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C A McCulloch

Publications and source records attributed to C A McCulloch.

At least 73 records · Page 4Linked to original sources
PubMed1997

Tamoxifen attenuates glucocorticoid actions on bone formation in vitro.

Tamoxifen is a synthetic estrogen analog which may regulate osteogenesis in vivo by virtue of its antiglucocorticoid properties. We have examined tamoxifen regulation of glucocorticoid-induced osteogenesis in two different in vitro bone systems: the chicken periosteal osteogenesis model (CPO) and rat bone marrow stromal cells (RBMC). Hormone uptake studies were conducted with the osteosarcoma cell line, ROS 17/2.8. In the CPO model, alkaline phosphatase (AP) activity and collagen synthesis were stimulated by the glucocorticoid dexamethasone (Dex; 0.1 microM). These Dex-mediated effects were inhibited by increasing concentrations of tamoxifen (10-100 microM). Similarly, in the RBMC model, Dex-dependent (0.01 microM Dex) mineralized tissue formation and AP activity were blocked by tamoxifen (0.1 microM). Although tamoxifen inhibited Dex-mediated increases of AP activity in ROS 17/2.8 cells, it did not inhibit uptake of 3H-Dex or of 3H-estrogen. Northern analyses showed that tamoxifen did not affect messenger RNAs (mRNAs) for AP. Tamoxifen did seem to reduce mRNA for collagen type I, but not bone sialoprotein, osteopontin, and osteocalcin. Dex-induced increases for all proteins mRNAs in the RBMC model were not reduced by tamoxifen. Similarly, tamoxifen had no effects on cellular proliferation. We conclude that tamoxifen has no direct effect on gene expression of bone-related proteins of osteoblastic cells. Further, in the ROS 17/2.8 cell line, the antiglucocorticoid properties of tamoxifen do not appear to be mediated through either Dex or estrogen receptors.

Alkaline Phosphatase↗
PubMed1997

Calcium ions and tyrosine phosphorylation interact coordinately with actin to regulate cytoprotective responses to stretching.

The actin-dependent sensory and response elements of stromal cells that are involved in mechanical signal transduction are poorly understood. To study mechanotransduction we have described previously a collagen-magnetic bead model in which application of well-defined forces to integrins induces an immediate (< 1 second) calcium influx. In this report we used the model to determine the role of calcium ions and tyrosine-phosphorylation in the regulation of force-mediated actin assembly and the resulting change in membrane rigidity. Collagen-beads were bound to cells through the focal adhesion-associated proteins talin, vinculin, alpha 2-integrin and beta-actin, indicating that force application was mediated through cytoskeletal elements. When force (2 N/m2) was applied to collagen beads, confocal microscopy showed a marked vertical extension of the cell which was counteracted by an actin-mediated retraction. Immunoblotting showed that force application induced F-actin accumulation at the bead-membrane complex but vinculin, talin and alpha 2-integrin remained unchanged. Atomic force microscopy showed that membrane rigidity increased 6-fold in the vicinity of beads which had been exposed to force. Force also induced tyrosine phosphorylation of several cytoplasmic proteins including paxillin. The force-induced actin accumulation was blocked in cells loaded with BAPTA/AM or in cells preincubated with genistein, an inhibitor of tyrosine phosphorylation. Repeated force application progressively inhibited the amplitude of force-induced calcium ion flux. As force-induced actin reorganization was dependent on calcium and tyrosine phosphorylation, and as progressive increases of filamentous actin in the submembrane cortex were correlated with increased membrane rigidity and dampened calcium influx, we suggest that cortical actin regulates stretch-activated cation permeable channel activity and provides a desensitization mechanism for cells exposed to repeated long-term mechanical stimuli. The actin response may be cytoprotective since it counteracts the initial force-mediated membrane extension and potentially strengthens cytoskeletal integrity at force-transfer points.

Actins↗
PubMed1996

Dependence of fibroblast migration on actin severing activity of gelsolin.

Gelsolin nucleates actin filament assembly, blocks the fast-exchanging ends of actin filaments, and severs filaments, processes that may play an important role in cell motility. We studied the relationship between cell migration, gelsolin content, and actin severing activity in human gingival fibroblasts. These cells were keratin negative and desmin negative but expressed vimentin and myosin II. Cells were separated by their ability to migrate in response to a chemoattractant stimulus. Northern analysis of mRNA, [35S]methionine incorporation into immunoprecipitated gelsolin, immunoblots of cell lysates, and quantitative confocal microscopy showed 1.4-2-fold higher levels of gelsolin in nonmigrant compared with migrant cells. Because the concentration of intracellular gelsolin did not appear to be a central determinant of cell migration, we assessed its requirement for motility. Cells that were electroinjected with a gelsolin antibody that inhibits actin severing by gelsolin in vitro showed a 72% reduction of the number of migrant cells compared with controls treated with an irrelevant antibody. Cells that were electroinjected with free gelsolin exhibited a 33% increase in migration compared with cells electroinjected with bovine serum albumin. Compared with nonmigrant cells, migrant cells contained abundant free gelsolin and exhibited gelsolin-dependent F-actin severing activity, which required Ca2+. Serum stimulation of cell migration required increases in [Ca2+]i because incubation with 3 microM 1,2-bis-(o-aminophenoxy)-ethane-N,N, N',N'-tetraacetic acid tetra(acetoxymethyl)-ester blocked calcium flux and cell migration. Serum also stimulated the recruitment of gelsolin into the supernatants of lysates from migrant but not from nonmigrant cells. In fibroblasts, gelsolin concentration alone does not apparently determine migratory capacity. Instead, the Ca2+-dependent actin severing activity of free gelsolin appears to be a major determinant of cell migration.

Actin Cytoskeleton↗
PubMed1996

TNF-alpha inactivation of collagen receptors: implications for fibroblast function and fibrosis.

TNF-alpha inhibits collagen synthesis and at high concentrations stimulates collagenase synthesis in fibroblasts. As fluid from chronic inflammatory lesions contains significant levels of TNF-alpha, it is puzzling why these lesions exhibit dense accumulations of disorganized collagen. In this study we determined if low concentrations of TNF-alpha may inhibit the collagen phagocytic pathway in fibroblasts and thereby contribute to fibrosis. Collagen phagocytosis was measured by flow cytometric assessment of internalized, fluorescent collagen beads. TNF-alpha induced a dose-dependent reduction (optimal dose: 40% at 10 ng/ml; p<0.001) in the proportion of phagocytic cells and a twofold reduction of the number of internalized beads per cell but did not alter the total number of vital cells. TNF-alpha reduced by twofold the degradation of collagen films. Fluid flow shear-force assays demonstrated that TNF-alpha caused a 72% reduction (p < 0.05) in strong binding of collagen-coated beads to cells indicating that TNF-alpha may inactivate receptors and inhibit collagen binding. Furthermore, TNF-alpha reduced cell contact area with collagen substrates by threefold and inhibited reattachment of trypsinized cells by fourfold. Although levels of collagen receptors were increased by TNF-alpha (53% increase in alpha(2) (beta)1 integrin; p<0.001, 20% increase in alpha(1)beta(1)), the receptors were inactivated by the cytokine. The reduced phagocytic activity of TNF-alpha-treated cells was restored to control levels by treatment with the integrin-activating Abs A16G6 and JBS2. TNF-alpha inhibited focal adhesion formation and phosphotyrosine staining in focal adhesions. These effects were replicated by the tyrosine kinase inhibitor genistein, which also inhibited phagocytosis. Collectively, these data indicate that TNF-alpha inhibits adherence and phagocytosis of collagen. These effects are mediated by a reduction in the strength of alpha(2)beta(1) integrin binding to collagen, possibly through tyrosine kinases in focal adhesions. At low concentrations of TNF-alpha (10 ng/ml) that are found in the periphery of chronic inflammatory lesions, we suggest that inhibition of the collagen phagocytic pathway may contribute to fibrosis.

Actins↗
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

Periodontal ligament cell population: the central role of fibroblasts in creating a unique tissue.

BACKGROUND: Fibroblasts are the predominant cells of the periodontal ligament (PL) and have important roles in the development, function, and regeneration of the tooth support apparatus. Biological processes initiated during the formation of the PL contribute to the long-lasting homeostasic properties exhibited by PL fibroblast populations. DEVELOPMENT: The formation of the PL is likely controlled by epithelial-mesenchymal and epithelial hard tissue interactions, but the actual mechanisms that contribute to the development of cellular lineages in the PL are unknown. Fibroblasts in the normally functioning PL migrate through the tissue along collagen fibres to cementum and bone and in an apico-coronal direction during tooth eruption. ADULT TISSUE: Cell kinetic experiments have shown that PL fibroblasts comprise a renewal cell system in steady-state and the progenitors can generate multiple types of more differentiated, specialized cells. Progenitor cell populations of the PL are enriched in locations adjacent to blood vessels and in contiguous endosteal spaces. In normally functioning periodontal tissues, there is a relatively modest turnover of cells in which apoptotic cell death balances proliferation. Large increases of cell formation and cell differentiation occur after application of orthodontic forces or wounding. As PL cells comprise multiple cellular phenotypes, it has been postulated that after wounding, the separate phenotypes repopulating the site will ultimately dictate the tissue form and type. CONCLUSIONS: PL fibroblasts play an essential role in responses to mechanical force loading of the tooth by remodelling and repairing effete or damaged matrix components. In consideration of the important roles played by fibroblasts in PL homeostasis, they could be described as "the architect, builder, and caretaker" of the periodontal ligament.

Adult↗
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

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

Relationship of clonogenic capacity to plating efficiency and vital dye staining of human periodontal ligament cells: implications for tooth replantation.

The survival rate of avulsed permanent teeth following replantation is affected primarily by the duration of the extra-alveolar period and the nature of the storage conditions. These factors are believed to strongly affect the viability of periodontal ligament (PL) cells but in vitro assays of cell viability based on vital dye assays are only weakly correlated with the tooth survival rate after replantation. The aim of the present study was to examine the relative dependence of cell membrane integrity, attachment and clonogenic capacity of human PL cells on the temperature and duration of the extra-alveolar period and the type of storage medium. Twenty-four premolar teeth were extracted for orthodontic reasons from 9 patients 11-18 years of age. Teeth were maintained at 4 degrees C or 23 degrees C for 15, 30, 60 or 120 min in either milk or dry conditions. Cell membrane integrity was determined by BCECF/AM dye inclusion. Plating efficiency was determined by measurement of cell attachment at 3 and 6 h. The clonogenic capacity of progenitor cells was estimated by limiting dilution and colony counts. For all assays teeth stored in milk at 4 degrees C showed the highest percentages of BCECF positive, attached cells with clonogenic capacity. Increased storage time (15-120 min) was associated with a 50% relative reduction of BCECF staining and a 5-fold relative reduction of cell attachment regardless of storage conditions. However, the clonogenic capacity of progenitor cells decreased 25-fold over the same duration of storage. These data demonstrate that in vitro assays of clonogenic capacity are much more sensitive to extra-oral storage time and storage conditions than dye inclusion or cell attachment. We suggest that in comparison with in vitro measures of cell membrane integrity, the clonogenic capacity of PL cells is more closely liked to tooth survival rate, probably reflecting the capacity of PL progenitor cells to recolonize the root surface after replantation.

Adolescent↗
PubMed1996

Lymph and interstitial fluid dynamics in labial gingival tissues of sheep.

Lymphatic drainage and circulation in periodontal tissues have been cited as important components of host defence and pathogenic mechanisms, but quantitative data are sparse because of the technical difficulties associated with small animal lymphatic studies. However, the lymphatic vessels draining the periodontal tissues and surrounding region are sufficiently large in sheep to permit surgical placement of lymphatic catheters. Consequently, lymph and recirculating lymphocytes can be continuously collected and this permits the quantitative assessment of local immune responses in these tissues. We have studied the lymphatic drainage pathways from the labial gingival tissues in sheep by two methods. First, in a series of anatomical studies (n = 6), a complex of Evan's blue dye and albumin was injected into the labial gingival tissues. One hour after injection the animals were sacrificed and the submandibular and cervical regions were dissected to expose the stained lymphatics. This anatomical study demonstrated 2 major drainage pathways: 1) cervical lymph ducts and; 2) efferent prescapular lymphatics. Secondly, to compare the relative importance of these two drainage pathways, radiolabeled protein (125I-albumin) was injected directly into the gingival tissues and its appearance in the cervical and prescapular lymph was measured (n = 7). Despite the technical difficulties encountered in the experiments, data collected showed that over 7.5 h, 64.7% of the injected protein was recovered in the prescapular and cervical lymph vessels (31.8 +/- 6.5% and 32.9 +/- 8.5%, respectively). In addition, 11.9 +/- 2.1% of the injected protein was transported to the blood by routes not involving the cannulated cervical and prescapular lymph vessels. With most of the remaining radiolabeled protein (17.9 +/- 4.9%) recovered from the injection site, we were able to account for approximately 95% of the injected protein. This study suggests that the lymph drainage from this region in the sheep model could provide one of the best described closed and contained systems and thus, could be a useful system for future continuous monitoring of inflammatory responses during experimental periodontal diseases.

Albumins↗
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

Interleukin-1-induced calcium flux in human fibroblasts is mediated through focal adhesions.

Interleukin-1 (IL-1) is an important mediator of inflammation and also modulates fibroblast metabolism. To assess mechanisms of IL-1-induced signal transduction and calcium flux, early passage human fibroblasts were loaded with fura2/AM. Cells grown on coverslips exhibited dose-dependent [Ca2+]i responses that were maximal at 10(-8) M IL-1 beta with time to maximum flux of 50 s. Cells incubated with anti-Type 1-IL-1 receptor antibody exhibited a 45 nM increase in [Ca2+]i above baseline but demonstrated no calcium response after IL-1 beta treatment. Incubation with EGTA (5 mM) or thapsigargin (1 microM) caused 75% and 37% reductions, respectively, in the IL-1-induced [Ca2+]i increase, suggesting that extracellular Ca2+ predominates in IL-1-stimulated calcium flux. Cells in suspension did not exhibit [Ca2+]i responses to IL-1 beta. The relationship between [Ca2+]i signaling and focal adhesions was examined by plating cells on fibronectin or poly-L-lysine, conditions that either permitted or blocked the formation of focal adhesions. Cells on fibronectin exhibited co-distribution of immunostaining for talin, vinculin, IL-1 receptor, and focal adhesion kinase (pp125fak) in focal adhesions and demonstrated [Ca2+]i responses with 10(-8) M IL-1 beta. Cells on poly-L-lysine or cells in suspension did not exhibit co-distribution of pp125fak, IL-1 receptor, and focal adhesion proteins and did not exhibit calcium flux. The dependence of IL-1-stimulated [Ca2+]i responses on tyrosine kinases was examined first by treating cells with genistein, a selective inhibitor of tyrosine kinases. Genistein (100 microM) completely blocked [Ca2+]i responses to 10(-8) M IL-1, whereas its inactive analogue genistin was not inhibitory. Second, fibroblasts lysates were immunoprecipitated with an antiphosphotyrosine antibody and the lysates were Western-blotted with an anti-pp125fak antibody. Cells grown on fibronectin and stimulated with IL-1 exhibited tyrosine phosphorylation of pp125fak whereas untreated cells or cells grown on poly-L-lysine and treated with IL-1 showed no reaction. Fibroblasts electroinjected with anti-pp125fak monoclonal antibody showed no [Ca2+], response, whereas cells treated with an irrelevant antibody exhibited a normal [Ca2+]i response. Collectively, these data indicate that fibroblasts require substrate attachment and clustering of IL-1 receptors to focal adhesions for IL-1-induced [Ca2+]i responses. Calcium fluxes are mediated through tyrosine kinases whose substrates include pp125fak. These studies therefore demonstrate that activation of intracellular signaling pathways by IL-1 is dependent on IL-1 receptor-cytoskeletal protein interactions.

Calcium↗
PubMed1995

Probing glucocorticoid-dependent osteogenesis in rat and chick cells in vitro by specific blockade of osteoblastic differentiation with progesterone and RU38486.

Glucocorticoids and sex-steroids can modulate osteogenesis in vivo and in vitro. Although the effects of glucocorticoids on bone cells in vitro have been described in detail, the role of sex-steroids is not as well defined. We examined whether sex-steroids influence bone metabolism indirectly by regulating glucocorticoid effects on bone. Interactions of the sex-steroid progesterone or its analog RU38486 with the glucocorticoid dexamethasone (dex) were studied in functional assays of osteogenesis. Three osteoblastic models were evaluated: (1) the rat bone marrow stromal cell (RBMC) nodule system; (2) the chick periosteal osteogenesis (CPO) model; and (3) ROS 17/2.8 cells. RU38486, progesterone, and unlabelled dex competitively inhibited 3H-dex uptake by ROS 17/2.8 cells as well as its (3H-dex) binding to cytosol preps. Both RU38486 and progesterone inhibited dex-induced increases in alkaline phosphatase in CPO cultures, in RBMC cultures, and in ROS 17/2.8 cells. Dex-induced decreases in cell proliferation in ROS 17/2.8 cells were reversed by RU38486 but dex-induced increases in proliferation in the CPO model were not affected. In CPO cultures, dex-induced increases in collagen synthesis were inhibited completely by RU38486 and progesterone. Dex-dependent nodule formation in the RBMC was blocked by RU38486. Both RU38486 and dex mediated reduction of calcium uptake in the CPO model but did not affect mineralized tissue area. The data indicate that RU38486 and progesterone competitively inhibit dex-mediated stimulation of osteogenesis in vitro; this inhibition is exerted on early but not late stage differentiation events of osteoprogenitor cells.

Animals↗
PubMed1995

Concanavalin A induced apoptosis in fibroblasts: the role of cell surface carbohydrates in lectin mediated cytotoxicity.

Cell surface carbohydrates play important regulatory roles during development and in tissue homeostasis by mediating cellular signalling. Concanavalin A (con A) cross links mannose residues on cell surfaces. We used con A to examine the role of cell surface carbohydrates in apoptosis. Balb/c 3T3 (3T3) and diploid human gingival fibroblasts (HGF) incubated with con A (50 micrograms/mL) exhibited rounding, reduction of cell size, loss of cytoskeletal definition, nuclear condensation, and ultrastructural changes consistent with apoptotic cell death. However oligonucleosomal DNA fragmentation was not observed. The sugar methyl-alpha-D-mannopyranoside (MADM) competes for binding sites with con A but failed to induce apoptosis. Cell survival assays after con A treatment demonstrated a concentration dependent (5-500 micrograms/mL) loss of cell viability in 3T3 and HGF cells which was blocked by MADM. The relationship between cell surface binding of con A and cell death was studied in 3T3 cells labelled with varying concentrations of FITC-con A. Flow cytometry and cell survival analyses revealed that smaller cells with lower total cell surface FITC-con A binding sites were approximately 8 times more susceptible to cell death than larger cells with higher number of binding sites. This suggests a positive relationship between cell size, number of con A binding sites, and cell death. Flow cytometric cell cycle analysis of HGF cells showed a 46% reduction in the proportion of G1 phase cells but there was little change in the relative proportions of S and G2+M phase cells. 3H-thymidine labelling of con A treated cells showed a five-fold decrease (chi sq; P < 0.05) in the percentage of labelled cells, indicating blockade of cell cycle transit into S. Thus, cell death occurred predominantly in G1, possibly due to inhibition of protein synthesis which in turn prevented entry of cells into S phase. 35S-methionine uptake in con A treated cells was significantly reduced (approximately 42% con A 50 micrograms/mL; approximately 67% con A 500 micrograms/mL; P < 0.05) compared to untreated controls indicating inhibition of de novo protein synthesis. SDS-PAGE of total cellular proteins confirmed reduction of predominantly lower molecular mass proteins after con A treatment. Con A treatment (50 micrograms/mL) induced a 25% reduction of free intracellular calcium ion concentration over 30 min suggesting that calcium dependent enzymes and translational mechanisms may be inhibited. Collectively, these results indicate that con A binding of cell surface carbohydrates can induce apoptotic cell death in fibroblastic cells due in part to protein synthesis inhibition.

3T3 Cells↗
PubMed1995

Induction of cytoskeletal rearrangements and loss of volume regulation in epithelial cells by Treponema denticola.

The early responses of oral epithelial cells to the adhesion of the oral spirochaete Treponema denticola were studied as a model of microbial perturbation of the plasma membrane. KB cell (ATCC CCL 17) monolayers were incubated with T. denticola (ATCC 35405) in alpha-MEM (minimal essential medium) for periods of 1-4 h at 37 degrees C without serum. Control cultures were exposed to bacteria-conditioned alpha-MEM without serum or bacteria or to alpha-MEM alone. At the end of each incubation, detached and attached epithelial cells were harvested and analysed separately. Compared with controls, T. denticola induced in 25% of cells a two-fold, time-dependent increase of detachment by 4 h. Detached cells in both T. denticola-exposed and control cultures exhibited 25% reductions in modal diameter, did not exclude propidium iodide, did not readhere, and did not form colonies. In T. denticola-exposed cultures, a larger subset (75%) of cells remained attached to the substratum, demonstrated no significant reduction of colony-forming efficiency and excluded propidium iodide. However, these cells exhibited a 21% reduction in diameter (p < 0.05), a 60% decrease of F-actin (p < 0.001), and a 74% reduction in the proportion expressing desmoplakin II (p < 0.01) after exposure to T. denticola. Flow cytometry showed a small (14%) but significant (p < 0.001) reduction in mean fluorescence intensity due to keratin expression in T. denticola-treated cultures. Exposure of cells to anisosmotic media demonstrated that, in contrast to controls, cultures challenged by bacteria failed to undergo compensatory volume regulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
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↗