Osteoprotegerin production by human osteoblast lineage cells is stimulated by vitamin D, bone morphogenetic protein-2, and cytokines

Rheumatoid arthritis(RA) is a chronic, deforming and destructive arthritis of unknown etiology. For the medical treatment of RA, NSAID has been the first choice of drug. Recently it has been known that early use of DMARD may result in clinical remission. Understanding of the pivotal role of cytokines and adhesion molecules for the rheumatoid joint destruction enabled us to target these cytokines and molecules as therapeutic measures. Monoclonal antibodies were produced against the cytokines and adhesion molecules such as IL-1, IL-6, IL-6R, TNF-alpha, as well as CD4 molecules. Clinical use of these monoclonal antibodies was found to be effective for rheumatoid arthritis. However these therapeutic measures have several disadvantages such as transient efficacy and side effect.     

Cloning of mouse diastrophic dysplasia sulfate transporter gene induced during osteoblast differentiation by bone morphogenetic protein-2

Although intensive studies have been directed at understanding osteoblastic differentiation, the molecular mechanisms are still unclear. In this study, we describe a cDNA that encodes a sulfate transporter that was cloned as a gene induced in osteoblast precursor cells in association with osteoblastic differentiation. Based on the fact that bone morphogenetic protein-2 (BMP-2) induces osteoblastic phenotypes in immature mouse fibroblastic C3H10T1/2 cells, we performed a subtraction hybridization between BMP-2-treated and untreated cells, and have isolated one clone (designated as st-ob for sulfate transporter in osteoblast) induced by BMP-2 that is constantly expressed in osteoblastic cells. The deduced amino acid sequence and proposed structure of st-ob are mostly identical to those of the human diastrophic dysplasia sulfate transporter gene product (DTDST). St-ob mRNA was abundantly expressed in the thymus, testis, calvaria and osteoblastic MC3T3-E1 cells, whereas its expression was faint in C3H10T1/2 cells. Expression of st-ob in C3H10T1/2 cells was increased by transforming growth factor-beta1 (TGF-beta1), retinoic acid and dexamethasone as well as BMP-2. Furthermore, BMP-2 increased sulfate incorporation in C3H10T1/2 cells about twice as high as the baseline level. Osteoblasts actively take up sulfate to synthesize proteoglycans, which are one of the major components of the extracellular matrix of bone and cartilage. The present study demonstrates that st-ob induced during osteoblastic differentiation is an important phenotype of osteoblasts for characterizing their function.     

Inhibition of protein kinase C suppresses megakaryocytic differentiation and stimulates erythroid differentiation in HEL cells

The bisindolylmaleimide, GF109203X (2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide ), a highly selective inhibitor of protein kinase C (PKC), was used to test the role of this enzyme in phorbol ester-induced megakaryocytic differentiation of HEL cells. Treatment of these cells with 10 nmol/L phorbol 12-myristate 13-acetate (PMA) for 3 days caused a complete inhibition of proliferation and a threefold increase in the surface expression of glycoprotein (GP) IIIa, a marker of megakaryocytic differentiation that forms part of the fibrinogen receptor complex, GPIIb/IIIa. A similar effect was observed with phorbol 12,13-dibutyrate, but not with the biologically inactive derivative PMA-4-O-methyl ether. The PMA-induced increase in GPIIIa expression was completely inhibited by GF109203X in a dose-dependent manner (IC50 = 0.5 mumol/L), with a maximal effect at 2.5 to 5.0 mumol/L. GF109203X also blocked the inhibitory effect of PMA on cell growth and inhibited PMA-stimulated phosphorylation of the 47-kD PKC substrate, pleckstrin. Incubation of HEL cells with 25 mumol/L hemin for 3 days caused a fourfold to fivefold increase in expression of the erythroid differentiation marker, glycophorin A. In contrast to the inhibitory effect of GF109203X on GPIIIa expression, hemin induction of glycophorin A was enhanced by this compound. Furthermore, GF109203X alone caused a dose-dependent increase in glycophorin A expression, and induced hemoglobinization. Consistent with these changes, Northern blot analysis revealed that GF109203X treatment reduced the steady-state level of GPIIb mRNA and increased those for glycophorin A and gamma-globin. These results suggest that PKC may act as a developmental switch controlling erythroid/megakaryocytic differentiation.     

Radiographic analysis of regenerated bone around endosseous implants in the canine using recombinant human bone morphogenetic protein-2

A selective and sensitive HPLC method was developed for the determination of U-39968E in rat plasma. The assay involved solid-phase extraction of the analyte and the internal standard and precolumn derivatization with cyclohexane-1,3-dione reagent before injection on to the HPLC column. The samples were chromatographed on a Spherisorb S5 CN column (25 cm x 4.6 mm i.d.) with a mobile phase containing acetonitrile-trifluoroacetic acid-water (17:0.2:83, v/v/v) at a flow rate of 1.5 ml min-1. The column eluent was monitored by flourescence detection with excitation at 272 nm and emission at 320 nm. The assay is linear over the range 4-759 ng ml-1. The relative standard deviation at the limit of quantification, 4 ng ml-1, was 7.1%. This method was successfully applied to the determination of U-89968E in rat plasma during pharmacokinetic studies.     

Immunohistochemical detection of bone morphogenetic protein-2 and transforming growth factor beta-1 in tracheopathia osteochondroplastica

The purpose of this study was to develop objective assessment instruments for use in psychomotor skill training and to test the instruments for interobserver reliability. Two checklist style instruments, one for suturing and one for endotracheal intubation, were developed through a process of review of standard texts, consultation with local experts and field testing. Following development they were used by paired examiners in an Objective Structured Clinical Examination (OSCE) setting to test the instruments for interobserver reliability. A total of 88 final year medical students were recruited from the five Ontario medical schools to participate as examinees. The checklists worked well within the practical constraints of a 10 minute OSCE station and demonstrated a high level of interobserver reliability with Kappa scores of 0.65 for the suturing checklist and 0.71 for the intubation checklist Furthermore, the Kappa scores for individual checklist items served to identify items which demonstrated poor interobserver reliability and thus highlighted them for review.     

Opposing effects by glucocorticoid and bone morphogenetic protein-2 in fetal rat bone cell cultures

Glucocorticoid in excess produces bone loss in vivo. Consistent with this, it reduces the stimulatory effect of transforming growth factor beta (TGF-beta) on collagen synthesis in osteoblast-enriched cultures in vitro, where it also suppresses TGF-beta binding to its type I receptors. Analogous studies with bone morphogenetic protein-2 (BMP-2) show directly opposite results. These findings prompted us to assess the effect of glucocorticoid on BMP-2 activity in cultured bone cells, and whether either agent had a dominant influence on TGF-beta binding or function. BMP-2 activity was retained in part in osteoblast-enriched cultures pre-treated or co-treated with cortisol, and was fully evident when glucocorticoid exposure followed BMP-2 treatment. In addition, BMP-2 suppressed the effects of cortisol on TGF-beta activity, on TGF-beta binding, and on gene promoter activity directed by a glucocorticoid sensitive transfection construct. While BMP-2 also alters the function of less-differentiated bone cells, it only minimally prevented cortisol activity in these cultures. Our studies indicate that BMP-2 can oppose certain effects by cortisol on differentiated osteoblasts, and may reveal useful ways to diminish glucocorticoid-dependent bone wasting.     

Histamine potently suppresses human IL-12 and stimulates IL-10 production via H2 receptors

IL-12 and IL-10, respectively, stimulate Th1 and Th2 immune responses. The development of some allergic reactions, infections, and tumors are associated with excessive histamine production and a shift toward Th2 responses. Here we address the possibility that this association is causally linked, at least in part, to modulation of IL-12 and IL-10 production by histamine. We report that histamine dose-dependently inhibited the secretion of human IL-12 (p70) and increased the production of IL-10 in LPS-stimulated whole blood cultures. These effects of histamine were antagonized by cimetidine, an H2 receptor antagonist, but not by selective H1 and H3 receptor blockers, and were mimicked by an H2 receptor agonist. The effects of histamine on IL-12 and IL-10 secretion were independent of endogenous secretion of IL-10 or exogenous addition of IL-12, while Ro 20-1724, a phosphodiesterase inhibitor, potentiated the effects of histamine on IL-12 and IL-10 production, implicating cAMP in its actions. Similar modulatory effects of histamine on IL-12 and IL-10 production, which were reversed by the H2 antagonist cimetidine, were observed in PBMC and isolated monocytes stimulated by Staphylococcus aureus Cowan strain 1 and LPS, respectively. Thus, histamine, via stimulation of H2 receptors on peripheral monocytes and subsequent elevation of cAMP, suppresses IL-12 and stimulates IL-10 secretion, changes that may result in a shift of Th1/Th2 balance toward Th2-dominance. This may represent a novel mechanism by which excessive secretion of histamine potentiates Th2-mediated allergic reactions and contributes to the development of certain infections and tumors normally eliminated by Th1-dependent immune mechanisms.     

Involvement of CD44 in matrix metalloproteinase-2 regulation in human melanoma cells

CD44 is a family of cell-surface-adhesion proteins that are thought to play an important role in cancer invasion and metastasis. However, the specific mechanisms by which CD44 expression modulates invasion or metastasis are not well understood. In the current study, we have demonstrated that treatment of human melanoma cells with a CD44 MAb, F10-44-2, induces up-regulation of matrix metalloproteinase-2 (MMP-2) protein and mRNA. Moreover, treatment of melanoma cells with MAb F10-44-2 enhances their migration through gelatin-coated membranes and invasion through reconstituted basement membranes. Treatment of melanoma cells with several known CD44 ligands, including hyaluronate, extracellular-matrix proteins, and osteopontin, did not induce MMP-2 production. CD44 binding by F10-44-2 MAb results in induction of MMP-2 expression, which is associated with enhanced cell migration and invasion. These findings have several implications for investigations into tumor metastasis, development, and lymphocyte function.     

Recombinant human bone morphogenetic protein (rhBMP) induced heterotopic bone development in vivo and in vitro

In vivo, recombinant human bone morphogenetic protein (rhBMP-2) with deactivated bone matrix as a carrier, implanted in muscle in adult rates, induced development of heterotopic bone, including bone marrow. The volume of bone was proportional to the dose of rhBMP-2 in a range of 0.2-150 microg. In vitro, in response to 50-microgram dose range, subcutis- and brain-derived outgrowths differentiated into loosely woven connective tissues composed of spindle-shaped fibroblasts, adipocytes, and cartilage. Muscle-derived connective tissues cultivated first in culture media supplemented with 50 microgram of rhBMP-2 for 72 hr, then enclosed in a diffusion chamber, and immediately transplanted into a rectus abdominous muscle pouch in an autogenic rat for 28 days, induced cartilage development on the inside and transmembrane hetertoptic bone development including bone marrow on the outside. These experiments are interpreted to show that muscle derived connective tissue cells have the competence of embryonic cells to develop de novo in response to BMP in postfetal life.     

Remodeling of collagen matrix by human tumor cells requires activation and cell surface association of matrix metalloproteinase-2

We assessed the functional significance of tumor cell-associated matrix metalloproteinase (MMP)-2 in extracellular matrix remodeling compared with that of the soluble enzyme by evaluating the contraction of three-dimensional collagen lattices by human glioma U251.3 and fibrosarcoma HT-1080 cell lines. In this model, the constitutive synthesis and activation of the MMP-2 proenzyme were modulated by stable transfections of tumor cells with cDNA encoding membrane type 1-MMP (MT1-MMP). The efficiency of transfected cells in contracting collagen lattices was shown to be dependent on the MT1-MMP-mediated activation of MMP-2 accompanied by cell surface association of activated MMP-2, on the cell-matrix interactions controlled by collagen-specific integrins, and on the integrity of actin and microtubule cytoskeletons. Each one of these mechanisms was essential but was not sufficient by itself in accomplishing gel contraction by MT1-MMP-transfected cells. Both MMP-2 activation and gel contraction by transfected glioma cells were inhibited by tissue inhibitor of metalloproteinase (TIMP)-2 and the recombinant COOH-terminal domain of MMP-2. However, the kinetics and mechanisms of their inhibitory effects were different, because TIMP-2 and the COOH-terminal domain of MMP-2 preferentially inhibited the MT1-MMP-dependent and autocatalytic steps of MMP-2 activation, respectively. By contrast, TIMP-1, an efficient inhibitor of soluble MMP-2 activity, failed to affect gel contraction. In addition, soluble MMP-2 activated by either organomercurials or cells was not able to induce the contraction of collagen lattices when added to transfected cells. Therefore, soluble activated MMP-2, sensitive to TIMP-1 inhibition, does not mediate collagen gel contraction by tumor cells, whereas the activity of cell surface-associated MMP-2 plays a critical role in remodeling of the extracellular matrix in vitro. These mechanisms of functional and spatial regulation of MMP-2 may also be applicable to different aspects of tissue reorganization in vivo, including cell migration and invasion, angiogenesis, and wound healing.     

Ambient pressure stimulates immortalized human aortic endothelial cells to increase DNA synthesis and matrix metalloproteinase 1 (tissue collagenase) production

In the present study, we investigated the effect of ambient pressure on [3H]-thymidine incorporation and on the production of matrix metalloproteinase 1 (tissue collagenase/proMMP-1) using human aortic endothelial cells immortalized with simian virus 40 (SE-1). Incubation of cells at ambient pressures of 50 and 100 mmHg for 24 h slightly increased [3H]-thymidine incorporation when directly compared with normal culture conditions. The amount of [3H]-thymidine incorporated in SE-1 reached a maximum at 150 mmHg, while a further increase in pressure to 200 mmHg decreased incorporation. The same ambient pressure slightly stimulated human aortic intimal smooth muscle cells (SMC) to increase [3H]-thymidine incorporation but not medial SMC. Immunoblot analysis also showed that ambient pressure, ranging from 50 to 200 mmHg, like 12-O-tetradecanoyl-phorbol-13-acetate stimulated SE-1 to produce proMMP-1, an effect not seen with either intimal or medial SMC. The amount of proMMP-1 produced also reached a maximum level at 150 mmHg. We postulate that human endothelial cells are ambient pressure sensitive and that relatively lower ambient pressures play an important role in the growth of endothelial cells, while higher pressures injure endothelial cells, resulting in the initiation of atherosclerosis. This cell line may prove useful in the investigation of both the physiological and pathological roles of blood pressure on endothelial cell function.     

Immunolocalization and messenger RNA expression of bone morphogenetic protein-6 in human benign and malignant prostatic tissue

Skeletal metastases are common in advanced prostate cancer, causing considerable morbidity, and they are usually osteoblastic in nature with no clear explanation for this phenomenon. Bone morphogenetic proteins (BMPs) induce bone formation in vivo, and preliminary work showed a possible association between BMPs and prostatic skeletal metastases; differential expression favors BMP-6 as a potential new marker and mediator of osteosclerotic deposit formation. We investigated BMP-6 mRNA and protein expression by in situ hybridization and immunohistochemistry in malignant and benign prostates from 40 men. BMP-6 mRNA expression was detected exclusively in malignant epithelial cells in 20 of 21 patients (95%) with metastases and in 2 of 11 patients (18%) with localized cancer, and it was absent in 8 benign samples. Immunostaining for BMP-6 was predominantly cytoplasmic and was present in all primary tumors with established metastases and in 4 of 11 (36%) organ-confined cancers. In benign prostatic hyperplasia, basal cells and areas of basal cell hyperplasia were positive for BMP-6 by immunohistochemistry. The results suggest a close association between BMP-6 expression in primary malignant prostatic tissue and skeletal metastases. BMP-6 may be responsible, in part, for the osteoblastic changes in metastatic lesions secondary to prostate cancer.     

Transforming growth factor-beta, osteogenin, and bone morphogenetic protein-2 inhibit intercellular communication and alter cell proliferation in MC3T3-E1 cells

Intercellular communication by gap junctions has been implicated to function in the control of cell growth and differentiation in osseous tissues-processes which are regulated, in part, by peptide growth factors, including transforming growth factor-beta (TGF-beta) and the bone morphogenetic proteins (BMPs). Using the osteoblastic cell line MC3T3-E1, we tested the hypothesis that the effects of TGF-beta and BMPs on cell proliferation may be correlated to changes in intercellular communication. In a series of proliferation assays, MC3T3-E1 cells were cultured in the presence of bone morphogenetic protein-2 (BMP-2) or TGF-beta for up to 48 hr. Proliferation of cells during the linear log phase (days 2 to 4) was assessed by 3H-thymidine (3H-TdR) incorporation. After times ranging from 6 to 48 hr, BMP-2 significantly inhibited uptake of 3H-TdR at doses of 50-800 ng/ml. Similarly, TGF-beta inhibited uptake of 3H-TdR at doses of 2-32 ng/ml. In a separate group of experiments, intercellular communication through gap junctions was demonstrated by cell-cell transfer of the fluorescent tracer, lucifer yellow, after microinjection. One series of experiments showed that the gap junctional intercellular communication (GJIC) of cells, incubated for 48 hr in the presence of the higher dose of osteogenin (OG) (5.0 vs. 0.5 microgram/ml) or higher dose of TGF-beta (2.0 vs. 0.2 ng/ml), was significantly inhibited compared to control. In another series of experiments, time and dose dependent effects of BMP-2 and TGF-beta on GJIC were investigated. In the time course experiments (3, 6, 12, 24, and 48 hr), TGF-beta (2.0 ng/ml) demonstrated a statistically significant effect in inhibiting GJIC as early as 6 hr, while BMP-2 (50 ng/ml) inhibited GJIC after 24 and 48 hr of treatment. The dose-dependent effects of BMP-2 and TGF-beta on cell couplings, determined at 48 hr, showed significant inhibitory effects with BMP-2 at 25 and 50 ng/ml and with TGF-beta at 2 and 4 ng/ml. The cell count results and injection study performed at 12 hr, at a fixed cell density, confirmed that the inhibitory effect was not due to differences in cell density. The 50% effective inhibitory concentrations (EC50) calculated for BMP-2 and TGF-beta at 48 hr, showed no dose correlation between proliferation and GJIC, suggesting that these two events are independent occurrences. Additionally, marked morphological change was observed in the cells treated with TGF-beta. The observation may suggest that TGF-beta may have effects upon cytoskeletal elements in osseous tissues.     

The spatial and temporal immunolocalization of TGF-beta 1 and bone morphogenetic protein-2/-4 in phallic bone formation in inbred Sprague Dawley male rats

Classical and artificial xenodiagnostic techniques made with Dipetalogaster maximus of first stage were performed simultaneously in 57 patients with chronic T. cruzi infection (22 male and 35 female patients, aged 7-80 years). With the exception of two patients with megaoesophagus, all had two previous positive serological reaction and a further test was done at the time of the examination. The patients came from the outpatient department of the university hospital or were resident in Mambaí, Goiás. Of the 57 patients, 24 (42%) had a positive xenodiagnoses. Of a total of 114 tests performed, 36(32%) were positive. Comparing the two xenodiagnostic techniques, no significant advantage was apparent statistically (p = 0.42), but the artificial technique has advantages because the blood is offered for triatomines through a device while in the classical technique, the triatomines suck through the patient's skin.     

Bone morphogenetic protein 2 suppresses the transformed phenotype and restores actin microfilaments of human lung carcinoma A549 cells

We compared the endotracheal intubating conditions after rocuronium, using the "timing principle," with those after succinylcholine. The timing principle entails administration of a single bolus dose of nondepolarizing muscle relaxant, followed by an induction drug at the onset of clinical weakness. Forty-five patients were randomly assigned to three groups. Patients allocated to Groups 1 and 2 received rocuronium 0.6 mg/kg. At the onset of clinical weakness (onset of ptosis), anesthesia was induced with thiopental 4-6 mg/kg; intubation was accomplished after 45 s in Group 1 and after 60 s in Group 2. Patients in Group 3 received vecuronium (0.01 mg/kg) 3 min before the administration of thiopental and succinylcholine 1.5 mg/kg, and their tracheas were intubated 60 s later by a blind anesthesiologist. Intubating conditions were assessed according to a grading scale and were either good (5 patients in Groups 1 and 2, 4 patients in Group 3) or excellent (10 patients in Groups 1 + 2, 11 patients in Group 3) in all patients. Patients were interviewed postoperatively, and all were satisfied with the induction of anesthesia. We conclude that rocuronium 0.6 mg/kg provides good to excellent intubating conditions 45 and 60 s after the induction of anesthesia using the timing principle. Implications: We compared the ease with which a breathing tube could be placed in patients using three techniques. The standard technique (succinylcholine) was compared with two others in which a muscle-relaxing drug (rocuronium) was administered just before the anesthetic drug (so-called timing principle). No difference among the techniques was observed.     

Bone morphogenetic protein-6 is expressed in nonparenchymal liver cells and upregulated by transforming growth factor-beta 1

Efficient selection of preimplantation transgenic embryos by an improved method after pronuclear injection of exogenous DNA is described. The method is based on subjecting DNA extracted from the embryos to restriction enzymes as well as the polymerase chain reaction (PCR). The incorporated procedure included recovery of the digested DNA with glassmilk before PCR, which markedly enhanced the rate of accurate detection of transgenic embryos. When exogenous DNA sequences in the mouse embryos were not integrated into the genome they were digested with both Dpn I and Bal 31, and subsequent PCR analysis generated DNA fragments of the injected DNA sequence in only 1.5% of cases examined. However, DNA extracted from mouse embryos containing the transgene sequences integrated into the genome evaded digestion by both enzymes and yielded transgene-specific PCR products in 68.6% of the embryos tested. When bovine embryos were used, sequences of the endogenous haemoglobin gene used as a control genomic DNA sequence were protected from enzyme digestion (PCR products in 70.5% of the embryos examined); by contrast, the non-integrated injected sequences were almost completely eliminated by the same treatment (PCR products in 1.4% of the embryos examined). It is suggested that this method might be useful for the selection of transgenic embryos before embryo transfer, thereby reducing the number of recipient females required.