Pro-oncogenic function of HIP-55/Drebrin-like (DBNL) through Ser269/Thr291-phospho-sensor motifs.

HIP-55 (HPK1-interacting protein of 55 kDa, also named DBNL, SH3P7, and mAbp1) is a multidomain adaptor protein that is critical for organ development and the immune response. Here, we report the coupling of HIP-55 to cell growth control through its 14-3-3-binding phospho-Ser/Thr-sensor sites. Using affinity chromatography, we found HIP-55 formed a complex with 14-3-3 proteins, revealing a new node in phospho-Ser/Thr-mediated signaling networks. In addition, we demonstrated that HIP-55 is required for proper cell growth control. Enforced HIP-55 expression promoted proliferation, colony formation, migration, and invasion of lung cancer cells while silencing of HIP-55 reversed these effects. Importantly, HIP-55 was found to be upregulated in lung cancer cell lines and in tumor tissues of lung cancer patients. Upregulated HIP-55 was required to promote the growth of tumors in a xenograft animal model. However, tumors with S269A/T291A-mutated HIP-55, which ablates 14-3-3 binding, exhibited significantly reduced sizes, supporting a vital role of the HIP-55/14-3-3 protein interaction node in transmitting oncogenic signals. Mechanistically, HIP-55-mediated tumorigenesis activity appears to be in part mediated by antagonizing the tumor suppressor function of HPK1. Thus, the HIP-55-mediated oncogenic pathway, through S269/T291, may be exploited for the development of new therapeutic strategies.

In vivo suppression of microRNA-24 prevents the transition toward decompensated hypertrophy in aortic-constricted mice.

RATIONALE: During the transition from compensated hypertrophy to heart failure, the signaling between L-type Ca(2+) channels in the cell membrane/T-tubules and ryanodine receptors in the sarcoplasmic reticulum becomes defective, partially because of the decreased expression of a T-tubule-sarcoplasmic reticulum anchoring protein, junctophilin-2. MicroRNA (miR)-24, a junctophilin-2 suppressing miR, is upregulated in hypertrophied and failing cardiomyocytes. OBJECTIVE: To test whether miR-24 suppression can protect the structural and functional integrity of L-type Ca(2+) channel-ryanodine receptor signaling in hypertrophied cardiomyocytes. METHODS AND RESULTS: In vivo silencing of miR-24 by a specific antagomir in an aorta-constricted mouse model effectively prevented the degradation of heart contraction, but not ventricular hypertrophy. Electrophysiology and confocal imaging studies showed that antagomir treatment prevented the decreases in L-type Ca(2+) channel-ryanodine receptor signaling fidelity/efficiency and whole-cell Ca(2+) transients. Further studies showed that antagomir treatment stabilized junctophilin-2 expression and protected the ultrastructure of T-tubule-sarcoplasmic reticulum junctions from disruption. CONCLUSIONS: MiR-24 suppression prevented the transition from compensated hypertrophy to decompensated hypertrophy, providing a potential strategy for early treatment against heart failure.

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.

Proteome reference map and regulation network of neonatal rat cardiomyocyte.

AIM: To study and establish a proteome reference map and regulation network of neonatal rat cardiomyocyte. METHODS: Cultured cardiomyocytes of neonatal rats were used. All proteins expressed in the cardiomyocytes were separated and identified by two-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Biological networks and pathways of the neonatal rat cardiomyocytes were analyzed using the Ingenuity Pathway Analysis (IPA) program (www.ingenuity.com). A 2-DE database was made accessible on-line by Make2ddb package on a web server. RESULTS: More than 1000 proteins were separated on 2D gels, and 148 proteins were identified. The identified proteins were used for the construction of an extensible markup language-based database. Biological networks and pathways were constructed to analyze the functions associate with cardiomyocyte proteins in the database. The 2-DE database of rat cardiomyocyte proteins can be accessed at http://2d.bjmu.edu.cn. CONCLUSION: A proteome reference map and regulation network of the neonatal rat cardiomyocytes have been established, which may serve as an international platform for storage, analysis and visualization of cardiomyocyte proteomic data.

α1A-adrenergic receptor induces activation of extracellular signal-regulated kinase 1/2 through endocytic pathway.

G protein-coupled receptors (GPCRs) activate mitogen-activated protein kinases through a number of distinct pathways in cells. Increasing evidence has suggested that endosomal signaling has an important role in receptor signal transduction. Here we investigated the involvement of endocytosis in α(1A)-adrenergic receptor (α(1A)-AR)-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2). Agonist-mediated endocytic traffic of α(1A)-AR was assessed by real-time imaging of living, stably transfected human embryonic kidney 293A cells (HEK-293A). α(1A)-AR was internalized dynamically in cells with agonist stimulation, and actin filaments regulated the initial trafficking of α(1A)-AR. α(1A)-AR-induced activation of ERK1/2 but not p38 MAPK was sensitive to disruption of endocytosis, as demonstrated by 4°C chilling, dynamin mutation and treatment with cytochalasin D (actin depolymerizing agent). Activation of protein kinase C (PKC) and C-Raf by α(1A)-AR was not affected by 4°C chilling or cytochalasin D treatment. U73122 (a phospholipase C [PLC] inhibitor) and Ro 31-8220 (a PKC inhibitor) inhibited α(1B)-AR- but not α(1A)-AR-induced ERK1/2 activation. These data suggest that the endocytic pathway is involved in α(1A)-AR-induced ERK1/2 activation, which is independent of G(q)/PLC/PKC signaling.

Proteomic profiling reveals comprehensive insights into adrenergic receptor-mediated hypertrophy in neonatal rat cardiomyocytes.

Myocardial adrenergic receptors (ARs) play important roles in cardiac hypertrophy. However, the detailed molecular mechanism of AR-mediated cardiac hypertrophy remains elusive to date. To gain full insight into how ARs are involved in the regulation of cardiac hypertrophy, protein expression profiling was performed with comparative proteomics approach on neonatal rat cardiomyocytes. Forty-six proteins were identified as differentially expressed in hypertrophic cardiomyocytes induced by AR stimulation. To better understand the biological significance of the obtained proteomic data, we utilized the ingenuity pathway analysis tool to construct biological networks and analyze function and pathways that might associate with AR-mediated cardiac hypertrophy. Pathway analysis strongly suggested that ROS may be involved in the development of AR-mediated cardiac hypertrophy, which was then confirmed by further experimentation. The results showed that a marked increase in ROS production was detected in AR-mediated cardiac hypertrophy and blocking of ROS production significantly inhibited AR-mediated cardiac hypertrophy. We further proved that the ROS production was through NADPH oxidase or the mitochondrial electron transport chain and this ROS accumulation resulted in activation of extracellular signal-regulated kinase 1/2 leading to AR-mediated cardiac hypertrophy. These experimental results support the hypothesis, from the ingenuity pathway analysis, that AR-mediated cardiac hypertrophy is associated with the dysregulation of a complicated oxidative stress-regulatory network. In conclusion, our results provide a basis for understanding the detailed molecular mechanisms of AR-mediated cardiac hypertrophy.

The gp130/STAT3 signaling pathway mediates beta-adrenergic receptor-induced atrial natriuretic factor expression in cardiomyocytes.

beta-Adrenergic receptor (beta-AR)-induced cardiac remodeling is closely linked with the re-expression of the atrial natriuretic factor (ANF) gene. However, the exact molecular mechanism of this response remains elusive. Here, we demonstrate that the beta-AR agonist isoproterenol potently evokes the tyrosine phosphorylation of STAT3 and increases its transcriptional activity in an extracellularly regulated kinase 1/2 and glycoprotein (gp)130 signaling-dependent manner in rat cardiomyocytes. Interestingly, both specific silencing of signal transducers and activators of transcription 3 (STAT3) expression by lentivirus-mediated RNA interference and antagonism of gp130 signaling lead to significant inhibition of isoproterenol-stimulated ANF expression. Together, these results indicate that gp130/STAT3 signaling has an essential role in ANF expression by beta-AR stimulation.

Nuclear Ca2+ sparks and waves mediated by inositol 1,4,5-trisphosphate receptors in neonatal rat cardiomyocytes.

Dynamic nuclear Ca(2+) signals play pivotal roles in diverse cellular functions including gene transcription, cell growth, differentiation, and apoptosis. Here we report a novel nuclear Ca(2+) regulatory mechanism mediated by inositol 1,4,5-trisphosphate receptors (IP(3)Rs) around the nucleus in developing cardiac myocytes. Activation of IP(3)Rs by alpha(1)-adrenergic receptor (alpha(1)AR) stimulation or by IP(3) application (in saponin-permeabilized cells) increases Ca(2+) spark frequency preferentially in the region around the nucleus in neonatal rat ventricular myocytes. A nuclear enrichment of IP(3)R distribution supports the higher responsiveness of Ca(2+) release in this particular region. Strikingly, we observed "nuclear Ca(2+)waves" that engulf the entire nucleus without spreading into the bulk cytosol. alpha(1)AR stimulation enhances the occurrence of nuclear Ca(2+) waves and confers them the ability to trigger cytosolic Ca(2+) waves via IP(3)R-dependent pathways. This finding accounts, at least partly, for a profound frequency-dependent modulation of global Ca(2+) oscillations during alpha(1)AR stimulation. Thus, IP(3)R-mediated Ca(2+) waves traveling in the nuclear region provide active, autonomous regulation of nuclear Ca(2+) signaling, which provides for not only the local signal transduction, but also a pacemaker to drive global Ca(2+) transient in the context of alpha(1)AR stimulation in developing cardiac myocytes.

Receptor subtype involved in alpha 1-adrenergic receptor-mediated Ca2+ signaling in cardiomyocytes.

AIM: The enhancement of intracellular Ca2+ signaling in response to alpha 1-adrenergic receptor (alpha 1-AR) stimulation is an essential signal transduction event in the regulation of cardiac functions, such as cardiac growth, cardiac contraction, and cardiac adaptation to various situations. The present study was intended to determine the role(s) of the alpha 1-AR subtype(s) in mediating this response. METHODS: We evaluated the effects of subtype-specific agonists and antagonists of the alpha 1- AR on the intracellular Ca2+ signaling of neonatal rat ventricular myocytes using a confocal microscope. RESULTS: After being cultured for 48 h, the myocytes exhibited spontaneous local Ca2+ release, sparks, and global Ca2+ transients. The activation of the alpha 1-AR with phenylephrine, a selective agonist of the alpha 1-AR, dose-dependently increased the frequency of Ca2+ transients with an EC50 value of 2.3 micromol/L. Blocking the alpha 1A-AR subtype with 5-methylurapidil (5-Mu) inhibited the stimulatory effect of phenylephrine with an IC(50) value of 6.7 nmol/L. In contrast, blockade of the alpha 1B-AR and alpha 1D-AR subtypes with chloroethylclonidine and BMY 7378, respectively, did not affect the phenylephrine effect. Similarly, the local Ca2+ spark numbers were also increased by the activation of the alpha 1-AR, and this effect could be abolished selectively by 5-Mu. More importantly, A61603, a novel selective alpha 1A-AR agonist, mimicked the effects of phenylephrine, but with more potency (EC(50) value =6.9 nmol/L) in the potentiation of Ca2+ transients, and blockade of the alpha 1A-AR by 5-Mu caused abolishment of its effects. CONCLUSION: These results indicate that alpha 1-adrenergic stimulation of intracellular Ca2+ activity is mediated selectively by the alpha 1A-AR.

Real-time detection of alpha1A-AR movement stimulated by phenylephrine in single living cells.

AIM: To investigate the movement of alpha(1A)-adrenergic receptors(alpha(1A)-AR) stimulated by agonist, phenylephrine (PE), and the dynamics of receptor movement in real time in single living cells with millisecond resolution. METHODS: We labeled alpha(1A)-AR using the monoclonal, anti-FLAG (a kind of tag) antibody and Cy3-conjugated goat anti-mouse IgG and recorded the trajectory of their transport process in living HEK293A cells stimulated by agonist, PE, and then analyzed their dynamic properties. RESULTS: The specific detection of alpha(1A)-AR on the surface of living HEK293A-alpha(1A) cells was achieved. alpha(1A)-AR internalize under the stimulation of PE. After the cells were stimulated with PE for 20 min, apparent colocalization was found between alpha(1A)-AR and F-actins. After 40 min stimulation of PE, trajectories of approximate linear motion in HEK293A-alpha(1A) cells were recorded, and their velocity was calculated. CONCLUSION: The specific labeling method on the living cell surface provides a convenient means of real-time detection of the behavior of surface receptors. By this method we were able to specifically detect alpha(1A)-AR and record the behavior of individual particles of receptors with 50 ms exposure time in real time in single living cells.

alpha1-adrenergic receptors activate AMP-activated protein kinase in rat hearts.

To test the hypothesis that AMP-activated protein kinase (AMPK) is possibly the downstream signaling molecule of certain subtypes of adrenergic receptor (AR) in the heart, we evaluated AMPK activation mediated by ARs in H9C2 cells, a rat cardiac source cell line, and rat hearts. The AMPK-alpha subunit and the phosphorylation level of Thr(172)-AMPK-alpha subunit were subjected to Western blot analysis. Osmotic minipumps filled with norepinephrine (NE), phenylephrine (PE) or vehicle [0.01% (W/V) vitamin C solution] were implanted into male Sprague-Dawley rats subcutaneously. The pumps delivered NE or PE continuously at the rate of 0.2 mg/kg per hour. After 7-day infusion, the activity of AMPK was examined following immunoprecipitation with anti-AMPK-alpha antibody. At the cellular level, we found that NE elevated AMPK phosphorylation level in a dose- and time-dependent manner, with the maximal effect at 10 micromol/L NE after 10-minute treatment. This effect was insensitive to propranolol, a specific beta-AR antagonist, but abolished by prazosin, an alpha(1)-AR antagonist, suggesting that alpha(1)-AR but not beta-AR mediated the phosphorylation of AMPK. Moreover, the results from rat models of 7-day-infusion of AR agonists demonstrated that the activity of AMPK was significantly higher in NE (7.4-fold) and PE (6.0-fold) infusion groups than that in the vehicle group (P<0.05, n=6). On the other hand, no obvious cardiac hypertrophy and tissue fibrosis changes were observed in PE-infused rats. Taken together, our results demonstrate that alpha(1)-AR stimulation enhances the activity of AMPK, indicating an important role of alpha(1)-AR stimulation in the regulation of AMPK in the heart. Understanding the activation of AMPK mediated by alpha(1)-AR might have clinical implications in the therapy of heart failure.

Heterogeneous transportation of alpha1B-adrenoceptor in living cells.

The heterogeneous motion of alpha(1B)-adrenoceptor (alpha(1B)-AR) was visualized in living cells with BODIPY-labeled antagonist of AR by single molecule fluorescence microscopy at high spatial resolution. The moving trajectory was reconstructed by precise localization (better than 20 nm) with a least-square fit of a two-dimensional Gaussian point spread function to each single spot. Trajectory analysis revealed two apparent groups of movements: directed motion and hindered motion. The directed motion had speeds higher than 0.1 mum/s. The histogram of diffusion coefficients of the hindered motion showed distinction between the cell membrane and the cytoplasm: the diffusion coefficient was lower near the cell membrane than in the internal cytoplasm, suggesting that alpha(1B)-AR was located or trapped in different networks, which was consistent with the natural distribution of cytoskeleton in living cells. These results suggested that the heterogeneity in the motion of alpha(1B)-AR in living cell might be associated with different localizations of cell skeleton proteins in the cell, which could provide molecular insight of AR regulation in living cells.