as determined by quantitation of affinity-purified radiolabeled protein and by enzymography. TIMP biosynthesis was analyzed by immunoprecipitation and quantitated by functional assays for biologically active TIMP following fractionation of the conditioned media. During the first 24 h TGF-beta had little apparent effect on TIMP activity in the medium although the TIMP mRNA transcript was induced 1.3-1.4-fold. Subsequently, TIMP levels were increased 1.7-fold relative to control cells on day 4. This was accompanied by a 2.4-fold increase in TIMP mRNA, indicating that the regulation of TIMP mRNA and protein levels may be a secondary response to TGF-beta. In comparison, the synthesis of the Mr 48,000 PAI-1, analyzed by [35S] methionine labeling and immunoprecipitation, was elevated greater than 10-fold by TGF-beta at all time points with the highest levels occurring at day 2. Thus, the effects of TGF-beta on procollagenase, 72-kDa progelatinase, TIMP, and PAI-1 were selective and showed temporal differences.(ABSTRACT TRUNCATED AT 250 WORDS)

Carrier Proteins↗
PubMed1989

Characterization of multiple forms of small collagenous apatite-binding proteins in bone.

A number of small (Mrs 25-28 kDa) collagenous apatite-binding (SCAB) proteins that stain blue with 'Stains-All' have been isolated from fetal porcine bone by sequential extractions with 4M GuHCl (G1), followed by 0.5M EDTA (E), and again with 4M GuHCl (G2). Following purification under dissociative conditions, two types of SCAB proteins both with approximately one-third of their structure being collagenous, were identified in the EDTA extract. One type, which appears to be a novel protein, was revealed in two forms (SCABs 1 and 2, Mrs 25 and 28 kDa) that were recognized by a monoclonal antibody (MBP-322). The second type, SCAB 3, was also present in two forms; one form (SCAB 3a) having a lower affinity for hydroxyapatite than the other (SCAB 3b). These proteins were resistant to CNBr and displayed the chemical and immunochemical properties of the alpha 1 pN-propeptide of type I collagen. A third form of the propeptide (G2-28K) was a prominent component of the second 4M GuHCl extract. The chromatographic properties of serum alpha 1 (I) pN-propeptide were similar to SCAB 3a, indicating that SCAB 3b and G2-28K are post-translationally modified forms of the propeptide produced by bone cells. These propeptides may provide a link between the hydroxyapatite and collagen fibrils, and also have the potential to suppress collagen synthesis during bone resorption.

Animals↗