Differential effects of retinoic acid and growth factors on osteoblastic markers and CD10/NEP activity in stromal-derived osteoblasts

The effects of retinoic acid (RA) on the expression of osteoblastic-related cell makers was examined. A marrow and osteogenic cell line, MBA-15, was analyzed by Northern blotting for the expression of bone matrix proteins. These cells constituentively express mRNA encoding for procolllagen a₂ (1), osteonectin, osteopontin, biglycan and alkaline phosphatase (ALK-P). Gene expression was unchanged in response to RA triggering for 24hr. Furthermore, cell growth and enzymatic activities of ALK-P and neutral endopeptidase (CD10/NEP) were studied. These parameters were examined in MBA-15 and clonal populations representing different stages of differentiation. The cell's growth rate was unchanged, while ALK-P activity was greatly increased during the culture period under RA treatment in MBA-15 and in the clonal cell lines examined while CD10/NEP activity dispalyed a different pattern. MBA-15.4, a presosteoblast cell ine, exhibited an inhibition in CD10/NEP activity at the beginning of the culture period, reaching basal level with time. This activity was greatly increased over control level in MBA-15.6, a mature stage of osteoblasts. Furthermore, the response of cell lines to various growth factors was tested subsequent to priming the cultures with RA. A synergistic effect was monitored for ALK-P activity in MBA-15.4 and MBA-15.6 cells under rh-bone morphogenic protein (BMP-2) and purified osteogenin (BMP-3), and an antagonist effect was measured when cells were exposed to transforming growth factor β (TGFβ). Contrarily, BMP-2 and BMP-3 inhibited the CD10/NEP activity that had remained unchanged following priming of the cell with RA. Insulin-like growth factor 1 (IGF-1) and basic fibroblast growth factors (bFGF) did not affect either ALK-P nor CD10/NEP activities in both cloned cells. Cellular response to bone-seeking hormone, parathyroid hormone (PTH), and prostaglandin E₂ (PGE₂) was monitored by activation of intracellular cAMP. Treatment with RA caused a dramatic increase in MBA-15.6 cell responses to PTH and PGE₂ but no significant effects could be observed in other clonal lines.     

Phenotypic expression of marrow cells when grown on various substrata

Our aim was to study the role of various extracellular matrices (ECM) on growth and differentiation of marrow stromal cells in vitro. Morphology changes, gene expression, and enzymatic activities were monitored in stromal osteoblastic MBA-15 and adipocytic 14F1.1 cells. These stromal cells were plated on dishes precoated with different substrata, such as matrigel (basement membrane), collagen type I, and endothelial ECM, and compared with cells plated on protein-free dishes. Striking morphological differences were observed when the cells grew on these different substrata. Changes in cell shape and growth also led to differential mRNA expression and enzymatic activities. When MBA-15 cells were plated on collagen, there was a decrease in mRNA for alkaline phosphatase (ALK-P), osteopontin (OP), and osteonectin (ON), and an increase in mRNA for procollagen (I). A differential effect was noted on 14F1.1 cells, the mRNA for ALK-P increased, the expressions of OP and ON lowered, and no expression for procollagen (I) was monitored. MBA-15 cells cultured on matrigel had decreased mRNA for ALK-P and OP, while they had increased ON mRNA expression and remained unchanged for procollagen 1. No change in mRNA expression by 14F1.1 cells was monitored when cultured on matrigel. Functional enzymatic activities of ALK-P markedly decreased in MBA-15 cells cultured on various substrata, and increased or were unchanged in 14F1.1 cells. An additional enzyme, neutral endopeptidase (CD10/NEP), altered differentially in both cell types; this enzymatic activity increased or was unchanged when cells were cultured on these matrices. The results indicate a specific role for different ECM on various stromal cell types and their function. © 1996 Wiley-Liss, Inc.     

Marrow stroma-derived osteogenic clonal cell lines: Putative stages in osteoblastic differentiation

This report documents characterization of five osteogenic cell subpopulations of bone marrow stroma. The clonally derived cell lines were isolated from the parental line MBA-15 known to express osteoblastic-associated features in vitro and to form bone in vivo. The latter, presumably “arrested” at a particular stage along the osteogenic lineage, are useful models to study the processes involved in the differentiation of bone forming cells. The clones differ in their morphology, proliferation rate, quantities and distribution of extracellular matrix proteins, levels of alkaline phosphatase activity and activation of adenylate cyclase by parathyroid hormone and/or prostaglandin E. These properties have been retained during prolonged growth and subculturing through many passages. MBA-15.4 is a presumptive preosteoblast with a fibroblast-like appearance; it proliferates rapidly, synthesizes equal amounts of collagen and noncollagenous proteins, and produces constitutively low levels of alkaline phosphatase. This clone has PGE₂-stimulated adenylate cyclase activity and a very low constitutive response to PTH. On the other hand, MBA-15.6 has a large polygonal morphology with limited proliferative potential, synthesizes twice as much noncollagenous proteins as collagen, has high alkaline phosphatase activity, and responds strongly to PTH. The characteristics of the other clones place them between these two categories. The effects of 10^?7 M dexamethasone or 10^?12–10^?8 M 1,25 dihydroxyvitamin D₃ on growth and differentiation further strengthen the variance between these clones. The different in vitro characteristics of the various clones were directly reflected in their bone formation ability in vivo. When transplanted under the renal capsule, MBA-15.33 formed a thick fibrous tissue, MBA-15.4 formed small foci of bone, and MBA-15.6 formed massive woven bone at the same period of time. © 1993 Wiley-Liss, Inc.     

THE EXPRESSION OF ESTROGEN RECEPTOR AND ESTROGEN EFFECT IN MBA-15 MARROW STROMAL OSTEOBLASTS

MBA-15, a marrow stromal-derived cell line, was shown to express an estrogen receptor. This finding was confirmed byin situhybridization and receptor binding assay. An exposure to estrogen (10^?12–10^?6m ) in a dose response manner resulted in a decrease of cell proliferation as measured by MTT assay. Cell function was measured by enzymatic activities of two osteoblastic markers, CD10/NEP and alkaline phosphatase. These enzymatic activities were elevated following the estrogen treatment. This model enabled direct evaluation of the estrogen effect on stromal osteoblast cells.     

Cultured mouse marrow stromal cell lines. II. Distinct subtypes differing in morphology, collagen types, myelopoietic factors, and leukemic cell growth modulating activities

A series of stromal cell lines were studied for their growth properties, electron microscopic morphology, cytochemical profile, collagen types, production of myelopoietic factors, and modulation of leukemic cell growth. Three cell types were identified in addition to the previously described macrophages (14M and 14M1) and preadipocytes (14F). MBA-1 cells were found to be fibroblasts by their ability to synthesize collagen types I and III, while the cell line MBA-13 shared properties in common with both fibroblasts and endothelial cells (collagen types I, III, IV, V). The third cell type, represented by the stromal cell line MBA-2, produced mainly collagen types IV and V and exhibited junctional complexes between adjacent cells. All of the cell lines tested produced and secreted a macrophage-colony-stimulating factor, CSF-1. MBA-2 and to a lesser extent, MBA-13, produced an additional activity resistant to anti-CSF-1 antiserum. Trypsin extraction of outer surface components from two clones of the MBA-2 cell line (MBA-2.1 and MBA-2.4) yielded high molecular weight factor(s) that specifically inhibited the growth of a plasmacytoma cell line (MPC-11). Such inhibitory activity was not detected in other stromal cell lines. It is possible that this variability in the nature of stromal cell lines represents corresponding diversity of cell types comprising the hematopoietic microenvironment in vivo.     

Human bone marrow osteoprogenitors express estrogen receptor-alpha and bone morphogenetic proteins 2 and 4 mRNA during osteoblastic differentiation.

Understanding the mechanisms that control the proliferation and commitment of human stem cells into cells of the osteogenic lineage for the preservation of skeletal structure is of basic importance in bone physiology. This study examines some aspects of the differentiation in vitro of human bone marrow fibroblastic cells cultured in the absence (basal media) or presence of 1nM dexamethasone and 50 micrograms/ml ascorbate for 6, 10, 14, and 21 days. Northern blot analysis and in situ hybridisation with digoxygenin-labelled riboprobes for Type I collagen, osteocalcin, bone morphogenetic proteins 2 (BMP-2), and 4 (BMP-4) and the estrogen receptor alpha (ERalpha), together with immunocytochemical analysis of ERalpha expression and histochemical staining of alkaline phosphatase was performed. In basal media, alkaline phosphatase activity and collagen expressions were detected at day 6, ERalpha from day 10 and osteocalcin from day 10. In the presence of dexamethasone and ascorbate, cell proliferation and alkaline phosphatase were markedly stimulated over 10 to 14 days with a dramatic increase in the temporal expression of Type I collagen, ERalpha, and osteocalcin mRNAs in these cultures. Northern blot analysis showed cells cultured in basal media, expressed the highest levels of the mRNA for each marker protein at day 14, whereas in the presence of ascorbate and dexamethasone, the highest levels for alkaline phosphatase, ERalpha, osteocalcin, BMP-2, and BMP-4 were observed at day 21. ERalpha, BMP-2, and BMP-4 expression were found to correlate temporally with induction of the osteoblast phenotype as determined by alkaline phosphatase, collagen, and osteocalcin expression. These results give additional information on the development of the osteoblast phenotype from early fibroblastic stem cells and on the biological factors involved in this process. These studies suggest a role for estrogen and BMP-2 and -4 in the differentiation of osteoprogenitor cells.     

Direct inhibition of neutral endopeptidase in vasopeptidase inhibitor-mediated amelioration of cardiac remodeling in rats with chronic heart failure

Vasopeptidase inhibitors possess dual inhibitory actions on neutral endopeptidase (NEP) and angiotensin-converting enzyme (ACE) and have beneficial effects on cardiac remodeling. However, the contribution of NEP inhibition to their effects is not yet fully understood. To address the role of cardiac NEP inhibition in the anti-remodeling effects of a vasopeptidase inhibitor, we examined the effects of omapatrilat on the development of cardiac remodeling in rats with left coronary artery ligation (CAL) and those on collagen synthesis in cultured fibroblast cells. In vivo treatment with omapatrilat (30 mg/kg/day for 5 weeks) inhibited cardiac NEP activity in rats with CAL, which was associated with a suppression of both cardiac hypertrophy and collagen deposition. In cultured cardiac fibroblasts, omapatrilat (10^–7~10^–5 M) inhibited NEP activity and augmented the ANP-induced decrease in [³H]-proline incorporation. ONO-BB, an active metabolite of the NEP selective inhibitor ONO-9902, also augmented the ANP-induced response, whereas captopril, an ACE inhibitor, did not. The angiotensin I-induced increase in [³H]-proline incorporation was prevented by omapatrilat and captopril, but not by ONO-BB. The results suggest that vasopeptidase inhibitor suppressed cardiac remodeling in the setting of chronic heart failure, possibly acting through the direct inhibition of cardiac NEP. Vasopeptidase inhibitors may have therapeutic advantages over the classical ACE and NEP inhibitors alone with respect to the regression of cardiac fibrosis.     

Dexamethasone Enhances Osteogenic Differentiation of Bone Marrow- and Muscle-Derived Stromal Cells and Augments Ectopic Bone Formation Induced by Bone Morphogenetic Protein-2

We evaluated whether dexamethasone augments the osteogenic capability of bone marrow-derived stromal cells (BMSCs) and muscle tissue-derived stromal cells (MuSCs), both of which are thought to contribute to ectopic bone formation induced by bone morphogenetic protein-2 (BMP-2), and determined the underlying mechanisms. Rat BMSCs and MuSCs were cultured in growth media with or without 10-7 M dexamethasone and then differentiated under osteogenic conditions with dexamethasone and BMP-2. The effects of dexamethasone on cell proliferation and osteogenic differentiation, and also on ectopic bone formation induced by BMP-2, were analyzed. Dexamethasone affected not only the proliferation rate but also the subpopulation composition of BMSCs and MuSCs, and subsequently augmented their osteogenic capacity during osteogenic differentiation. During osteogenic induction by BMP-2, dexamethasone also markedly affected cell proliferation in both BMSCs and MuSCs. In an in vivo ectopic bone formation model, bone formation in muscle-implanted scaffolds containing dexamethasone and BMP-2 was more than two fold higher than that in scaffolds containing BMP-2 alone. Our results suggest that dexamethasone potently enhances the osteogenic capability of BMP-2 and may thus decrease the quantity of BMP-2 required for clinical application, thereby reducing the complications caused by excessive doses of BMP-2.Highlights: 1. Dexamethasone induced selective proliferation of bone marrow- and muscle-derived cells with higher differentiation potential. 2. Dexamethasone enhanced the osteogenic capability of bone marrow- and muscle-derived cells by altering the subpopulation composition. 3. Dexamethasone augmented ectopic bone formation induced by bone morphogenetic protein-2.     

Bone marrow-derived stromal cell line expressing osteoblastic phenotype in vitro and osteogenic capacity in vivo

Marrow stroma has been shown to have osteogenic potential. Here we report the characterization of a unique stromal cell line derived from mouse bone marrow (MBA-15), which expresses osteoblastic phenotype in vitro and forms bone in vivo. More than 70% of cells in culture were histochemically positive for alkaline phosphatase. The enzyme levels were enhanced threefold when cultures were treated with dexamethasone. Gel electrophoresis of [3H]-proline-labeled cultures showed that MBA-15 cells produced only type I collagen. These cells were responsive to PTH, as indicated by a 50-fold increase in intracellular cAMP. Prostaglandin E2, but not calcitonin, stimulated cAMP up to 70-fold. When cultures were grown to confluence and fed daily with ascorbic acid and beta-glycerophosphate, the cells formed a Von Kossa positive, thick extracellular matrix, shown to contain hydroxyapatite crystals. MBA-15 cells produced mineralized bone when implanted in diffusion chambers. These results indicate that the MBA-15 cell line possesses osteoblastic features in vitro and osteogenic capacity in vivo.     

Extracellular matrix modulates mesangial cell apoptosis and mRNA expression of cathepsin-B and tissue transglutaminase

Mesangial matrix is a dynamic structure which modulates mesangial cell function. Since accumulation of matrix precedes the development of focal glomerulosclerosis, we studied the effect of different matrices on mesangial cell (MC) apoptosis. Suspended mesangial cells became apoptotic in a time dependent manner. Collagen type III did not modulate MC apoptosis when compared to cells grown on plastic. MCs grown on Matrigel, collagen type I and IV showed an increased number of apoptotic cells when compared to MCs grown on plastic. DNA end-labeling further confirmed these observations. MCs grown on Matrigel showed enhanced (P < 0.05) mRNA expression for tissue transglutaminase (TTG) and cathepsin-B. Mesangial cells grown on Matrigel also showed enhanced expression of superoxide dismutase (SOD). We conclude that mesangial cells require attachment to the matrix for their survival and alteration of the quality of matrix modulates mesangial cell apoptosis. J. Cell. Biochem. 68:22–30, 1998. © 1998 Wiley-Liss, Inc.     

Gluconeogenesis in rat liver parenchymal cells in primary culture: Permissive effect of the glucocorticoids on glucagon stimulation of gluconeogenesis

Primary cultures of parenchymal cells isolated from adult rat liver by a collagenase perfusion procedure and maintained as a monolayer in a serum-free culture medium were used to study glucoeogenesis and the role that the glucocorticoids play in the control of this pathway. These cells carried out gluconeogenesis from three-carbon precursors (alanine and lactate) in response to glucagon and dexamethasone added alone or in combination. Maximum glucose production was observed with cells pretreated for several hours with dexamethasone and glucagon prior to addition of substrate and glucagon (8- to 12-fold increase over basal glucose production). Half-maximum stimulation of gluconeogenesis was seen with 3.6 × 10^?10 M glucagon and 3.6 × 10^?8 M dexamethasone. Maximum stimulation was oberved with 10^?7 M glucagon and 10^?6 M dexamethasone. The length of time of dexamethasone pretreatment was found to be important in demonstrating the effect of glucocorticoids on glucagon-stimulated gluconeogenesis. Treeatment of cells with dexamethasone for 2 hours did not result in an increase in glucose production over identical experimental conditions in the absence of dexamethasone, wherease pretreatment for 5 hours (1.2-fold increase) or 15 hours (1.7-fold increase) did result in an increase in glucose production. The results establish that the adult rat liver parenchymal cells in primary culture are a valid model system to study hepatic gluconeogenesis. In addition, we have established directly that the glucocorticoids amplify the glucagon stimulation of gluconeogenesis.     

Identification of neuropeptide-degrading enzymes in the pancreas.

Neutral endopeptidase (NEP) and aminopeptidase M (APM) were identified in the pancreas by enzymatic assays and Western blotting. The NEP activity, assessed by the phosphoramidon- and DL-thiorphan-inhibitable degradation of glutaryl-Ala-Ala-Phe-4-methoxy-2-naphthylamine, was 28.8 pmol/h/micrograms of pancreatic membrane protein and 124 pmol/h/10(6) pancreatic acinar cells. The APM enzymatic activity, assessed by the actinonin- and amastatin-inhibitable degradation of Ala-4-methoxy-2-naphthylamine, was 633 pmol/h/micrograms pancreatic membrane protein and 17.4 nmol/h/10(6) pancreatic acinar cells. Proteins corresponding to NEP (95 kDa) and APM (140 kDa) were identified in membranes by Western blotting. Both NEP and APM on acinar cells may degrade neuropeptides and regulate their effects on exocrine secretion.     

The effect of various substrates on cell attachment and differentiation of 3T3-F442A preadipocytes

The influence of extracellular matrix (Matrigel), collagen, and polylysine substrates on cell attachment and differentiation in 3T3-F442A preadipocytes was investigated. In comparison to an uncoated-polystyrene substrate, a concentrated Matrigel substrate (100 microg/cm2) markedly increased intracellular lipid level by about 30%, whereas a lower density Matrigel (10 microg/cm2) accelerated the differentiation rate but did not increase the amount of lipid 21 days after addition of adipogenic factors. Preadipocytes on the collagen surface differentiated less extensively than cells on the polystyrene. Polylysine did not effectively support attachment for either differentiated or undifferentiated cells. These results suggest that Matrigel provides the most suitable environment for both cell adhesion and differentiation for 3T3-F442A cells. This is in contrast to a previous report that extracellular matrix (from corneal endothelial cells) was detrimental to differentiation of 3T3-F442A cells.     

Proliferation and differentiation of mouse uterine epithelial cells in primary serum-free culture: Estradiol-17β suppresses uterine epithelial proliferation cultured on a basement membrane-like substratum

Summary The effects of different substrata and estradiol-17β(E₂) on proliferation and differentiation of mouse uterine epithelial cells was examined in a serum-free primary culture system. When cultured on rat-tail collagen gels, the epithelial cells rapidly increased in number to form a simple squamous cell layer that exhibited a relatively undifferentiated state (a few short microvilli, no secretory granules, and poorly developed endoplasmic reticulum). Addition of E₂ into the culture medium did not affect the proliferation of epithelial cells on collagen gel. Uterine epithelial cells grown on a reconstituted basement membrane-like substratum (Matrigel) formed a simple columnar/cuboidal cell layer exhibiting fully developed characteristics (many long microvilli, many secretory granules, and fully developed endoplasmic reticulum). Examination of epithelial proliferation by counting substratum-attached cell number revealed only a slow increase in cell growth on Matrigel, and E₂ did not significantly affect it. However, measurement of proliferating cells by labeling cells with 5-bromo-2′-deoxyuridine revealed that cells on Matrigel were replicating and that E₂ (10⁻⁷ to 10⁻¹¹ M) actually significantly suppressed epithelial proliferation. However, there was not an effect of E₂ on total cell number, indicating that the cells in control medium replicate faster and detach more readily from the substratum than those in E₂-supplemented medium on Matrigel. Thus, it is probable that E₂ significantly reduces the rate of cell detachment from the substratum, which may mimic the in vivo condition where significant decrease in apoptosis or cell death is induced by E₂.     

A novel composite scaffold for cardiac tissue engineering

Summary One approach to the engineering of functional cardiac tissue for basic studies and potential clinical use involves bioreactor cultivation of dissociated cells on a biomaterial scaffold. Our objective was to develop a scaffold that is (1) highly porous with large intereconnected pores (to facilitate mass transport), (2) hydrophilic (to enhance cell attachment), (3) structurally stable (to withstand the shearing forces during bioreactor cultivation), (4) degradable (to provide ultimate biocompatibility of the tissue graft), and (5) elastic (to enable transmission of contractile forces). The scaffold of choice was made as a composite of poly(Dl-lactide-co-caprolactone), poly(Dl-lactide-co-glycolide) (PLGA), and type I collagen, with open interconnected pores and the average void volume of 80±5%. Neonatal rat heart cells suspended in Matrigel were seeded into the scaffold at a physiologically high density (1.35×10⁸ cells/cm³) and cultivated for 8 d in cartridges perfused with culture medium or in orbitally mixed dishes (25 rpm); collagen sponge (Ultrafoam^⋆m) and PLGA sponge served as controls. Construct cellularity, presence of cardiac markers, and contractile properties were markedly improved in composite scaffolds as compared with both controls.     

Bone morphogenetic protein-2 stimulates alkaline phosphatase activity and collagen synthesis in cultured osteoblastic cells, MC3T3-E1

The activities of three bone morphogenetic proteins (BMPs), BMP-1, BMP-2 and BMP-3, on alkaline phosphatase activity, collagen synthesis and DNA synthesis were studied in cultured osteoblastic cells, MC3T3-E1. Treatment of cells with BMP-2 for 48 h induces an increase in cellular alkaline phosphatase activity. This stimulatory effect is evident at a concentration as low as 20 ng/ml of BMP-2 and becomes greater with increasing doses of BMP-2. The BMP-2-induced increase in alkaline phosphatase activity is enhanced by the presence of beta-estradiol, dexamethasone or 1 alpha, 25(OH)2D3. BMP-2 and BMP-3 slightly but significantly stimulate collagen synthesis. None of the BMPs stimulates DNA synthesis in MC3T3-E1 cells at doses tested. These results indicate that BMPs act directly on osteoblastic cells and stimulate the expression of the osteoblastic phenotypes.     

Osteogenic inhibition by rat periodontal ligament cells: modulation of bone morphogenic protein-7 activity in vivo

Periodontal ligament width is precisely maintained throughout the lifetime of adult mammals but the biological mechanisms that inhibit ingrowth of bone into this soft connective tissue are unknown. As bone morphogenic proteins strongly stimulate osteogenesis and can induce ectopic bone formation in vivo, we tested the hypothesis that topical application of this powerful osteogenic agent will overwhelm the osteogenic inhibitory mechanisms of periodontal ligament cells and induce ankylosis. Wounds through the alveolar bone and periodontal ligament were created in 45 male Wistar rats. Defects were filled with either a collagen implant or collagen plus bone morphogenic protein (BMP-7), or were left unfilled (controls). Three animals per time period were killed on days 2, 5, 10, 21 and 60 after surgery for each wound type. Cellular proliferation and clonal growth in periodontal tissues were assessed by ³H-thymidine labeling 1 h before death, followed by radioautography. Cellular differentiation of soft and mineralizing connective tissue cell populations was determined by immunohistochemical staining of α-smooth muscle actin, osteopontin and bone sialoprotein. In regenerating periodontium, BMP-7 induced abundant bone formation by 21 days (2.5-fold greater than controls or collagen implant only; P<0.001), but by day 60 the volume of the newly formed bone had returned to baseline levels and was similar for all groups. Independent of the type of treatment, periodontal ligament width was unchanged throughout the experimental period (P>0.05). Animals treated with BMP-7 implants showed greatly increased cellular proliferation in the periodontal ligament adjacent to the wound site and in the regenerating alveolar bone at days 5 and 10 after wounding compared to the other treatment groups (P<0.005). Animals in the BMP-7 group exhibited similar spatial and temporal staining patterns for α-smooth muscle actin, osteopontin and bone sialoprotein as controls. Collectively, these data show that BMP-7 promoted the proliferation of precursor cells in the periodontal ligament but did not induce osteogenic differentiation in this compartment. Consequently a powerful osteogenic stimulus like BMP-7 cannot significantly perturb the mechanisms that regulate periodontal ligament width and maintain periodontal homeostasis. Received: 2 March 1998 / Accepted: 16 June 1998     

The antagonistic effect of dexamethasone and insulin on alpha-fetoprotein secretion by cultured H4-II-E-C3 cells derived from the Reuber H-35 hepatoma.

Non-confluent monolayers of H4-II-E-C3 cells were maintained in serum-free media. Dexamethasone alone (5 × 10^?7M) stimulated α-fetoprotein secretion 2- to 4-fold while insulin alone (8.7 × 10^?8M) inhibited α-fetoprotein secretion by 20%. When dexamethasone (5 × 10^?7 to 5 × 10^?9M) and insulin (8.7 × 10^?8 to 8.7 × 10^?11M) were added simultaneously, insulin diminished the stimulatory effect of dexamethasone. When α-fetoprotein secretion was elevated by dexamethasone and the medium was replaced by media containing either insulin or no hormones, the rate of α-fetoprotein secretion diminished more rapidly with the insulin-supplemented medium. Alone or in combination, insulin and dexamethasone had little effect on albumin secretion.     

Dexamethasone counteracts the anti‐osteoclastic,but not the anti‐leukemic,activity of TNF‐related apoptosis inducing ligand (TRAIL)

We have analyzed the effect of the synthetic glucocorticoid dexamethasone, used alone or in combination with recombinant TRAIL, on in vitro osteoclastic differentiation of peripheral blood‐derived macrophages cultured in the presence of macrophage‐colony stimulating factor (M‐CSF) + RANKL for 12–14 days. Dexamethasone exhibited different effects based on the concentration used. Indeed, while at 10^?7 M dexamethasone reduced the number of mature osteoclasts, at 10^?8 M showed no significant effects and at 10^?9 M significantly increased the number of mature osteoclasts, with respect to cells cultured with only M‐CSF + RANKL. On the other hand, the addition in culture of recombinant TRAIL inhibited the output of mature osteoclasts induced by M‐CSF + RANKL. However, the presence of dexamethasone (10^?8 or 10^?9 M) into the culture medium significantly counteracted the anti‐osteoclastic activity of TRAIL. In order to ascertain whether dexamethasone, might also interfere with the anti‐leukemic activity of TRAIL, the degree of apoptosis induced by TRAIL was evaluated in several myeloid (OCI, MOLM, HL‐60) and lymphoid (SKW6.4, MAVER, BJAB) leukemic cell lines. The levels of TRAIL‐triggered apoptosis were not significantly different between leukemic cells cultured in the absence or presence of dexamethasone. Concerning the molecular mechanism mediating the dexamethasone‐suppression of the TRAIL activity in pre‐osteoclasts, but not in leukemic cells, we found that dexamethasone induced a significant down‐regulation of the surface levels of TRAIL‐R2 in cells of the osteoclastic lineage but not in leukemic cells. The ability of dexamethasone to counteract the TRAIL pathway envisions a novel mechanism mediating the pro‐osteoclastic activity of dexamethasone in vivo. J. Cell. Physiol. 222: 357–364, 2010. © 2009 Wiley‐Liss, Inc.