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.

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.

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.

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

Vertically aligned, laterally spaced nanoscale titanium nanotubes were grown on a titanium surface by anodization, and the growth of chondroprogenitors on the resulting surfaces was investigated. Surfaces bearing nanotubes of 70 to 100 nm in diameter were found to trigger the morphological transition to a cortical actin pattern and rounded cell shape (both indicative of chondrocytic differentiation), as well as the up-regulation of type II collagen and integrin beta4 protein expression through the down-regulation of Erk activity. Inhibition of Erk signaling reduced stress fiber formation and induced the transition to the cortical actin pattern in cells cultured on 30-nm-diameter nanotubes, which maintained their fibroblastoid morphologies in the absence of Erk inhibition. Collectively, these results indicate that a titanium-based nanotube surface can support chondrocytic functions among chondroprogenitors, and may therefore be useful for future cartilaginous applications.

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.

p53-independent induction of G1 arrest and p21WAF1/CIP1 expression by ascofuranone, an isoprenoid antibiotic, through downregulation of c-Myc.

Ascofuranone has been shown to have antitumor activity, but the precise molecular mechanism by which it inhibits the proliferation of cancer cells remains unclear. Here, we study the effects of ascofuranone on cell cycle progression in human cancer cells and find that ascofuranone induces G(1) arrest without cytoxicity with upregulation of p53 and p21(WAF1/CIP1) while downregulating c-Myc and G(1) cyclins. Chromatin immunoprecipitation assay and RNA interference studies with cells deficient in p53 and p21 show that ascofuranone induces p21(WAF1/CIP1) expression and subsequent G(1) arrest through the release of p21(WAF1/CIP1) promoter from c-Myc-mediated transcriptional repression, independent of p53. Ascofuranone-induced p21(WAF1/CIP1) associates with CDK2 and prevents CDK2-cyclin E complex formation, leading to the inactivation of E2F transcriptional activity. These results suggest that ascofuranone upregulates p21(WAF1/CIP1) through p53-independent suppression of c-Myc expression, leading to cytostatic G(1) arrest. Thus, ascofuranone represents a unique natural antitumor compound that targets c-Myc independent of p53.

TGF-beta3 inhibits chondrogenesis by suppressing precartilage condensation through stimulation of N-cadherin shedding and reduction of cRREB-1 expression.

Transforming growth factor-beta (TGF-beta) plays crucial roles in controlling cell differentiation and maintaining tissue integrity. Previously we reported that TGF-beta3 treatment decreased the mRNA expression of the gap junction protein, connexin 43 as well as cell number, which lead to the inhibition of chondrogenic condensation in cultured chick leg bud mesenchymal cells. The present study demonstrates that TGF-beta3 can induce cleavage in the ectodomain of neuronal cadherin (N-cadherin) at the initiation stage of chondrogenesis and reduce cell numbers, cellular adhesion and the expression level of connexin 43. Differential displayed PCR (DD-PCR) comparison of adherent- and non-adherent chick leg chondrogenic progenitor cells showed increased expression of the chick ras-responsive element binding transcription factor, cRREB-1, in adherent cells. In chick leg bud mesenchymal cells, cRREB-1 transcription was inhibited by TGF-beta3 at the early stage of chondrogenesis. Small interfering RNA (siRNA)-mediated knockdown of cRREB-1 reduced cell numbers, cellular adhesion, and the expression level of connexin 43 resulting in the inhibition of precartilage condensation. Taken together, these findings indicate that TGF-beta3 mediates the inhibitory signal necessary for precartilage condensation by stimulating N-cadherin shedding and reducing cRREB-1 expression levels.

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.

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.

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.

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.

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

Curcumin is a well known natural polyphenol product isolated from the rhizome of the plant Curcuma longa, anti-inflammatory agent for arthritis by inhibiting synthesis of inflammatory prostaglandins. However, the mechanisms by which curcumin regulates the functions of chondroprogenitor, such as proliferation, precartilage condensation, cytoskeletal organization or overall chondrogenic behavior, are largely unknown. In the present report, we investigated the effects and signaling mechanism of curcumin on the regulation of chondrogenesis. Treating chick limb bud mesenchymal cells with curcumin suppressed chondrogenesis by stimulating apoptotic cell death. It also inhibited reorganization of the actin cytoskeleton into a cortical pattern concomitant with rounding of chondrogenic competent cells and down-regulation of integrin beta1 and focal adhesion kinase (FAK) phosphorylation. Curcumin suppressed the phosphorylation of Akt leading to Akt inactivation. Activation of Akt by introducing a myristoylated, constitutively active form of Akt reversed the inhibitory actions of curcumin during chondrogenesis. In summary, for the first time, we describe biological properties of curcumin during chondrogenic differentiation of chick limb bud mesenchymal cells. Curcumin suppressed chondrogenesis by stimulating apoptotic cell death and down-regulating integrin-mediated reorganization of actin cytoskeleton via modulation of Akt signaling.

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.

Integrin signaling and cell spreading alterations by rottlerin treatment of chick limb bud mesenchymal cells.

Endochondral skeletal development begins with the formation of a cartilaginous template where mesenchymal cells aggregate and increase in density prior to their overt differentiation into chondrocytes. Prechondrogenic condensation, in which mesenchymal cells aggregate, requires cell migration and proliferation. However, the molecular mechanisms promoting this aggregation remain to be elucidated. Here, we report that rottlerin suppresses migration and cell surface expression of integrin beta1 in chondrogenic progenitors. Perturbation of integrin beta1 function using an anti-integrin beta1 blocking antibody suppressed the migration of wing bud mesenchymal cells. Furthermore, phosphorylation levels of Src and focal adhesion kinase (FAK) were decreased by rottlerin treatment. Cell treatment with PP2, an inhibitor of Src family kinase, or electroporation of FAK specific siRNA, suppressed cell migration in a wound-healing assay. Cells treated with rottlerin showed decreased phosphorylation of Akt, independent of PKCdelta inhibition. In addition, an Akt inhibitor suppressed the migration of chick limb bud mesenchymal cells. Taken together, our results point to the novel finding that rottlerin may act as a negative regulator for cell migration, an essential step for prechondrogenic condensation, by regulating integrin beta1 signaling at focal adhesion complexes via modulation of Akt activity.

Enhanced ex vivo expansion of human adipose tissue-derived mesenchymal stromal cells by fibroblast growth factor-2 and dexamethasone.

In the present study, we investigated the ex vivo expansion of human adipose tissue-derived mesenchymal stromal cells (ATSCs) to identify factors that promoted efficient expansion while preserving stem cell potential. We examined several growth factors and steroids, and found that the combination of a low concentration of fibroblast growth factor-2 (FGF-2) (1 ng/mL) and dexamethasone (DEX) or betamethasone (BET) enhanced the proliferation of ATSCs by approximately 30-60% as compared to control. Enhanced proliferation under these conditions was confirmed using ATSCs isolated from three independent donors. ATSCs that were expanded in the presence of FGF-2 and DEX for 5 days were capable of differentiating into either osteoblastic or adipogenic cells, and the cells were positive for the mesenchymal stem cell markers such as CD29, CD44, CD90, CD105, and CD146, suggesting that the stem cell potential of the ATSCs was preserved. Analysis of signaling pathway revealed that tyrosine phosphorylation of Src kinase was dramatically increased in response to FGF-2 and DEX, suggesting the involvement of Src-dependent pathways in the stimulatory mechanism of proliferation of ATSCs by FGF-2 and DEX. Moreover, Src family kinase inhibitors (SU6656 and Src kinase inhibitor I) substantially reduced the FGF-2 and DEX-induced proliferation of ATSCs. SU6656 also inhibited the osteogenic and adipogenic differentiation of ATSCs. The results of the current study demonstrate that FGF-2 in combination with DEX stimulates the proliferation and osteoblastic and adipogenic differentiation of ATSCs through a Src-dependent mechanism, and that FGF-2 and DEX promote the efficient ex vivo expansion of ATSCs.

Integrity of the cortical actin ring is required for activation of the PI3K/Akt and p38 MAPK signaling pathways in redifferentiation of chondrocytes on chitosan.

Cell shape alterations and accompanying cytoskeletal changes have diverse effects on cell function. We have already shown that dedifferentiated chondrocytes have a round cell morphology and undergo redifferentiation when cultured on chitosan membrane. In the present study, we investigate the role of the cytoskeleton in chondrocyte redifferentiation. Chondrocytes obtained from a micromass culture of chick limb bud mesenchymal cells were subcultured four times. Immunofluorescence analysis of F-actin showed cortical distribution of the actin cytoskeleton upon subculture of dedifferentiated chondrocytes on chitosan membrane. Treatment with cytochalasin D disrupted the cortical actin ring formed during cultivation of chondrocytes on the chitosan membrane, and inhibited chondrocyte redifferentiation. Moreover, cytochalasin D inhibited the phosphorylation of Akt and p38 mitogen activated protein kinase (MAPK), induced during redifferentiation on chitosan membrane. LY294002, an inhibitor of phosphatidylinositol-3-OH-kinase (PI3K), suppressed chondrocyte redifferentiation. These findings suggest that integrity of the actin cytoskeleton is a crucial requirement for PI3K/Akt and p38 MAPK in chondrocyte redifferentiation.

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.

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.