Aline Delalandre

Glucocorticoid receptor regulates expression of L-selectin and CD11/CD18 on human neutrophils

BACKGROUND Recent studies have raised the hypothesis that glucocorticoids could diminish the ability of endothelial cells to direct leukocyte traffic into inflamed tissues by inhibiting expression of the adhesion molecules endothelial-leukocyte adhesion molecule-1 and intercellular adhesion molecule-1. The aim of the present study was to investigate whether glucocorticoids also regulate the expression of L-selectin and CD11/CD18 integrins on human neutrophil granulocytes. METHODS AND RESULTS Incubation of human whole blood with platelet-activating factor (PAF, 1 mumol/L) evoked downregulation of L-selectin and upregulation of CD11/CD18 adhesion receptors on neutrophils as measured by flow cytometry. While dexamethasone (0.1 nmol/L to 100 mumol/L) did not affect expression of adhesion molecules on resting neutrophils, it attenuated the PAF-induced changes in L-selectin and CD18 expression in a time- and concentration-dependent fashion with IC50 values of 31 and 13 nmol/L, respectively. These effects of dexamethasone were completely aborted by RU-486 (10 mumol/L), which blocks transcriptional activation of the glucocorticoid receptor, and by the protein synthesis inhibitor cycloheximide (35.5 mumol/L). Dexamethasone, up to a concentration of 1 mumol/L, neither affected significantly the release of granule enzymes nor interfered with PAF binding to its membrane receptors. CONCLUSIONS Our results show that glucocorticoids at clinically relevant concentrations exert specific actions on expression of adhesion molecules on activated neutrophils, which are mediated through ligation of glucocorticoid receptors and induction of protein synthesis, and suggest a novel mechanism by which anti-inflammatory corticosteroids may inhibit leukocyte accumulation.

Local leptin production in osteoarthritis subchondral osteoblasts may be responsible for their abnormal phenotypic expression

IntroductionLeptin is a peptide hormone with a role in bone metabolism and rheumatic diseases. The subchondral bone tissue plays a prominent role in the pathophysiology of osteoarthritis (OA), related to abnormal osteoblast (Ob) differentiation. Although leptin promotes the differentiation of Ob under normal conditions, a role for leptin in OA Ob has not been demonstrated. Here we determined if endogenous leptin produced by OA Ob could be responsible for the expression of the abnormal phenotypic biomarkers observed in OA Ob.MethodsWe prepared primary normal and OA Ob from subchondral bone of tibial plateaus removed for knee surgery of OA patients or at autopsy. We determined the production of leptin and of the long, biologically active, leptin receptors (OB-Rb) using reverse transcriptase-polymerase chain reaction, ELISA and Western blot analysis. We determined the effect of leptin on cell proliferation by BrdU incorporation and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, and we determined by Western blot analysis phospho 42/44 MAPK (p42/44 Erk1/2) and phospho p38 levels. We then determined the effect of the addition of exogenous leptin, leptin receptor antagonists, inhibitors of leptin signaling or siRNA techniques on the phenotypic features of OA Ob. Phenotypic features of Ob were determined by measuring alkaline phosphatase activity (ALP), osteocalcin release (OC), collagen type 1 production (CICP) and of Transforming Growth Factor-β1 (TGF-β1).ResultsLeptin expression was increased approximately five-fold and protein levels approximately two-fold in OA Ob compared to normal. Leptin stimulated its own expression and the expression of OB-Rb in OA Ob. Leptin dose-dependently stimulated cell proliferation of OA Ob and also increased phosphorylated p42/44 Erk1/2 and p38 levels. Inactivating antibodies against leptin reduced ALP, OC, CICP and TGF-β1 levels in OA Ob. Tyrphostin (AG490) and piceatannol (Pce), inhibitors of leptin signaling, reproduced this effect. Inhibition of endogenous leptin levels using siRNA for leptin or inhibiting leptin signaling using siRNA for OB-Rb expression both reduced ALP and OC about 60%. Exogenous leptin addition stimulated ALP, yet this failed to further increase OC or CICP.ConclusionsThese results suggest that abnormal production of leptin by OA Ob could be responsible, in part, for the elevated levels of ALP, OC, collagen type 1 and TGF-β1 observed in these cells compared to normal. Leptin also stimulated cell proliferation, and Erk 1/2 and p38 signaling. Taken together, these data suggest leptin could contribute to abnormal osteoblast function in OA.

Altered mineralization of human osteoarthritic osteoblasts is attributable to abnormal type I collagen production.

OBJECTIVE Bone tissue in osteoarthritis (OA) is composed of abundant undermineralized osteoid matrix. The aim of this study was to investigate the mechanisms responsible for this abnormal matrix, using in vitro OA subchondral osteoblasts. METHODS Primary normal and OA osteoblasts were prepared from tibial plateaus. Phenotype was determined by alkaline phosphatase activity, and osteocalcin, osteopontin, prostaglandin E2 (PGE2), and transforming growth factor beta1 (TGFbeta1) were assessed by enzyme-linked immunosorbent assay. Expression of COL1A1 and COL1A2 was determined by real-time polymerase chain reaction. The production of type I collagen was determined by the release of its C-terminal propeptide and Western blot analysis. In vitro mineralization was evaluated by alizarin red staining. Inhibition of TGFbeta1 expression was performed using a small interfering RNA technique. RESULTS Mineralization of OA osteoblasts was reduced compared with mineralization of normal osteoblasts, even in the presence of bone morphogenetic protein 2 (BMP-2). Alkaline phosphatase and osteocalcin levels were elevated in OA osteoblasts compared with normal osteoblasts, whereas osteopontin levels were similar. The COL1A1-to-COL1A2 messenger RNA ratio was 3-fold higher in OA osteoblasts compared with normal osteoblasts, and the production of collagen by OA osteoblasts was increased. Because TGFbeta1 inhibits BMP-2-dependent mineralization, and because TGFbeta1 levels are approximately 4-fold higher in OA osteoblasts than in normal osteoblasts, inhibiting TGFbeta1 levels in OA osteoblasts corrected the abnormal COL1A1-to-COL1A2 ratio and increased alizarin red staining. CONCLUSION Elevated TGFbeta1 levels in OA osteoblasts are responsible, in part, for the abnormal ratio of COL1A1 to COL1A2 and for the abnormal production of mature type I collagen. This abnormal COL1A1-to-COL1A2 ratio generates a matrix that blunts mineralization in OA osteoblasts.

Dual Role for Nitric Oxide in the Regulation of Plasma Volume and Albumin Escape During Endotoxin Shock in Conscious Rats

To assess the role of nitric oxide (NO) produced by the constitutive (cNOS) and inducible NO synthase (iNOS) in the regulation of vascular functions, we compared the effects of aminoguanidine, a relatively selective inhibitor of iNOS, and NG-nitro-L-arginine methyl ester (L-NAME), a nonselective NOS inhibitor on blood pressure, plasma volume, and albumin escape during the early and delayed phases of endotoxin shock in conscious, chronically catheterized rats. Red blood cell volume and plasma volume were determined by using chromium-51-tagged erythrocytes and iodine-125-labeled albumin, respectively. Injection of lipopolysaccharide (LPS) 10 mg/kg i.v. resulted in a fall in blood pressure, hemoconcentration, and increased total-body albumin escape, which is reflected by a 25% reduction in plasma volume. When LPS was injected into animals pretreated with L-NAME (7.4 mumol/kg i.v. 15 minutes before LPS), losses in plasma volume and albumin escape were significantly greater than in rats that received LPS alone, despite that L-NAME attenuated the hypotensive action of LPS. Aminoguanidine pretreatment (162 mumol/kg) had no effect on the early responses to LPS, whereas it was as potent as L-NAME in reversing hypotension when injected 70 minutes after LPS. Aminoguanidine treatment also prevented further losses in plasma volume and markedly attenuated total-body and organ albumin escape rates elicited by LPS. L-NAME produced only a slight attenuation of LPS-induced losses in plasma volume and albumin escape in most organs studied, whereas it potentiated albumin extravasation in the lung. These results demonstrate that inhibition of cNOS potentiates, whereas inhibition of iNOS markedly attenuates, losses in plasma volume and albumin escape elicited by LPS, and suggest that selective inhibitors of iNOS may be more effective than nonselective inhibitors of all forms of NOS in the therapy of septic shock.

Elevated Dickkopf-2 levels contribute to the abnormal phenotype of human osteoarthritic osteoblasts.

The Wnt signaling pathway is crucial for osteogenesis and regulates terminal osteoblast differentiation. Although osteoarthritic (OA) osteoblasts show an abnormal phenotype and poor in vitro mineralization, the mechanism leading to this situation still remains unknow. Recent evidence indicates that Wnt signaling may be altered in OA osteoblasts. In this study we determined whether an alteration of the Wnt/β‐catenin signaling pathway is responsible for the abnormal phenotype of OA osteoblasts. Expression of the Wnt signaling antagonist Dickkopf‐1 (DKK1) was similar in normal and OA osteoblasts, whereas DKK2 expression was higher in OA osteoblasts than in normal osteoblasts. OA osteoblasts showed a decrease of Wnt3a‐dependent Wnt/β‐catenin signaling, measured by the TOPflash reporter assay and by Western blot analysis, compared with normal osteoblasts. Correcting DKK2 levels in OA osteoblasts by siRNA techniques enhanced Wnt/β‐catenin signaling. Elevated DKK2 levels could be explained by elevated transforming growth factor β1 (TGF‐β1) in OA osteoblasts, and exogenous TGF‐β1 increased DKK2 expression in normal osteoblasts, whereas ablating TGF‐β1 expression in OA osteoblasts reduced DKK2 expression. Inhibiting TGF‐β1 or DKK2 expression corrected the abnormal phenotype of OA osteoblasts. In vitro mineralization of OA osteoblasts also was increased by DKK2 siRNA. We conclude that elevated TGF‐β1 levels in OA osteoblasts can stimulate DKK2 expression, which, in turn, is responsible, at least in part, for their abnormal phenotype.

The shunt from the cyclooxygenase to lipoxygenase pathway in human osteoarthritic subchondral osteoblasts is linked with a variable expression of the 5-lipoxygenase-activating protein

Osteoarthritis (OA) is characterized by articular cartilage degradation and hypertrophic bone changes with osteophyte formation and abnormal bone remodeling. Two groups of OA patients were identified via the production of variable and opposite levels of prostaglandin E2 (PGE2) or leukotriene B4 (LTB4) by subchondral osteoblasts, PGE2 levels discriminating between low and high subgroups. We studied whether the expression of 5-lipoxygenase (5-LO) or 5-LO-activating protein (FLAP) is responsible for the shunt from prostaglandins to leukotrienes. FLAP mRNA levels varied in low and high OA groups compared with normal, whereas mRNA levels of 5-LO were similar in all osteoblasts. Selective inhibition of cyclooxygenase-2 (COX-2) with NS-398-stimulated FLAP expression in the high OA osteoblasts subgroup, whereas it was without effect in the low OA osteoblasts subgroup. The addition of PGE2 to the low OA osteoblasts subgroup decreased FLAP expression but failed to affect it in the high OA osteoblasts subgroup. LTB4 levels in OA osteoblasts were stimulated about twofold by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) plus transforming growth factor-β (TGF-β), a situation corresponding to their effect on FLAP mRNA levels. Treatments with 1,25(OH)2D3 and TGF-β also modulated PGE2 production. TGF-β stimulated PGE2 production in both OA osteoblast groups, whereas 1,25(OH)2D3 alone had a limited effect but decreased the effect of TGF-β in the low OA osteoblasts subgroup. This modulation of PGE2 production was mirrored by the synthesis of COX-2. IL-18 levels were only slightly increased in a subgroup of OA osteoblasts compared with normal; however, no relationship was observed overall between IL-18 and PGE2 levels in normal and OA osteoblasts. These results suggest that the shunt from the production of PGE2 to LTB4 is through regulation of the expression of FLAP, not 5-LO, in OA osteoblasts. The expression of FLAP in OA osteoblasts is also modulated differently by 1,25(OH)2D3 and TGF-β depending on their endogenous low and high PGE2 levels.

Low sirtuin 1 levels in human osteoarthritis subchondral osteoblasts lead to abnormal sclerostin expression which decreases Wnt/β-catenin activity.

INTRODUCTION Wnt/β-catenin (cWnt) signaling plays a key role in osteogenesis by promoting the differentiation and mineralization of osteoblasts, activities altered in human osteoarthritic subchondral osteoblast (OA Ob). Sclerostin (SOST) has been shown to alter cWnt signaling. Sirtuin 1 (SIRT1) acts as a novel bone regulator and represses SOST levels in Ob. However the role of SIRT1 and SOST in OA Ob remains unknown. Herein, we explored the role played by SIRT1 and SOST on the abnormal mineralization and cWnt signaling in OA Ob. METHODS Primary human normal and OA Ob were prepared from tibial plateaus. SOST levels were evaluated by immunohistochemistry, the expression and production of genes by qRT-PCR and WB analysis. Their inhibitions were performed using siRNA. cWnt signaling was measured by the TOPflash TCF/lef luciferase reporter assay. Mineralization was determined by alizarin red staining. RESULTS SOST levels were significantly increased in OA Ob compared to normal and were linked with elevated TGF-β1 levels in these cells. SIRT1 expression was significantly reduced in OA Ob compared to normal yet not modified by TGF-β1. Specific inhibition of SIRT1 increased TGF-β1 and SOST expressions in OA Ob, while stimulating SIRT1 activity with β-Nicotinamide mononucleotide reduced the expression of TGF-β1 and SOST, and increased mineralization in OA Ob. Resveratrol also reduced SOST expression in OA Ob. Reduced cWnt signaling, β-catenin levels, and mineralization in OA Ob were all corrected via reducing SOST expression. CONCLUSION These data indicate that high level of SOST is responsible, in part, for the reduced cWnt and mineralization of human OA Ob, which in turn is linked with abnormal SIRT1 levels in these pathological cells.

Hyaluronic acid reverses the abnormal synthetic activity of human osteoarthritic subchondral bone osteoblasts

The underlying mechanisms responsible for both cartilage loss and subchondral bone changes in osteoarthritis (OA) remain unknown. It is becoming recognized that the extracellular matrix influences the metabolism of cells both in vivo and in vitro and can modify their responses to external stimuli. Indeed, the glycosaminoglycan/proteoglycan matrix is of major importance for the proliferation and/or differentiation of a number of cells. Here, we determined the potential role of hyaluronic acid (HA) of increasing molecular weight (MW) to alter the expression of metabolic markers and cytokine production by human osteoarthritic (OA) subchondral osteoblasts (Ob). Both 1,25(OH)(2)D(3)-induced alkaline phosphatase activity (ALPase) and osteocalcin release were increased in OA Ob when compared to normal. HA reduced osteocalcin release in OA Ob at MW of 300 and above, whereas HA failed to significantly modify ALPase. Parathyroid hormone (PTH) stimulated cyclic AMP (cAMP) formation by OA Ob. HA had a biphasic effect on this PTH-dependent activity, totally inhibiting cAMP formation at MW of 300 and 800. HA of increasing MW progressively reduced the levels of Prostaglandin E(2) (PGE(2)) and interleukin-6 (IL-6) produced by OA Ob. Interestingly, urokinase plasminogen activator (uPA) and and PA inhibitor-1 (PAI-1) levels were not significantly affected by HA of increasing MW; however, the PAI-1 to uPA ratio showed a slight, yet nonsignificant increase. Surprisingly, uPA activity was increased in OA Ob under the same conditions. Last, HA had no effect on the production of insulin-like growth factor-1 by these cells. Our data suggest that high MW HA can modify cellular parameters in OA Ob that are increased when compared to normal. The effect of HA on inflammatory mediators, such as PGE(2) and IL-6, and on uPA activity is more striking at higher MW as well. Taken together, these results could suggest that HA of increasing MW has positive effects on OA Ob by modifying their biological synthetic capacities.

R‐spondins are newly recognized players in osteoarthritis that regulate Wnt signaling in osteoblasts

OBJECTIVE Clinical and in vitro studies suggest that altered osteogenesis or bone remodeling is involved in the progression and/or onset of osteoarthritis (OA). Wnt signaling plays a key role in osteogenesis via the canonical Wnt/β-catenin signaling pathway. Two of the R-spondins, Rspo-1 and Rspo-2, a family of 4 proteins unrelated to other Wnt ligands that act as Wnt agonists, are present in bone tissues. The purpose of this study was to investigate the potential role of Rspo-1 and Rspo-2 in OA osteoblasts. METHODS Primary human normal and OA osteoblasts were prepared from tibial plateaus. The expression of Rspo-1 and Rspo-2 was evaluated by quantitative reverse transcription-polymerase chain reaction analysis. Western blot analysis was used to determine Rspo-2, β-catenin, and phospho-β-catenin levels. Wnt/β-catenin signaling was evaluated using the TOPflash T cell factor (TCF)/lymphoid enhancer factor (LEF) luciferase reporter assay. Mineralization was evaluated by alizarin red staining. RESULTS The expression of Rspo-1 was similar in normal and OA osteoblasts, whereas the expression and production of Rspo-2 were reduced in OA osteoblasts due to elevated levels of transforming growth factor β1 in these cells. The reduced Wnt-3a-dependent TOPflash TCF/LEF luciferase reporter activity in OA osteoblasts as compared to normal osteoblasts was corrected by the addition of recombinant human Rspo-2. Wnt-3a-dependent β-catenin levels were also corrected in OA osteoblasts by Rspo-2 addition. Wnt-3a alone increased the mineralization of OA osteoblasts, which was further increased by Rspo-2. CONCLUSION Reduced Rspo-2 levels in OA osteoblasts are responsible, at least in part, for their reduced Wnt/β-catenin signaling and abnormal mineralization. As Rspo-2 is a secreted soluble protein, this could lead to potential new avenues of treatment of OA.

Beneficial effect of resveratrol on phenotypic features and activity of osteoarthritic osteoblasts

BackgroundOsteoarthritis (OA) is a complex disease, which affects multiple tissues, namely the subchondral bone, articular cartilage and synovial membrane. Alterations of the subchondral bone include an increased, yet under mineralized osteoid matrix, abnormal osteoblast cell phenotype including elevated alkaline phosphatase (ALP) activity, increased release of osteocalcin (OC) and transforming growth factor β-1 (TGF-β1). Previous studies have demonstrated an inhibition of the canonical Wnt signaling (cWnt) pathway in OA osteoblasts (Ob). As resveratrol (RSV) has been shown to upregulate the Wnt signaling pathway in different cell systems, we hypothesized that RSV could be beneficial for OA Ob.MethodWe prepared primary human Ob using the subchondral bone plate of tibial plateaus of OA patients undergoing total knee arthroplasty, or tibial plateaus of normal individuals at autopsy. Sirtuin 1 (Sirt1) expression in normal and OA subchondral bone tissue was evaluated by immunohistochemical analysis. Expression of genes was evaluated by qRT-PCR and protein production by western blot analysis. ALP activity and osteocalcin secretion were evaluated respectively with substrate hydrolysis and enzyme immunoassay. Mineralization levels were evaluated with alizarin red staining. Wnt/β-catenin signaling was evaluated by target gene expression using the TOPflash TCF/lef luciferase reporter assay and intracellular signaling using β-catenin levels in western blot analysis. Extracellular signal-regulated kinase (Erk)1/2 and the Smad1/5/8 pathways were evaluated by western blot analysis.ResultsSirt1 expression and production were reduced in OA subchondral bone tissue compared to normal tissue. RSV upregulated Sirt1 and its activity, and reduced the expression of leptin. RSV increased Erk1/2 phosphorylation in OA Ob; however, it had no effect on Smad 1/5/8 phosphorylation. RSV had little effect on cell proliferation and only slightly affected the Bax/Bcl2 ratio. The expression of Runx2/Cbfa1 and peroxisome proliferator-activated receptor (PPAR)γ were not affected by increasing doses of RSV. The endogenous increased ALP activity and OC release observed in OA Ob compared to normal Ob were partly corrected only for ALP at high RSV levels but not for OC release. In contrast, RSV increased the mineralization of OA Ob. Moreover, whereas Wnt3a stimulates the Wnt/β-catenin pathway in these cells, RSV further increased the response to Wnt3a.ConclusionThese data indicate that RSV promotes Sirt1 levels, inhibits the endogenous expression of leptin by OA osteoblasts and can promote the Wnt/β-catenin and Erk1/2 signaling pathways, which are altered in these cells.