David Hum

Regulation of the IGFBP-5 and MMP-13 genes by the microRNAs miR-140 and miR-27a in human osteoarthritic chondrocytes

BackgroundMMP-13 and IGFBP-5 are important factors involved in osteoarthritis (OA). We investigated whether two highly predicted microRNAs (miRNAs), miR-140 and miR-27a, regulate these two genes in human OA chondrocytes.MethodsGene expression was determined by real-time PCR. The effect of each miRNA on IGFBP-5 and MMP-13 expression/production was evaluated by transiently transfecting their precursors (pre-miRNAs) and inhibitors (anti-miRNAs) into human OA chondrocytes. Modulation of IGFBP-5, miR-140 and miR-27a expression was determined upon treatment of OA chondrocytes with cytokines and growth factors.ResultsIGFBP-5 was expressed in human chondrocytes with its level significantly lower (p < 0.04) in OA. Five computational algorithms identified miR-140 and miR-27a as possible regulators of MMP-13 and IGFBP-5 expression. Data showed that both miRNAs were expressed in chondrocytes. There was a significant reduction (77%, p < 0.01) in miR-140 expression in OA compared to the normal chondrocytes, whereas miR-27a expression was only slightly decreased (23%). Transfection with pre-miR-140 significantly decreased (p = 0.0002) and with anti-miR-140 significantly increased (p = 0.05) IGFBP-5 expression at 24 hours, while pre-miR-27a did not affect either MMP-13 or IGFBP-5. Treatment with anti-miR-27a, but not with anti-miR-140, significantly increased the expression of both MMP-13 (p < 0.05) and IGFBP-5 (p < 0.01) after 72 hours of incubation. MMP-13 and IGFBP-5 protein production followed the same pattern as their expression profile. These data suggest that IGFBP-5 is a direct target of miR-140, whereas miR-27a down-regulates, likely indirectly, both MMP-13 and IGFBP-5.ConclusionThis study is the first to show the regulation of these miRNAs in human OA chondrocytes. Their effect on two genes involved in OA pathophysiology adds another level of complexity to gene regulation, which could open up novel avenues in OA therapeutic strategies.

Biochemistry and physiology of the natriuretic peptide receptor guanylyl cyclases

Guanylyl cyclases (GC) exist as soluble and particulate, membrane-associated enzymes which catalyse the conversion of GTP to cGMP, an intracellular signalling molecule. Several membrane forms of the enzyme have been identified up to now. Some of them serve as receptors for the natriuretic peptides, a family of peptides which includes atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP), three peptides known to play important roles in renal and cardiovascular physiology. These are transmembrane proteins composed of a single transmembrane domain, a variable extracellular natriuretic peptide-binding domain, and a more conserved intracellular kinase homology domain (KHD) and catalytic domain. GC-A, the receptor for ANP and BNP, also named natriuretic peptide receptor-A or -1 (NPR-A or NPR-1), has been studied widely. Its mode of activation by peptide ligands and mechanisms of regulation serve as prototypes for understanding the function of other particulate GC. Activation of this enzyme by its ligand is a complex process requiring oligomerization, ligand binding, KHD phosphorylation and ATP binding. Gene knockout and genetic segregation studies have provided strong evidence for the importance of GC-A in the regulation of blood pressure and heart and renal functions. GC-B is the main receptor for CNP, the latter having a more paracrine role at the vascular and venous levels. The structure and regulation of GC-B is similar to that of GC-A. This chapter reviews the structure and roles of GC-A and GC-B in blood pressure regulation and cardiac and renal pathophysiology.

Characterization of a cGMP-Response Element in the Guanylyl Cyclase/Natriuretic Peptide Receptor A Gene Promoter

Previous studies have shown that atrial natriuretic peptide (ANP) can inhibit transcription of its receptor, guanylyl cyclase A, by a mechanism dependent on cGMP and have suggested the presence of a putative cGMP-response element (cGMP-RE) in the Npr1 gene promoter. To localize and characterize the putative cis-acting element, we have subcloned a 1520-bp fragment of the rat Npr1 promoter in an expression vector containing the luciferase reporter gene. Several fragments, generated by exonuclease III-directed deletions, were transiently transfected into cells to measure their promoter activity. Deletion from −1520 to −1396 of a 1520-bp-long Npr1 promoter led to a 5-fold increase in luciferase activity. Subsequent deletion to the position −1307 resulted in a decrease of luciferase activity by 90%. Preincubation of cells with 100 nM of ANP or 100 μM 8-bromo-cGMP inhibited luciferase activity of the 1520-bp and 1396-bp-long fragments, but not the activity of the 1307-bp fragment, suggesting that the cGMP-RE is localized between positions −1396 and −1307. The cGMP regulatory region was narrowed by gel shift assays and footprinting to position −1372 to −1354 from the transcription start site of Npr1 and indicated its interaction with transcriptional factor(s). Cross-competition experiments with mutated oligonucleotides led to the definition of a consensus sequence (−1372 A a A tRKaN TTC a A c A K T Y −1354) for the novel cGMP-RE, which is conserved in the human (75% identity) and mouse (95% identity) Npr1 promoters.

NFAT3 and TGF-β/SMAD3 regulate the expression of miR-140 in osteoarthritis.

IntroductionMicroRNAs (miRNAs) down-regulate their target genes. The intronic miR-140, present in the WW domain containing E3 ubiquitin protein ligase 2 (WWP2) gene, decreases the expression of genes that play detrimental roles in osteoarthritis (OA). As the expression level of miR-140 is significantly decreased in human OA chondrocytes, we investigated its regulation in those cells.MethodsGene expression in human chondrocytes was determined by quantitative polymerase chain reaction (qPCR) and gene silencing was done in OA chondrocytes by transient transfection with specific small interfering RNAs (siRNAs). Binding sites of the miR-140 regulatory sequence (rsmiR-140) were identified by mutagenesis and chromatin immunoprecipitation (ChIP) in OA chondrocytes. The effects of translocation on OA chondrocytes were determined by immunocytochemistry and qPCR.ResultsIn contrast to miR-140, the expression of WWP2 was similar in both normal and OA cells, suggesting that miR-140 has an additional level of regulation. rsmiR-140 showed activity and predicted binding sites for nuclear matrix transcription factor 4 (NMP4), myc-associated zinc (MAZ), nuclear factor of activated T-cells (NFAT), and mothers against decapentaplegic homolog 3 (SMAD3). Silencing NFAT3 (P ≤0.01) and SMAD3 (P ≤0.05) differentially regulated miR-140 independently of WWP2. Silencing NFAT5 decreased both miR-140 and WWP2 (P ≤0.003 and P ≤0.05, respectively). NFAT3 activation increased and transforming growth factor-β (TGF-β) decreased rsmiR-140 activity. Mutagenesis of rsmiR-140 and ChIP assays identified binding sites at which NFAT3 (activator) and SMAD3 (repressor) directly regulated miR-140. TGF-β interfered with NFAT3 translocation, and subsequently with miR-140 expression.ConclusionsThis is the first study to provide evidence of a regulatory mechanism of miR-140 independent of WWP2, and new and differential roles for NFAT3 and SMAD3 in the OA process in the regulation of miR-140 transcription. Such knowledge could advance therapeutic strategies targeting OA.

In vivo bone-specific EphB4 overexpression in mice protects both subchondral bone and cartilage during osteoarthritis

OBJECTIVE In vitro activation of the receptor EphB4 positively affects human osteoarthritis (OA) articular cell metabolism. However, the specific in vivo role of this ephrin receptor in OA remains unknown. We investigated in mice the in vivo effect of bone-specific EphB4 overexpression on OA pathophysiology. METHODS Morphometric, morphologic, and radiologic evaluations were performed on postnatal day 5 (P5) mice and on 10-week-old mice. Knee OA was induced surgically by destabilization of the medial meniscus (DMM) in 10-week-old male EphB4 homozygous transgenic (EphB4-Tg) and wild-type (WT) mice. Medial compartment evaluations of cartilage were performed using histology and immunohistochemistry, and evaluations of subchondral bone using histomorphometry, osteoclast staining, and micro-computed tomography. RESULTS There was no obvious phenotype difference in skeletal development between EphB4-Tg mice and WT mice at P5 or at 10 weeks. At 8 and 12 weeks post-DMM, the EphB4-Tg mice demonstrated significantly less cartilage alteration in the medial tibial plateau and the femoral condyle than did the WT mice. This was associated with a significant reduction of aggrecan and type II collagen degradation products, type X collagen, and collagen fibril disorganization in the operated EphB4-Tg mice. The medial tibial subchondral bone demonstrated a significant reduction in sclerosis, bone volume, trabecular thickness, and number of tartrate-resistant acid phosphatase-positive osteoclasts at both times assessed post-DMM in the EphB4-Tg mice than in the WT mice. CONCLUSION This is the first in vivo evidence that bone-specific EphB4 overexpression exerts a protective effect on OA joint structural changes. The findings of this study stress the in vivo importance of subchondral bone biology in cartilage integrity.

TA Repeat Variation, Npr1 Expression, and Blood Pressure. Impact of the Ace Locus

Abstract—The activity of the atrial natriuretic peptide receptor (Npr1) is altered in spontaneously hypertensive rats (SHR) in relation to its mRNA levels, suggesting abnormal transcriptional control in hypertension. A single-stranded conformational polymorphism caused by a repetitive dinucleotide segment of 10 TA in BN-Lx and of 40 TA in SHR was localized at position −943 relative to the transcription start site of the Npr1 gene, downstream of a putative cGMP-regulatory region, and was the only sequence difference noted between the two strains. Transient transfections of −1520 to −920 Npr1 promoter-SV40-luciferase fusion vector showed that the construct from BN-Lx stimulated the SV40 promoter, whereas that from SHR slightly inhibited it. In contrast to the BN-Lx construct, the activity of the SHR fragment was refractory to downregulation by atrial natriuretic peptide. Genotype-phenotype correlation studies in recombinant inbred strains (RIS) derived from BN-Lx and SHR crosses revealed significant correlations of the TA repeat with basal guanylyl cyclase activity and Npr1 mRNA levels. The correlations were heightened by a locus on chromosome 10 containing the Ace gene. The highest basal guanylyl cyclase activity and Npr1 mRNA values were found in RIS with both genes (Npr1/ Ace) of BN genotypes, whereas the lowest were recorded in RIS, with the SHR genotypes at both loci. This was inversely correlated with diastolic blood pressure in these strains. In conclusion, the longer TA repeat unit in the promoter of Npr1 of SHR, in tandem with a putative cGMP responsive element, regulates the transcription of the Npr1 gene with consequences on diastolic blood pressure.

The unfolded protein response genes in human osteoarthritic chondrocytes: PERK emerges as a potential therapeutic target.

BackgroundThe unfolded protein response (UPR) is activated following an endoplasmic reticulum (ER) stress. The aim of this study was to investigate the global expression of UPR genes in human OA chondrocytes in induced (I)-UPR conditions, and to explore the regulation and role of the UPR genes in homeostatic (H)-UPR conditions in human normal and OA chondrocytes.MethodsGene expression was determined by PCR array and qPCR. Protein production in cartilage was determined by immunohistochemistry, gene silencing by specific siRNAs, and gene regulation by treating chondrocytes with cytokines and growth factors associated with cartilage pathobiology.ResultsSeveral UPR genes, among them ERN1, PERK, and CREB3L2 were downregulated in OA compared to normal chondrocytes at both the mRNA and protein levels, but the ER stress response triggered by thapsigargin or tunicamycin treatment was similar in normal and OA chondrocytes. The activation of ER stress sensors (phosphorylated PERK, cleavage of ATF6B, and the spliced mRNA forms of XBP1) was not significantly increased in OA chondrocytes/cartilage. PDGF-BB and IL-6 significantly downregulated the expression of ERN1, PERK, and CREB3L2, but not that of ATF6B. Silencing experiments done under conditions of no ER stress (physiological conditions) revealed that decreasing ERN1 expression led to decreased COL2a1, MMP-13, ADAMTS4 and ADAMTS5 expression, while decreasing CREB3L2 and ATF6B led to decreased ADAMTS5 and ADAMTS4 expression, respectively. Importantly, the downregulation of PERK expression increased COL1a1 and suppressed COL2a1 expression.ConclusionsAlthough the level of ER stress is not significantly increased in OA chondrocytes, these cells respond strongly to an acute ER stress despite the decreased expression of ERN1, PERK, and CREB3L2. Emerging findings revealed for the first time that these genes play a role in cartilage biology in conditions where an acute ER stress response is not triggered and OA is not characterized by an overall basal activation of the ER stress response. Importantly, these findings identify PERK as a potential target for new OA treatment avenues.

Hsp90β and p130cas: novel regulatory factors of MMP-13 expression in human osteoarthritic chondrocytes

Background: Human osteoarthritic (OA) chondrocytes were previously classified into L (low)- and H (high)-OA according to matrix metalloproteinase-13 (MMP-13) basal levels and interleukin 1β (IL1β) inducibility. In H-OA chondrocytes, the regulatory proteins p130cas and nuclear matrix protein 4 (NMP4) acting on the MMP-13 promoter were identified. Objective: To identify regulators of MMP-13 expression/production in human L-OA chondrocytes, to determine their effect on the expression of other MMPs and the effect of IL1β on these molecules. Methods: The identification of the L-OA chondrocyte proteins interacting specifically with the AGRE site of the MMP-13 promoter was performed by mass spectrometry. Heat shock protein 90β (Hsp90β), p130cas and NMP4 small interfering RNAs (siRNAs) were transfected into L-OA chondrocytes and incubated with or without IL1β. Gene expression was determined by real-time PCR, MMP-1 and MMP-13 production by ELISA, and signalling pathway activation by western blotting and ELISA. Results: Hsp90β was identified as a protein of the L-OA/AGRE-specific complex. Silencing p130cas and Hsp90β significantly increased MMP-13 expression (about four- and twofold, respectively) and production. sip130cas affected to a lesser extent MMP-1 expression (twofold) and production. siNMP4 showed no effect. Expression of MMP-2, -3, -9 and -14 was unaffected. Silencing both Hsp90β and p130cas had a significant additive effect on MMP-13, but not on MMP-1 expression, the level of which was similar to that with sip130cas alone. IL1β decreased p130cas and Hsp90β expression/production, indicating another pathway by which this cytokine upregulates MMP expression. The IL1β-triggered signalling pathways responsible for MMP upregulation were unaffected in the silenced cells. Conclusion: This study illustrates the complex regulation of MMP-13 by showing the inhibitory effect of the two cytoplasmic molecules, p130cas and Hsp90β, in L-OA chondrocytes.

The in vivo effect of prophylactic subchondral bone protection of osteoarthritic synovial membrane in bone-specific Ephb4-overexpressing mice

Osteoarthritis (OA) is characterized by progressive joint destruction, including synovial membrane alteration. EphB4 and its ligand ephrin-B2 were found in vitro to positively affect OA subchondral bone and cartilage. In vivo in an experimental mouse model overexpressing bone-specific Ephb4 (TgEphB4), a protective effect was found on both the subchondral bone and cartilage during OA. We investigated in the TgEphB4 mouse model the in vivo effect on synovial membrane during OA. Knee OA was surgically induced by destabilization of the medial meniscus (DMM). Synovial membrane was evaluated using histology, histomorphometry, IHC, and real-time PCR. Compared to DMM-wild-type (WT) mice, DMM-TgEphB4 mice had a significant decrease in synovial membrane thickness, vascular endothelial growth factor, and the profibrotic markers fibrin, type 1 procollagen, type 3 collagen, connective tissue growth factor, smooth muscle actin-α, cartilage oligomeric matrix protein, and procollagen-lysine, and 2-oxoglutarate 5-dioxygenase 2. Moreover, factors known to modulate transforming growth factor-β signaling, transforming growth factor receptor 1/ALK1, phosphorylated Smad-1, and heat shock protein 90β were significantly decreased in DMM-TgEphB4 compared with DMM-WT mice. Ephb4 overexpression also exhibited a protective effect on synovial membrane thickness of aged (24-month-old) mice. Overexpression of bone-specific Ephb4 clearly demonstrated prevention of the development and/or progression of fibrosis in OA synovial membrane, reinforcing the hypothesis that protecting the subchondral bone prophylactically and during OA reduces the pathologic changes in other articular tissues.

THU0473 Diacerein Reduces Antagonists of WNT Enabling this System's Activity in Human Osteoarthritic Subchondral Bone Osteoblasts

Background Clinical and in vitro studies suggest that altered subchondral bone remodeling is involved in the progression and/or onset of osteoarthritis (OA). Human OA subchondral bone osteoblasts display an abnormal metabolism, including alterations in the Wnt/β-catenin signaling pathway. Objectives As therapeutic strategies aimed at modifying the metabolism of subchondral bone may be indicated in the treatment of OA, we investigated diacerein, an anthraquinone, which has been shown to positively impact the abnormal subchondral bone metabolism in OA. Here, we further explored the effect of this drug, and its active metabolite rhein, on elements of the Wnt system in human OA subchondral bone osteoblasts. Methods Therapeutic concentrations of diacerein/rhein (5-20 μg/ml) were used to assess their effects on the levels of β-catenin and the Wnt antagonists Dickkopf (DKK)-1 and -2. The β-catenin expression, as well as its nuclei translocation, was assessed after 20 hours of incubation using mRNA and Western blot. DKK-1 and -2 expressions were determined after 20 hours of incubation, and DKK-1 protein production after 48 hours in the culture medium using a specific ELISA. Results The RNA level of β-catenin expression was increased in diacerein (p≤0.01) and rhein (p≤0.014) treated cells. For the β-catenin nuclei translocation, the Wnt ligand most potent in bone tissue was added at a concentration of 100 ng/ml, and increased this activity by only 50%. Both diacerein and rhein increased β-catenin nuclei translocation (diacerein, p≤0.04; rhein, p≤0.06). DKK-1 and DKK-2 mRNA expressions were dose-dependently decreased with both drugs (p≤0.009). Compared to DKK-1, the expression level of DKK-2 demonstrated a more marked decrease (90% vs. 60%). Both drugs also markedly and significantly decreased DKK-1 protein levels (p≤0.02) in a dose-dependent manner. Conclusions Diacerein and rhein positively impact the abnormal Wnt system in OA by increasing the level of β-catenin in the nuclei. This effect is likely mediated via a decrease in the DKK-1 and -2. This study infers that these drugs may be appropriate for positively modulating the abnormal metabolism of human OA subchondral bone osteoblasts. Disclosure of Interest None declared