Opposing actions of extracellular signal-regulated kinase (ERK) and signal transducer and activator of transcription 3 (STAT3) in regulating microtubule stabilization during cardiac hypertrophy.

Excessive proliferation and stabilization of the microtubule (MT) array in cardiac myocytes can accompany pathological cardiac hypertrophy, but the molecular control of these changes remains poorly characterized. In this study, we examined MT stabilization in two independent murine models of heart failure and revealed increases in the levels of post-translationally modified stable MTs, which were closely associated with STAT3 activation. To explore the molecular signaling events contributing to control of the cardiac MT network, we stimulated cardiac myocytes with an α-adrenergic agonist phenylephrine (PE), and observed increased tubulin content without changes in detyrosinated (glu-tubulin) stable MTs. In contrast, the hypertrophic interleukin-6 (IL6) family cytokines increased both the glu-tubulin content and glu-MT density. When we examined a role for ERK in regulating cardiac MTs, we showed that the MEK/ERK-inhibitor U0126 increased glu-MT density in either control cardiac myocytes or following exposure to hypertrophic agents. Conversely, expression of an activated MEK1 mutant reduced glu-tubulin levels. Thus, ERK signaling antagonizes stabilization of the cardiac MT array. In contrast, inhibiting either JAK2 with AG490, or STAT3 signaling with Stattic or siRNA knockdown, blocked cytokine-stimulated increases in glu-MT density. Furthermore, the expression of a constitutively active STAT3 mutant triggered increased glu-MT density in the absence of hypertrophic stimulation. Thus, STAT3 activation contributes substantially to cytokine-stimulated glu-MT changes. Taken together, our results highlight the opposing actions of STAT3 and ERK pathways in the regulation of MT changes associated with cardiac myocyte hypertrophy.

c-Jun N-terminal kinase/c-Jun inhibits fibroblast proliferation by negatively regulating the levels of stathmin/oncoprotein 18.

The JNKs (c-Jun N-terminal kinases) are stress-activated serine/threonine kinases that can regulate both cell death and cell proliferation. We have developed a cell system to control JNK re-expression at physiological levels in JNK1/2-null MEFs (murine embryonic fibroblasts). JNK re-expression restored basal and stress-activated phosphorylation of the c-Jun transcription factor and attenuated cellular proliferation with increased cells in G1/S-phase of the cell cycle. To explore JNK actions to regulate cell proliferation, we evaluated a role for the cytosolic protein, STMN (stathmin)/Op18 (oncoprotein 18). STMN, up-regulated in a range of cancer types, plays a crucial role in the control of cell division through its regulation of microtubule dynamics of the mitotic spindle. In JNK1/2-null or c-Jun-null MEFs or cells treated with c-Jun siRNA (small interfering RNA), STMN levels were significantly increased. Furthermore, a requirement for JNK/cJun signalling was demonstrated by expression of wild-type c-Jun, but not a phosphorylation-defective c-Jun mutant, being sufficient to down-regulate STMN. Critically, shRNA (small hairpin RNA)-directed STMN down-regulation in JNK1/2-null MEFs attenuated proliferation. Thus JNK/c-Jun regulation of STMN levels provides a novel pathway in regulation of cell proliferation with important implications for understanding the actions of JNK as a physiological regulator of the cell cycle and tumour suppressor protein.

Reduced SOCS1 Expression in Lung Fibroblasts from Patients with IPF Is Not Mediated by Promoter Methylation or Mir155.

The interleukin (IL)-6 family of cytokines and exaggerated signal transducer and activator of transcription (STAT)3 signaling is implicated in idiopathic pulmonary fibrosis (IPF) pathogenesis, but the mechanisms regulating STAT3 expression and function are unknown. Suppressor of cytokine signaling (SOCS)1 and SOCS3 block STAT3, and low SOCS1 levels have been reported in IPF fibroblasts and shown to facilitate collagen production. Fibroblasts and lung tissue from IPF patients and controls were used to examine the mechanisms underlying SOCS1 down-regulation in IPF. A significant reduction in basal SOCS1 mRNA in IPF fibroblasts was confirmed. However, there was no difference in the kinetics of activation, and methylation of SOCS1 in control and IPF lung fibroblasts was low and unaffected by 5'-aza-2'-deoxycytidine' treatment. SOCS1 is a target of microRNA-155 and although microRNA-155 levels were increased in IPF tissue, they were reduced in IPF fibroblasts. Therefore, SOCS1 is not regulated by SOCS1 gene methylation or microRNA155 in these cells. In conclusion, we confirmed that IPF fibroblasts had lower levels of SOCS1 mRNA compared with control fibroblasts, but we were unable to determine the mechanism. Furthermore, although SOCS1 may be important in the fibrotic process, we were unable to find a significant role for SOCS1 in regulating fibroblast function.

MicroRNA Signatures in Malignant Pleural Mesothelioma Effusions.

Malignant pleural mesothelioma (MPM) is an incurable cancer of the pleura that can be difficult to diagnose. Biomarkers for an easier and/or earlier diagnosis are needed. Approximately 90% of MPM patients develop a pleural effusion (PE). PEs are ideal sources of biomarkers as the fluid would almost always require drainage for diagnostic and/or therapeutic reasons. However, differentiating MPM PE from PE caused by other diseases can be challenging. MicroRNAs are popular biomarkers given their stable expression in tissue and fluid. MicroRNAs have been analysed in PE cytology samples for the diagnosis of MPM but have not been measured in frozen/fresh PE. We hypothesise that microRNAs expressed in PE are biomarkers for MPM. TaqMan OpenArray was used to analyse over 700 microRNAs in PE cells and supernatants from 26 MPMs and 21 other PE-causing diseases. In PE cells, combining miR-143, miR-210, and miR-200c could differentiate MPM with an area under the curve (AUC) of 0.92. The three-microRNA signature could also discriminate MPM from a further 40 adenocarcinomas with an AUC of 0.9887. These results suggest that the expression of miR-143, miR-210, and miR-200c in PE cells might provide a signature for diagnosing MPM.

Gene expression profiling reveals complex changes following MEK-EE expression in cardiac myocytes.

The activation of the MEK/ERK pathway has been implicated in the proliferative growth of many tissues, however in the heart it has been linked with hypertrophic growth of the individual cardiac myocytes. We have explored the transcriptional consequences of prolonged ERK1/2 activation in cardiac myocytes following the adenoviral overexpression of a constitutively active form of MEK, MEK-EE. Analysis of microarray data obtained using full rat genome arrays showed >2000 gene expression changes in response to MEK-EE overexpression for 24h. We observed similar numbers of genes upregulated and downregulated. The genes were involved in diverse processes including cell structure, metabolism and intracellular signalling. There were also changes in the pro- and ani-apoptotic genes as well as downregulation of the antioxidant enzymes, Mn superoxide dismutase, catalase and thioredoxin 2. Our results reveal the complexity of transcriptional changes that follow the activation of the ERK signalling pathway in these cells and suggest that activation of this MAPK pathway impinges on diverse cellular functions.

Changes in the transcriptional profile of cardiac myocytes following green fluorescent protein expression.

Green fluorescent protein (GFP) and its multiple forms, such as enhanced GFP (EGFP), have been widely used as marker proteins and for tracking purposes in many biological systems, including the heart and cardiac cell systems. Despite some concerns on its toxicity under certain circumstances, GFP remains amongst the most reliable and easy-to-use markers available. Using rat full genome DNA microarrays, we have investigated the broader consequences of adenoviral-driven GFP expression in cardiac myocytes. In our transcriptional profiling analysis, we set a threshold of a twofold change. We removed possible changes resulting from adenoviral infection by comparison with transcriptional profiles of cardiac myocytes with adenoviral-driven expression of an unrelated protein, the kinase MEK. Our analysis revealed changes in the expression of 212 genes. Of these genes, 174 were upregulated and 38 were downregulated following GFP expression. Many of these genes remain unannotated, but an evaluation of those with described functions for their resulting proteins indicated that many were involved in processes, including responses to stimuli/stress and signal transduction. Our analysis thus indicates the broader consequences of GFP expression in altering gene expression profiles in cardiac cells. Care should therefore be taken when using GFP expression as a control in gene expression studies.

Targeting microRNA to improve diagnostic and therapeutic approaches for malignant mesothelioma.

Malignant mesothelioma is an aggressive and often fatal cancer associated with asbestos exposure. The disease originates in the mesothelial lining of the serosal cavities, most commonly affecting the pleura. Survival rates are low as diagnosis often occurs at an advanced stage and current treatments are limited. Identifying new diagnostic and therapeutic targets for mesothelioma remains a priority, particularly for the new wave of victims exposed to asbestos through do-it-yourself renovations and in countries where asbestos is still mined and used. Recent advances have demonstrated a biological role for the small but powerful gene regulators microRNA (miRNA) in mesothelioma. A number of potential therapeutic targets have been identified. MiRNA have also become popular as potential biomarkers for mesothelioma due to their stable expression in bodily fluid and tissues. In this review, we highlight the current challenges associated with the diagnosis and treatment of mesothelioma and discuss how targeting miRNA may improve diagnostic, prognostic and therapeutic approaches.

Loss of miR-223 and JNK Signaling Contribute to Elevated Stathmin in Malignant Pleural Mesothelioma.

UNLABELLED: Malignant pleural mesothelioma (MPM) is often fatal, and studies have revealed that aberrant miRNAs contribute to MPM development and aggressiveness. Here, a screen of miRNAs identified reduced levels of miR-223 in MPM patient specimens. Interestingly, miR-223 targets Stathmin (STMN1), a microtubule regulator that has been associated with MPM. However, whether miR-223 regulates STMN1 in MPM and the functions of miR-223 and STMN1 in this disease are yet to be determined. STMN1 is also regulated by c-Jun N-terminal kinase (JNK) signaling, but whether this occurs in MPM and whether miR-223 plays a role are unknown. The relationship between STMN1, miR-223, and JNK was assessed using MPM cell lines, cells from pleural effusions, and MPM tissue. Evidence indicates that miR-223 is decreased in all MPM tissue compared with normal/healthy tissue. Conversely, STMN1 expression was higher in MPM cell lines when compared with primary mesothelial cell controls. Following overexpression of miR-223 in MPM cell lines, STMN1 levels were reduced, cell motility was inhibited, and tubulin acetylation induced. Knockdown of STMN1 using siRNAs led to inhibition of MPM cell proliferation and motility. Finally, miR-223 levels increased while STMN1 was reduced following the re-expression of the JNK isoforms in JNK-null murine embryonic fibroblasts, and STMN1 was reduced in MPM cell lines following the activation of JNK signaling. IMPLICATIONS: miR-223 regulates STMN1 in MPM, and both are in turn regulated by the JNK signaling pathway. As such, miR-223 and STMN1 play an important role in regulating MPM cell motility and may be therapeutic targets.

Increased circulating miR-625-3p: a potential biomarker for patients with malignant pleural mesothelioma.

INTRODUCTION: We investigated the ability of cell-free microRNAs (miRNAs) in plasma and serum to serve as a biomarker for malignant mesothelioma (MM). METHODS: Using miRNA microarrays, we profiled plasma samples from MM patients and healthy controls. miRNAs with significantly different abundance between cases and controls were validated in a larger series of MM patients and in an independent series of MM patients using quantitative real-time polymerase chain reaction. Levels of candidate miRNAs were also quantified in MM tumor samples. RESULTS: We compared cell-free miRNA profiles in plasma from MM patients with healthy controls. Reviewing 90 miRNAs previously reported to be associated with MM, we found that the levels of two miRNAs, miR-29c* and miR-92a, were elevated in plasma samples from MM patients. In addition, we identified 15 novel miRNAs present at significantly higher levels in the plasma of MM patients. Further analysis of candidate miRNAs by real time-quantitative polymerase chain reaction confirmed that one of them, miR-625-3p, was present in significantly higher concentration in plasma/serum from MM patients and was able to discriminate between cases and controls, in both the original and the independent series of patients. MiR-625-3p was also found to be up-regulated in tumor specimens from a group of 18 MM patients, who underwent extrapleural pneumonectomy. CONCLUSION: Our data confirm the potential of miR-29c* and miR-92a as candidate tumor markers and reveal that miR-625-3p is a promising novel diagnostic marker for MM.

Contrasting actions of prolonged mitogen-activated protein kinase activation on cell survival.

Activation of the ERK mitogen-activated protein kinase pathway has been implicated in pro-survival and cellular protective mechanisms, so that chronic ERK activation may be a useful therapeutic strategy. Here, we further explored the consequences of prolonged ERK activation following expression of constitutively active form of MEK, MEK-EE, in cardiac myocytes. We confirmed that chronic MEK-EE overexpression halved myocyte death following glucose deprivation, but surprisingly this was not associated with preserved intracellular ATP levels. Whilst activities of a number of antioxidant enzymes were not altered upon MEK-EE expression, paradoxically Cu/Zn superoxide dismutase activity was almost halved upon MEK-EE expression. When we then exposed myocytes to the superoxide generator menadione, we observed significantly higher death of MEK-EE expressing myocytes. Pre-incubation with U0126 inhibited menadione-induced death. Our results are the first to show that MEK-ERK signalling can act to increase or decrease cell survival, the outcome depending on the form of stress stimulus encountered.