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J Sodek

Publications and source records attributed to J Sodek.

At least 73 records · Page 4Linked to original sources
PubMed1994

Carbonic anhydrase II mRNA expression in individual osteoclasts under "resorbing" and "nonresorbing" conditions.

Rabbit osteoclasts can be transformed from a nonresorbing state to a resorbing state by transferring them from culture medium at pH 7.5 to one at pH 6.5. We evaluated whether expression of mRNA for carbonic anhydrase (CA-II) could be used as an indicator of the state of activity of individual osteoclasts. A cDNA probe to rabbit carbonic anhydrase II (CA-II) was prepared and used for in situ hybridization analysis of osteoclasts isolated from neonatal rabbit long bones. Quantitation by grain counting revealed heterogeneity within the osteoclast population: osteoclasts with a "compact" (rounded, less spread) morphology expressed higher levels of CA-II mRNA than "spread" osteoclasts with similar numbers of nuclei. When maintained at pH 6.5 for 6 h, the level of CA-II mRNA was increased significantly in osteoclasts of both morphologies compared with those in parallel cultures maintained at pH 7.5. These results were confirmed by quantitating CA-II mRNA using the polymerase chain reaction (PCR). Oligonucleotide primers specific for rabbit CA-II were synthesized and used to amplify CA-II cDNA transcribed from mRNA prepared from single or small numbers (one to eight cells) of osteoclasts that were collected with a micromanipulator. This generated a approximately 510 bp PCR product, corresponding to the predicted size of the CA-II fragment encompassed by the primers. For quantitation, CA-II mRNA levels were compared with the levels of a approximately 900 bp actin fragment that was coamplified in the same reaction mixture or amplified separately in a duplicate sample of the reaction mixture.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1994

Characterization of the human bone sialoprotein (BSP) gene and its promoter sequence.

Bone sialoprotein (BSP) is a major structural protein of the bone matrix that is specifically expressed by fully-differentiated osteoblasts. To characterize the gene and to study the tissue-and differentiation stage-specific regulation of BSP gene transcription we have isolated and partially sequenced two overlapping genomic fragments which span the complete human BSP gene and its promoter region. The approximately 15 kb gene comprises seven exons of 82 bp, 68 bp, 51 bp, 78 bp, 63 bp, 159 bp and 2.5 kb (1-7, respectively), separated by six introns of approximately 3 kb, 92 bp, 95 bp, approximately 3 kb, approximately 0.5 kb and approximately 4.5 kb. All of the intron-exon boundaries defining the splice sites conform to the consensus sequence of: AG at the 3' splice site; and GT at the 5' splice site, except the 3' splice site of exon 1. The first exon encodes the 5'-UTR, the second exon the signal sequence and the first two amino acids, exons 3 and 4 the Tyr-and Phe-rich amino terminus, and exon 5 the first segment of polyglutamic acid. Exon 7 encodes over half of the protein including a second polyglutamic acid segment, the RGD cell attachment motif, the sulphated tyrosine-rich C-terminus and the 3'-UTR. The promoter region is characterized by an inverted TATA-like sequence (TTTATA), nts -28 to -23 from the transcriptional start site (+1), and an inverted CCAAT box (ATTGG) at -54 to -50. Analysis of chimeric constructs fused to a CAT reporter gene indicate that the presence of both the inverted TATA-like sequence and CCAAT elements are required for basal promoter activity. Comparison of the human BSP promoter with the rat BSP promoter (Li and Sodek, 1993) reveal that the nature and position of the inverted TATA-like sequence and CCAAT box together with an Ap1 (-148 to -142), CRE (-122 to -116) and a homeobox-binding site (-200 to -191) have been conserved. A putative Glucocorticoid Response Unit (GRU) consisting of a Glucocorticoid Response Element (GRE) and an overlapping direct repeat (DR5) similar to the retinoic acid receptor element (RARE) is present at -1038 to -1022. These studies have defined the structure of the human BSP gene and have identified novel transcriptional elements in the promoter that may be involved in the developmentally regulated, tissue specific expression of this gene.

Amino Acid Sequence↗
PubMed1994

Bone sialoprotein mRNA expression and ultrastructural localization in fetal porcine calvarial bone: comparisons with osteopontin.

Bone sialoprotein (BSP) and osteopontin (OPN) are two major non-collagenous proteins in bone that have similar biochemical properties and can mediate cell attachment through an RGD (Arg-Gly-Asp) motif that recognizes the vitronectin receptor. To facilitate evaluations of the biological functions of BSP and OPN in bone formation, affinity-purified rabbit polyclonal antibodies against porcine BSP and OPN were used, together with a high-resolution protein A-gold immunocytochemical technique to reveal the ultrastructural localization of these proteins in undermineralized sections of 50-day fetal porcine calvarial bone. In addition, 35S-labelled antisense riboprobes were prepared to demonstrate the cellular expression of BSP and OPN in the same tissues using in situ hybridization. Immunolocalization for both BSP and OPN revealed the highest density of gold particles associated with electron-dense organic material found at the mineralization front and in 'cement lines'. Labelling was also observed in the mineralized matrix over electron-dense material between collagen fibrils. In the osteoid of newly-formed bone, immunogold labelling for BSP and OPN was associated with loci of mineralization, which were often characterized by feathery clusters of fine needle-like crystals. Results of in situ hybridization on the same tissues demonstrated that BSP mRNA expression was restricted to differentiated osteoblasts with particularly strong signals evident at sites of de novo bone formation. More moderate expression of BSP was observed in 'older' osteoblasts and in some of the newly-entrapped osteocytes. Although expression of OPN mRNA was also observed in osteoblasts and osteocytes, the level of hybridization was similar for most bone cells and not markedly stronger than the signal observed in some stromal cells. While it is evident from these and other studies that both BSP and OPN are associated with bone formation, the differences observed in cellular expression indicate distinct roles for these proteins in bone formation.

Amino Acid Sequence↗
PubMed1993

Cloning and characterization of the rat bone sialoprotein gene promoter.

To study the transcriptional regulation of the rat bone sialoprotein (BSP) gene, the nucleotide sequence of a approximately 1 kb HindIII/KpnI subfragment from a genomic clone containing the 5' flanking sequence, exon 1 and part of intron 1 was determined and the transcription start site defined. This region includes an inverted TATA element (nt -24 to -19), an inverted CCAAT box, a homeobox-binding site, a putative 1,25-dihydroxyvitamin D3 response element (VDRE) sequence overlapping the inverted TATA sequence, and a novel 18 nt palindrome that may control the tissue-specific transcription of the BSP gene. The shortest promoter sequence capable of directing bacterial chloramphenicol acetyltransferase reporter gene expression included the inverted TATA element and the inverted CCAAT box. However, the promoter activity was down-regulated by 1,25-dihydroxyvitamin D3, indicating that the unique VDRE-like sequence overlapping the TATA element is functional. Thus the rat BSP gene promoter is characterized by novel cis-acting elements that may be involved in hormone- and tissue-specific regulation of transcription.

Animals↗
PubMed1993

Bone sialoprotein in developing porcine dental tissues: cellular expression and comparison of tissue localization with osteopontin and osteonectin.

Bone sialoprotein (BSP) is a highly sulphated and glycosylated phosphoprotein that is a major constituent of bone and other mineralized connective tissues. Although BSP can mediate cell attachment through an RGD sequence and binds selectively to hydroxyapatite, its precise function in mineralized tissues is unknown. To provide insights into its possible function, affinity-purified polyclonal antibodies directed against porcine BSP were used to demonstrate the histological distribution of this protein in developing porcine mandibular alveolar bone and the associated tooth tissues from 35- and 50-day fetuses. In addition, a porcine cRNA probe was used to determine the cellular expression of BSP in the same tissues by in situ hybridization. Immunoreactivity to BSP protein was restricted to the cells and matrix of the mineralized tissues of alveolar bone and dentine. In dentine, BSP was localized to the odontoblasts and their processes and to the peritubular dentine. In the alveolar bone, immunoreactivity for BSP was evident in osteoblastic cells and osteocytes and in the bone matrix; the older bone stained more strongly than newly formed bone. In addition, BSP appeared to be concentrated in the reversal lines of the rapidly remodelling bone. The distribution of BSP in these tissues revealed distinct differences when compared to osteopontin and SPARC/osteonectin, which are also prominent non-collagenous proteins of mineralized tissues. Most notable was the localization of osteopontin and especially osteonectin in non-mineralizing tissues. The immunoreactivity of osteoblasts and osteocytes for BSP in bone was consistent with the high levels of BSP mRNA revealed by in situ hybridization. However, much lower levels of hybridization were evident in the odontoblasts of developing mandibular molars. These studies demonstrate that BSP is expressed during the early formation of dentine and alveolar bone and that the protein accumulates in the peritubular dentine and bone matrix.

Alveolar Process↗
PubMed1993

Influence of an intermittent compressive force on matrix protein expression by ROS 17/2.8 cells, with selective stimulation of osteopontin.

The purpose of this study was to determine the response of bone cells to physical stress. Intermittent compressive force (ICF) was applied to 13 kPa to subconfluent ROS 17/2.8 cells at 18 cycles/min. After 48 h of this application, the cells were labelled with [35S]-methionine or [32PO4]. Application of ICF over this time did not alter the synthesis of type I collagen, fibronectin or bone SPARC (osteonectin) compared to that of control cells. However, the activity of alkaline phosphatase was increased 1.5-fold, and the synthesis of a 32PO4-labelled, 75-kDa phosphoprotein, recognized as osteopontin by immunoprecipitation with specific antibodies, was increased 1.4-fold. Also, an increase in osteopontin mRNA starting within 12h of ICF application was observed. The selective increase in osteopontin expression associated with ICF may be important in the remodelling of bone tissues during growth and development and in response to functional forces.

Alkaline Phosphatase↗
PubMed1993

Developmental expression of osteopontin (OPN) mRNA in rat tissues: evidence for a role for OPN in bone formation and resorption.

Osteopontin (OPN) is a 34-kDa, highly-phosphorylated glycoprotein with cell attachment properties that is a prominent constituent of the bone matrix. To aid in elucidating the function of this protein we have studied the cellular expression of OPN mRNA during the formation, growth and maturation of rat calvarial (membranous) and tibial (endochondral) bone. From Northern hybridization analysis OPN expression was demonstrated in the kidney and gravid uterus as well as in bone tissues. Compared to collagen, the expression of OPN was low in early bone formation but increased subsequently and reached peak levels in 14-day-old bone. However, both the collagen and OPN mRNAs decreased markedly thereafter and remained low in young adult bone. From in situ hybridization studies using a [35S]-labelled rat OPN cRNA probe, OPN mRNA was localized to osteoblastic cells in newly-forming calvariae, jaw bones, and in the metaphyseal and periosteal bone of the tibia. In contrast to bone sialoprotein (BSP), which is expressed almost exclusively by osteoblasts at sites of de novo bone formation, OPN transcripts were present in cells lining both endosteal and periosteal bone surfaces, and in osteocytes. Moreover, expression of OPN persisted during the subsequent growth and remodelling of both membranous and endochondral bone and was expressed at particularly high levels by bone cells and hypertrophic chondrocytes at sites of osteoclastic resorption. In the more mature bone of young adult rats OPN expression was significantly reduced but remained detectable in bone cells lining periosteal and endosteal surfaces and in the primary and secondary spongiosa of the tibia. These studies on the developmental expression of OPN support the concept of a multifunctional role for OPN in bone formation and remodelling. Thus, the expression of OPN by osteoblasts early in bone development is consistent with a role for this protein in the formation of bone matrix, whereas the peak expression of OPN later in bone development, together with high expression at sites of rapid remodelling, indicate that OPN deposited on the surface of mineralized connective tissues may provide a template for osteoclastic resorption.

Animals↗
PubMed1993

Characterization of porcine bone sialoprotein: primary structure and cellular expression.

Bone sialoprotein (BSP) is a highly glycosylated and sulphated phosphoprotein that is a major non-collagenous protein of bone. To further characterize the porcine protein and to study its expression during bone formation BSP cDNA clones were isolated from a porcine bone cDNA library. The primary sequence of the protein was derived from the nucleotide sequence of the largest cDNA insert and from the amino-terminal amino acid sequence determined by the automated Edman degradation procedure. When compared with sequences obtained from the human and rat BSPs 74% and 64% of the amino acids, respectively, were identical and a further 11% and 17%, respectively, were conservative replacements. Moreover, 60% of the amino acids in a concensus sequence derived from the primary sequences of mammalian BSPs were conserved with 16% conservative replacements. The two stretches of polyglutamic acid, through which the protein is capable of binding to hydroxyapatite, and an RGD motif that mediates cell attachment are retained in conserved sequences as are a number of potential sites of serine, threonine and tyrosine phosphorylation, glycosylation and tyrosine sulphation. Secondary structure prediction and hydrophilicity analysis indicate that the nascent BSP has an open flexible structure with the potential to form significant amounts of alpha-helix and some beta-sheet. In situ hybridization of fetal porcine bone with cRNA probes to porcine BSP mRNA shows that BSP is specifically expressed in differentiated osteoblasts on the surface of newly-forming bone trabeculae with especially high levels of hybridization at sites of de novo bone formation. The highly conserved features of BSP and its restricted distribution indicate an important role for this sialoprotein in the formation of bone.

Amino Acid Sequence↗
PubMed1992

Characterization of the promoter region of the porcine opn (osteopontin, secreted phosphoprotein 1) gene. Identification of positive and negative regulatory elements and a 'silent' second promoter.

Osteopontin (secreted phosphoprotein-1, Opn) is a phosphorylated glycoprotein expressed by transformed cells, macrophages, activated T-lymphocytes, specialized epithelial cells and bone cells that is characteristically enriched in milk and in the mineralized matrix of bone. The synthesis of Opn by bone cells is regulated by glucocorticoids and growth factors, which promote bone formation, and by the osteotropic hormone calcitriol (1,25-dihydroxycholecalciferol) and retinoic acid, which mediate bone resorption, indicating a bifunctional role for this protein in bone remodelling. To study the transcriptional regulation of the opn gene, two genomic clones (10 and 15 kb) encoding the opn gene were isolated from a porcine liver genomic library cloned into lambda phage. From the 15-kb clone a 4-kb EcoRI fragment containing the first two exons and 2.6 kb of the 5' flanking region of the opn gene was sequenced, and the transcriptional start site determined by primer extension analysis and S1 nuclease mapping. To identify the opn promoter, chimeric chloramphenicol acetyltransferase constructs were prepared using fragments from the first intron and the 5' flanking region of the opn gene. Transient transfection of porcine bone cells with these constructs showed strong promoter activity located within 74 bp upstream from the transcription initiation site. Within this region a TATA sequence, TTTAAA, was identified at positions -26 to -31. However, the highest transcription rate was observed in a construct extending 180 bp upstream that included a CCGCCC Sp1 binding sequence (-63 to -68), and an AP1 site (-74 to -80). Further upstream in the 5' flanking region and within the first intron of the opn, a number of consensus sequences could be identified. Chimeric constructs containing a GGGTCAtatGGTTCA direct repeat consensus sequence for a vitamin D3 response element located at nucleotides -2245 to -2259 responded to the addition of 0.1 microM calcitriol by a 2.5-fold stimulation of transcription, although a greater than 2-fold increase was also observed in shorter constructs -180 to -905 lacking such a consensus sequence. Promoter activity was also exhibited by a region containing a TTTAAA sequence in the first intron that corresponded to the putative promoter site reported for mouse opn in macrophages (Miyazaki, Y., Setoguchi, M., Yoshida, S., Higuchi, Y., Akizuki, S. & Yamamoto, S. (1990) J. Biol. Chem. 265, 14432-14438).(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1992

Development expression of bone sialoprotein mRNA in rat mineralized connective tissues.

Bone sialoprotein (BSP) is a phosphorylated and sulfated glycoprotein that is a major noncollagenous protein of bone and other mineralizing connective tissues. BSP is characterized by the presence of several polyglutamic acid segments and an RGD motif that mediates cell attachment through a vitronectin-like receptor. Although the precise function of BSP is unknown, the expression of BSP in conjunction with bone formation in vitro indicates a role for this protein in the biomineralization of connective tissues. In this study we used Northern hybridization and in situ hybridization to determine the tissue-specific and developmental expression of BSP during embryogenesis and growth of rat tissues. Analysis of tissues obtained from 13, 17, and 21 day fetuses, and from 4-, 14-, and 100-day-old animals indicates that BSP mRNA expression is restricted to cells actively forming the mineralizing tissues of bone, dentin and cementum. BSP mRNA transcripts were first evident in fully differentiated osteoblasts of 17 day fetal tissues at sites of de novo intramembranous and endochondral bone formation, with maximal expression observed at 21 days of gestation. Thereafter, BSP mRNA levels decreased markedly, and in adult bone hybridization was detected only in the primary spongiosa of long bones. In comparison, mRNAs for osteopontin (OPN), alkaline phosphatase (ALP), and osteocalcin (OC) peaked at 4-14 days postpartum before declining. In the tibiae, Northern hybridization revealed a second peak of mRNA for BSP, ALP, and OPN at 14 days, reflecting an increased osteogenic activity due to the formation of the secondary centers of ossification in the epiphyseal cartilage. In situ hybridization also revealed BSP mRNA in hypertrophic chondrocytes at sites of bone formation, in odontoblasts of the incisor during dentinogenesis, and in cementoblasts during cementogenesis. In view of the restricted distribution and temporal changes in the expression of BSP mRNA that we observed together with the chemical properties of BSP, we believe that this protein has a specific role in mediating the initial stages of connective tissue mineralization.

Alkaline Phosphatase↗
PubMed1992

Temporal studies on the tissue compartmentalization of bone sialoprotein (BSP), osteopontin (OPN), and SPARC protein during bone formation in vitro.

To study the role of noncollagenous proteins in bone formation, the synthesis and tissue distribution of BSP (bone sialoprotein), OPN (osteopontin) and SPARC (secreted protein acidic and rich in cysteine) were analyzed using pulse-chase and continuous labeling protocols during bone formation by cultures of rat calvarial cells. Following a 1 h labeling period with [35S]methionine or [35SO4], radiolabeled BSP was rapidly lost from the cells and appeared transiently in the culture medium and in a 4 M GuHCl extract (G1) of the mineralized tissue. Coinciding with the loss of BSP from these compartments, radiolabeled BSP increased in demineralizing, 0.5 M EDTA extracts (E) of the bone, in a subsequent GuHCl extract (G2), and in a bacterial collagenase digest (CD fraction) of the extracted tissue, over a 24 h chase period. In comparison, the 55 kDa form of OPN, with a small amount of the 44 kDa OPN, was secreted almost entirely into the culture medium. Most of the 44 kDa OPN, together with some 55 kDa OPN, accumulated rapidly in the E extract but could not be detected in either G extract or in the CD fraction. SPARC appeared transiently in the G1 extract, but was otherwise quantitatively secreted into the culture medium from where it was lost by complexing and/or degradation. When cultures were continuously labeled over a 12 day period with [35S]methionine, radiolabeled BSP and 44 kDa OPN accumulated in the E extract together with a small amount of SPARC. Some radiolabeled BSP also accumulated in the G2 extract. From the relative incorporation of [35SO4] over the same time period, a time-dependent loss in sulphate from the BSP was evident. Using a 24 h pulse-labeling protocol, the amount of radiolabeled BSP and OPN in the E extract and the BSP in the G2 extract were not altered significantly over a 12-day chase period. These studies demonstrate that the 44 kDa OPN and most of the BSP are rapidly bound to the hydroxyapatite crystals where they may regulate crystal formation and growth during bone formation. Some BSP is deposited in the osteoid and appears to become masked by the formation of hydroxyapatite, indicating a potential role for this protein in epitactic nucleation of hydroxyapatite crystal formation.

Animals↗
PubMed1992

Gingival crevicular fluid gelatinase and its relationship to periodontal disease in human subjects.

Collagenolytic enzymes released by neutrophils are associated with the destruction of periodontium in periodontal diseases. Measurement of these enzymes in gingival crevicular fluid (GCF) could be used to test for periodontal diseases and thereby simplify diagnosis. To test this hypothesis, gelatinase (MMP-9) was analyzed in GCF samples with a simple assay system. GCF was collected by a mouthrinse method from 10 patients with gingivitis (G); 10 well-treated and maintained periodontitis patients (TP) without detectable loss of attachment; and 9 patients with recurrent loss of periodontal attachment (greater than 2 mm) and/or abscess formation (RP). Clinical measurements including tooth mobility (MOB) and gingival attachment level (GAL) were made monthly for a maximum of 10 months. Active and latent forms of gelatinase were measured by a functional assay using gelatin substrate-gel enzymography and the activities were quantified by laser densitometry. Reproducibility analysis demonstrated that the assay (inter-gel, inter-assay, inter-scan) and diurnal variations were small compared to biological variation. The presence of active gelatinase was detected in 97.8% of TP samples, 86.4% of RP samples, but in only 11.4% of G samples. In addition, the mean active gelatinase activity was found to be significantly higher (p less than 0.001) in the RP (71,006 U) than the TP (43,814 U) groups, both of which were higher (p less than 0.001) than the G group (2824 U). During periods of attachment loss, samples from the RP group exhibited a 2-fold increase of mean active gelatinase activity (129,414 U).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1992

Matrix metalloproteinases in periodontal tissue remodelling.

Inflammation of the periodontium leads to connective tissue degradation and eventual tooth loss. The regulation of matrix metalloproteinases (MMPs) has been studied to determine their role in these processes and also during tissue remodelling. Analysis of gingival crevicular fluid has revealed the presence of collagenase and gelatinase that, in the acute stages of periodontal disease, are derived predominantly from polymorphonuclear leukocytes. These MMPs appear to be intimately associated with tissue destruction since the levels of the active forms of these enzymes obtained from either crevicular fluid or mouthrinse samples correlate with tissue destruction and, therefore, provide a sensitive means of demonstrating disease activity. Transforming growth factor-beta, an important regulator of connective tissue remodelling, has been implicated in the rapid remodelling of periodontal tissues. TGF-beta promotes tissue matrix formation by stimulating both the synthesis of matrix proteins (collagen, fibronectin and SPARC) and proteinase inhibitors (TIMP, PAI-1) and by decreasing the synthesis of MMPs, but not the 72 kDa-gelatinase. Nuclear run-on analyses have shown that TGF-beta reduces collagenase and stromelysin synthesis by suppressing gene transcription without altering mRNA stabilities. In contrast, the transcription of the gelatinase and TIMP genes was increased by TGF-beta, which also increased gelatinase mRNA stability. Remodelling of alveolar bone involves interaction between osteoblasts and osteoclasts. Osteoblasts, under the influence of osteotropic hormones (vit D3, PTH and retinoic acid), produce MMPs which appear to function in the removal of soft tissue that precludes access of osteoclasts to the mineralized tissue surface. Rat osteoblastic cells produce MMPs with activity on native collagen, native collagen 3/4-fragments and gelatin and, in addition, two forms of TIMP activity. The 3/4-collagen endopeptidase, purified to apparent homogeneity, also has significant collagenase and gelatinase activities and an amino terminal sequence almost identical to human 72 kDa-gelatinase. The production of this enzyme was stimulated by TGF-beta, which suppresses bone resorption, and by osteotropic hormones which stimulate bone resorption, supporting a bifunctional role for the gelatinase in connective tissue remodelling. Although there is strong evidence for the involvement of MMPs in the resorption of bone and in the inflammation-mediated destruction of periodontal tissues, the role of MMPs in the remodelling of mature soft connective tissues remains equivocal.

Alveolar Bone Loss↗
PubMed1991

Transcriptional and post-transcriptional regulation of 72-kDa gelatinase/type IV collagenase by transforming growth factor-beta 1 in human fibroblasts. Comparisons with collagenase and tissue inhibitor of matrix metalloproteinase gene expression.

The temporal aspects and mechanisms of the regulation of the matrix metalloproteinase (MMP) 72-kDa gelatinase/type IV collagenase (MMP-2) by transforming growth factor-beta 1 (TGF-beta 1) were investigated in early passage human gingival fibroblasts and compared with the regulation of the genes for collagenase (MMP-1) and TIMP, the tissue inhibitor of MMPs. Northern hybridization analyses revealed that 1.0 ng/ml TGF-beta 1 increased the abundance of MMP-2 mRNA/cell approximately 1.5-fold at 24 h, an increase similar to that observed in the level of [35S]methionine pulse-labeled MMP-2 at 24 h (1.9-fold). At 48 and 72 h, the increase in MMP-2 mRNA abundance remained elevated by 1.5-2.2-fold on a per cell basis whereas TIMP mRNA levels were elevated by up to 3.3-fold. In contrast, the relative levels of collagenase mRNA were reduced by 66-75%. The changes in the MMP-2, collagenase, and TIMP mRNA concentrations in response to TGF-beta 1 were blocked by cycloheximide indicating that protein synthesis was required to mediate the effects of TGF-beta 1 on these mRNA levels. TGF-beta 1 was also found to increase the half-life of the MMP-2 mRNA from approximately 46 to approximately 150 h but did not alter the stability of TIMP mRNA (t1/2 approximately 60 h). Nuclear run-off transcription assays revealed that MMP-2 gene transcription was increased approximately 5-fold 7 h following TGF-beta 1-treatment but returned to control levels by 24 h. In comparison, increased TIMP gene transcription was only detectable after 24 h whereas collagenase gene transcription, although low in control cells, was undetectable at 24 h. Gene transcription, mRNA levels, and message stability of the genes for the extracellular matrix proteins type I collagen and fibronectin were also increased by TGF-beta 1. Thus, the similarity in the control of MMP-2, alpha 1 (I) procollagen, and fibronectin expression at the transcriptional and post-transcriptional levels indicates that these genes may share regulatory elements. In comparison, TGF-beta 1 reduced the level of collagenase mRNA and increased the level of TIMP mRNA as a result of altered transcriptional activities, through pathways that required protein synthesis, and without changes in mRNA stability.

Blotting, Northern↗
PubMed1991

Regulation of the expression of a secreted acidic protein rich in cysteine (SPARC) in human fibroblasts by transforming growth factor beta. Comparison of transcriptional and post-transcriptional control with fibronectin and type I collagen.

Transforming growth factor beta (TGF-beta) and secreted protein acidic rich cysteine (SPARC) have been associated with the rapid remodeling of connective tissues that occurs in wound healing and developmental processes. To study the temporal and mechanistic aspects of TGF-beta-regulated extracellular-protein gene expression in human fibroblasts, confluent cells were pulse labeled for 30 min with [35S]methionine at various times following the single addition of 1.0 ng/ml TGF-beta. After a 4-h chase period, specific radiolabeled media proteins were isolated by either immunoprecipitation or affinity chromatography and quantitated. Stimulation of SPARC synthesis was first apparent 5 h after addition of TGF-beta, reached a maximum (3.5-fold increase) at 24 h and persisted for at least 96 h. A similar temporal response to TGF-beta was observed for the extracellular matrix proteins collagen and fibronectin. In contrast, TGF-beta induced a strong (greater than sixfold increase at 9 h after addition of TGF-beta), but transient stimulation of the synthesis of endothelial-type plasminogen activator inhibitor. Northern blot analysis showed that SPARC mRNA levels were increased by TGF-beta in parallel with increase in SPARC synthesis; a maximum 3.9-fold increase in SPARC mRNA being reached at 24 h. Similarly, the levels of both collagen and fibronectin mRNA were increased by TGF-beta treatment. In each case the stimulation of mRNA was blocked by the presence of the translation inhibitor, cycloheximide. Stability of SPARC mRNA (half-life of approximately 50 h) was not significantly altered by TGF-beta. In contrast, the stability of collagen and fibronectin mRNA were both increased in the presence of TGF-beta; the increased stability being pronounced in less dense cells. In addition to effects on stability, transcription of the collagen and fibronectin genes was increased 7 h after TGF-beta addition, but returned to control levels by 24 h. However, transcription of the SPARC gene was unaffected by TGF-beta at both time points and, together with the stability data, indicates that TGF-beta regulates SPARC expression via a nuclear post-transcriptional mechanism. Differential regulation of gene expression by TGF-beta in a precise temporal pattern via transcriptional and post-transcriptional pathways may be an important aspect of the response of fibroblast cells in a wound environment.

Animals↗
PubMed1991

Biosynthesis of bone proteins [SPP-1 (secreted phosphoprotein-1, osteopontin), BSP (bone sialoprotein) and SPARC (osteonectin)] in association with mineralized-tissue formation by fetal-rat calvarial cells in culture.

To determine the relationship between the expression of bone proteins and the formation of mineralized-tissue matrix, the biosynthesis of non-collagenous bone proteins was studied in cultures of fetal-rat calvarial cells, which form mineralized nodules of bone-like tissue in the presence of beta-glycerophosphate. The temporal pattern of protein synthesis in both mineralizing and non-mineralizing cultures was studied by metabolic labelling with [35S]methionine, 35SO4(2-) or 32PO4(3-) over a 5-day period. After a 24 h labelling period, the culture media were harvested and the cell layers extracted sequentially with aq. 0.5 M-NH3, followed by 4 M-guanidinium chloride (GdmCl), 0.5 M-EDTA and a second extraction with 4 M-GdmCl. Protein associated with collagenous bone matrix was analysed after digestion with bacterial collagenase. On the basis of [35S]methionine labelling, the major proteins extracted from the mineralizing matrix were secreted phosphoprotein-1 (SPP-1; osteopontin), bone sialoprotein (BSP) and a 14 kDa phosphoprotein. The presence of SPP-1 and BSP in the conditioned media of both mineralizing and non-mineralizing cultures and their incorporation into the mineralizing nodules indicated that these proteins associate with preformed mineral crystals. However, some BSP was also present in GdmCl extracts and, together with a 35 kDa sulphated protein, was released from a bacterial-collagenase digestion of the tissue residue in both non-mineralizing and mineralizing cultures. Two forms of sulphated SPP-1 were identified, a highly phosphorylated 44 kDa species being the predominant form in the mineralized matrix. The BSP was more highly sulphated but less phosphorylated than SPP-1. Bone SPARC (secreted protein, acid and rich in cysteine) protein (osteonectin) was present almost entirely in the conditioned media and did not incorporate 32PO4(3-) or 35SO4(2-). The SPP-1 and the 14 kDa protein were susceptible to thrombin digestion, the 44 kDa SPP-1 being specifically cleaved into 28 and 26 kDa fragments. The fragments were labelled uniformly with [35S]methionine, but the 28 kDa fragment incorporated more 35SO4(2-), but less 32PO4(3-), than the 26 kDa fragment. These studies demonstrate that SPP-1 and BSP are the major osteoblast-derived bone proteins to bind to the bone mineral. That BSP also binds to the collagenous bone matrix indicates a potential role for this protein in linking the hydroxyapatite with collagen.

Animals↗
PubMed1991

Regulation of transformation-sensitive secreted phosphoprotein (SPPI/osteopontin) expression by transforming growth factor-beta. Comparisons with expression of SPARC (secreted acidic cysteine-rich protein).

Secreted phosphoprotein I (SPPI; osteopontin), a highly phosphorylated form of which has been associated with cell transformation, is one of the major phosphorylated proteins in bone. Populations of rat bone cells derived from fetal calvariae, neonatal parietal bone and a rat osteosarcoma cell line (ROS 17/2.8) produce several forms of the protein, the major forms having apparent molecular masses of 55 and 44 kDa by SDS/PAGE on 15% (w/v) cross-linked gels and of 60 and 56 kDa on 10% gels. Northern blot analysis of SPPI mRNA using total cellular RNA revealed a single 1.5 kb mRNA species, indicating that the nascent protein chains of these phosphoproteins are identical. On treatment of the cells with transforming growth factor-beta (TGF-beta; 1 ng/ml), the levels of SPPI mRNA and the synthesis of the 55 kDa phosphoprotein, but not of the 44 kDa phosphoprotein, were increased by 1.8-4.5-fold in the normal osteoblastic cells, the stimulation first being evident at 3 h and reaching a maximum at 12 h. In the transformed ROS 17/2.8 cells, TGF-beta did not alter significantly the SPPI mRNA level or the synthesis of either the 55 kDa or the 44 kDa SPPI over the 24 h period studied. By comparison, neither the steady-state levels of SPARC (secreted protein, acidic, rich in cysteine) mRNA nor the synthesis of SPARC protein were affected significantly by the addition of TGF-beta to any of the osteoblastic bone cells. The half-lives for SPPI and SPARC mRNAs in the osteoblastic calvarial cells were calculated to be 18 h and greater than 50 h respectively, in both the presence and the absence of TGF-beta. Since the stability of the mRNA was unchanged by TGF-beta and the increased expression of SPPI mRNA could be blocked by cycloheximide, TGF-beta appears to increase transcription of the SppI gene indirectly by stimulating the synthesis of a protein that promotes transcription. These results demonstrate that several forms of SPPI are synthesized constitutively by bone cells, and that there are clear differences in the regulation of SppI gene expression by TGF-beta in normal bone cells compared with the tumorigenic ROS 17/2.8 cells. The differential responses of normal osteoblastic cells to TGF-beta in the expression of SPPI and the selective stimulation of specific forms of the SPPI protein may be important in bone repair and remodelling.

Animals↗