Osteoarthritic cartilage chondrocytes alter subchondral bone osteoblast differentiation via MAPK signalling pathway involving ERK1/2

Osteoarthritic subchondral bone is characterized by abnormal bone density and enhanced production of bone turnover markers, an indication of osteoblast dysfunction. Several studies have proposed that pathological changes in articular cartilage influence the subchondral bone changes, which are typical of the progression of osteoarthritis; however, direct evidence of this has yet to be reported. The aim of the present study was to investigate what effects articular cartilage cells, isolated from normal and osteoarthritic joints, may have on the subchondral bone osteoblast phenotype, and also the potential involvement of the mitogen activated protein kinase (MAPK) signalling pathway during this process. Our results suggest that chondrocytes isolated from a normal joint inhibited osteoblast differentiation, whereas chondrocytes isolated from an osteoarthritic joint enhanced osteoblast differentiation, both via a direct and indirect cell interaction mechanisms. Furthermore, the interaction of subchondral bone osteoblasts with osteoarthritic chondrocyte conditioned media appeared to significantly activate ERK1/2 phosphorylation. On the other hand, conditioned media from normal articular chondrocytes did not affect ERK1/2 phosphorylation. Inhibition of the MAPK–ERK1/2 pathways reversed the phenotype changes of subchondral bone osteoblast, which would otherwise be induced by the conditioned media from osteoarthritic chondrocytes. In conclusion, our findings provide evidence that osteoarthritic chondrocytes affect subchondral bone osteoblast metabolism via an ERK1/2 dependent pathway.

Synergism between Wnt3a and heparin enhances osteogenesis via a phosphoinositide 3-kinase/Akt/RUNX2 pathway

A new strategy has emerged to improve healing of bone defects using exogenous glycosaminoglycans by increasing the effectiveness of bone-anabolic growth factors. Wnt ligands play an important role in bone formation. However, their functional interactions with heparan sulfate/heparin have only been investigated in non-osseous tissues. Our study now shows that the osteogenic activity of Wnt3a is cooperatively stimulated through physical interactions with exogenous heparin. N-Sulfation and to a lesser extent O-sulfation of heparin contribute to the physical binding and optimal co-stimulation of Wnt3a. Wnt3a-heparin signaling synergistically increases osteoblast differentiation with minimal effects on cell proliferation. Thus, heparin selectively reduces the effective dose of Wnt3a needed to elicit osteogenic, but not mitogenic responses. Mechanistically, Wnt3a-heparin signaling strongly activates the phosphoinositide 3-kinase/Akt pathway and requires the bone-related transcription factor RUNX2 to stimulate alkaline phosphatase activity, which parallels canonical beta-catenin signaling. Collectively, our findings establish the osteo-inductive potential of a heparin-mediated Wnt3a-phosphoinositide 3-kinase/Akt-RUNX2 signaling network and suggest that heparan sulfate supplementation may selectively reduce the therapeutic doses of peptide factors required to promote bone formation.

Menstrual effluent induces epithelial-mesenchymal transitions in mesothelial cells

Background Menstrual effluent affects mesothelial cell (MC) morphology. We evaluated whether these changes were consistent with epithelial-mesenchymal transitions (EMT). Methods Monolayer cultures of MC were incubated overnight in conditioned media, prepared from cells isolated form menstrual effluent, with or without kinase and ATP inhibitors. Changes in cell morphology were monitored using time-lapse video microscopy and immunohistochemistry. Effects on the expression of EMT-associated molecules were evaluated using real-time RT-PCR and/or Western blot analysis. Results Incubation in conditioned media disrupted cell-cell contacts, and increased MC motility. The changes were reversible. During the changes the distribution of cytokeratins, fibrillar actin and α-tubulin changed. Sodium azide, an inhibitor of ATP production, and Genistein, a general tyrosine kinase inhibitor, antagonized these effects. Wortmannin, a phosphatidylinositol 3-kinase inhibitor, and SU6656, an Src tyrosine kinase inhibitor, only partially antagonized the effect. The expression of Snail and vimentin was markedly up-regulated, whereas the expression of E-cadherin was decreased and cytokeratins were altered. Conclusions In MC, menstrual effluent initiates a reversible, energy-dependent transition process from an epithelial to a mesenchymal phenotype. Involvement of the (Src) tyrosine kinase signalling pathway and the changes in the expression of cytokeratins, Snail, vimentin and E-cadherin demonstrate that the morphological changes are EMT.

Sulfated glycosaminoglycans mediate the effects of FGF2 on the osteogenic potential of rat calvarial osteoprogenitor cells

Fibroblast growth factor-2 (FGF2) is a powerful promoter of bone growth. We demonstrate here that brief exposure to FGF2 enhances mineralized nodule formation in cultured rat osteoprogenitor cells due to an expansion of cells that subsequently mineralize. This mitogenic effect is mediated via sulfated glycosaminoglycans (GAGs), FGFR1, and the extracellular signal-regulated kinase (ERK) pathway. The GAGs involved in this stimulation are chondroitin sulfates (CS) rather than heparan sulfates (HS). However, continuous FGF2 treatment reduces alkaline phosphatase (ALP) activity, downregulates collagen Ialpha1 (ColIalpha1) and FGFR3 expression, upregulates the expression and secretion of osteopontin (OPN) and inhibits mineralization. The inhibitory effects of FGF2 on FGFR3 expression and ALP activity are also mediated by the ERK pathway, although the effects of FGF2 on ColIalpha1 and OPN expression are mediated by GAGs and PKC activity. Thus short-term activation of FGF2/FGFR1 promotes osteoprogenitor proliferation and subsequent differentiation, while long-term activation of FGF2 signaling disrupts mineralization by modulating osteogenic marker expression. This study thus establishes the central role of sulfated GAGs in the osteogenic progression of osteoprogenitors.

Influence of cytochrome P-450 inhibitors on the inhalative uptake of methyl chloride and methylene chloride in male B6C3F1 mice

Dichloromethane (DCM) is thought to be metabolized in vivo by two independent pathways: a glutathione (GSH) dependent pathway that yields CO2 and a cytochrome P-450 mediated one that yields both CO and CO2 (Gargas et al 1986). With a physiologically based pharmacokinetic (PB-PK) model, Andersen et al (1987) calculate the quantitative parameters for both metabolic pathways. Using the kinetic parameters thus obtained and the results of two carcinogenicity studies with rodents (Serota et al 1986; NTP 1985), the authors then estimate the tumour risk for humans.

Species differences in acrylonitrile metabolism and toxicity between experimental animals and humans based on observations in human accidental poisonings

The high acute toxicity of acrylonitrile may be a result of its intrinsic biological reactivity or of its metabolite cyanide. Intravenous N-acetylcysteine has been recommended for treatment of accidental intoxications in acrylonitrile workers, but such recommendations vary internationally. Acrylonitrile is metabolized in humans and experimental animals via two competing pathways; the glutathione-dependent pathway is considered to represent an avenue of detoxication whilst the oxidative pathway leads to a genotoxic epoxide, cyanoethylene oxide, and to elimination of cyanide. Cases of acute acrylonitrile overexposure or intoxication have occurred within persons having industrial contact with acrylonitrile; the route of exposure was by inhalation and/or by skin contact. The combined observations lead to the conclusion of a much higher impact of the oxidative metabolism of acrylonitrile in humans than in rodents. This is confirmed by differences in the clinical picture of acute life-threatening intoxications in both species, as well as by differential efficacies of antidotes. A combination of N-acetylcysteine with sodium thiosulfate seems an appropriate measure for antidote therapy of acute acrylonitrile intoxications. Clinical observations also highlight the practical importance of human individual susceptibility differences. Furthermore, differential adduct monitoring, assessing protein adducts with different rates of decay, enables the development of more elaborated biological monitoring strategies for the surveillance of workers with potential acrylonitrile contact.

Estrogen receptor α regulates area-adjusted bone mineral content in late pubertal girls

Context: Whether the action of estrogen in skeletal development depends on estrogen receptor α as encoded by the ESR1 gene is unknown. Objectives: The aim of this study was to establish whether the gain in area-adjusted bone mineral content (ABMC) in girls occurs in late puberty and to examine whether the magnitude of this gain is related to ESR1 polymorphisms. Design: We conducted a cross-sectional analysis. Setting: The study involved the Avon Longitudinal Study of Parents and Children (ALSPAC), a population-based prospective study. Participants: Participants included 3097 11-yr-olds with DNA samples, dual x-ray absorptiometry measurements, and pubertal stage information. Outcomes: Outcome measures included separate prespecified analyses in boys and girls of the relationship between ABMC derived from total body dual x-ray absorptiometry scans and Tanner stage and of the interaction between ABMC, Tanner stage, and ESR1 polymorphisms. Results: Total body less head and spinal ABMC were higher in girls in Tanner stages 4 and 5, compared with those in Tanner stages 1, 2, and 3. In contrast, height increased throughout puberty. No differences were observed in ABMC according to Tanner stage in boys. For rs2234693 (PvuII) and rs9340799 (XbaI) polymorphisms, differences in spinal ABMC in late puberty were 2-fold greater in girls who were homozygous for the C and G alleles, respectively (P = 0.001). For rs7757956, the difference in total body less head ABMC in late puberty was 50% less in individuals homozygous or heterozygous for the A allele (P = 0.006). Conclusions: Gains in ABMC in late pubertal girls are strongly associated with ESR1 polymorphisms, suggesting that estrogen contributes to this process via an estrogen receptor α-dependent pathway.

Gonadotropin-induced ovarian cancer cell migration and proliferation require extracellular signal-regulated kinase 1/2 activation regulated by calcium and protein kinase Cδ

The gonadotropin hypothesis proposes that elevated serum gonadotropin levels may increase the risk of epithelial ovarian cancer (EOC). We have studied the effect of treating EOC cell lines (OV207 and OVCAR-3) with FSH or LH. Both gonadotropins activated the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase 1/2 (ERK1/2) pathway and increased cell migration that was inhibited by the MAPK 1 inhibitor PD98059. Both extra- and intracellular calcium ion signalling were implicated in gonadotropin-induced ERK1/2 activation as treatment with either the calcium chelator EGTA or an inhibitor of intracellular calcium release, dantrolene, inhibited gonadotropin-induced ERK1/2 activation. Verapamil was also inhibitory, indicating that gonadotropins activate calcium influx via L-type voltage-dependent calcium channels. The cAMP/protein kinase A (PKA) pathway was not involved in the mediation of gonadotropin action in these cells as gonadotropins did not increase intracellular cAMP formation and inhibition of PKA did not affect gonadotropin-induced phosphorylation of ERK1/2. Activation of ERK1/2 was inhibited by the protein kinase C (PKC) inhibitor GF 109203X as well as by the PKCδ inhibitor rottlerin, and downregulation of PKCδ was inhibited by small interfering RNA (siRNA), highlighting the importance of PKCδ in the gonadotropin signalling cascade. Furthermore, in addition to inhibition by PD98059, gonadotropin-induced ovarian cancer cell migration was also inhibited by verapamil, GF 109203X and rottlerin. Similarly, gonadotropin-induced proliferation was inhibited by PD98059, verapamil, GF 109203X and PKCδ siRNA. Taken together, these results demonstrate that gonadotropins induce both ovarian cancer cell migration and proliferation by activation of ERK1/2 signalling in a calcium- and PKCδ-dependent manner.

Osteopontin is a downstream effector of the PI3-kinase pathway in melanomas that is inversely correlated with functional PTEN

The tumor suppressor PTEN antagonizes phosphatidylinositol 3-kinase (PI3K), which contributes to tumorigenesis in many cancer types. While PTEN mutations occur in some melanomas, their precise mechanistic consequences have yet to be elucidated. We sought to identify novel downstream effectors of PI3K using a combination of genomic and functional tests. Microarray analysis of 53 melanoma cell lines identified 610 genes differentially expressed (P<0.05) between wild-type lines and those with PTEN aberrations. Many of these genes are known to be involved in the PI3K pathway and other signaling pathways influenced by PTEN. Validation of differential gene expression by qRT-PCR was performed in the original 53 cell lines and an independent set of 18 melanoma lines with known PTEN status. Osteopontin (OPN), a secreted glycophosphoprotein that contributes to tumor progression, was more abundant at both the mRNA and protein level in PTEN mutants. The inverse correlation between OPN and PTEN expression was validated (P<0.02) by immunohistochemistry using melanoma tissue microarrays. Finally, treatment of cell lines with the PI3K inhibitor LY294002 caused a reduction in expression of OPN. These data indicate that OPN acts downstream of PI3K in melanoma and provides insight into how PTEN loss contributes to melanoma development.

Cyclin-dependent kinase-mediated phosphorylation of RBP1 and pRb promotes their dissociation to mediate release of the SAP30·mSin3·HDAC transcriptional repressor complex

Eukaryotic cell cycle progression is mediated by phosphorylation of protein substrates by cyclin-dependent kinases (CDKs). A critical substrate of CDKs is the product of the retinoblastoma tumor suppressor gene, pRb, which inhibits G1-S phase cell cycle progression by binding and repressing E2F transcription factors. CDK-mediated phosphorylation of pRb alleviates this inhibitory effect to promote G1-S phase cell cycle progression. pRb represses transcription by binding to the E2F transactivation domain and recruiting the mSin3·histone deacetylase (HDAC) transcriptional repressor complex via the retinoblastoma-binding protein 1 (RBP1). RBP1 binds to the pocket region of pRb via an LXCXE motif and to the SAP30 subunit of the mSin3·HDAC complex and, thus, acts as a bridging protein in this multisubunit complex. In the present study we identified RBP1 as a novel CDK substrate. RBP1 is phosphorylated by CDK2 on serines 864 and 1007, which are N- and C-terminal to the LXCXE motif, respectively. CDK2-mediated phosphorylation of RBP1 or pRb destabilizes their interaction in vitro, with concurrent phosphorylation of both proteins leading to their dissociation. Consistent with these findings, RBP1 phosphorylation is increased during progression from G 1 into S-phase, with a concurrent decrease in its association with pRb in MCF-7 breast cancer cells. These studies provide new mechanistic insights into CDK-mediated regulation of the pRb tumor suppressor during cell cycle progression, demonstrating that CDK-mediated phosphorylation of both RBP1 and pRb induces their dissociation to mediate release of the mSin3·HDAC transcriptional repressor complex from pRb to alleviate transcriptional repression of E2F.

Increased expression of cyclin-dependent kinase inhibitor 2 (CDKN2A) gene product P16INK4A in ovarian cancer is associated with progression and unfavourable prognosis

Paraffin sections from 190 epithelial ovarian tumours, including 159 malignant and 31 benign epithelial tumours, were analysed immunohistochemically for expression of cyclin-dependent kinase inhibitor 2 (CDKN2A) gene product p16INK4A (p16). Most benign tumours showed no p16 expression in the tumour cells, whereas only 11% of malignant cancers were p16 negative. A high proportion of p16-positive tumour cells was associated with advanced stage and grade, and with poor prognosis in cancer patients. For FIGO stage 1 tumours, a high proportion of p16-positive tumour cells was associated with poorer survival, suggesting that accumulation of p16 is an early event of ovarian tumorigenesis. In contrast to tumour cells, high expression of p16 in the surrounding stromal cells was not associated with the stage and grade, but was associated with longer survival. When all parameters were combined in multivariate analysis, high p16 expression in stromal cells was not an independent predictor for survival, indicating that low p16 expression in stromal cells is associated with other markers of tumour progression. High expression of p16 survival in the stromal cells of tumours from long-term survivors suggests that tumour growth is limited to some extent by factors associated with p16 expression in the matrix.

An extracellular signal-regulated kinase 2 survival pathway mediates resistance of human mesothelioma cells to asbestos-induced injury

We hypothesized that normal human mesothelial cells acquire resistance to asbestos-induced toxicity via induction of one or more epidermal growth factor receptor (EGFR) - linked survival pathways (phosphoinositol-3-kinase/AKT/ mammalian target of rapamycin and extracellular signal - regulated kinase [ERK] 1/2) during simian virus 40 (SV40) transformation and carcinogenesis. Both isolated HKNM-2 mesothelial cells and a telomerase-immortalized mesothelial line (LP9/TERT-1) were more sensitive to crocidolite asbestos toxicity than an SV40 Tag-immortalized mesothelial line (MET5A) and malignant mesothelioma cell lines (HMESO and PPM Mill). Whereas increases in phosphorylation of AKT (pAKT) were observed in MET5A cells in response to asbestos, LP9/TERT-1 cells exhibited dose-related decreases in pAKT levels. Pretreatment with an EGFR phosphorylation or mitogen-activated protein kinase kinase 1/2 inhibitor abrogated asbestos-induced phosphorylated ERK (pERK) 1/2 levels in both LP9/TERT-1 and MET5A cells as well as increases in pAKT levels in MET5A cells. Transient transfection of small interfering RNAs targeting ERK1, ERK2, or AKT revealed that ERK1/2 pathways were involved in cell death by asbestos in both cell lines. Asbestos-resistant HMESO or PPM Mill cells with high endogenous levels of ERKs or AKT did not show dose-responsive increases in pERK1/ERK1, pERK2/ERK2, or pAKT/AKT levels by asbestos. However, small hairpin ERK2 stable cell lines created from both malignant mesothelioma lines were more sensitive to asbestos toxicity than shERK1 and shControl lines, and exhibited unique, tumor-specific changes in endogenous cell death - related gene expression. Our results suggest that EGFR phosphorylation is causally linkedto pERK and pAKT activation by asbestos in normal and SV40 Tag - immortalized human mesothelial cells. They also indicate that ERK2 plays a role in modulating asbestos toxicity by regulating genes critical to cell injury and survival that are differentially expressed in human mesotheliomas.

Induction of epithelial-mesenchymal transition (EMT) in breast cancer cells is calcium signal dependent

Signals from the tumor microenvironment trigger cancer cells to adopt an invasive phenotype through epithelial-mesenchymal transition (EMT). Relatively little is known regarding key signal transduction pathways that serve as cytosolic bridges between cell surface receptors and nuclear transcription factors to induce EMT. A better understanding of these early EMT events may identify potential targets for the control of metastasis. One rapid intracellular signaling pathway that has not yet been explored during EMT induction is calcium. Here we show that stimuli used to induce EMT produce a transient increase in cytosolic calcium levels in human breast cancer cells. Attenuation of the calcium signal by intracellular calcium chelation significantly reduced epidermal growth factor (EGF)- and hypoxia-induced EMT. Intracellular calcium chelation also inhibited EGF-induced activation of signal transducer and activator of transcription 3 (STAT3), while preserving other signal transduction pathways such as Akt and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. To identify calcium-permeable channels that may regulate EMT induction in breast cancer cells, we performed a targeted siRNA-based screen. We found that transient receptor potential-melastatin-like 7 (TRPM7) channel expression regulated EGF-induced STAT3 phosphorylation and expression of the EMT marker vimentin. Although intracellular calcium chelation almost completely blocked the induction of many EMT markers, including vimentin, Twist and N-cadherin, the effect of TRPM7 silencing was specific for vimentin protein expression and STAT3 phosphorylation. These results indicate that TRPM7 is a partial regulator of EMT in breast cancer cells, and that other calcium-permeable ion channels are also involved in calcium-dependent EMT induction. In summary, this work establishes an important role for the intracellular calcium signal in the induction of EMT in human breast cancer cells. Manipulation of calcium-signaling pathways controlling EMT induction in cancer cells may therefore be an important therapeutic strategy for preventing metastases.

Epidermal growth factor promotes MDA-MB-231 breast cancer cell migration through a phosphatidylinositol 3'-kinase and phospholipase C-dependent mechanism

Epidermal growth factor receptor (EGFR) levels predict a poor outcome in human breast cancer and are most commonly associated with proliferative effects of epidermal growth factor (EGF), with little emphasis placed on motogenic responses to EGF. We found that MDA-MB-231 human breast cancer cells elicited a potent chemotactic response despite their complete lack of a proliferative response to EGF. Antagonists of EGFR ligation, the EGFR kinase, phosphatidylinositol 3'-kinase, and phospholipase C, but not the mitogen- activated protein kinases (extracellular signal-regulated protein kinase 1 and 2), blocked MDA-MB-231 chemotaxis. These findings suggest that EGF may influence human breast cancer progression via migratory pathways, the signaling for which appears to be dissociated, at least in part, from the proliferative pathways.

Atmospheric gas plasma–induced ROS production activates TNF-ASK1 pathway for the induction of melanoma cancer cell apoptosis

Atmospheric gas plasmas (AGPs) are able to selectively induce apoptosis in cancer cells, offering a promising alternative to conventional therapies that have unwanted side effects such as drug resistance and toxicity. However, the mechanism of AGP-induced cancer cell death is unknown. In this study, AGP is shown to up-regulate intracellular reactive oxygen species (ROS) levels and induce apoptosis in melanoma but not normal melanocyte cells. By screening genes involved in apoptosis, we identify tumor necrosis factor (TNF)-family members as the most differentially expressed cellular genes upon AGP treatment of melanoma cells. TNF receptor 1 (TNFR1) antagonist-neutralizing antibody specifically inhibits AGP-induced apoptosis signal, regulating apoptosis signal-regulating kinase 1 (ASK1) activity and subsequent ASK1-dependent apoptosis. Treatment of cells with intracellular ROS scavenger N-acetyl-l-cysteine also inhibits AGP-induced activation of ASK1, as well as apoptosis. Moreover, depletion of intracellular ASK1 reduces the level of AGP-induced oxidative stress and apoptosis. The evidence for TNF-signaling dependence of ASK1-mediated apoptosis suggests possible mechanisms for AGP activation and regulation of apoptosis-signaling pathways in tumor cells.