Salvianolic acid B exerts vasoprotective effects through the modulation of heme oxygenase-1 and arginase activities.

Salvia miltiorrhiza (Danshen), a traditional Chinese herbal medicine, is commonly used for the prevention and treatment of cardiovascular disorders including atherosclerosis. However, the mechanisms responsible for the vasoprotective effects of Danshen remain largely unknown. Salvianolic acid B (Sal B) represents one of the most bioactive compounds that can be extracted from the water-soluble fraction of Danshen. We investigated the effects of Danshen and Sal B on the inflammatory response in murine macrophages. Danshen and Sal B both induced the expression of heme oxygenase-1 (HO-1) and inhibited nitric oxide (NO) production and inducible NO synthase (iNOS) expression in lipopolysaccharide (LPS)-activated RAW 264.7 cells. Inhibition of HO activity using Sn-protoporphyrin-IX (SnPP) abolished the inhibitory effect of Sal B on NO production and iNOS expression. Sal B increased macrophage arginase activity in a dose-dependent manner and diminished LPS-inducible tumor necrosis factor-α production. These effects were also reversed by SnPP. These data suggest that HO-1 expression plays an intermediary role in the anti-inflammatory effects of Sal B. In contrast to the observations in macrophages, Sal B dose-dependently inhibited arginase activity in murine liver, kidney, and vascular tissue. Furthermore, Sal B increased NO production in isolated mouse aortas through the inhibition of arginase activity and reduction of reactive oxygen species production. We conclude that Sal B improves vascular function by inhibiting inflammatory responses and promoting endothelium-dependent vasodilation. Taken together, we suggest that Sal B may represent a potent candidate therapeutic for the treatment of cardiovascular diseases associated with endothelial dysfunction.

MicroRNA-142-3p regulates TGF-ß3-mediated region-dependent chondrogenesis by regulating ADAM9.

Position-dependent chondrogenesis is regulated by processes that are both common to and differ among all limb types and limb skeletal elements. Despite intrinsic differences between wing and leg bud mesenchyme, the exact regulatory molecules and mechanisms involved in these processes have not been elucidated. Here, we show the limb type-specific role of TGF-ß3 during chondrogenic differentiation of chick limb mesenchymal cells. Exposure of wing cells to TGF-ß3 stimulated chondrogenic differentiation, whereas in leg bud mesenchymal cells, TGF-ß3 induced apoptotic cell death via G2M arrest. Consistent with a limb type-specific effect of TGF-ß3 on chondrogenic differentiation, we found different levels of miR-142-3p induction. Inhibition of miR-142-3p via PNA-based antisense oligonucleotides (ASOs) markedly promoted cell migration and precartilage condensation, while exogenous induction of miR-142-3p reduced cell survival and increased cell death. Overexpression of ADAM9 significantly reduced chondrogenic differentiation via downregulation of cell migration and cell survival and upregulation of apoptotic cell death. Limb type-specific expression levels of ADAM9 induced by TGF-ß3 were observed. Collectively, this study demonstrates that differential induction of miR-142-3p is involved in the limb type-specific effect of TGF-ß3 on wing vs. leg mesenchymal cells through direct modulation of ADAM9 transcription.

4-O-methylascochlorin, methylated derivative of ascochlorin, stabilizes HIF-1α via AMPK activation.

Chemopreventive or anticancer agents induce cancer cells to apoptosis through the activation of adenosine AMP-activated protein kinase (AMPK), which plays a major role as energy sensors under ATP-deprived condition or ROS generation. In this study, we compared the effects of ascochlorin (ASC), from the fungus Ascochyta viciae, and its derivatives on AMPK activity. We also examined a regulatory mechanism for hypoxia-inducible factor-1α (HIF-1α) stabilization in response to 4-O-methylascochlorin (MAC). We found that AMPK activation was mainly involved with MAC, but not ASC and 4-O-carboxymethylascochlorin (AS-6), indicating that the substitution of 4-O-methyl group from 4-O-hydroxyl group of ASC is important in the activation of AMPK and the expression of HIF-1α. MAC-stabilized HIF-1α via AMPK activation triggered by lowering the intracellular ATP level, not by ROS generation, increases glucose uptake and the expression of vascular endothelial growth factor (VEGF) and glucose transporter 1 (GLUT-1), major target genes of HIF-1α. Moreover, MAC-induced AMPK activity suppressed survival factors, including mTOR and ERK1/2 or translational regulators, including p70S6K and 4E-BP1. Our data suggest that AMPK is a key determinant of MAC-induced HIF-1α expression in response to energy stress, further implying its involvement in MAC-induced apoptosis.

Secretory phospholipase A2 promotes MMP-9-mediated cell death by degrading type I collagen via the ERK pathway at an early stage of chondrogenesis.

BACKGROUND INFORMATION: sPLA2 (secretory phospholipase A2) has been implicated in a wide range of cellular responses, including cell proliferation and ECM (extracellular matrix) remodelling. Even though ECM remodelling is an essential step for chondrogenesis, the expression and functions of sPLA2 during chondrogenesis have not been studied. RESULTS: In the present study, for the first time, we detect the secretion of sPLA2 during limb development and suggest that sPLA2 influences the proliferation and/or survival of limb mesenchymal cells. Treatment of wing bud mesenchymal cells with exogenous sPLA2 promoted cell death by activating MMP-9 (matrix metalloproteinase-9) and increasing type I collagen degradation. The additive chondro-inhibitory actions were induced by co-treatment of mp-BSA (p-aminophenyl-mannopyranoside-BSA), a known ligand of the mannose receptor. Chondro-inhibitory actions by sPLA2 were prevented by functional blocking of FcRY (chicken yolk sac IgY receptor), a mannose receptor family member that is the orthologue of the mammalian PLA2 (phospholipase A2) receptor and by inhibition of ERK (extracellular-signal-regulated kinase) activity. CONCLUSIONS: Taken together, our results suggest that elevated levels of sPLA2 secreted by wing bud mesenchymal cells promote type I collagen degradation by MMP-9 in a manner typical of receptor-mediated signalling and that these events lead to cell death.

Correction: Curcumin inhibits cellular condensation and alters microfilament organization during chondrogenic differentiation of limb bud mesenchymal cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Correction: TiO2 nanotube stimulate chondrogenic differentiation of limb mesenchymal cells by modulating focal activity.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Alterations in the temporal expression and function of cadherin-7 inhibit cell migration and condensation during chondrogenesis of chick limb mesenchymal cells in vitro.

Endochondral bone formation requires a complex interplay among immature mesenchymal progenitor cells to form the cartilaginous anlagen, which involves migration, aggregation and condensation. Even though condensation of chondrogenic progenitors is an essential step in this process, its mechanism(s) has not been well studied. Here, we show that cadherin-7 plays a central role in cellular condensation by modulating cell motility and migration. In this study, many mesenchymal cells failed to migrate, and precartilage condensation was inhibited, after knockdown of endogenous cadherin-7 levels. Exposure of mesenchymal cells to SB203580 (a specific inhibitor of p38MAPK), LiCl (an inhibitor of GSK-3beta) or overexpression of beta-catenin resulted in inhibition of cadherin-7 levels and, subsequently, suppression of cell migration. Collectively, our results suggest that cadherin-7 controls cell migration in chick limb bud mesenchymal cells, and that p38MAPK and GSK signals are responsible for regulating cadherin-7-mediated cell migration.

Transforming growth factor-beta3-induced Smad signaling regulates actin reorganization during chondrogenesis of chick leg bud mesenchymal cells.

Endochondral ossification is characterized by a significant interdependence between cell shape and cytoskeletal organization that accompanies the onset of chondrogenic signaling. However, the mechanisms mediating these interactions have not been well studied. Here, treatment with transforming growth factor (TGF)-beta3 at a later stage of chondrogenesis led to activation of Smad-2 signaling and the formation of intense stress fibers, which resulted in suppressing chondrogenic differentiation of leg bud mesenchymal cells. Moreover, specific siRNA knockdown of Smad-2 reduced TGF-beta3-induced stress fibers via physical interactions with beta-catenin. In conclusion, our results indicate that TGF-beta3-induced Smad signaling, in conjunction with beta-catenin, is involved in the reorganization of the actin cytoskeleton into a cortical pattern with a concomitant rounding of cells. J. Cell. Biochem. (c) 2009 Wiley-Liss, Inc.This article was published online on 28 May 2009. An error was subsequently identified. This notice is included in the online and print versions to indicate that both have been corrected 8 June 2009.

Distribution of TGF-beta isoforms and signaling intermediates in corneal fibrotic wound repair.

In this study, temporal and spatial distribution of three TGF-beta isoforms and their downstream signaling pathways including pSmad2 and p38MAPK were examined during fibrotic wound repair. In normal chick corneas, TGF-beta1, -2, and -3 were weakly detected in Bowman's layer (BL). In healing corneas, TGF-beta1 was primarily deposited in the fibrin clot and the unwounded BL. TGF-beta2 was highly expressed in healing epithelial and endothelial cells, and numerous active fibroblasts/myofibroblasts. TGF-beta3 was mainly detected in the unwound region of basal epithelial cells. alpha-Smooth muscle actin (alpha-SMA) was initially appeared in the posterior region of repairing stroma at day 3, and was detected in the entire healing stroma by day 7. Notably, alpha-SMA was absent in the central region of healing stroma by day 14, and its staining pattern was similar to those of TGF-beta2 and p38MAPK. By contrast, pSmad2 was mainly detected in the fibroblasts. In normal cornea, laminin was mainly detected in both epithelial basement membrane (BM) and Descemet's membrane (DM). By contrast to reconstitution of the BM in the wound region, the DM was not repaired although endothelial layer was regenerated, indicating that high levels of TGF-beta2 were released into the posterior region of healing stroma on day 14. High levels of alpha-SMA staining, shown in cultured repair stromal cells from healing corneas on day 14 and in TGF-beta2 treated normal stromal cells, were significantly reduced by p38MAPK inhibition. Collectively, this study suggests that TGF-beta2-mediated myofibroblast transformation is mediated, at least partly, by the p38MAPK pathway in vivo.

BMP-2 treatment of C3H10T1/2 mesenchymal cells blocks MMP-9 activity during chondrocyte commitment.

Members of both the Wnt and bone morphogenetic protein (BMP) families of signaling molecules have been implicated in the regulation of cartilage development. We explored the underlying mechanism of BMP-2-induced chondrocyte commitment of C3H10T1/2 cells. Treating cells with exogenous BMP-2 was tied to chondrocyte commitment by inhibiting matrix metalloproteinase-9 activity (MMP-9: 92 kDa type IV collagenase/gelatinase B). Glycogen synthase kinase (GSK)-3beta inhibition by its specific inhibitor blocked BMP-2-induced chondrocyte commitment by stimulating MMP-9 activity. These findings indicate that the downregulation of MMP-9 by BMP-2 is associated with chondrocyte commitment, and that the GSK-3beta signaling pathway is involved in this process.

TGF-beta3 inhibits chondrogenesis of cultured chick leg bud mesenchymal cells via downregulation of connexin 43 and integrin beta4.

Transforming growth factor beta (TGF-beta) is a multifunctional cytokine that regulates a number of biological responses including chemotaxis, cell cycle progression, differentiation, and apoptosis of cells. Even though temporal and spatial expression of TGF-beta3 suggests its role in chick limb development, it is not well characterized how TGF-beta3 regulates chondrogenic differentiation of limb bud mesenchymal cells. In this study, differential display polymerase chain reaction (DD-PCR) screening and reverse transcription PCR analysis revealed that the mRNA expression of the gap junction protein, connexin 43 (Cx43), was significantly decreased during the first treatment of TGF-beta3 for 24 h in cultured chick leg bud mesenchymal cells. Treatment of these cells with lindane, a general gap junction blocker, or expression of dominant negative Cx43 increased apoptotic cell death and decreased the level of integrin beta4 protein, in a manner similar to that observed when these cells were exposed to TGF-beta3. Similarly, exposure of cultured leg chondroblasts to a functional blocking antibody against integrin-beta4 induced an increase in apoptosis. Treatment of cells with TGF-beta3 decreased the membrane translocation of PKC-alpha, leading to activation of ERK. The increase in apoptotic cell death triggered by TGF-beta3 and dominant negative Cx43 was blocked by inhibition of ERK but increased by inhibition of PKC. Collectively, these data indicate that, in cultured chick leg bud mesenchyme cells, TGF-beta3 treatment downregulates Cx43 and induces apoptotic cell death via downregulation of integrin beta4, activation of ERK and suppression of PKC-alpha activation.

Suppression of ADAM 10-induced Delta-1 shedding inhibits cell proliferation during the chondro-inhibitory action of TGF-beta3.

Although transforming growth factors (TGFs) are implicated in the process of endochondral ossification, which is initiated by the differentiation of mesenchymal cells into chondrocytes, it is not clear how TGF-beta 3 regulates the chondrogenic differentiation of limb bud mesenchymal cells. Here, differential display polymerase chain reaction (DD-PCR) screening and RT-PCR analysis revealed that transcripts of A Disintegrin And Metalloprotease 10 (ADAM 10) decreased during the chondro-inhibitory action of TGF-beta 3 on cultured chick leg bud mesenchymal cells. Electroporation of ADAM 10 morpholino antisense oligonucleotides inhibited the ectodomain shedding of delta-1, and cell proliferation and subsequent precartilage condensation, in a manner similar to that caused by TGF-beta3. The suppression of mesenchymal cell proliferation induced by TGF-beta 3 and ADAM 10 morpholino antisense oligonucleotides was reversed by activation of ADAM 10 with phorbol 12-myristate 13-acetate (PMA) or knockdown of Notch-1 with siRNA. Collectively, these data indicate that, in cultured chick leg bud mesenchyme cells, TGF-beta 3 downregulates ADAM 10 and inhibits cell proliferation and subsequent precartilage condensation by inhibiting the ectodomain shedding of delta-1, and that this results in the activation of Notch signaling.

Redifferentiation of dedifferentiated chondrocytes on chitosan membranes and involvement of PKCalpha and P38 MAP kinase.

To investigate the effects of chitosan on the redifferentiation of dedifferentiated chondrocytes, we used chondrocytes obtained from a micromass culture system. Micromass cultures of chick wing bud mesenchymal cells yielded differentiated chondrocytes, but these dedifferentiated during serial monolayer subculture. When the dedifferentiated chondrocytes were cultured on chitosan membranes they regained the phenotype of differentiated chondrocytes. Expression of protein kinase C (PKC) increased during chondrogenesis, decreased during dedifferentiation, and increased again during redifferentiation. Treatment of the cultures with phorbol 12-myristate 13-acetate (PMA) inhibited redifferentiation and down-regulated PKC. In addition, the expression of p38 mitogen-activated protein (MAP) kinase increased during redifferentiation, and its inhibition suppressed redifferentiation. These findings establish a culture system for producing chondrocytes, point to a new role of chitosan in the redifferentiation of dedifferentiated chondrocytes, and show that PKC and p38 MAP kinase activities are required for chondrocyte redifferentiation in this model system.

Wnt-5a is involved in TGF-beta3-stimulated chondrogenic differentiation of chick wing bud mesenchymal cells.

In this study we investigated whether signalling by TGF-beta3 and Wnt-5a cross-talk during chondrogenic differentiation of chick wing mesenchyme. Using differential display polymerase chain reaction screening, we found the expression of Wnt-5a to be significantly increased during transforming growth factor-beta3 (TGF-beta3)-induced precartilage condensation in mesenchyme micromass cultures. Transfection of cells with a Wnt-5a expression construct promoted precartilage condensation and chondrogenesis in micromass cultures, similar to that observed when chondrogenic-competent cells were exposed to TGF-beta3. Overexpression of Wnt-5a or treatment with TGF-beta3 stimulated the activation of protein kinase C-alpha (PKC-alpha) and p38 mitogen-activated protein kinase (MAPK), both positive regulators of chondrogenic differentiation. Inactivation of PKC-alpha and p38 MAPK by specific inhibitors abrogated chondrogenesis stimulated by both TGF-beta3 and Wnt-5a. Similarly, partial reduction in TGF-beta3-induced Wnt-5a expression by small interfering RNA resulted in decreased activities of PKC-alpha and p38 MAPK, and abolished the chondro-stimulatory effect of TGF-beta3. Collectively, these findings indicate that Wnt-5a, a non-canonical Wnt, can mediate the chondro-stimulatory effect of TGF-beta3 through upregulation of PKC-alpha and p38MAPK signaling.

BMP-2-enhanced chondrogenesis involves p38 MAPK-mediated down-regulation of Wnt-7a pathway.

The bone morphogenetic protein (BMP) family has been implicated in control of cartilage development. Here, we demonstrate that BMP-2 promotes chondrogenesis by activating p38 mitogen-activated protein kinase (MAPK), which in turn downregulates Wnt-7a/b-catenin signaling responsible for proteasomal degradation of Sox9. Exposure of mesenchymal cells to BMP-2 resulted in upregulation of Sox9 protein and a concomitant decrease in the level of b-catenin protein and Wnt-7a signaling. In agreement with this, the interaction of Sox9 with b-catenin was inhibited in the presence of BMP-2. Inhibition of the p38 MAPK pathway using a dominant negative mutant led to sustained Wnt-7a signaling and decreased Sox9 expression, with consequent inhibition of precartilage condensation and chondrogenic differentiation. Moreover, overexpression of b-catenin caused degradation of Sox9 via the ubiquitin/26S proteasome pathway. Our results collectively indicate that the increase in Sox9 protein resulting from downregulation of b-catenin/Wnt-7a signaling is mediated by p38 MAPK during BMP-2 induced chondrogenesis in chick wing bud mesenchymal cells.

Inactivation of NADP(+)-dependent isocitrate dehydrogenase by nitric oxide.

Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and oxidative damage is one of the primary functions of NADP(+)-dependent isocitrate dehydrogenase (ICDH) through to supply NADPH for antioxidant systems. NO donors such as S-nitrosothiols, diethylamine NONOate, spermine NONOate, and 3-morpholinosydnomine N-ethylcarbamide (SIN-1)/superoxide dismutase inactivated ICDH in a dose- and time-dependent manner. The inhibition of ICDH by S-nitrosothiol was partially reversed by thiol, such as dithiothreitol or 2-mercaptoethanol. Loss of enzyme activity was associated with the depletion of the cysteine-reactive 5,5'-dithiobis-(2-nitrobenzoate) and the loss of fluorescent probe N,N'-dimethyl-N(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethyleneamine accessible thiol groups. Using electrospray ionization mass spectrometry with tryptic digestion of protein, we found that nitric oxide forms S-nitrosothiol adducts on Cys305 and Cys387. These results indicate that S-nitrosylation of cysteine residues on ICDH is a mechanism involving the inactivation of ICDH by NO. The structural alterations of modified enzyme were indicated by the changes in protease susceptibility and intrinsic tryptophan fluorescence. When U937 cells were incubated with 200 microM SNAP for 1 h, a significant decrease in both cytosolic and mitochondrial ICDH activities were observed. Furthermore, stimulation with lipopolysaccharide significantly decreased intracellular ICDH activity in RAW 264.7 cells, and this effect was blocked by NO synthase inhibitor N(omega)-methyl-L-arginine. This result indicates that ICDH was also inactivated by endogenous NO. The NO-mediated damage to ICDH may result in the perturbation of cellular antioxidant defense mechanisms and subsequently lead to a pro-oxidant condition.

Interleukin-1beta stimulates matrix metalloproteinase-2 expression via a prostaglandin E2-dependent mechanism in human chondrocytes.

IL-1beta is known promote cyclooxygenase-2 (COX- 2) and matrix metalloproteinase-2 (MMP-2) expression. This study focuses on the characterization of the signaling cascade associated with IL-1beta-induced matrix metalloproteinase-2 (MMP-2) regulation in human chondrocytes. The decrease in collagen levels in the conditioned media was prevented by a broad spectrum MMP inhibitor, suggesting that IL-1beta promotes the proteolytic process leading to MMP-2 activation. IL-1beta-related MMP-2 expression was found to be dependent on prostaglandin E2 (PGE2) production. In addition, the induction of COX-2 and MMP-2 was inhibited by the pretreatment of chondrocytes with a SB203580 or Ro 31-8220, indicating the involvement of protein kinase C (PKC) or p38 mitogen-activated protein kinase (MAPK). However, there is no cross-talk between PKC and p38 MAPK in the IL-1beta-induced MMP-2 activation. Taken together, these results demonstrated that IL-1beta induces MMP-2 expression through the PGE2-dependent mechanism in human chondrocytes.

Expression of p21WAF1 is dependent on the activation of ERK during vitamin E-succinate-induced monocytic differentiation.

Vitamin E-succinate (VES) induced monocvtic differentiation of HL-60 human leukemia cells. Treatment with VES increased the nitroblue tetrazolium reduction activity, and the expression of monocyte specific cell surface antigen, CD14 and c-fms. During the monocytic differentiation of HL-60 cells that were induced by VES, the phosphorylation of extracellular signal-regulated protein kinase (ERK) was increased by 12 h and then gradually decreased to a level that was similar to that of the control. However, the phosphorylation levels of p38 and JNK, as well as the expression levels of ERK, p38, and JNK, were unchanged by the VES treatment. Treatment with VES also induced hypophosphorylation of the retinoblastoma protein and an increase of the p21WAF1 protein level. VES-induced ERK phosphorylation was abolished by the ERK inhibitor, PD98059, which resulted in a remarkable prevention of VES-induced monocytic differentiation. Inhibition of the ERK activity by PD98059 also diminished the VES-induced p21WAF1 protein expression, but did not change the phosphorylation state of the retinoblastoma protein. Collectively, these data suggest that the ERK signaling pathway mediates the up-regulation of the p21xWAF1 expression that is induced by VES, which is required for monocytic differentiation of HL 60 cells.

Receptor activator of nuclear factor-kappaB is induced by a rottlerin-sensitive and p38 MAP kinase-dependent pathway during monocyte differentiation.

Receptor activator of nuclear factor-kappaB (RANK) plays a central role in the regulation of osteoclast differentiation and activation, but the mechanisms underlying its expression remain to be elucidated. In the present study we showed that expression of RANK was strongly induced by phorbol-12-myristate-13-acetate (PMA) during monocyte differentiation of U937 cells, and was enhanced by concomitant treatment with vitamin D3. Induction was dramatically inhibited by protein kinase C (PKC) inhibitors such as rottlerin and Gö6983, but not by Gö6976. Interestingly, rottlerin, a selective inhibitor of PKCdelta, reduced PMA-induced RANK expression while having no effect on CD11b expression. However overexpression of wild type PKCdelta, or a kinase-inactive mutant, did not affect PMA-induction of RANK, suggesting that rottlerin inhibits PMA-induced expression of RANK via a PKCdelta-independent mechanism. Rottlerin also inhibited PMA-induced phosphorylation of p38 mitogen-activated protein kinase (p38MAPK), and the p38 MAPK inhibitor SB203580 inhibited induction of RANK. Rottlerin and SB203580 also substantially reduced RANK mRNA expression in mouse BMM cells stimulated with macrophage colony stimulating factor (M-CSF). Together, these results demonstrate that expression of RANK is dependent upon a rottlerin-sensitive and p38MAPK-dependent pathway during monocyte differentiation.