Fn Synthesis Research Articles

A novel Cdc42-YAP-fibronectin signaling axis regulates ameloblast differentiation during early enamel formation.

Enamel formation is a developmental event governed by intricate molecular signal pathways. Cdc42 is proven to regulate enamel development yet its underlying role and molecular mechanism in early amelogenesis remain elusive. The extracellular matrix of tooth germ basement membrane is critical for the regulation of ameloblast differentiation. Present study investigated whether Cdc42 influences amelogenesis by affecting ECM synthesis and how Cdc42 regulates ameloblasts differentiation. Epithelial-specific knockout of Cdc42 (Cdc42-cKO) mice model was employed to study the ECM expression including Fibronectin (Fn) and amelogenesis markers. Cdc42-cKO mice results in retarded ameloblast differentiation and enamel matrix decrease. Fn synthesis in the enamel organ and basal membrane was totally diminished along with Cdc42 knockdown. YAP acting as the Cdc42 downstream transcription factor, its distribution in ameloblasts was synchronously attenuated by Cdc42 knockdown and nuclear localization progressively decreased with tooth germ development. Cdc42 unidirectionally controls the Fn synthesis via YAP regulation. Overall, ameloblast differentiation inhibition by silencing of Cdc42 was successfully rescued by YAP activation. We demonstrated that Cdc42 as an initiator, mediated downstream pathway through transcriptional activator YAP, thereby affecting ameloblast differentiation by controlling Fn synthesis. The Cdc42-YAP-Fn signaling axis are elucidated to act critical role during the early amelogenesis.

18F]Fluspidine-A PET Tracer for Imaging of σ1 Receptors in the Central Nervous System.

σ1 receptors play a crucial role in various neurological and neurodegenerative diseases including pain, psychosis, Alzheimer's disease, and depression. Spirocyclic piperidines represent a promising class of potent σ1 receptor ligands. The relationship between structural modifications and σ1 receptor affinity and selectivity over σ2 receptors led to the 2-fluoroethyl derivative fluspidine (2, Ki = 0.59 nM). Enantiomerically pure (S)-configured fluspidine ((S)-2) was prepared by the enantioselective reduction of the α,β-unsaturated ester 23 with NaBH4 and the enantiomerically pure co-catalyst (S,S)-24. The pharmacokinetic properties of both fluspidine enantiomers (R)-2 and (S)-2 were analyzed in vitro. Molecular dynamics simulations revealed very similar interactions of both fluspidine enantiomers with the σ1 receptor protein, with a strong ionic interaction between the protonated amino moiety of the piperidine ring and the COO- moiety of glutamate 172. The 18F-labeled radiotracers (S)-[18F]2 and (R)-[18F]2 were synthesized in automated syntheses using a TRACERlab FX FN synthesis module. High radiochemical yields and radiochemical purity were achieved. Radiometabolites were not found in the brains of mice, piglets, and rhesus monkeys. While both enantiomers revealed similar initial brain uptake, the slow washout of (R)-[18F]2 indicated a kind of irreversible binding. In the first clinical trial, (S)-[18F]2 was used to visualize σ1 receptors in the brains of patients with major depressive disorder (MDD). This study revealed an increased density of σ1 receptors in cortico-striato-(para)limbic brain regions of MDD patients. The increased density of σ1 receptors correlated with the severity of the depressive symptoms. In an occupancy study with the PET tracer (S)-[18F]2, the selective binding of pridopidine at σ1 receptors in the brain of healthy volunteers and HD patients was shown.

Biological Effects of Shikonin in Human Gingival Fibroblasts via ERK 1/2 Signaling Pathway.

Shikonin, an active ingredient of Lithospermum erythrorhizon, exerts anti-inflammatory and antibacterial effects, and promotes wound healing. We investigated whether shikonin stimulated gingival tissue wound healing in human gingival fibroblasts (hGF). In addition, we evaluated the effects of shikonin on the mitogen-activated protein kinase (MAPK) signaling pathway, which has an important role in wound healing. hGF were subjected to primary culture using gingiva collected from patients. The cells were exposed to/treated with Shikonin at concentrations ranging from 0.01 to 100 μM. The optimal concentration was determined by cell proliferation and migration assays. Type I collagen and fibronectin synthesis, the gene expression of vascular endothelial growth factor (VEGF) and FN, and the phosphorylation of Extracellular signal-regulated kinase (ERK) 1/2 were investigated. Identical experiments were performed in the presence of PD98059 our data suggest, a specific ERK 1/2 inhibitor. Shikonin significantly promoted hGF proliferation and migration. Shikonin (1 µM) was chosen as the optimal concentration. Shikonin promoted type I collagen and FN synthesis, increased VEGF and FN expression, and induced ERK 1/2 phosphorylation. These changes were partially suppressed by PD98059. In conclusion, Shikonin promoted the proliferation, migration, type I collagen and FN synthesis, and expression of VEGF and FN via ERK 1/2 signaling pathway in hGFs. Therefore, shikonin may promote periodontal tissue wound healing.

649 Hypoxia-pretreatment as a possible approach to stimulate wound healing potentials of dermal fibroblasts in treatment of chronic skin ulcers

The development of new therapeutic approaches to chronic wounds hasn’t lost its relevance. In non-healing diabetic ulcers (diabetic foot) auto-transplantation (a common treatment approach) is not effective due to impaired fibroblast functionality and shifts in synthesis of ECM proteins (COL1, COL3, FN) and certain growth factors. It was shown that HIF1 (hypoxia-induced factor) is a key regulator of ECM remodeling by fibroblasts through control of collagen prolyl hydroxylase (P4HA1, P4HA2 – both required for collagen deposition) and lysylhydroxylase (PLOD2 – modulates collagen fibers strength) expression. Therefore, the use of hypoxia-stimulated fibroblasts (sFbs) may represent a reasonable treatment alternative in the maintenance of chronic diabetic wounds. Here, we evaluated the expression levels of wound healing-related genes and proteins in sFbs after 24h, 48h and 72h exposure to 1% O2. After a short-term (24/48h) exposure to hypoxia, sFbs exhibited a marked increase in expression of a number of genes as compared to 21% O2 culture: Hif1a – 7.4 and 16.9 fold respectively; Vegf1a – 21/60; Egln1 – 9/9; Egln2 – 6.5/5; Egln3 – 53/110; COL1 – 2.4/3; Fn – 3.3/1.7; P4HAa1 - 5.8/7.8; P4ha2 – 6.6/8.5; and Plod2 - 9/25 fold. In contrast, with a longer exposure to hypoxia (72h), activation was significantly diminished (Hif1a – 2 fold; Vegf1a – 6; Egln3 – 2.4) or completely abrogated (Egln1, Egln2, COL1, Fn, P4ha1, P4ha2, and Plod2). Importantly, at protein level, we observed only COLI (2 fold) stimulation by hypoxia. The stimulation of COLIII and Fn synthesis in sFb was detected only after a 6-18h period of their re-oxygenation. Based on our results, we propose that short-term stimulation of dermal fibroblasts (for example, immobilized on a biopolymer scaffold) by hypoxia could be beneficial for chronic diabetic wound treatment. (NRC Kurchatov Institute intramural grant supported research).

FibronectinEDA promotes chronic cutaneous fibrosis through Toll-like receptor signaling.

Scleroderma is a progressive autoimmune disease affecting multiple organs. Fibrosis, the hallmark of scleroderma, represents transformation of self-limited wound healing into a deregulated self-sustaining process. The factors responsible for maintaining persistent fibroblast activation in scleroderma and other conditions with chronic fibrosis are not well understood. Toll-like receptor 4 (TLR4) and its damage-associated endogenous ligands are implicated in immune and fibrotic responses. We now show that fibronectin extra domain A (Fn(EDA)) is an endogenous TLR4 ligand markedly elevated in the circulation and lesional skin biopsies from patients with scleroderma, as well as in mice with experimentally induced cutaneous fibrosis. Synthesis of Fn(EDA) was preferentially stimulated by transforming growth factor-β in normal fibroblasts and was constitutively up-regulated in scleroderma fibroblasts. Exogenous Fn(EDA) was a potent stimulus for collagen production, myofibroblast differentiation, and wound healing in vitro and increased the mechanical stiffness of human organotypic skin equivalents. Each of these profibrotic Fn(EDA) responses was abrogated by genetic, RNA interference, or pharmacological disruption of TLR4 signaling. Moreover, either genetic loss of Fn(EDA) or TLR4 blockade using a small molecule mitigated experimentally induced cutaneous fibrosis in mice. These observations implicate the Fn(EDA)-TLR4 axis in cutaneous fibrosis and suggest a paradigm in which aberrant Fn(EDA) accumulation in the fibrotic milieu drives sustained fibroblast activation via TLR4. This model explains how a damage-associated endogenous TLR4 ligand might contribute to converting self-limited tissue repair responses into intractable fibrogenesis in chronic conditions such as scleroderma. Disrupting sustained TLR4 signaling therefore represents a potential strategy for the treatment of fibrosis in scleroderma.

Fibronectin (FN) decreases glomerular lesions and synthesis of tumour necrosis factor-alpha (TNF-alpha), platelet-activating factor (PAF) and FN in proliferative glomerulonephritis.

We have studied the effect of therapy with plasma FN on glomerular synthesis of PAF, TNF-alpha and FN, in experimental proliferative glomerulonephritis. Glomerular PAF, TNF-alpha and FN production were increased in rats with nephritis. Peak glomerular PAF production preceded, while peak glomerular TNF-alpha bioactivity coincided with maximal proteinuria. Rats treated with FN (5 mg/kg per 48 h) for 15 days had less proteinuria, glomerular and interstitial cell infiltration and glomerular PAF, TNF-alpha and FN synthesis than non-treated rats. In order to characterize further the mechanisms of action of FN, healthy rats were injected with either FN or saline. Peripheral blood mononuclear cells and neutrophils from healthy rats injected with FN secreted less TNF-alpha and PAF, respectively, than those obtained from saline-treated rats. Our data suggest that the beneficial effect of FN may be related to decreased number of glomerular leucocytes and decreased synthesis of inflammatory mediators and extracellular matrix.

Increased intrathecal synthesis of fibronectin in bacterial and carcinomatous meningitis

Immunoreactive fibronectin (Fn) was quantified in paired cerebrospinal fluid (CSF) and serum samples from patients with bacterial meningitis (n = 46), tick-borne encephalitis (TBE) (n = 6), HIV infection (n = 6), Guillain-Barré syndrome (n = 5), carcinomatous meningitis (n = 11), multiple sclerosis (n = 15), disk disease (n = 11), and controls (n = 28). A highly significant elevation of CSF Fn was found in bacterial meningitis, TBE, and carcinomatous meningitis. There were no significant differences in serum Fn between any of the groups. An Fn index to estimate the rate of intrathecal Fn synthesis reached the highest value in bacterial meningitis. Our findings suggest that CSF Fn may be an indicator of adequate host reaction and tissue repair. For diagnostic purposes, the determination of CSF Fn probably does not add much to routine CSF laboratory tests.

Formation and activation of fibroblast spheroids depend on fibronectin–integrin interaction

Clustering of fibroblasts into spheroids induces a massive proinflammatory, proteolytic and growth-factor response, named nemosis, which promotes tumor cell invasiveness and differentiation of leukemia cells. We have now sought to investigate mechanisms leading to the formation of multicellular spheroids and subsequent activation of fibroblasts (nemosis). Cell lines either lacking fibronectin expression (FN −/−) or expressing FN with a mutated integrin-binding site (FN RGE/RGE) were unable to form compact spheroids. Furthermore, inhibition of FN synthesis by siRNA or functional inhibition of FN or its integrins impaired spheroid formation (α5, β1) and quenched fibroblast activation (αV). The integrin ligand GRGDSP hexapeptide interfered with spheroid formation and induced activation of fibroblasts. Surprisingly, a 70 kDa FN fragment, which prevents deposition of FN matrix but does not interfere with FN–integrin interaction, prevented spheroid formation only marginally and did not block the activation. Our results present a new mechanism of fibroblast activation, which is initiated by interaction of FN with its integrin receptors.

Protein Kinase Cϵ Mediates Polymeric Fibronectin Assembly on the Surface of Blood-borne Rat Breast Cancer Cells to Promote Pulmonary Metastasis

Malignant breast cancer cells that have entered the blood circulation from primary mammary fat pad tumors or are grown in end-over-end suspension culture assemble a characteristic, multi-globular polymeric fibronectin (polyFn) coat on their surfaces. Surface polyFn is critical for pulmonary metastasis, presumably by facilitating lung vascular arrest via endothelial dipeptidylpeptidase IV (CD26). Here, we show that cell-surface polyFn assembly is initiated by the state of suspension, is dependent upon the synthesis and secretion of cellular Fn, and is augmented in a dose- and time-dependent manner by plasma Fn. PolyFn assembly is regulated by protein kinase Cepsilon (PKCepsilon), which translocates rapidly and in increasing amounts from the cytosol to the plasma membrane and is phosphorylated. PolyFn assembly is impeded by select inhibitors of this kinase, i.e. bisindolylmaleimide I, Ro-32-0432, Gö6983, and Rottlerin, by the phorbol 12-myristate 13-acetate-mediated and time-dependent loss of PKCepsilon protein and decreased plasma membrane translocation, and more specifically, by stable transfection of lung-metastatic MTF7L breast cancer cells with small interfering RNA-PKCepsilon and dominant-negative PKCepsilon constructs (e.g. RD-PKCepsilon). The inability to assemble a cell surface-associated polyFn coat by knockdown of endogenous Fn or PKCepsilon impedes cancer cells from metastasis to the lungs. The present studies identify a novel regulatory mechanism for polyFn assembly on blood-borne breast cancer cells and depict its effect on pulmonary metastasis.

Prostaglandin E2 Stimulates Fibronectin Expression through EP1 Receptor, Phospholipase C, Protein Kinase Cα, and c-Src Pathway in Primary Cultured Rat Osteoblasts

Fibronectin (Fn) is involved in the early stages of bone formation, and prostaglandin E (PGE) is an important factor regulating osteogenesis. Here we found that PGE(2) enhanced extracellular Fn assembly in rat primary osteoblasts, as shown by immunofluorescence staining and enzyme-linked immunosorbent assay. PGE(2) also increased the protein levels of Fn by using Western blotting analysis. By using pharmacological inhibitors or activators or genetic inhibition by the EP receptor, antisense oligonucleotides revealed that the EP(1) receptor but not other PGE receptors is involved in PGE(2)-mediated up-regulation of Fn. At the mechanistic level, Ca(2+) chelator (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester)), phosphatidylinositol-phospholipase C inhibitor (U73122), or Src inhibitor (PP2) attenuated the PGE(2)-induced Fn expression. Protein kinase C (PKC) inhibitor (GF109203X) also inhibited the potentiating action of PGE(2). Furthermore, treatment with antisense oligonucleotides of various PKC isoforms, including alpha, beta, epsilon, and delta, demonstrated that alpha isozyme plays an important role in the enhancement action of PGE(2) on Fn assembly. Flow cytometry and reverse transcription-PCR showed that PGE(2) and 17-phenyl trinor PGE(2) (EP(1)/EP(3) agonist) increased the surface expression and mRNA level of alpha5 or beta1 integrins. Fn promoter activity was enhanced by PGE(2) and 17-phenyl trinor PGE(2) in cells transfected with pGL2F1900-Luc. Cotransfection with dominant negative mutants of PKCalpha or c-Src inhibited the potentiating action of PGE(2) on Fn promoter activity. Local administration of PGE(2) or 17-phenyl trinor PGE(2) into the metaphysis of the tibia via the implantation of a needle cannula significantly increased the Fn and alpha5beta1 integrin immunostaining and bone volume of secondary spongiosa in tibia. Taken together, our results provided evidence that PGE(2) increased Fn and promoted bone formation in rat osteoblasts via the EP(1)/phospholipase C/PKCalpha/c-Src signaling pathway.

Enhancement of fibronectin synthesis and fibrillogenesis by BMP-4 in cultured rat osteoblast.

The skeletal extracellular matrix produced by osteoblasts contains the glycoprotein fibronectin (Fn), which regulates the adhesion, differentiation, and function of osteoblasts. Fn fibrillogenesis is involved in the process of bone mineralization. Bone morphogenetic proteins (BMPs) can be isolated from organic bone matrix and are able to initiate de novo cartilage and bone formation. In this study, the effect of BMP-4 on Fn fibrillogenesis in cultured rat osteoblasts was examined. BMP-4 enhanced Fn synthesis and extracellular Fn assembly in primary cultured osteoblasts. In addition, the extracellular assembly of Fn from exogenously applied soluble human Fn was also increased by BMP-4. It has been reported that alpha5beta1 integrin is related to Fn fibrillogenesis. The synthesis of both alpha5 and beta1 integrins was upregulated by BMP-4. Immunocytochemistry showed that the clustering of alpha5 and beta1 integrins was also increased by BMP-4. BMP-4 increased fibril formation of Fn and the adhesion of osteoblasts onto Fn matrix, which was inhibited by disintegrin triflavin and Gly-Arg-Gly-Asp-Ser (GRGDS) peptide. Phosphorylation of extracellular signal-regulated kinase (ERK) and focal adhesion kinase (FAK) was increased by BMP-4. Enhancement of extracellular Fn fibrillogenesis and the mRNA expression of beta1 integrin by BMP-4 were inhibited by ERK kinase (MEK) inhibitor PD98059. These results suggest that the enhancement of extracellular Fn fibrillogenesis by BMP-4 in cultured osteoblasts is related to the increase of the synthesis of Fn and clustering of alpha5 and beta1 integrins. ERK is involved in the signaling pathway of BMP-4 in regulating Fn fibrillogenesis in osteoblasts.

Regulation of fibronectin fibrillogenesis by protein kinases in cultured rat osteoblasts.

Fibronectin (Fn) plays an important role in the regulation of adhesion, migration, and maturation of osteoblasts. Fn fibrillogenesis is involved in the process of bone mineralization. To elucidate the regulatory role of protein kinases in the formation of fibrillar Fn matrix, Fn synthesis and assembly were examined in cultured osteoblasts. Osteoblasts assembled the endogenously released soluble Fn into immobilized form on the substratum in a time-dependent manner. Both 12-O-tetradecanoylphorbol-13 acetate (TPA) and forskolin increased the synthesis of Fn. However, the extracellular assembly of Fn fibril from both endogenously released and exogenously applied soluble Fn was increased by TPA but decreased by forskolin. Protein kinase C (PKC) inhibitors, such as H7, Ro 318220, and Gö 6976, inhibited Fn fibrillogenesis. These results suggest that the dynamic of Fn fibrillogenesis is differentially regulated by the activation of PKC and protein kinase A (PKA). Both classic and novel isoforms of PKC are involved in the action of TPA in osteoblasts. It has been reported that alpha5beta1 integrin is related to Fn fibrillogenesis. Immunocytochemistry and flow cytometry showed that TPA and forskolin increased and inhibited, respectively, the clustering and surface expression of alpha5 integrins. TPA and forskolin did not affect protein levels of alpha5 integrins. The Western blot and reverse transcriptase-polymerase chain reaction showed that protein and mRNA levels of beta1 integrins also were not affected by TPA and forskolin. These results suggest that TPA and forskolin may affect the surface expression of alpha5beta1 integrins. cAMP response element-binding protein phosphorylation is involved in the action of forskolin but not that of TPA. Our results suggest that PKC activation enhanced Fn fibrillogenesis, whereas PKA activation inhibited extracellular Fn fibrillogenesis in primary cultured osteoblasts. Cytosolic Fn synthesis and extracellular Fn assembly may be differentially regulated by the activation of PKA.

Transcriptional repression of the human fibronectin gene in laryngeal squamous cell carcinoma cells.

The aim of the experiments was to analyze the mRNA expression pattern and verify the repression of FN gene expression in laryngeal squamous cell carcinoma (SCC) cells in comparison with benign mucosal keratinocytes. Messenger RNA from SCC cells and benign keratinocytes was reverse transcribed and subjected to PCR following differential display (DD) analysis of the amplicons. Northern hybridization was carried out to confirm the reduction of the FN-mRNA expression in both laryngeal SCC cells and larynx carcinoma biopsies, in contrast to adjacent normal mucosa. Quantitation of protein synthesis was performed with homogenates of fresh tumor biopsies and their normal phenotypes, as well as of benign keratinocytes and laryngeal SCC cell lines, respectively, using ELISA. In the liposome-mediated transient transfection assay, FN promoter activity was analyzed by linking the FN promoter sequence to the chloramphenicol acetyltransferase (CAT) reporter gene. Transfection efficacy was monitored by co-transfection with pGL3 control vector. A 191 bp mRNA fragment revealing a 99% homology with the human FN-mRNA was detected, the expression of which was repressed 20 times as much in SCC cells as compared to benign phenotypes. Northern hybridization confirmed the distinctly reduced expression of FN-mRNA in both laryngeal SCC cells and larynx carcinoma biopsies, in contrast to adjacent normal mucosa. The quantitation experiments showed a correlation between the range of FN synthesis and the expression of FN-mRNA in cell lines and the biopsies which were used. The 1.28 kb FN gene promoter drove expression of the CAT reporter gene, which was similar to the FN-mRNA expression showed by DD and Northern hybridization. The mechanisms leading to the low level of FN in many tumors have not yet been sufficiently investigated. Our findings suggest that the decrease of FN in laryngeal SCC cells is transcriptionally regulated.

Staphylococcal fibronectin binding protein interacts with heat shock protein 60 and integrins: role in internalization by epithelial cells.

We reported previously that internalization of Staphylococcus aureus by nonprofessional phagocytes involves an interaction between fibronectin (Fn) binding protein (FnBP) and the host cell, resulting in signal transduction, tyrosine kinase activity, and cytoskeletal rearrangement (K. Dziewanowska, J. M. Patti, C. F. Deobald, K. W. Bayles, W. R. Trumble, and G. A. Bohach, Infect. Immun. 67:4673-4678, 1999). The goal of the present study was to identify the host molecules responsible for uptake of the organism through an interaction with FnBP. First, Fn was required for internalization. Addition of small amounts of exogenous Fn stimulated the uptake of S. aureus by HEp-2 cells, which are deficient in Fn synthesis. Fn antibodies blocked internalization of the organism by MAC-T cell monolayers, a bovine epithelial cell line which expresses Fn. Second, a monoclonal antibody (MAb) specific for beta(1) integrins dramatically reduced S. aureus invasion, suggesting that the formation of a Fn bridge linking the host cell beta(1) integrin and FnBP precedes internalization. However, ligand blotting of cell membrane proteins with a functional fragment of FnBP consistently identified an additional approximately 55-kDa receptor on both human and bovine epithelial cells. This protein was purified and identified by N-terminal microsequencing as heat shock protein 60 (Hsp60). The interaction between FnBP and Hsp60 also occurred when the whole cells were used. Cell membrane localization of Hsp60 was confirmed by biotinylation with an agent nonpermeable to the cell membrane. Pretreatment of epithelial cells with a MAb specific for eukaryotic Hsp60 significantly reduced internalization of S. aureus. Combined, these results suggest that the FnBP binds directly to both Hsp60 and Fn and is linked to beta(1) integrins through a Fn bridge. The simultaneous involvement of Fn and two host cell ligands, beta(1) integrins and Hsp60, suggests that FnBP is a multifunctional adhesin that mediates internalization in a manner similar to that proposed for OpaA, the Neisseria gonorrhoeae FnBP homolog (J. P. M. van Putten, T. D. Duensing, and R. L. Cole, Mol. Microbiol. 29:369-379, 1998).

Glucocorticoid Effects in the Human Placenta: Evidence That Dexamethasone-Mediated Inhibition of Fibronectin Expression in Cytotrophoblasts Involves a Protein Intermediate1

Oncofetal fibronectin is an extracellular matrix protein that is suggested to play an important role in regulating adherence at uterine-placental interfaces. The purpose of the present study was to elucidate a mechanism through which glucocorticoids (GCs) inhibit the synthesis of FN in human placenta as part of their matrix-suppressive action near parturition. We observed that treatment of cytotrophoblasts isolated from human term placentas for 48 h with 10(-7) mol/L dexamethasone (DEX) down-regulated levels of FN expression to 13-19% of control levels in immunoprecipitation, Northern blotting, and enzyme-linked immunosorbent assay experiments. Conversely, GC treatment increased FN expression in placental fibroblasts to 164-310% of control levels in Northern blotting and enzyme-linked immunosorbent assay procedures, suggesting that GC-mediated suppression of FN expression is specific to cytotrophoblasts. Results indicated that the DEX-mediated suppression of FN expression in cytotrophoblasts was not mediated through changes in the stability of FN messenger ribonucleic acid (mRNA). Run-on transcription assays using isolated nuclei suggested that GC treatment did not markedly affect transcription of the FN gene in cytotrophoblasts. To test whether the GC-mediated suppression of FN expression was mediated through a protein intermediate, levels of FN mRNA were examined by Northern blotting in cells treated for 48 h with and without 10(-7) mol/L DEX and cycloheximide (CHX; 125 ng/mL). We observed that CHX treatment increased FN expression in DEX-treated cells to 91% of control values. We noted that whereas the presence of 100-300 ng/mL CHX reversed the DEX-mediated inhibition of FN mRNA expression in cytotrophoblasts, it did not alter the overall rates of protein synthesis in DEX-treated and control cells. These data suggest that suppression of FN mRNA expression by GC in cytotrophoblasts requires de novo protein synthesis and is mediated through a short lived intermediate, the synthesis of which is inhibited at low concentrations of CHX. Thus, GC-induced protein intermediates may influence uterine-placental adherence by modulating levels of oncofetal FN at sites of uterine-placental contact.

Effects of Staphylococcus aureus protein A on the production of primary host defense factors against S. aureus infection

In vitro examination was carried out to investigate the effects of protein A of Staphylococcus aureus (S. aureus) on the production of fibronectin (Fn) and the third component of complement (C3) by macrophages and that of CD11b, CD49e and H2O2 by granulocytes. Fn production of cultured murine peritoneal macrophages (M phi ) increased significantly (P < 0.01) by treatment for 4 hr at 37 degrees C with recombinant PA (rPA) and the supernatant of overnight culture of S. aureus Cowan I strain (CoCS) (p < 0.01), not that of Wood 46 strain (WoCS), in comparison with that of control. The activity of rPA was inhibited strongly in the presence of CYH (1.0 microgram/ml). The production of C3 by cultured M phi did not increased by treatment for 4 hr at 37 degrees C with rPA, CoCS and WoCS. In these cells treated with CoCS and WoCS, however, the production increased by cultivation in serum free medium for a further 20 hr at 37 degrees C after the treatment. But increase was not found in rPA treated cells. On the other hand, the production of Mac-1, VLA-5 and H2O2 by granulocytes did not increase by treatment with rPA and CoCS. These results show that rPA and PA in CoCS are major components which stimulates Fn synthesis and secretion by cultured M phi some component(s) contained in the supernatant of overnight culture of S. aureus increased the production C3 by cultured M phi , and the supernatant may not contain stimulators to induce production of CD49e, CD11b and H2O2. Increase of Fn production of M phi by PA stimulation may play an important role in the primary host defense against S. aureus infection.

Opposing actions of transforming growth factor-beta and glucocorticoids in the regulation of fibronectin expression in the human placenta.

Alterations in the expression of extracellular matrix (ECM) proteins in the placenta and fetal membranes have been linked to parturition whether occurring before or at term. In the present study, we examined the individual and combined effects of transforming growth factor (TGF)-beta and dexamethasone (DEX) on the expression of oncofetal fibronectin (onfFN), i.e. a major ECM protein synthesized by placenta, in cytotrophoblasts isolated from human term placentas to establish a model system from which to evaluate the actions of positive and negative regulators of ECM protein expression in the human placenta. Cytotrophoblasts were maintained for 21=62 h in medium supplemented with 4% charcoal-stripped calf serum in the presence or absence of TGF-beta (2 ng/mL) and DEX (10(-7) mol/L). Levels of onfFN in culture media were determined by immunoassay. TGF-beta treatment alone induced approximately a 150% increase in media levels of onfFN after 21 and 45 h of culture when compared with control, whereas DEX treatment alone reduced levels of onfFN to 15% of control levels. Media levels of onfFN in cells treated with both TGF-beta and DEX were 40-90% of control levels. Similarly, treatment of cells with TGF-beta alone promoted a 100-250% increase in rates of FN synthesis and levels of FN messenger ribonucleic acid, whereas DEX treatment alone reduced these indices of FN expression to approximately 10% of control levels. In cells treated with TGF-beta and DEX, levels of ECM protein synthesis and FN messenger ribonucleic acid were between 30 and 100% of control values. Similar patterns of regulation of FN expression by TGF-beta and DEX were observed when experiments were carried out in serum-free medium. Our results suggest that during pregnancy, TGF-beta and glucocorticoids may be important opposing physiological regulators of placental ECM protein expression.

Protein kinase C signals thromboxane induced increases in fibronectin synthesis and TGF-β bioactivity in mesangial cells

Previous studies have demonstrated that thromboxane (TX) stimulates matrix protein synthesis in mesangial cells (MC), and that this action is signalled by receptor mediated activation of protein kinase C (PKC). In the present study, we examined the hypothesis that activation of PKC by TX signals increases in transforming growth factor beta (TGF-beta) bioactivity, which in turn induces enhanced matrix protein synthesis. In cultured rat MC, the TXA2/prostaglandin endoperoxide analogue U-46619, but not exogenous human platelet TGF-beta 1, activated PKC as reflected by enhanced in situ phosphorylation of MARCKS protein, an endogenous substrate of PKC. U-46619 and TGF-beta 1 stimulated fibronectin (Fn) synthesis in MC, as shown by [35S]methionine incorporation into immunoprecipitable Fn. Pan-specific rabbit anti-TGF-beta antibody blocked the increases in Fn synthesis induced by exogenous TGF-beta and those induced by U-46619 at 24 to 72 hours after addition. Anti-TGF-beta antibody did not block the small increases in FN synthesis observed six hours after addition of U-46619, suggesting that this acute response was not dependent on TGF-beta. Anti-TGF-beta antibody also failed to block activation of PKC by U-46619. U-46619 and 50 nM of the PKC agonist phorbol dibutyrate (PDBu) significantly increased both the active fraction and total (latent plus active) TGF-beta in MC culture media, as assayed with the mink lung epithelial cell bioassay system.(ABSTRACT TRUNCATED AT 250 WORDS)

Role for Transforming Growth Factor β in Thromboxane-Induced Increases in Mesangial Cell Fibronectin Synthesis

Thromboxane A2 (TXA2) has been implicated in the pathogenesis of progressive glomerulosclerosis and stimulates the synthesis of matrix protein by mesangial cells (MCs). This study examined the role of transforming growth factor beta (TGF-beta) in the mediation of the action of the stable TXA2/prostaglandin (PG) endoperoxide analog U-46619 to stimulate fibronectin (Fn) synthesis in cultured rat MC. Exogenous TGF-beta increased Fn synthesis by MC in a concentration- and time-dependent fashion, as reflected by incorporation of [35S]methionine into immunoprecipitable Fn. Submaximal concentrations of TGF-beta (1-2.5 pmol/l) increased Fn synthesis two- to threefold, a response comparable in magnitude to that observed with a maximal stimulatory concentration (1 mumol/l) of U-46619. Anti-TGF-beta antibody, but not isotypic IgG, blocked the increases in Fn synthesis induced by both U-46619 and exogenous TGF-beta. Endogenous TGF-beta bioactivity in MC culture media, assessed by the mink lung epithelial cell system, was significantly increased by 1 mumol/l U-46619 (1.7 +/- 0.3 pmol/l) compared with that of control media (0.6 +/- 0.1 pmol/l, P < 0.05). Total (active plus latent) TGF-beta bioactivity, assayed after heat activation of latent TGF-beta, was also significantly higher in media of MCs cultured with U-46619 (45 +/- 4 pmol/l) compared with control (24 +/- 4 pmol/l). Thus, U-46619 increased endogenous TGF-beta bioactivity to a level sufficient to account for the enhancement of Fn synthesis observed with U-46619, as reflected by the Fn synthetic response to exogenous TGF-beta.(ABSTRACT TRUNCATED AT 250 WORDS)

Thromboxane stimulation of mesangial cell fibronectin synthesis is signalled by protein kinase C and modulated by cGMP

Thromboxane (TX) has been implicated in the pathogenesis of glomerulosclerosis in several models of glomerular injury. In the present study, we examined the role of the protein kinase C (PKC) signalling system in expression of the action of the TXA2/PGH2 analogue U-46619 to stimulate fibronectin (Fn) synthesis in cultured rat mesangial cells (MC), and the influence of cGMP on this MC response. U-46619 activated PKC and enhanced Fn synthesis in MC in a time and concentration dependent fashion. Both responses to U-46619 were blocked by GF 109203X, a selective inhibitor of PKC activity, as well as by calphostin C and staurosporine, PKC inhibitors structurally distinct from GFX. Down-regulation of PKC by prior sustained exposure of MC to 0.5 microM phorbol myristate acetate similarly blocked increases in Fn synthesis induced by U-46619. The TXA2/PGH2 receptor antagonist Sq-29548 also prevented activation of PKC and stimulation of Fn synthesis by U-46619, consistent with transduction of these responses via specific high affinity TXA2/PGH2 receptors on MC. Addition of exogenous 8-Br-cGMP or stimulation of endogenous cGMP generation with atrial natriuretic peptide (ANP) suppressed both U-46619 activation of PKC and stimulation of Fn synthesis. cGMP did not alter TXA2/PGH2 receptor number of affinity in MC, but significantly suppressed phorbol ester activation of PKC. Thus, cGMP inhibition of U-46619 actions is expressed at steps distal to TX receptor binding and may involve effects at and proximal to activation of PKC. Interactions between the PKC and cGMP cellular signalling systems may be important determinants of MC matrix protein production in response to TX.