Biomedical subjects

J Sodek

Publications and source records attributed to J Sodek.

At least 55 records · Page 3Linked to original sources

PubMed1996

Osteopontin and bone sialoprotein expression in regenerating rat periodontal ligament and alveolar bone.

BACKGROUND: Osteopontin (OPN) and bone sialoprotein (BSP) are differentially expressed over time in bone formation and remodelling. We have examined the expression of these proteins as phenotypic markers in studies of periodontal ligament (PL) and alveolar bone (AB) regeneration. METHODS: Window wounds (0.6 mm in diameter through alveolar bone) with either preservation or extirpation of PL were prepared under general anaesthesia in 30 Wistar male rats. Animals were killed on days 1, 3, 7, 14, and 28 after surgery, and OPN and BSP were detected with mouse monoclonal antirat antibodies. RESULTS: In regenerating alveolar bone, OPN was first detected on day 3, prior to the expression of BSP. However, only OPN could be detected in the PL where it localized to the border with the AB. Compared to wounds with PL extirpation, wounds with preservation of PL exhibited BSP staining within the AB compartment of the wound site in a significantly shorter period of time (day 7) and exhibited intense OPN and BSP staining by day 28. CONCLUSIONS: These studies show that early OPN and BSP expression and bone regeneration are enhanced by preservation of the PL and that during wound healing the PL contains cells that transiently express some osteoblastic protein markers but not mineralization.

Animals↗

PubMed1996

Role of integrins in regulation of collagen phagocytosis by human fibroblasts.

Phagocytosis of collagen fibrils by fibroblasts is an important pathway for degradation of extracellular matrix in mature connective tissues. To study regulatory mechanisms in phagocytosis, 2-microns fluorescent beads coated with either collagen (COL) or bovine serum albumin (BSA) were incubated with human gingival fibroblasts in vitro. For these studies single cell suspensions were prepared by trypsinization, and bead internalization and collagen receptor expression were assessed by flow cytometry. After 3-h incubations, up to 8-fold more cells internalized COL beads than BSA-coated beads. Increased collagen coating concentration was associated with elevated proportions of cells that internalized COL beads, and was observed also in the presence of competing fibronectin-coated beads. The number of beads per cell and the percent of phagocytic cells increased proportionally with higher bead loadings. At > 4 beads per cell a maximum of approximately 80% of cells were phagocytic. Cells reacted with mAbs against the alpha 1, alpha 2, and alpha 3 integrin subunits were, respectively, 5%, 98% and 93% positively stained above background controls. All cells that internalized COL beads exhibited alpha 2 staining but there were large proportions of phagocytic cells that were not stained for alpha 1. In unfixed cells, bead internalization caused an immediate reduction of surface staining of membrane-bound alpha 2 by approximately 55% which returned to control levels within 3 h, indicating that cell-surface alpha 2 was internalized by phagocytosis. Preincubation of cells with up to 8 COL beads per cell reduced the proportion of phagocytic cells and the number of internalized beads after a second COL bead incubation 4 h later. To assess the relationship between the percent of phagocytic cells and alpha 2 integrin levels, serum starvation and cycloheximide experiments were conducted. Compared to controls, serum starvation for 24 h induced a 3.2-fold increase of cells internalizing COL beads but did not alter alpha 2 staining levels. In contrast, 3 h cycloheximide treatment reduced alpha 2 staining to 60% of control levels and this treatment also inhibited COL bead internalization. GRGDTP peptide as well as mAbs against the alpha 1 and alpha 2 subunits significantly reduced internalization of COL beads by 1.8 to 2.6-fold, whereas GRGESP peptide and alpha 3 mAb exerted no effect. Internalization of BSA beads was not affected by any of these treatments. Collectively, these data indicate that the alpha 2 integrin, along with other, as yet unidentified components, is likely involved in COL bead internalization. The alpha 2 integrin subunit is rapidly recycled or synthesized following a phagocytic load. In contrast, the alpha 1 integrin is not directly required for phagocytosis but may regulate the internalization step.

Amino Acid Sequence↗

PubMed1996

Effects of osteogenic protein-1 (OP-1, BMP-7) on bone matrix protein expression by fetal rat calvarial cells are differentiation stage specific.

Bone morphogenetic proteins (BMPs) are a group of cytokines that are characterized by their ability to stimulate osteoblast differentiation and bone formation. However, the influence of BMPs on osteoblastic cells at different stages of differentiation is not known. Since bone matrix proteins are differentially regulated during bone formation we have studied the effects of recombinant human osteogenic protein-1 (rhOP-1; BMP-7) on the expression of these proteins by fetal rat calvarial cells (FRCCs) at discrete stages of osteoblast differentiation. Continuous administration of rhOP-1 to FRCCs, beginning at confluence (day 7), produced a dose-dependent increase in the number, size and mineralization of bone-like nodules formed in the presence of vitamin C and beta-glycerophosphate. Within 9 h of administration, rhOP-1 stimulated a 3-fold increase in OPN mRNA which was reflected in a comparable increase in the low phosphorylated, 55 kDa form of osteopontin. In contrast, changes in type 1 collagen, alkaline phosphatase and bone sialoprotein mRNAs followed the differentiation of preosteoblastic cells, and were increased 2-, 4- and 5-fold, respectively, after 8 days (day 15). When administered at intermediate stages of osteoblast differentiation (days 12, 15 and 18) BSP remained refractory to rhOP-1 whereas the ALP was increased almost 2-fold, independent of the constitutive levels of mRNA expression. To determine the effects on osteoblasts, FRCCs were first grown to the bone nodule-forming stage (day 21) before rhOP-1 was administered. Only modest, transient increases in the expression of ALP and OPN mRNAs were evident whereas OC expression was increased more than 3-fold. In contrast, collagen type 1 and BSP mRNA levels were not changed significantly. These results suggest that rhOP-1 increases bone formation by promoting osteoblastic differentiation, as indicated by the increased number of bone forming colonies and by increasing the number of osteoblastic cells in the colonies, but not by increasing matrix production by individual osteoblasts. It is also evident that the regulation of bone matrix proteins by rhOP-1 is dependent upon the differentiated state of the cell.

Animals↗

PubMed1996

Expression of rat bone sialoprotein promoter in transgenic mice.

Bone sialoprotein (BSP) is a major protein of the mineralized bone extracellular matrix that has been implicated in the nucleation of hydroxyapatite crystals. Our previous studies have demonstrated that BSP mRNA is expressed by differentiated osteoblasts, odontoblasts, and cementoblasts involved in de novo mineralized tissue formation in a tissue-specific and developmentally regulated manner. To determine the basis of the selective expression of the BSP gene, we have generated four transgenic mouse lines in which 2.7 kb of the rat BSP promoter ligated to a luciferase reporter gene has been stably integrated into the mouse genome. Assays of luciferase activities in 5-day-old animals has revealed consistently high levels in bone tissues with negligible activities in various other organs including kidney, liver, stomach, intestine, and spleen. In some animals, variable expression was observed in brain and skin. Temporal analyses revealed the highest luciferase expression in neonatal bones, with expression decreasing markedly with subsequent growth and development, as observed previously for the endogenous gene in rats. Immunohistochemical analysis of luciferase activity and in situ hybridization of luciferase mRNA in bone tissues show that differentiated osteoblasts express the highest levels of luciferase, consistent with the induction of endogenous gene expression. These studies demonstrate that the regulation of the BSP gene during osteoblastic differentiation, together with its tissue-specific, developmentally regulated expression, is primarily mediated within the 2.7 kb region of the promoter.

Animals↗

PubMed1996

Relationship of cellular proliferation to expression of osteopontin and bone sialoprotein in regenerating rat periodontium.

Cellular repopulation was studied in a model in which adjacent mineralising and soft connective tissue matrices are regenerated. Window wounds were created through alveolar bone, with either preservation or removal of periodontal ligament, in 30 male Wistar rats. Three animals per time period were killed on days 1, 3, 7, 14, and 28 after surgery for each wound type. Cellular proliferation in alveolar bone and periodontal ligament was assessed by 3H-thymidine labelling 1 h before death, followed by radioautographic analysis. Cellular differentiation was determined by the temporal expression of the bone-related markers osteopontin and bone sialoprotein, using immunohistochemical methods. In regenerating periodontium, osteopontin was expressed earlier than bone sialoprotein, which was restricted to alveolar bone. After wounding, transient expression of osteopontin was detected in the periodontal ligament at days 1 and 3. In general, wounding induced fivefold higher proliferation and clonal growth of periodontal ligament cells compared to the unwounded (control) side. Combined immunostaining and radioautography demonstrated colocalisation of osteopontin in sites with high numbers of labelled cells in both nascent periodontal ligament and regenerating alveolar bone at days 3 and 7. In contrast, bone sialoprotein, which appeared in regenerating alveolar bone on days 14-28 after wounding, was expressed much later than the peak of cellular proliferation. We conclude that (1) the intact periodontal ligament influences cell proliferation and osteopontin expression; (2) osteopontin is an early marker of periodontal tissue regeneration that is temporally and spatially associated with intensive cell proliferation and migration in osteogenic and periodontal ligament cell populations; and (3) bone sialoprotein is expressed after proliferation at sites of mineralising bone formation.

Animals↗

PubMed1996

AP-1 regulation of the rat bone sialoprotein gene transcription is mediated through a TPA response element within a glucocorticoid response unit in the gene promoter.

Bone sialoprotein (BSP), a protein which has been implicated in the initial mineralization of newly-formed bone, provides an early phenotypic marker for differentiated osteoblasts. BSP expression is induced by glucocorticoids in association with osteoblast differentiation, and a glucocorticoid response element (GRE) overlapping a putative TRE (TPA, 12-O-tetradecanoyl-phorbol 13-acetate, response element) site has been identified in the rat BSP promoter (Ogata et al., 1995). Since AP-1 and the glucocorticoid receptor have a central role in regulating cell proliferation and differentiation, we have studied AP-1 activity, stimulated by 100 ng/ml TPA in normal fetal rat calvarial cells and in transformed rat osteosarcoma cells (ROS 17/2.8). A transient induction of both c-fos and c-jun mRNAs by TPA was observed in both cell populations, together with an associated suppression of BSP mRNA in the fetal rat calvarial cells. Rat BSP promoter constructs, transiently transfected into ROS 17/2.8 cells, were used to show that TPA suppressed transcription of a luciferase construct (-938/+60; pLUC6) that included the GRE/TRE, but not transcription of shorter contructs lacking this element. Notably, suppression of pLUC6 transcription by TPA was abrogated in the presence of the synthetic glucocorticoid, dexamethasone. Gel mobility shift analyses were performed using two double-stranded synthetic oligonucleotides. These encompassed the TRE and either the distal pair of GRE half-sites (-936/ -910; GRE3) or the proximal pair of GRE half-sites (-925/-899; GRE 4) that comprise the GRE/AP-1 element. The assay showed binding of both AP-1 complexes and recombinant c-Jun homodimers. Additionally, either the c-Jun or glucocorticoid receptor could displace its counterpart from the GRE/TRE but not from consensus GRE and TRE oligonucleotides, indicating that the abrogation of AP-1-mediated gene suppression by glucocorticoids could involve competitive binding. These studies, therefore, have identified a glucocorticoid response unit through which c-Fos and c-Jun can suppress the expression of BSP in proliferating pre-osteoblastic cells and through which glucocorticoids can ameliorate the effects of AP-1 and promote osteoblast differentiation and the associated expression of BSP.

Animals↗

PubMed1996

Regulation of bone sialoprotein and osteopontin mRNA expression by dexamethasone and 1,25-dihydroxyvitamin D3 in rat bone organ cultures.

Bone sialoprotein (BSP) and osteopontin (OPN) are prominent components of the extracellular matrix of mineralized connective tissues that have been implicated in the formation and remodelling of bone. Although these proteins have similar biochemical properties and are expressed by bone cells during bone formation it has been suggested that they have different functions and that their expression is regulated independently by hormones and cytokines. The precise role of these proteins has, however, yet to be firmly established. Since steroid hormones strongly influence the formation of bone we have analyzed the effects of glucocorticoids and 1,25 dihydroxyvitamin D3 (1,25-(OH)2D3) on the expression of BSP and OPN mRNAs in developing rat bone in vitro using in situ hybridization. In these studies it has been possible to identify the nature and spatial distribution of the cells that respond to these hormones by changes in sialoprotein expression. When cultured in the presence of the synthetic glucocorticoid, dexamethasone (dex), expression of BSP mRNA by hypertrophic cartilage cells in the tibiae and mandible was dramatically increased as were the number of responding cells indicating that glucocorticoids promote differentiation of hypertrophic cartilage cells as well as osteoblasts. Dexamethasone also stimulated a marked (> 5-fold) increase in OPN expression by osteoblasts and cells lining endosteal and periosteal bone surfaces. In contrast to dex, 1,25-(OH)2D3 suppressed BSP expression in osteoblastic cells whereas OPN expression was strongly (> 5-fold) stimulated in all three cultured bone tissues. Histological examination of the tissues showed that cell viability was retained over the culture period. However, in the presence of 1,25-(OH)2D3 considerable resorption of the tissue was evident, with cement and reversal lines being prominent. The increased expression of BSP and OPN by dex is consistent with the stimulation of bone formation by glucocorticoids, whereas the differential effects of 1,25-(OH)2D3 on BSP and OPN may reflect a stimulation of bone remodelling.

Animals↗

PubMed1996

Characterization of the bone sialoprotein (BSP) gene promoter.

Bone sialoprotein is a 34 kDa phosphorylated and sulphated glycoprotein that is essentially unique to mineralizing connective tissues. Recent studies on the developmental expression of BSP mRNA and the temporo-spatial appearance of the protein during bone formation in vivo and in vitro have demonstrated that BSP is expressed by differentiated osteoblasts and that it may function in the initial nucleation of hydroxyapatite crystals in de novo bone formation. To study the cell-specific regulation of BSP we have isolated genomic clones that encompass the BSP promoter regions of both the human and rat genes. These promoters are characterized by a highly conserved region (BSP Box) that extends upstream from the transcription start site to nt -370. Within this region the immediate promoter is further characterized by a unique inverted TATA box and an inverted CCAAT box, both of which are required for basal transcriptional activity. The TATA box is overlapped by a vitamin D3 response element (VDRE) which appears to mediate vitamin D suppression of BSP gene transcription by competing with the TATA-binding protein (TBP) for occupancy of the site of the pre-initiation complex formation. Mutation of the inverted TATA box into a normal TATA sequence increases transcription slightly but does not affect the functionality of the VDRE indicating that the orientation of the TATA box is not critical for these functions. Further upstream an AP-1 site, overlapped by a steroid hormone response-like sequence, mediates down-regulation of BSP transcription induced by TPA that is abrogated by a complex interaction between Jun and the glucocorticoid receptor protein induced by dexamethasone. Thus, the characterization of approximately 3 kb of the BSP promoter and approximately 2 kb of the first intron has revealed several sites of transcriptional regulation that are important in regulating BSP expression and, consequently, bone formation.

Animals↗

PubMed1996

Dexamethasone stimulates luciferase gene expression through the rat bone sialoprotein gene promoter in transgenic mice.

Bone sialoprotein (BSP) is expressed by differentiated osteoblasts during the initial formation and mineralization of bone matrix. Studies using transgenic mice harboring 2.7 kb of the rat BSP promoter linked to a luciferase reporter gene have shown luciferase activity in bone and other mineralized tissues while most soft tissues tested expressed a much lower level of the reporter gene. To study regulation of the transgene, mice were administered dexamethasone (dex) by intramuscular injection. After 4 h and 24 h, various tissues were dissected from the treated mice as well as from untreated transgenic littermates. Luciferase assays showed that dex stimulated expression of the transgene significantly. In bone tissues, dex increased the average luciferase activity 1.6- to 11-fold compared with control tissues from untreated transgenic mice. The luciferase activity in lung, liver and kidney remained at a low level and showed no increase with dex treatment. In some animals, however, the luciferase activity in brain and skin was also increased after dex administration. These experiments indicate that a transgene comprising 2.7 kb of the rat BSP promoter linked to a luciferase reporter is regulated in a tissue and developmental stage-dependent manner and that glucocorticoid-induced stimulation of BSP gene expression may be mediated within this region of the promoter.

Animals↗

PubMed1996

Influence of osteogenic protein-1 (OP-1;BMP-7) and transforming growth factor-beta 1 on bone formation in vitro.

The bone morphogenetic proteins (BMPs) and transforming growth factor-beta s (TGF-beta s), are a group of structurally related proteins which have been shown to stimulate bone formation in vivo. Since these proteins are concentrated in the organic matrix of bone and would be released during bone resorption, they are likely to have a profound effect on the remodeling bone and may provide a link between bone resorption and bone formation. We are using primary cultures of fetal rat calvarial cells (FRCC) to study the independent and combined effects of OP-1/BMP-7 and TGF-beta 1 on bone cells at different stages of differentiation in order to identify responding cell populations and target genes. We have confirmed prior reports that OP-1 stimulates, while TGF-beta 1 inhibits, osteogenic differentiation in this system. The increase in both number and size of the mineralized nodules induced by OP-1 was accompanied by increased expression of alkaline phosphatase and type I collagen with an induction of bone sialoprotein (BSP) suggesting that OP-1 stimulates both differentiation and clonal expansion of osteoblastic cells. Interestingly, TGF-beta 1 abrogated OP-1 induced nodule formation. Despite these opposing effects on osteogenic differentiation, TGF-beta 1 (Wrana et al, 1991) and OP-1 both stimulated a rapid induction of osteopontin (OPN) mRNA in confluent FRCC cultures enriched in pre-osteoblastic cells. In contrast, when OP-1 was added to nodule-forming cultures which are enriched in osteoblastic cells, there was only a weak induction of OPN. Moreover, while the expression of one marker for mature osteoblasts (BSP) was refractory to OP-1, another (osteocalcin) was markedly stimulated. Thus OP-1 has selective effects on bone matrix protein expression that are dependent on the differentiated state of the cells.

Alkaline Phosphatase↗

PubMed1995

An inverted TATA box directs downstream transcription of the bone sialoprotein gene.

The orientation of the TATA box is thought to direct downstream transcription of eukaryotic genes by RNA polymerase II. However, the putative TATA box in the promoter of the bone sialoprotein (BSP) gene, which codes for a tissue-specific and developmentally regulated bone matrix protein, is inverted (5'-TTTATA-3') relative to the consensus TATA box sequence (5'-TATAAA-3') and is overlapped by a vitamin D3-response element. Here we show that the inverted TATA sequence in the rat BSP gene binds to recombinant TATA-box-binding protein (TBP) with an affinity similar to that observed with the consensus TATA box, and site-directed point mutations in the inverted TATA sequence (mutating TTTATA into TCTCTA) abrogate both TBP binding and BSP promoter activity. However, when the inverted TATA sequence is changed to a canonical TATAAA, the TBP- and vitamin D3 receptor-binding properties together with the BSP promoter activity are retained. In addition, we found that the TBP is required to reconstitute in vitro transcription driven by the BSP promoter. These studies, which have revealed a naturally occurring inverted TATA box that can bind TBP and direct downstream transcription, demonstrate that the orientation of the TATA box does not determine the direction of transcription in higher eukaryotic genes. Consequently, the inverted TATA box that is conserved in the human, rat and mouse BSP gene promoters will provide an excellent in vivo model to investigate the polarity of the transcription factor IID-DNA complex and its relation to downstream transcription.

Animals↗

PubMed1995

Glucocorticoid regulation of bone sialoprotein (BSP) gene expression. Identification of a glucocorticoid response element in the bone sialoprotein gene promoter.

Glucocorticoids modulate the development and growth of many organs through interactions with a specific intracellular receptor (glucocorticoid receptor) that regulates gene transcription through a cognate element, the glucocorticoid response element (GRE), in the promoter of target genes. In bone formation glucocorticoids stimulate osteoblast differentiation and the formation of bone matrix. Recent studies have demonstrated that the induction of the bone sialoprotein (BSP) gene is associated with osteoblast differentiation and de novo bone formation. To determine the molecular pathways of glucocorticoid regulation of BSP expression, we have analyzed the effects of the synthetic glucocorticoid, dexamethasone, on the expression of the BSP by bone cells in vitro. At 10 nM, dexamethasone induced BSP expression in association with bone tissue formation by confluent fetal rat calvarial cells and adult rat marrow cells and also stimulated BSP expression up to sixfold in osteoblastic cells (UMR 106-6 and ROS 17/2.8 cells). Most of the stimulation was blocked by cycloheximide, indicating direct and indirect mechanisms of BSP gene regulation. Nuclear 'run-on' transcription analysis revealed an up to twofold increase in transcription corresponding to the increase in mRNA that was unaffected by cycloheximide. Analysis of BSP mRNA in the presence of a transcription inhibitor (5,6-dichloro-1-beta-D-ribofuanosyl benzimidazole) by Northern hybridization revealed that the stability of the BSP mRNA was not significantly altered by dexamethasone, indicating that the major, indirect, stimulation of BSP expression involves a nuclear post-transcriptional mechanism. To study the direct effects of dexamethasone, nucleotide sequence analysis of the rat BSP promoter was extended upstream to position -2992 and downstream to +2282 in the first intron. Transient transfection analyses, using various rat BSP promoter constructs linked to a luciferase reporter gene, and gel mobility shift assays were used to identify a putative glucocorticoid response unit comprising three GRE half-sites and a putative AP-1 site, located within positions -906 to -931 upstream from the translation start site of the BSP gene promoter. BSP transcription was stimulated approximately 1.5-fold by dexamethasone through this GRE, indicating that its direct effects are mediated by glucocorticoid receptor binding to this site. These studies, therefore, have identified both indirect and direct pathways of glucocorticoid regulation of BSP gene expression, the direct effects being mediated by a GRE in the rat BSP promoter through which the effects of glucocorticoids on BSP gene transcription appear to be regulated.

Animals↗

PubMed1995

Regulation of osteopontin expression in osteoblasts.

Osteopontin (OPN) is a prominent bone matrix protein that is synthesized by osteoblastic cells. To elucidate the function of OPN in bone we studied the regulated expression of the rat OPN protein during bone formation in vivo and in vitro. OPN mRNA is expressed by preosteoblastic cells early in bone formation, but the highest expression is observed in mature osteoblasts at sites of bone remodelling. A low-phosphorylated, 55-kDa form of OPN is produced by the preosteoblastic cells, whereas osteoblasts produce a highly phosphorylated, 44-kDa protein; the two forms of OPN corresponding to pp69 and pp62 in transformed rat cells. The synthesis of the 55-kDa OPN correlates with the formation of a 'cement' matrix that is synthesized prior to bone deposition, whereas the 44-kDa OPN synthesized by osteoblasts associates rapidly with hydroxyapatite, possibly regulating crystal growth, and may also provide a substratum for osteoclast attachment. Expression of OPN mRNA is upregulated by growth and differentiation factors (PDGF, EGF, TGF-beta and BMP-7/OP-1) and by mechanical stress, which promote bone formation, as well as by osteotropic hormones (retinoic acid and vitamin D3), which can promote bone resorption and remodelling. However, OPN mRNA is down-regulated by bisphosphonates, which abrogate bone resorption. Regulation of OPN expression is, therefore, consistent with a multiplicity of functions for OPN that involve specific structural motifs in both the synthesis and resorption of bone.

Amino Acid Sequence↗

PubMed1995

Synthesis of noncollagenous extracellular matrix proteins during development of mineralized nodules by rat periodontal ligament cells in vitro.

To characterize the mineralized nodules produced by rat periodontal ligament (PDL) cells in vitro, we have studied the synthesis and distribution of mineralized tissue proteins at various stages of nodule formation. PDL cells were obtained from coagulum in the socket at 2 days after tooth extraction and cultured in Dulbecco's Modified Eagles Medium (DMEM) containing 10% fetal bovine serum and antibiotics. Confluent cells were grown in the presence of ascorbic acid (50 micrograms/ml), dexamethasone (5 microM), and beta-glycerophosphate (10 mM) for 3 weeks. Four stages showing distinct morphological characteristics during development of mineralized nodules were identified. Protein synthesis and deposition of proteins into the matrix were studied during these stages by metabolic labeling with [35S]methionine for 24 hours. Large quantities of SPARC (secreted protein, acidic and rich in cysteine) were synthesized by confluent cells but decreased during the progress of mineralized nodule formation. Two forms of osteopontin (OPN) (67 kDa and 61 kDa) were synthesized in comparable quantities by confluent cells; OPN and bone sialoprotein (BSP) were induced by dexamethasone and represented the major proteins in the mineralized matrix. The 67 kDa form of OPN was the predominant species in the mineralized matrix. Both OPN and BSP were localized by immunogold electron microscopy on globular as well as fused electron-dense structures at sites of tissue mineralization.

Animals↗

PubMed1995

Evidence of a direct relationship between neutrophil collagenase activity and periodontal tissue destruction in vivo: role of active enzyme in human periodontitis.

To assess the temporal relationship between periodontal tissue destruction and the activity of collagenase, exudate from inflamed periodontal tissues was collected and latent and active collagenase activities were measured by a functional assay in a longitudinal cohort study. Comparisons were made between human subjects with either: 1) inflammation with a previous history of progressive loss of connective tissue and bone support (n = 14); 2) inflammation and previous history of bone loss but now clinically stable (n = 27); or 3) inflammation and no loss of bone support (n = 17). Experiments using specific enzyme inhibitors, blocking antibodies and SDS-PAGE fluorograph to identify the pattern of collagen substrate degradation demonstrated that the collagenase activity was derived from neutrophils and not from bacteria or other host cells. Active collagenase activity pooled from 6 sites per subject was respectively 5 and 6-fold higher in the group with progressive loss of connective tissue compared to the groups with either inflamed tissues alone or with inflammation and previous bone loss. In contrast, latent collagenase was increased up to 2 fold higher in the group with inflammation but no bone loss compared to the group with progressive lesions. Moreover, the ratio of active to total collagenase activity was 50% higher in the group with progressive lesions. Although in all subjects successive measurements of site-specific active collagenase 1 month apart demonstrated wide variation (r < 0.50), only in sites with progressive periodontal destruction was there significant increase of active collagenase with time (1.28 x 10(-4) collagenase units per day). There were also sharp elevations in active enzyme level at the time of detection of loss of connective tissue attachment in specific sites of 8 subjects. At the time of detection of connective tissue attachment loss, there was an overall 40% increase of pooled active collagenase activity in all subjects with progressive loss of connective tissue compared to pre-breakdown sampling times. These data provide strong in vivo evidence for a direct role of active neutrophil collagenase in the pathological destruction of periodontal connective tissue.

Adult↗

PubMed1995

Regulation of bone sialoprotein gene transcription by steroid hormones.

During the initial formation of bone, dentine and cementum in tooth morphogenesis, fully differentiated osteoblasts, odontoblasts and cementoblasts express bone sialoprotein (BSP), a mineralized tissue-specific acidic glycoprotein that has been implicated in the nucleation of hydroxyapatite crystal growth. The expression of BSP is regulated by steroid hormones that modulate mineralized tissue formation. Thus, the transcription of the BSP gene is induced by glucocorticoids in association with osteoblast differentiation and glucocorticoids also stimulate the expression of BSP in differentiated osteoblasts. In contrast, however, vitamin D3 suppresses bone formation and abrogates the expression of BSP. Our studies, using the osteoblastic cell lines ROS 17/2.8 and UMR 106-06, have revealed that the glucocorticoid (10(-8) M dexamethasone; dex) effect on BSP mRNA involves both direct and indirect pathways. To determine the molecular basis of the direct pathway on transcriptional regulation of the BSP we have isolated and characterized the promoter regions of both the human and rat BSP genes. The promoters are characterized by a highly conserved region (BSP box) encompassing the immediate promoter region, which includes a unique inverted TATA box overlapped by a putative (DR3) vitamin D3 response element (VDRE). Possible glucocorticoid response elements are present approximately 1 kb and approximately 1.4 kb further upstream. Transient transfection analysis of chimeric constructs linked to a luciferase reporter gene have shown Dex-stimulated expression in constructs that include one or both GREs, whereas vit D3 suppresses expression in a short construct that includes the VDRE.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Temporal changes in matrix protein synthesis and mRNA expression during mineralized tissue formation by adult rat bone marrow cells in culture.

To characterize the bone-like tissue produced by rat bone marrow cells (RBMC) from young adult femurs, the synthesis of bone proteins and the expression of their mRNA were studied in vitro. RBMC plated at a density of 5 x 10(3) cells/cm2 and grown in the presence of 10(-8) M dexamethasone (Dex) and 10 mM beta-glycerophosphate (beta-GP) produced mineralized bone nodules, which were first evident at day 3 and increased markedly to day 13. However, in the absence of dexamethasone, few mineralized nodules were observed. The formation of mineralized nodules was reflected by the uptake of 45Ca, which also increased markedly to day 13. Analysis of bone protein expression by Northern and slot-blot hybridizations revealed an increase in mRNA levels of collagen type I (Col I), osteonectin/SPARC (ON), alkaline phosphatase (ALP), osteopontin (OPN), bone sialoprotein (BSP), and osteocalcin (OC) during the formation of mineralized nodules. Whereas the Col I, ON, ALP, and OPN mRNAs were expressed before the formation of mineralized nodules was evident and were also expressed at various levels in the absence of Dex, the expression of BSP and OC mRNA was induced in the bone-forming cultures. The expression of BSP mRNA was correlated temporally with bone tissue formation, reaching maximal levels on day 16. In contrast, OC mRNA was expressed later and, following induction, increased over the 28 day culture period. Production of matrix proteins during the rapid formation of the bone tissue appeared to reflect the levels of the respective mRNAs. However, whereas some of the collagen and almost all of the SPARC were secreted into the culture medium, virtually all of the OPN and most of the BSP were extracted from the mineralized tissue matrix with EDTA. Some OPN and BSP were present in the medium, especially early in the culture, and a significant amount of BSP was also found associated with the collagenous tissue matrix. These studies point to the importance of Col I, ALP, OPN, and BSP, but not ON or OC, in the initial formation of bone tissue.

Alkaline Phosphatase↗

PubMed1994

Carboxyl-terminal parathyroid hormone peptide (53-84) elevates alkaline phosphatase and osteocalcin mRNA levels in SaOS-2 cells.

Previous findings in our laboratory have shown that hPTH-(53-84) stimulates alkaline phosphatase activity in dexamethasone-treated ROS 17/2.8 cells. In the present study, we examined the effects of hPTH-(53-84) and hPTH-(1-34) on the expressions of alkaline phosphatase, osteocalcin, and collagen type I mRNA levels in the human osteosarcoma cell line SaOS-2. The effect of hPTH-(53-84) on alkaline phosphatase and osteocalcin message levels was dose dependent (ANOVA, p < 0.005 and p < 0.001, respectively), with significant stimulation observed at 10 nM. Treatment with 10 nM hPTH-(53-84) for 24 h resulted in significant 2- and 1.4-fold increases in mRNA levels for alkaline phosphatase and osteocalcin, respectively (p < 0.05), but had no effect on collagen type I expression. The increased alkaline phosphatase mRNA levels was associated with a 1.5-fold increase in enzyme activity (p < 0.05). In contrast, under similar incubation conditions, hPTH-(1-34) had no significant effects on alkaline phosphatase or osteocalcin mRNA levels. On the other hand, hPTH-(1-34) had dose-dependent stimulatory effects on collagen type I mRNA levels (ANOVA, p < 0.001), 10 nM hPTH-(1-34) stimulating collagen type I expression 1.6-fold (p < 0.05). The results indicate that carboxyl-terminal hPTH-(53-84) has direct and unique biologic effects in human osteoblast-like cells in culture.

Alkaline Phosphatase↗