Prostaglandin E2 stimulates insulin-like growth factor binding protein-4 expression and synthesis in cultured human articular chondrocytes: Possible mediation by Ca++ -calmodulin regulated processes

Insulin-like growth factor-1, IGF-1, is believed to be an important anabolic modulator of cartilage metabolism whose action is mediated by high affinity cell surface receptors and bioactivity and bioavailability regulated, in part, by IGF-1 binding proteins (IGFBPs). Prostaglandin E₂ (PGE₂) stimulates collagen and proteoglycan synthesis in cartilage via an autocrine feedback loop involving IGF-1. We determined whether the eicosanoid could regulate IGFBP-4, a major form expressed by chondrocytes and, as such, act as a modifier of IGF-1 action at another level. Using human articular chondrocytes in high-density primary culture, Western and Western ligand blotting to measure secreted IGFBP-4 protein, and Northern analysis to monitor IGFBP-4 mRNA levels, we demonstrated that PGE₂ provoked a 2.7 ± 0.3- and 3.8 ± 0.5- (n = 3) fold increase in IGFBP-4 mRNA and protein, respectively. This effect was reversed by the Ca⁺⁺ channel blocker, verapamil, and the Ca⁺⁺/calmodulin inhibitor, W-7. The Ca⁺⁺ ionophore, ionomycin, mimicked the effects of PGE₂. The phorbol ester, PMA, which activated phospholipid-dependent protein kinase C (PKC) in chondrocytes, had no effect on IGFBP-4 production. Cyclic AMP mimetics and PKA activators, IBMX, and Sp-cAMP, inhibited the expression of the binding protein as did the PGE₂ secretagogue, interleukin-1β (IL-β). The inhibitory effect of the latter cytokine was mediated by a erbstatin/genistein (tyrosine) sensitive kinase. Dexamethasone, an inhibitor of cyclooxygenase (COX-2) expression and PGE₂ synthesis, down-regulated control, constitute levels of IGFBP-4 mRNA and protein, eliminating the previously demonstrated possibility of cross-talk between glucocorticoid receptor (GR) and PGE₂-receptor signalling pathways. The results suggest that extracellular signals control IGFBP-4 production by a number of different transducing networks with changes in Ca⁺⁺ and calmodulin activity exerting a strong positive influence, possibly maintaining the constitutivity of IGFBP-4 synthesis under basal conditions. PGE₂ activation of the IGF-1/IGFBP axis may play a pivotal role in the metabolism of cartilage and possibly connective tissues in general. Eicosanoid biosynthesis may be a rate-limiting step in cartilage repair processes. J. Cell. Biochem. 65:408–419. © 1997 Wiley-Liss, Inc.     

Bone morphogenetic protein-4 expression characterizes inductive boundaries in organs of developing zebrafish

 We have cloned and examined the expression pattern of zebrafish bone morphogenetic protein-4 (BMP4) as a start to evaluating signals which might participate in the fashioning of organ systems in this genetically tractable species. The predicted sequence of the mature zebrafish protein is more than 75% identical to that of other vertebrates and 66% identical to Drosophila decapentaplegic (Dpp). As in other species, BMP4 is expressed ventrally during gastrulation, but the zebrafish is unusual in having an additional dorsal domain of expression. Subsequent BMP4 expression is especially prominent in sensory organs, fin buds, and in the gut, kidney, and heart. In all these sites, it becomes particularly enriched in regions of inductive demarcations. For example, expression initially extends through the entire heart tube but then becomes limited to the boundaries between cardiac chambers (sinus venosus-atrial junction, atrio-ventricular junction, and aortic root) prior to cushion formation. In early pectoral fin development, BMP4 is at first expressed uniformly but then becomes restricted to the mesenchyme subjacent to the apical ectodermal ridge. This suggests that among its roles in development, BMP4 serves as a signal in primordial outgrowth and also as a signal demarcating the borders within organs or structures where subspecializations occur. Received: 13 January 1997 / Accepted: 3 April 1997     

Prostaglandin E2 up-regulates insulin-like growth factor binding protein-3 expression and synthesis in human articular chondrocytes by a c-AMP-independent pathway: Role of calcium and protein kinase A and C

Insulin-like growth factor-1, IGF-1, is believed to be an important anabolic modulator of cartilage metabolism and its bioactivity and bioavailability is regulated, in part, by IGF-1 binding protein 3 (IGFBP-3). Prostaglandin E₂ (PGE₂) stimulates IGF-1 production by articular chondrocytes and we determined whether the eicosanoid could regulate IGFBP-3 and, as such, act as a modifier of IGF-1 action at a different level. Using human articular chondrocytes in high density primary culture, Western and Western ligand blotting to measure secreted IGFBP-3 protein, and Northern analysis to monitor IGFBP-3 mRNA levels, we demonstrated that PGE₂ provoked a 3.9 ± 1.1 (n = 3) fold increase in IGFBP-3 mRNA and protein. This effect was reversed by the Ca⁺⁺ channel blockers, verapamil and nifedipine, and the Ca⁺⁺/calmodulin inhibitor, W-7. The Ca⁺⁺ ionophore, ionomycin, mimicked the effects of PGE₂ as did the phorbol ester PMA, which activates Ca⁺⁺-phospholipid-dependent protein kinase C (PKC). Cyclic AMP mimetics, such as forskolin, IBMX, Ro-20-1724, and Sp-cAMP, inhibited the expression and synthesis of the binding protein. PGE₂ did not increase the levels of cAMP or protein kinase A (PKA) activity in chondrocytes. The PGE₂ secretagogue, IL-1β, down-regulated control levels of IGFBP-3 which could be completely abrogated by pre-incubation with the tyrosine kinase inhibitor, erbstatin, and partially reversed (50 ± 8%) by KT-5720, a PKA inhibitor. These observations suggested that PGE₂ does not mediate the effect of its secretagogue and that IL-1β signalling in chondrocytes may involve multiple kinases of diverse substrate specificities. Dexamethasone down-regulated control, constitutive levels of IGFBP-3 mRNA and protein eliminating the previously demonstrated possibility of cross-talk between glucocorticoid receptor (GR) and PGE₂ receptor signalling pathways. Taken together, our results suggest that PGE₂ modulates IGFBP-3 expression, protein synthesis, and secretion, and that such regulation may modify human chondrocyte responsiveness to IGF-1 and influence cartilage metabolism. © 1996 Wiley-Liss, Inc.     

Bone morphogenetic protein-7 expression in human pyelonephritis

Bone morphogenetic protein 7 (BMP- 7) is a member of the transforming growth factor (TGF) beta superfamily and is involved in regeneration, repair, and development of specific tissues, for example kidney and skeleton. The experimental studies have shown its protective role against fibrotic processes. Tubulointerstitial changes are present in the pyelonephritic kidney which progresses to fibrosis. Renal fibrosis may lead to the loss of renal function. The aim of this study was to investigate BMP-7 expression in acute and chronic pyelonephritis in humans. Seven patients with acute pyelonephritis and 7 with chronic pyelonephritis were treated in Department of Nephrology Clinical Hospital, Rijeka. Tissue biopsy was taken and renal tissue was studied histopathologically by use of hematoxylin and eosin and scored for diagnosis of pyelonephritis. BMP-7 expression was studied by immunohistochemical staining. BMP-7 expression was observed in the tubular area of the pyelonephritic kidneys. The expression of BMP- 7 was stronger in the acute pyelonephritic group and less in the chronic pyelonephritic group of patients. The results imply that BMP-7 has a role in chronic pyelonephritis. Tubular BMP-7 expression had a negative correlation with fibrosis and tubular, atrophy. Our results are suggesting that BMP- 7 plays an important protective role in renal inflammatory diseases preventing greater damage and fibrosis.     

Bone morphogenetic protein expression in newborn rat kidneys after prenatal exposure to radiofrequency radiation

Effects of nonthermal radiofrequency radiation (RFR) of the global system of mobile communication (GSM) cellular phones have been as yet mostly studied at the molecular level in the context of cellular stress and proliferation, as well as neurotransmitter production and localization. In this study, a simulation model was designed for the exposure of pregnant rats to pulsed GSM-like RFR (9.4 GHz), based on the different resonant frequencies of man and rat. The power density applied was 5 microW/cm2, in order to avoid thermal electromagnetic effects as much as possible. Pregnant rats were exposed to RFR during days 1-3 postcoitum (p.c.) (embryogenesis, pre-implantation) and days 4-7 p.c. (early organogenesis, peri-implantation). Relative expression and localization of bone morphogenetic proteins (BMP) and their receptors (BMPR), members of a molecular family currently considered as major endocrine and autocrine morphogens and known to be involved in renal development, were investigated in newborn kidneys from RFR exposed and sham irradiated (control) rats. Semi-quantitative duplex RT-PCR for BMP-4, -7, BMPR-IA, -IB, and -II showed increased BMP-4 and BMPR-IA, and decreased BMPR-II relative expression in newborn kidneys. These changes were statistically significant for BMP-4, BMPR-IA, and -II after exposure on days 1-3 p.c. (P <.001 each), and for BMP-4 and BMPR-IA after exposure on days 4-7 p.c. (P <.001 and P =.005, respectively). Immunohistochemistry and in situ hybridization (ISH) showed aberrant expression and localization of these molecules at the histological level. Our findings suggest that GSM-like RFR interferes with gene expression during early gestation and results in aberrations of BMP expression in the newborn. These molecular changes do not appear to affect renal organogenesis and may reflect a delay in the development of this organ. The differences of relative BMP expression after different time periods of exposure indicate the importance of timing for GSM-like RFR effects on embryonic development.     

Bone morphogenetic protein-2 stimulates Runx2 acetylation

Runx2/Cbfa1/Pebp2aA is a global regulator of osteogenesis and is crucial for regulating the expression of bone-specific genes. Runx2 is a major target of the bone morphogenetic protein (BMP) pathway. Genetic analysis has revealed that Runx2 is degraded through a Smurf-mediated ubiquitination pathway, and its activity is inhibited by HDAC4. Here, we demonstrate the molecular link between Smurf, HDACs and Runx2, in BMP signaling. BMP-2 signaling stimulates p300-mediated Runx2 acetylation, increasing transactivation activity and inhibiting Smurf1-mediated degradation of Runx2. HDAC4 and HDAC5 dea-cetylate Runx2, allowing the protein to undergo Smurf-mediated degradation. Inhibition of HDAC increases Runx2 acetylation, and potentiates BMP-2-stimulated osteoblast differentiation and increases bone formation. These results demonstrate that the level of Runx2 is controlled by a dynamic equilibrium of acetylation, deacetylation, and ubiquitination. These findings have important medical implications because BMPs and Runx2 are of tremendous interest with regard to the development of therapeutic agents against bone diseases.     

Highly efficient baculovirus-mediated gene transfer into rat chondrocytes

To explore the potential of baculovirus serving as a gene delivery vector in tissue engineering of articular cartilage, the efficiencies of baculovirus-mediated gene delivery into primary rat chondrocytes were evaluated and the transduction protocol commonly employed by others (using concentrated virus at multiplicity of infection [MOI] 200 for 1 h) was found to be ineffective (<1%). Therefore, a modified protocol was adopted, which markedly enhanced the efficiency (68%). Optimization of the transduction parameters, such as incubation time (8 h), temperature (25 degrees C), and surrounding solutions (PBS), further increased the efficiency to 88% and prolonged the duration of expression to 21 days, suggesting that the cells previously considered nonpermissive to baculovirus transduction may be reexamined for their permissiveness using alternative transduction protocols. The elevated efficiency correlated well with increased virus uptake upon extended incubation time, as demonstrated by quantitative real-time polymerase chain reaction (Q-PCR). The Q-PCR also revealed the degradation of viral DNA over culture time. Although the virus transduction somewhat hindered the cell proliferation, growth rate could be restored in the long-term culture. More importantly, transduced cells could secrete articular cartilage-specific type II collagen and glycosaminoglycan as well as mock-transduced cells, confirming that normal differentiation state of rat chondrocytes is retained upon baculovirus transduction. Taken together, these data indicate that baculovirus is a safe and highly efficient gene delivery vehicle into rat chondrocytes.     

Bone morphogenetic protein-1 processes insulin-like growth factor-binding protein 3

The bone morphogenetic protein-1 (BMP1)-like metalloproteinases play key roles in extracellular matrix formation, by converting precursors into mature functional proteins involved in forming the extracellular matrix. The BMP1-like proteinases also play roles in activating growth factors, such as BMP2/4, myostatin, growth differentiation factor 11, and transforming growth factor β1, by cleaving extracellular antagonists. The extracellular insulin-like growth factor-binding proteins (IGFBPs) are involved in regulating the effects of insulin-like growth factors (IGFs) on growth, development, and metabolism. Of the six IGFBPs, IGFBP3 has the greatest interaction with the large pool of circulating IGFs. It is also produced locally in tissues and is itself regulated by proteolytic processing. Here, we show that BMP1 cleaves human and mouse IGFBP3 at a single conserved site, resulting in markedly reduced ability of cleaved IGFBP3 to bind IGF-I or to block IGF-I-induced cell signaling. In contrast, such cleavage is shown to result in enhanced IGF-I-independent ability of cleaved IGFBP3 to block FGF-induced proliferation and to induce Smad phosphorylation. Consistent with in vivo roles for such cleavage, it is shown that, whereas wild type mouse embryo fibroblasts (MEFs) produce cleaved IGFBP3, MEFs doubly null for the Bmp1 gene and for the Tll1 gene, which encodes the related metalloproteinase mammalian Tolloid-like 1 (mTLL1), produce only unprocessed IGFBP3, thus demonstrating endogenous BMP1-related proteinases to be responsible for IGFBP3-processing activity in MEFs. Similarly, in zebrafish embryos, overexpression of Bmp1a is shown to reverse an Igfbp3-induced phenotype, consistent with the ability of BMP1-like proteinases to cleave IGFBP3 in vivo.     

Bone morphogenetic protein-2 stimulates angiogenesis in developing tumors

Bone Morphogenetic Protein-2 (BMP-2) is highly overexpressed in the majority of patient-derived lung carcinomas. However, a mechanism revealing its role in cancer has not been established. Here we report that BMP-2 enhances the neovascularization of developing tumors. Recombinant BMP-2 stimulated blood vessel formation in tumors formed from A549 cells injected s.c. into thymic nude mice. Recombinant BMP-2 also enhanced angiogenesis in Matrigel plugs containing A549 cells in nude mice. The BMP-2 antagonist noggin abrogated BMP-2-induced angiogenic response. Furthermore, antisense transfection of BMP-2 cDNA resulted in a decrease in blood vessel formation in the Matrigel assays. BMP-2 induced tube formation in both human aortic endothelial cells (HAEC) and umbilical vein endothelial cells. BMP-2 also stimulated proliferation of HAEC. The ability of BMP-2 to activate endothelial cells was further demonstrated by its ability to phosphorylate Smad 1/5/8 and ERK-1/2 and to increase expression of Id1. This study reveals that BMP-2 enhanced the angiogenic response in developing tumors. Furthermore, these data suggest that BMP-2 stimulation of angiogenesis may involve the activation of endothelial cells.     

Bone morphogenetic protein-7 and interferon-alpha synergistically suppress hepatitis C virus replicon

Various cytokines contribute to control hepatitis C virus (HCV) viral replication. HCV subgenomic replicon systems have been developed, and cell-cycle-dependent replication has been reported. But the molecules involved in this processes is not totally elucidated. The aim of this study is to investigate the involvement of the bone morphogenetic protein (BMP)-7, a member of TGF-beta superfamily, to the in vitro HCV replication. BMP-7 dose-dependently suppressed the replication and protein expression from the HCV replicon in Huh7/Rep-Feo cells and was associated with cell-cycle arrest at the G1 phase. These results were consistent with the effect of TGF-beta in a previous study. Combination of BMP-7 and interferon-alpha showed a synergic decrease of HCV replication, and was more effective compared to the treatment with interferon-alpha alone. This synergistic effect was also present in HCV-JFH1 virus cell culture. While BMP-7 alone did not stimulate expression of the interferon-stimulated genes (ISGs), it augmented interferon-induced expression of the ISGs independently of the interferon-induced Jak/STAT pathway. Taken together, BMP-7 may constitute a novel molecule to suppress HCV replication.     

BmNPV chitinase gene deletion enhances foreign gene expression in a BmN cell system

Bombyx mori nucleopolyhedrovirus (BmNPV) is extensively being studied as an expression vector for heterologous gene expression in silkworm-derived cells as well as in the host larvae or pupae. BmNPV chitinase is necessary for liquefaction of the virus-infected host insect. The influence of chitinase on the efficiency of foreign gene expression was studied to provide a scientific basis for improving the BmNPV expression system. The BmNPV chitinase gene ( chiA ) was deleted and the expression level of the polyhedrin promoter controlling the lac Z gene in BmN cells was determined. Sodium dodecylsulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) showed that β-galactosidase accounted for approximately 6.9 and 7.7% of the total protein in BmN cells infected with the chiA deficient Bm lac Z⁺ chiA ⁻ at 3 and 4 days post infection, while the total protein was 3.2 and 4.2% in cells infected with Bm lac Z⁺. The relative β-galactosidase activities in Bm lac Z⁺ chiA ⁻-infected cells improved 2.33- and 1.56-fold compared to those of Bm lac Z⁺-infected cells at 3 and 4 days post infection. The results of the present study suggest that chitinase deletion could improve the lacZ expression level in the BmNPV-BmN cell expression system.