IMM-H007 attenuates isoprenaline-induced cardiac fibrosis through targeting TGFß1 signaling pathway.

Upon chronic stress, ß-adrenergic receptor activation induces cardiac fibrosis and leads to heart failure. The small molecule compound IMM-H007 has demonstrated protective effects in cardiovascular diseases via activation of AMP-activated protein kinase (AMPK). This study aimed to investigate IMM-H007 effects on cardiac fibrosis induced by ß-adrenergic receptor activation. Because adenosine analogs also exert AMPK-independent effects, we assessed AMPK-dependent and -independent IMM-H007 effects in murine models of cardiac fibrosis. Continual subcutaneous injection of isoprenaline for 7 days caused cardiac fibrosis and cardiac dysfunction in mice in vivo. IMM-H007 attenuated isoprenaline-induced cardiac fibrosis, diastolic dysfunction, α-smooth muscle actin expression, and collagen I deposition in both wild-type and AMPKα2-/- mice. Moreover, IMM-H007 inhibited transforming growth factor ß1 (TGFß1) expression in wild-type, but not AMPKα2-/- mice. By contrast, IMM-H007 inhibited Smad2/3 signaling downstream of TGFß1 in both wild-type and AMPKα2-/- mice. Surface plasmon resonance and molecular docking experiments showed that IMM-H007 directly interacts with TGFß1, inhibits its binding to TGFß type II receptors, and downregulates the Smad2/3 signaling pathway downstream of TGFß1. These findings suggest that IMM-H007 inhibits isoprenaline-induced cardiac fibrosis via both AMPKα2-dependent and -independent mechanisms. IMM-H007 may be useful as a novel TGFß1 antagonist.

Author Correction: Increased circulating ß2-adrenergic receptor autoantibodies are associated with smoking-related emphysema.

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

Prostaglandin E2 receptors differentially regulate the output of proinflammatory cytokines in myometrial cells from term pregnant women.

Prostaglandin (PG) E2 plays critical roles during pregnancy and parturition. Emerging evidence indicates that human labour is an inflammatory event. We sought to investigate the effect of PGE2 on the output of proinflammatory cytokines in cultured human uterine smooth muscle cells (HUSMCs) from term pregnant women and elucidate the role of subtypes of PGE2 receptors (EP1, EP2, EP3 and EP4). After drug treatment and/or transfection of each receptor siRNA, the concentrations of inflammatory secreting factors in HUSMCs culture medium were detected by the corresponding ELISA kits. The results showed that, PGE2 increased interleukin 6 (IL-6) and tumor necrosis factor alpha (TNFα) output, decreased chemokine (c-x-c motif) ligand 8 (CXCL8) output in a dose-dependent manner, but had no effect on IL-1ß and chemokine (c-c motif) ligand 2 (CCL-2) secretion of HUSMCs. EP1/EP3 agonist 17-phenyl-trinor-PGE2 stimulated IL-6 and TNFα whilst suppressing IL-1ß and CXCL8 output. The effects of 17-phenyl-trinor-PGE2 on IL-1ß and CXCL8 secretion were remained whereas its effect on IL-6 and TNFα output did not occur in the cells with EP3 knockdown. The stimulatory effects of 17-phenyl-trinor-PGE2 on IL-6 and TNFα were remained whereas the inhibitory effects of 17-phenyl-trinor-PGE2 on IL-1ß secretion was blocked in the cells with EP1 knockdown. Either of EP2 and EP4 agonists stimulated IL-1ß and TNFα output, which was reversed by EP2 and EP4 siRNA, respectively. The inhibitors of phospholipase C (PLC) and protein kinase C (PKC) blocked EP1/EP3 modulation of TNFα and CXCL8 output. PI3K inhibitor LY294002 and P38 inhibitor SB202190 blocked 17-phenyl-trinor-PGE2-induced IL-1ß and IL-6 output, respectively. The inhibitors of adenylyl cyclase and PKA prevented EP2 and EP4 stimulation of IL-1ß and TNFα output, whereas PLC and PKC inhibitors blocked EP2- and EP4-induced TNFα output but not IL-1ß output. Our data suggest that PGE2 receptors exhibit different effects on the output of various cytokines in myometrium, which can subtly modulate the inflammatory microenvironment in myometrium during pregnancy.

ß­estradiol alleviates hypertension­ and concanavalin A­mediated inflammatory responses via modulation of connexins in peripheral blood lymphocytes.

Gap junctions (GJs) formed by connexins (Cxs) in T lymphocytes have been reported to have important roles in the T lymphocyte­driven inflammatory response and hypertension­mediated inflammation. Estrogen has a protective effect on cardiovascular diseases, including hypertension and it attenuates excessive inflammatory responses in certain autoimmune diseases. However, the mechanisms involved in regulating the pro­inflammatory response are complex and poorly understood. The current study investigated whether ß­estradiol suppresses hypertension and pro­inflammatory stimuli­mediated inflammatory responses by regulating Cxs and Cx­mediated GJs in peripheral blood lymphocytes. Male, 16­week­old spontaneously hypertensive rats (SHR) and Wistar­Kyoto rats (WKY) rats were randomly divided into the following three groups: WKY rats, vehicle (saline)­treated SHRs, and ß­estradiol (20 µg/kg/day)­treated SHRs. ß­estradiol was administered subcutaneously for 5 weeks. Hematoxylin and eosin staining was performed to evaluate target organ injury. Flow cytometry and ELISA were used to measure the populations of T lymphocyte subtypes in the peripheral blood, and expression of Cx40/Cx43 in T cell subtypes, and pro­inflammation cytokines levels, respectively. ELISA, a dye transfer technique, immunofluorescence and immunoblotting were used to analyze the effect of ß­estradiol on pro­inflammatory cytokine secretion, Cx­mediated GJs and the expression of Cxs in concanavalin A (Con A)­stimulated peripheral blood lymphocytes isolated from WKY rat. ß­estradiol significantly decreased blood pressure and inhibited hypertension­induced target organ injury in SHRs. Additionally, ß­estradiol treatment significantly improved the immune homeostasis of SHRs, as demonstrated by the decreased percentage of cluster of differentiation (CD)4+/CD8+ T­cell subset ratio, reduced serum levels of pro­inflammatory cytokines and increased the percentage of CD4+CD25+ T cells. ß­estradiol also markedly reduced the expression of Cx40/Cx43 in T lymphocytes from SHRs. In vitro, ß­estradiol significantly suppressed the production of pro­inflammatory cytokines, reduced communication via Cx­mediated gap junctions and decreased the expression of Cx40/Cx43 in Con A­stimulated lymphocytes. These results indicate that ß­estradiol attenuates inflammation and end organ damage in hypertension, which may be partially mediated via downregulated expression of Cxs and reduced function of Cx­mediated GJ.

RNA-binding protein HuR regulates hsa-let-7c expression by its RNA recognition motif.

MicroRNAs (miRNAs) are small noncoding RNAs that control diverse cellular and developmental events through repression of large sets of target mRNAs. miRNAs expressions were mainly regulated at two levels: transcriptional and post-transcriptional. Transcriptional regulation of miRNA-encoding genes produce specific expression patterns of individual miRNA. However, the mechanism of post-transcriptional regulation of miRNAs remains largely unknown. The present study was aimed to clarify whether HuR, an evolutionary conserved AU-rich binding protein, could regulate miRNAs expressions. By means of a computational screen for AUUUA motifs within pri-miRNAs, we found that the downstream of hsa-let-7c but not hsa-miR-21 was enriched of AUUUA motifs. Then we transfected HuR and mutant HuR lacking RNA recognition motif 3 (RRM3) respectively into HEK293T cells. And HuR protein and miRNAs expressions were detected by Western blot and real-time PCR, respectively. The results showed that the overexpression of HuR promoted mature hsa-let-7c expression but not hsa-miR-21 expression. Furthermore, overexpression of HuR deletion mutant lacking RRM3 did not promote hsa-let-7c expression. These results suggest that RRM3 is crucial for HuR mediating mature hsa-let-7c expression. Collectively, these findings proposed a novel role of HuR in biogenesis of miRNAs, possibly by way of post-transcriptional regulation of miRNAs.

Increased circulating ß2-adrenergic receptor autoantibodies are associated with smoking-related emphysema.

Smoking is a dominant risk factor for chronic obstructive pulmonary disease (COPD) and emphysema, but not every smoker develops emphysema. Immune responses in smokers vary. Some autoantibodies have been shown to contribute to the development of emphysema in smokers. ß2-adrenergic receptors (ß2-ARs) are important targets in COPD therapy. ß2-adrenergic receptor autoantibodies (ß2-AAbs), which may directly affect ß2-ARs, were shown to be increased in rats with passive-smoking-induced emphysema in our current preliminary studies. Using cigarette-smoke exposure (CS-exposure) and active-immune (via injections of ß2-AR second extracellular loop peptides) rat models, we found that CS-exposed rats showed higher serum ß2-AAb levels than control rats before alveolar airspaces became enlarged. Active-immune rats showed increased serum ß2-AAb levels, and exhibited alveolar airspace destruction. CS-exposed-active-immune treated rats showed more extensive alveolar airspace destruction than rats undergoing CS-exposure alone. In our current clinical studies, we showed that plasma ß2-AAb levels were positively correlated with the RV/TLC (residual volume/total lung capacity) ratio (r = 0.455, p < 0.001) and RV%pred (residual volume/residual volume predicted percentage, r = 0.454, p < 0.001) in 50 smokers; smokers with higher plasma ß2-AAb levels exhibited worse alveolar airspace destruction. We suggest that increased circulating ß2-AAbs are associated with smoking-related emphysema.

Mechanical characterization of cervical squamous carcinoma cells by atomic force microscopy at nanoscale.

To investigate the nanoscale mechanical properties of exfoliated cervical epithelial cells from patients to further reveal the pathogenesis of cervical cancer and help early diagnose. Exfoliated cells were collected from nine patients with chronic cervicitis or CIN1(control group), 30 patients with CIN2-3 (CIN 2-3 group), and 13 patients with cervical cancer (cervical cancer group). Stiffness of the cells was determined by atomic force microscope (AFM). Expression of P16INK4A was studied by immunocytochemistry. Environmental scanning electron microscopy was performed to observe the surface microtopography of the exfoliated cells. Young's modulus was measured for cells exfoliated from control and patients with CIN 2-3 and cervical cancer by AFM. The results showed that with increasing cervical lesions, the Young's modulus of the exfoliated cervical cells increased (P < 0.05). The modulus of the exfoliated cells was significantly decreased in the three patients 1 year after the surgery compared with the value before the surgery. Expression of P16INK4A in the exfoliated cells had not been statistically significant. Squamous cells from cervical cancer group had dense and disordered microvilli without clear microridges compare to other groups. The Young's modulus is increased from the control group, to CIN2-3 and cervical cancer groups, suggesting that the stiffness of cervical epithelial cells increases gradually with increasing cervical lesions. The changes in the mechanical properties of the exfoliated cells occur earlier than the changes in cell morphology. Therefore, analysis of mechanical properties of the exfoliated cells may be used to aid early diagnosis of the cancer.

AMP-activated protein kinase α2 protects against liver injury from metastasized tumors via reduced glucose deprivation-induced oxidative stress.

It is well known that tumors damage affected tissues; however, the specific mechanism underlying such damage remains elusive. AMP-activated protein kinase (AMPK) senses energetic changes and regulates glucose metabolism. In this study, we examined the mechanisms by which AMPK promotes metabolic adaptation in the tumor-bearing liver using a murine model of colon cancer liver metastasis. Knock-out of AMPK α2 significantly enhanced tumor-induced glucose deprivation in the liver and increased the extent of liver injury and hepatocyte death. Mechanistically, we observed that AMPK α2 deficiency resulted in elevated reactive oxygen species, reduced mitophagy, and increased cell death in response to tumors or glucose deprivation in vitro. These results imply that AMPK α2 is essential for attenuation of liver injury during tumor metastasis via hepatic glucose deprivation and mitophagy-mediated inhibition of reactive oxygen species production. Therefore, AMPK α2 might represent an important therapeutic target for colon cancer metastasis-induced liver injury.

Growth differentiation factor (GDF)-15 blocks norepinephrine-induced myocardial hypertrophy via a novel pathway involving inhibition of epidermal growth factor receptor transactivation.

Accumulating evidence suggests that growth differentiation factor 15 (GDF-15) is associated with the severity and prognosis of various cardiovascular diseases. However, the effect of GDF-15 on the regulation of cardiac remodeling is still poorly understood. In this present study, we demonstrate that GDF-15 blocks norepinephrine (NE)-induced myocardial hypertrophy through a novel pathway involving inhibition of EGFR transactivation. Both in vivo and in vitro assay indicate that NE was able to stimulate the synthesis of GDF-15. The up-regulation of GDF-15 feedback inhibits NE-induced myocardial hypertrophy, including quantitation of [(3)H]leucine incorporation, protein/DNA ratio, cell surface area, and ANP mRNA level. Further research shows that GDF-15 could inhibit the phosphorylation of EGF receptor and downstream kinases (AKT and ERK1/2) induced by NE. Clinical research also shows that serum GDF-15 levels in hypertensive patients were significant higher than in healthy volunteers and were positively correlated with the thickness of the posterior wall of the left ventricle, interventricular septum, and left ventricular mass, as well as the serum level of norepinephrine. In conclusion, NE induces myocardial hypertrophy and up-regulates GDF-15, and this up-regulation of GDF-15 negatively regulates NE-induced myocardial hypertrophy by inhibiting EGF receptor transactivation following NE stimulation.

Profiling of microRNA-mRNA reveals roles of microRNAs in cervical cancer.

BACKGROUND: Cervical cancer is one of the most common malignant tumors in women. This study was designed to explore the expression profiles of microRNAs (miRNAs) and mRNAs and the gene regulation network in cervical tumorigenesis and to find candidate molecular markers and key tumorigenic genes in cervical cancer. METHODS: miRNAs and mRNAs expression microarrays were used to detect the expression of miRNAs and mRNAs in normal and cancer cervical tissues. TargetScan 5.0 database (UK) was used to predict the target genes of the miRNAs, analyze their intersection with differentially expressed mRNAs and negatively correlate the intersection with miRNAs. Bioinformatic approaches were used to analyze functions and pathways of the target genes and establish miRNA-gene network. RESULTS: Twenty-nine miRNAs and 2036 mRNAs were differentially expressed in normal and cervical tumor tissues. Among them, 13 miRNAs and 754 mRNAs were up-regulated in cervical tumor tissues and 16 miRNAs and 1282 RNA were down-regulated. The 327 target genes negatively related to miRNAs in the intersection were involved in functions and signal pathways. Down-regulated miRNAs targeted genes and up-regulated miRNAs targeted genes were involved in 415 and 163 functions, respectively, and in 37 and 17 significant pathways, respectively (P < 0.05, false discovery rate (FDR) < 0.05). We constructed the miRNAs-gene network and found that hsa-miR-15a, hsa-miR-106b and hsa-miR-20b were key nodes in the network. CONCLUSIONS: The differentially expressed miRNAs and mRNAs in cervical cancer and related miRNA-gene network have been identified. They play important roles in cervical tumorigenesis and are involved in many important biological functions and signal transduction pathways. These findings lay a foundation for research on the molecular mechanism of miRNAs in the pathogenesis of cervical cancer.

Mir-24 regulates junctophilin-2 expression in cardiomyocytes.

RATIONALE: Failing cardiomyocytes exhibit decreased efficiency of excitation-contraction (E-C) coupling. The downregulation of junctophilin-2 (JP2), a protein anchoring the sarcoplasmic reticulum to T-tubules, has been identified as a major mechanism underlying the defective E-C coupling. However, the regulatory mechanism of JP2 remains unknown. OBJECTIVE: To determine whether microRNAs regulate JP2 expression. METHODS AND RESULTS: Bioinformatic analysis predicted 2 potential binding sites of miR-24 in the 3'-untranslated regions of JP2 mRNA. Luciferase assays confirmed that miR-24 suppressed JP2 expression by binding to either of these sites. In the aortic stenosis model, miR-24 was upregulated in failing cardiomyocytes. Adenovirus-directed overexpression of miR-24 in cardiomyocytes decreased JP2 expression and reduced Ca(2+) transient amplitude and E-C coupling gain. CONCLUSIONS: MiR-24-mediated suppression of JP2 expression provides a novel molecular mechanism for E-C coupling regulation in heart cells and suggests a new target against heart failure.

Ultrastructural remodelling of Ca(2+) signalling apparatus in failing heart cells.

AIMS: The contraction of a heart cell is controlled by Ca(2+)-induced Ca(2+) release between L-type Ca(2+) channels (LCCs) in the cell membrane/T-tubules (TTs) and ryanodine receptors (RyRs) in the junctional sarcoplasmic reticulum (SR). During heart failure, LCC-RyR signalling becomes defective. The purpose of the present study was to reveal the ultrastructural mechanism underlying the defective LCC-RyR signalling and contractility. METHODS AND RESULTS: In rat models of heart failure produced by transverse aortic constriction surgery, stereological analysis of transmission electron microscopic images showed that the volume density and the surface area of junctional SRs and those of SR-coupled TTs were both decreased in failing heart cells. The TT-SR junctions were displaced or missing from the Z-line areas. Moreover, the spatial span of individual TT-SR junctions was markedly reduced in failing heart cells. Numerical simulation and junctophilin-2 knockdown experiments demonstrated that the decrease in junction size (and thereby the constitutive LCC and RyR numbers) led to a scattered delay of Ca(2+) release activation. CONCLUSIONS: The shrinking and eventual absence of TT-SR junctions are important mechanisms underlying the desynchronized and inhomogeneous Ca(2+) release and the decreased contractile strength in heart failure. Maintaining the nanoscopic integrity of TT-SR junctions thus represents a therapeutic strategy against heart failure and related cardiomyopathies.

Inhibition of the STAT3 signaling pathway is involved in the antitumor activity of cepharanthine in SaOS2 cells.

AIM: To investigate the molecular mechanisms underlying the antitumor activity of cepharanthine (CEP), an alkaloid extracted from Stephania cepharantha Hayata. METHODS: Human osteosarcoma cell line SaOS2 was used. MTT assay, Hoechst 33342 nuclear staining, flow cytometry, Western blotting and nude mouse xenografts of SaOS2 cells were applied to examine the antitumor activity of CEP in vitro and in vivo. The expression levels of STAT3 and its downstream signaling molecules were measured with Western blotting and immunochemistry analysis. The activity of STAT3 was detected based on the phosphorylation level of STAT3, luciferase gene reporter assay and translocation of STAT3 to the nucleus. RESULTS: Treatment of SaOS2 cells with CEP (2.5-20 µmol/L) inhibited the cell growth in a concentration- and time-dependent manner. CEP (10 µmol/L) caused cell cycle arrest at G(1) phase and induced apoptosis of SaOS2 cells. CEP (10 and 15 µmol/L) significantly decreased the expression of STAT3 in SaOS2 cells. Furthermore, CEP (5 and 10 µmol/L) significantly inhibited the expression of target genes of STAT3, including the anti-apoptotic gene Bcl-xL and the cell cycle regulators c-Myc and cyclin D1. In nude mouse xenografts of SaOS2 cells, CEP (20 mg·kg(-1)·d(-1), ip for 19 d) significantly reduced the volume and weight of the tumor. CONCLUSION: Our findings suggest that inhibition of STAT3 signaling pathway is involved in the anti-tumor activity of CEP.

Transcription factor Ap-1 mediates proangiogenic MIF expression in human endothelial cells exposed to Angiotensin II.

Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine associated with the atherosclerotic process and atherosclerotic plaque stability. MIF was shown to be highly expressed in advanced atherosclerotic lesions. Neutralizing MIF with a blocking antibody induced a regression of established atherosclerotic lesions. In this study, we investigated the mechanism underlying the proangiogenic effect of MIF in human umbilical vein endothelial cells (HUVECs). We showed that MIF induced the expression of angiogenesis-related genes in HUVECs. We also showed that MIF induced tube formation of HUVECs in vitro and in vivo. Angiotensin II (Ang II) could specifically up-regulate MIF expression in HUVECs. Using a luciferase reporter assay, we demonstrated that the AP-1 response element in the 5'-UTR of the MIF gene played a role in Ang II-induced MIF expression. Small hairpin RNA (shRNA) targeting c-Jun, a component of AP-1, and the AP-1 inhibitor CHX both efficiently inhibited MIF expression. The consistent result of electrophoretic mobility shift assay (EMSA) showed that Ang II specifically increased AP-1 activation in HUVECs. Our results suggest that AP-1 mediates Ang II-induced MIF expression which contributes to atherosclerotic plaque destabilization in human endothelial cells.

14-3-3 inhibits insulin-like growth factor-I-induced proliferation of cardiac fibroblasts via a phosphatidylinositol 3-kinase-dependent pathway.

1. Insulin-like growth factor (IGF)-I plays an important role in the pathogenesis of heart disease and has been shown to strongly induce the proliferation of cardiac fibroblasts (CFs). It remains unknown whether 14-3-3 proteins, which are associated the regulation of signal transduction, affect IGF-I-induced CF proliferation. 2. In the present study, we investigated the effects of 14-3-3 proteins on CF proliferation in response to IGF-I. Proliferation of CFs was determined by cell counting and a bromodeoxyuridine incorporation assay. Phosphorylation of signalling molecules was evaluated by western blottling. Activity of nuclear factor of activated T cells (NFAT) was examined using a dual luciferase reporter gene assay and immunofluorescence. 3. It was found that adenovirus-mediated transfection of YFP-R18 peptide (AdR18), a known inhibitor of 14-3-3, significantly enhanced IGF-I-induced CF proliferation. This potentiation arose from an increase in phosphorylation of phosphatidylinositol 3-kinase (PI3-K) and AKT (protein kinase B), inactivation of glycogen synthesis kinase (GSK) 3beta and increased NFAT activity. 4. Collectively, the results of the present study suggest that 14-3-3 proteins inhibit IGF-I-induced CF proliferation via a PI3-K-dependent NFAT signalling pathway. This finding may contribute to our understanding of the function of 14-3-3 proteins in the heart.

Regulation of gap-junction protein connexin 43 by beta-adrenergic receptor stimulation in rat cardiomyocytes.

AIM: beta-adrenergic receptor (beta-AR) agonists are among the most potent factors regulating cardiac electrophysiological properties. Connexin 43 (Cx43), the predominant gap-junction protein in the heart, has an indispensable role in modulating cardiac electric activities by affecting gap-junction function. The present study investigates the effects of short-term stimulation of beta-AR subtypes on Cx43 expression and gap junction intercellular communication (GJIC) function. METHODS: The level of Cx43 expression in neonatal rat cardiomyocytes (NRCM) was detected by a Western blotting assay. The GJIC function was evaluated by scrape loading/dye transfer assay. RESULTS: Stimulation of beta-AR by the agonist isoproterenol for 5 min induces the up-regulation of nonphosphorylated Cx43 protein level, but not total Cx43. Selective beta(2)-AR inhibitor ICI 118551, but not beta(1)-AR inhibitor CGP20712, could fully abolish the effect. Moreover, pretreatment with both protein kinase A inhibitor H89 and G(i) protein inhibitor pertussis toxin also inhibited the isoproterenol-induced increase of nonphosphorylated Cx43 expression. Isoproterenol-induced up-regulation of nonphosphorylated Cx43 is accompanied with enhanced GJIC function. CONCLUSION: Taken together, beta(2)-AR stimulation increases the expression of nonphosphorylated Cx43, thereby enhancing the gating function of gap junctions in cardiac myocytes in both a protein kinase A- and G(i)-dependent manner.Acta Pharmacologica Sinica (2009) 30: 928-934; doi: 10.1038/aps.2009.92.

Epinephrine increases phosphorylation of MAP-2c in rat pheochromocytoma cells (PC12 Cells) via a protein kinase C- and mitogen activated protein kinase-dependent mechanism.

Adrenoceptors mediate effects of endogenous catecholamines and have been shown to affect the neuronal development. Microtubule-associated protein-2 (MAP-2) is an important cytoskeleton protein whose phosphorylation in response to extracellular signal is involved in the regulation of neurite outgrowth and neuronal plasticity. The present study was designed to determine the effect of activation of adrenoceptor by epinephrine on MAP-2 phosphorylation in differentiation PC12 cells and, if so, to explore the mediating mechanism. We found that epinephrine could significantly increase the phosphorylation of MAP-2c at ser136 in a dose- and time-dependent manner in differentiated PC12 cells as well as microtubule arrays. Differentiated PC12 cells express alpha 2A-adrenoceptor, whose antagonists could block these mentioned effects of epinephrine, and clonidine which is the agonist of alpha 2-adrenoceptor could mimic the effect of epinephrine. Moreover phosphorylation of ERK and PKC was induced by epinephrine, and ERK and PKC specific inhibitors concentration-dependently prevented epinephrine-induced phosphorylation of MAP-2c at ser136. In addition, pretreatment of PC12 cells with epinephrine partly inhibited 30 microM nocodazole induced neurites retraction. These findings suggest that epinephrine induces phosphorylation of MAP-2c at ser136 through a alpha 2-adrenoceptor mediated, ERK/PKC-dependent signaling pathway, which may contribute to the stabilization of neurites.

AICAR inhibits proliferation and induced S-phase arrest, and promotes apoptosis in CaSki cells.

AIM: The aim of the present study was to determine the effect of 5-aminoimidazole-4-carboxamide-ribonucleoside (AICAR) on proliferation, cell cycle, and apoptosis in the human epithelial cervical cancer cell line CaSki cells. METHODS: Cell count and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay were used to determine cell proliferation and viability. Hoechst 33258 staining was conducted to distinguish the apoptotic cells. Cell cycle and Annexin-V/propidium iodide staining were analyzed by fluorescence-activated cell sorting (FACS). A Western blot assay was used to evaluate the expression of AKT (also known as protein kinase B), mammalian target of rapamycin (mTOR), p53, and extracellular signal-regulated kinase (ERK). RESULTS: AICAR (500 micromol/L) significantly inhibited the proliferation of CaSki cells treated for 24, 48, and 72 h as determined by cell count. The cells at the G1 and G2 phases were dramatically decreased while cells at the S phase were increased in response to AICAR treatment for 24, 48, and 72 h. The MTT assay showed less viable cells and Hoechst fluorescent staining showed more apoptotic cells upon AICAR stimulation. The results of the Annexin-V staining demonstrated a time-dependent increase of apoptosis in cells treated with AICAR for 24, 36, and 48 h. Furthermore, AICAR activated caspase-3 in a time-dependent manner. It was also found that AICAR inhibited the phosphorylation of AKT and mTOR, which are important kinases regulating cell growth and survival. AICAR stimulation obviously increased the expression of the tumor suppressor p53 and the phosphorylation of ERK. CONCLUSION: AICAR inhibited proliferation and induced S phase arrest and promoted apoptosis in CaSki cells, which might be mediated by the downregulation of the AKT/mTOR pathway and the upregulation of the p53/ERK pathway.

Crosstalk between signaling pathways of adrenoreceptors and signal transducers and activators of transcription 3 (STAT3) in heart.

Recently, there have been important advancements in our understanding of the signaling mechanisms of adrenoreceptors (AR) and signal transducers and activators of transcription 3 (STAT3). While their crucial roles in the pathological processes of the heart are well established, accumulating evidence suggests there is a complex pattern of crosstalk between these 2 signaling pathways. Moreover, the potential for crosstalk occurs at multiple levels in each signaling cascade and involves receptor transactivation, G proteins, small GTPases, cyclic adenosine 3',5'-monophosphate/protein kinase A, protein kinase C, scaffold/adaptor proteins, protein tyrosine kinases, and mitogen-activated protein kinases. In addition, post-translational modification (eg acetylation) of STAT3 may provide a link between STAT3 and AR signaling. In particular, crosstalk between these 2 systems in the heart would appear to be dependent upon the species/tissue studied, developmental stage, and eliciting stimulus. This at least partly accounts for the epigenetic effects on biological function that is mediated by the 2 signaling pathways. Elucidation of these mechanisms will provide new targets in the development of novel clinical strategies for heart disorders.