Adrenergic regulation of HCN4 channel requires protein association with β2-adrenergic receptor

β(1)- and β(2)-adrenergic receptors utilize different signaling mechanisms to control cardiac function. Recent studies demonstrated that β(2)-adrenergic receptors (β(2)ARs) colocalize with some ion channels that are critical for proper cardiac function. Here, we demonstrate that β(2)ARs form protein complexes with the pacemaker HCN4 channel, as well as with other subtypes of HCN channels. The adrenergic receptor-binding site was identified at a proximal region of the N-terminal tail of the HCN4 channel. A synthetic peptide derived from the β(2)AR-binding domain of the HCN4 channel disrupted interaction between HCN4 and β(2)AR. In addition, treatment with this peptide prevented adrenergic augmentation of pacemaker currents and spontaneous contraction rates but did not affect adrenergic regulation of voltage-gated calcium currents. These results suggest that the ion channel-receptor complex is a critical mechanism in ion channel regulation.     

Neural cell adhesion molecule potentiates the growth of murine melanoma via β-catenin signaling by association with fibroblast growth factor receptor and glycogen synthase kinase-3β

The neural cell adhesion molecule (NCAM) was recently shown to be involved in the progression of various tumors with diverse effects. We previously demonstrated that NCAM potentiates the cellular invasion and metastasis of melanoma. Here we further report that the growth of melanoma is obviously retarded when the expression of NCAM is silenced. We found that the proliferation of murine B16F0 melanoma cells, their colony formation on soft agar, and growth of transplanted melanoma in vivo are clearly inhibited by the introduction of NCAM siRNA. Interestingly, change of NCAM expression level is shown to regulate the activity of Wnt signaling molecule, β-catenin, markedly. This novel machinery requires the function of FGF receptor and glycogen synthase kinase-3β but is independent of the Wnt receptors, MAPK-Erk and PI3K/Akt pathways. In addition, NCAM is found to form a functional complex with β-catenin, FGF receptor, and glycogen synthase kinase-3β. Moreover, up-regulation of NCAM140 and NCAM180 appears more potent than NCAM120 in activation of β-catenin, suggesting that the intracellular domain of NCAM is required for facilitating the β-catenin signaling. Furthermore, the melanoma cells also exhibit distinct differentiation phenotypes with the NCAM silencing. Our findings reveal a novel regulatory role of NCAM in the progression of melanoma that might serve as a new therapeutic target for the treatment of melanoma.     

Cardiomyocyte lipids impair β-adrenergic receptor function via PKC activation

Normal hearts have increased contractility in response to catecholamines. Because several lipids activate PKCs, we hypothesized that excess cellular lipids would inhibit cardiomyocyte responsiveness to adrenergic stimuli. Cardiomyocytes treated with saturated free fatty acids, ceramide, and diacylglycerol had reduced cellular cAMP response to isoproterenol. This was associated with increased PKC activation and reduction of β-adrenergic receptor (β-AR) density. Pharmacological and genetic PKC inhibition prevented both palmitate-induced β-AR insensitivity and the accompanying reduction in cell surface β-ARs. Mice with excess lipid uptake due to either cardiac-specific overexpression of anchored lipoprotein lipase, PPARγ, or acyl-CoA synthetase-1 or high-fat diet showed reduced inotropic responsiveness to dobutamine. This was associated with activation of protein kinase C (PKC)α or PKCδ. Thus, several lipids that are increased in the setting of lipotoxicity can produce abnormalities in β-AR responsiveness. This can be attributed to PKC activation and reduced β-AR levels.     

Latent transforming growth factor β-binding protein-3 and fibulin-1C interact with the extracellular domain of the heparin-binding EGF-like growth factor precursor

Background  

The membrane-bound cell-surface precursor and soluble forms of heparin-binding epidermal growth factor-like growth factor (HB-EGF) contribute to many cellular developmental processes. The widespread occurrence of HB-EGF in cell and tissue types has led to observations of its role in such cellular and tissue events as tumor formation, cell migration, extracellular matrix formation, wound healing, and cell adherence. Several studies have reported the involvement of such extracellular matrix proteins as latent transforming growth factor β-binding protein, TGF-β, and fibulin-1 in some of these processes. To determine whether HB-EGF interacts with extracellular matrix proteins we used the extracellular domain of proHB-EGF in a yeast two-hybrid system to screen a monkey kidney cDNA library. cDNA clones containing nucleotide sequences encoding domains of two proteins were obtained and their derived amino acid sequences were evaluated.     

Rab1 interacts directly with the β2-adrenergic receptor to regulate receptor anterograde trafficking

Very little is understood about the trafficking of G protein-coupled receptors (GPCRs) from the endoplasmic reticulum (ER) to the plasma membrane. Rab guanosine triphosphatases (GTPases) are known to participate in the trafficking of various GPCRs via a direct interaction during the endocytic pathway, but whether this occurs in the anterograde pathway is unknown. We evaluated the potential interaction of Rab1, a GTPase known to regulate β2-adrenergic receptor (β2AR) trafficking, and its effect on export from the ER. Our results show that GTP-bound Rab1 interacts with the F(x)(6)LL motif of β2AR. Receptors lacking the interaction motif fail to traffic properly, suggesting that a direct interaction with Rab1 is required for β2AR anterograde trafficking.     

The β1-adrenergic receptor mediates extracellular signal-regulated kinase activation via Gαs

β-Adrenergic receptors can activate extracellular signal-regulated kinases (ERKs) via different mechanisms. In this study, we investigated the molecular mechanism of β1-adrenergic receptor (β1AR)-mediated ERK activation in African green monkey kidney COS-7 cells. Treatment of cells with isoproterenol (ISO), a β1AR selective agonist, induced phosphorylation of ERK1/2 in a dose-dependent manner. ISO-stimulated ERK phosphorylation was not influenced by the Gβγ inhibitor, βAR kinase carboxyl terminal (βARKct) or by the Gi inhibitor, pertussis toxin (PTX), but it was clearly abolished via inhibition of protein kinase A (PKA) with H89, or of mitogen-activated protein kinase kinase (MEK1) with PD98059, revealing that the Gαs subunit is involved in ERK regulation through the PKA/MEK1 pathway. We also tested the effect of the adenylate cyclase activator forskolin on ERK activation, and the result was identical to that of ISO stimulation. Moreover, pretreatment with the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor AG1478 or with the Src tyrosine kinase inhibitor PP2 did not affect ERK activation. These observations suggest a mechanism of β1AR-mediated ERK activity that involves the Gαs subunit, but not EGFR or Src tyrosine kinase.     

Basic fibroblast growth factor is a β-rich protein

The conformation of the 153-residue form of human basic fibroblast growth factor (bFGF) was studied with circular dichroism (CD) and sequence prediction methods. The far-UV CD spectrum with a minimum at 202 nm resembled that of an unordered polypeptide/protein or a protein rich in distorted antiparallel β-sheets. Analysis of the CD spectrum by the least-squares method of Changet al. (1978) and the CONTIN program of Provencher and Glöckner (1981) suggested that about one half of the molecule consisted of β-sheet and there was no α-helix. These estimates agreed with the prediction by the sequence method of Garnieret al. (1978) using decision constants based on CD results. bFGF had an unusual CD band at 187 nm, which disappeared upon ionization of Tyr side chains atpH 11.7. It also had another unusual property of irreversibly converting the CD spectrum to a helix-like one with a double minimum at 205 and 215 and a maximum at 189 nm upon heating the solution to above 55°C. The helicity was also enhanced in trifluoroethanol and in sodium dodecyl sulfate. The mutant bFGF in which cysteines 76 and 94 were replaced by serine residues had essentially the same properties as the wild-type.     

Strong oligomerization behavior of PDGFβ ?receptor transmembrane domain and its regulation by the juxtamembrane regions

The platelet-derived growth factor β-receptor (PDGFβR) represents an important subclass of receptor tyrosine kinase (RTK) thought to be activated by ligand-induced dimerization. Interestingly, the receptor is also activated by the bovine papillomavirus E5 oncoprotein, an interaction involving the transmembrane domains of both proteins and resulting in constitutive downstream signalling. This unique mode of activation along with emerging data for other RTKs raises important questions about the role of the PDGFβR transmembrane domain in signalling. To address this, we have investigated the murine PDGFβR transmembrane and juxtamembrane domains. We show for the first time the strong oligomerization behavior of PDGFβR transmembrane domain, forming dimers and trimers in natural membranes and detergents; and that these self-interactions are mediated by a leucine-zipper-like motif. The juxtamembrane regions are found to regulate these helix-helix interactions and select specifically for dimer formation. These data provide evidence that PDGFβR is able to form ligand-independent dimers, supporting similar observations in a number of other RTK's. A point mutant in the PDGFβR juxtamembrane domain previously shown to cause receptor activation was studied and yielded no change in oligomerization or folding, suggesting (in-line with observations of the c-Kit receptor) that it may moderate interactions with other regions of PDGFβR.     

β2-Adrenergic receptor-induced transactivation of epidermal growth factor receptor and platelet-derived growth factor receptor via Src kinase promotes rat cardiomyocyte survival

Chronic stimulation of the β-AR (adrenergic receptor) promotes apoptosis of cardiomyocytes, which is implicated in cardiac dysfunction. β1-AR and β2-AR are the main subtypes of β-AR that exert distinct effects on the survival of cardiomyocytes. To clarify the physiological roles of β1-AR and β2-AR in cardiomyocytes, the effects of β1-AR or β2-AR knockdown on the survival of H9c2 cardiomyocytes was investigated. Knockdown of β2-AR, but not β1-AR, suppressed the phosphorylation of EGFR (epidermal growth factor receptor) and PDGFR (platelet-derived growth factor receptor) induced by ISO (isoprenaline). The EGFR inhibitor, AG1478, attenuated ERK (extracellular-signal-regulated kinase) activation and partially decreased cell survival. Pretreatment with AG1296, a PDGFR inhibitor, abolished ISO-induced Akt (also known as protein kinase B) phosphorylation and led to a decrease in cell viability. In addition, the Src tyrosine kinase inhibitor, PP2, blocked ISO-mediated both Akt and ERK activation and heavily suppressed viability. Accordingly, in primary neonatal rat cardiomyocytes, the β2-AR inhibitor, but not the β1-AR inhibitor, abrogated the transactivation of EGFR and PDGFR, which was respectively related to Akt and ERK activation. The results show that β2-AR transactivates PDGFR and EGFR, thereby promoting survival of cardiomyocytes.     

Hyaluronan facilitates transforming growth factor-β1-dependent proliferation via CD44 and epidermal growth factor receptor interaction

Fibroblast proliferation is an early feature of progressive tissue fibrosis and is largely regulated by the cytokine transforming growth factor-β1 (TGF-β1). In the oral mucosa, fibroblasts have a unique phenotype and demonstrate healing with no fibrosis/scarring. Our previous studies show that whereas dermal fibroblasts proliferate in response to TGF-β1, oral fibroblasts have an antiproliferative response to this cytokine. Hyaluronan (HA) was directly linked to this TGF-β1-dependent response. The aim of this study was to understand the underlying mechanism through which HA regulates TGF-β-dependent responses. Using patient-matched oral and dermal fibroblasts, we show that TGF-β1-dependent proliferation is mediated through the HA receptor CD44, whereas the TGF-β1-mediated antiproliferative response is CD44-independent. Furthermore, overexpression of HAS2 (HA synthase-2) in oral cells modifies their response, and they subsequently demonstrate a proliferative, CD44-dependent response to TGF-β1. We also show that epidermal growth factor (EGF) and its receptor (EGFR) are essential for TGF-β1/HA/CD44-dependent proliferation. Increased HA levels promote EGFR and CD44 coupling, potentiating signal transduction through the MAPK/ERK pathway. Thus, in a HA-rich environment, late ERK1/2 activation results from EGFR/CD44 coupling and leads to a proliferative response to TGF-β1. In comparison, in a non-HA-rich environment, only early ERK1/2 activation occurs, and this is associated with an antiproliferative response to TGF-β1. In summary, HA facilitates TGF-β1-dependent fibroblast proliferation through promoting interaction between CD44 and EGFR, which then promotes specific MAPK/ERK activation, inducing cellular proliferation.     

Epidermal growth factor can signal via β-catenin to control proliferation of mesenchymal stem cells independently of canonical Wnt signalling

Bone marrow mesenchymal stem/stromal cells (MSCs) maintain bone homeostasis and repair through the ability to expand in response to mitotic stimuli and differentiate into skeletal lineages. Signalling mechanisms that enable precise control of MSC function remain unclear. Here we report that by initially examining differences in signalling pathway expression profiles of individual MSC clones, we identified a previously unrecognised signalling mechanism regulated by epidermal growth factor (EGF) in primary human MSCs. We demonstrate that EGF is able to activate β-catenin, a key component of the canonical Wnt signalling pathway. EGF is able to induce nuclear translocation of β-catenin in human MSCs but does not drive expression of Wnt target genes or T cell factor (TCF) activity in MSC reporter cell lines. Using an efficient Design of Experiments (DoE) statistical analysis, with different combinations and concentrations of EGF and Wnt ligands, we were able to confirm that EGF does not influence the Wnt/β-catenin pathway in MSCs. We show that the effects of EGF on MSCs are temporally regulated to initiate early “classical” EGF signalling mechanisms (e.g via mitogen activated protein kinase) with delayed activation of β-catenin. By RNA-sequencing, we identified gene sets that were exclusively regulated by the EGF/β-catenin pathway, which were distinct from classical EGF-regulated genes. However, subsets of classical EGF gene targets were significantly influenced by EGF/β-catenin activation. These signalling pathways cooperate to enable EGF-mediated proliferation of MSCs by alleviating the suppression of cell cycle pathways induced by classical EGF signalling.     

Cardiac pressure overload hypertrophy is differentially regulated by β-adrenergic receptor subtypes

In isolated myocytes, hypertrophy induced by norepinephrine is mediated via α(1)-adrenergic receptors (ARs) and not β-ARs. However, mice with deletions of both major cardiac α(1)-ARs still develop hypertrophy in response to pressure overload. Our purpose was to better define the role of β-AR subtypes in regulating cardiac hypertrophy in vivo, important given the widespread clinical use of β-AR antagonists and the likelihood that patients treated with these agents could develop conditions of further afterload stress. Mice with deletions of β(1), β(2), or both β(1)- and β(2)-ARs were subjected to transverse aortic constriction (TAC). After 3 wk, β(1)(-/-) showed a 21% increase in heart to body weight vs. sham controls, similar to wild type, whereas β(2)(-/-) developed exaggerated (49% increase) hypertrophy. Only when both β-ARs were ablated (β(1)β(2)(-/-)) was hypertrophy totally abolished. Cardiac function was preserved in all genotypes. Several known inhibitors of cardiac hypertrophy (FK506 binding protein 5, thioredoxin interacting protein, and S100A9) were upregulated in β(1)β(2)(-/-) compared with the other genotypes, whereas transforming growth factor-β(2), a positive mediator of hypertrophy was upregulated in all genotypes except the β(1)β(2)(-/-). In contrast to recent reports suggesting that angiogenesis plays a critical role in regulating cardiac hypertrophy-induced heart failure, we found no evidence that angiogenesis or its regulators (VEGF, Hif1α, and p53) play a role in compensated cardiac hypertrophy. Pressure overload hypertrophy in vivo is dependent on a coordination of signaling through both β(1)- and β(2)-ARs, mediated through several key cardiac remodeling pathways. Angiogenesis is not a prerequisite for compensated cardiac hypertrophy.     

Insulin-like growth factor 1 stimulation of androgen receptor activity requires β(1A) integrins

Despite the findings that β1 integrins play a vital role in the regulation of cell proliferation and survival, the mechanisms through which they operate and lead to cancer progression remain elusive. Previously, our laboratory has shown that β(1A) integrins support insulin-like growth factor 1 (IGFI)-mediated mitogenic and transforming activities. Here, we report that β(1A) integrins regulate basal levels of IGF-IR, although they are not critical for maintaining cancer cell morphology. Upon transfection of β(1A) siRNA and consequent downregulation of IGF-IR, we show inhibition of anchorage-independent growth of prostate cancer cells, a function which is dependent on IGF-IR expression. In addition, we demonstrate that IGFI-mediated activation of androgen receptor (AR), known to occur in prostate cancer cells, requires expression of β(1A) integrins as evaluated by luciferase reporter assays and immunoblotting analysis. Since β(1A) integrin levels are increased by R1881 or dihydrotestosterone (DHT), our results imply that β(1A) integrins support an androgen-enhanced feedback loop that regulates the expression of IGF-IR. β(1A) integrins also regulate inducible levels of IGF-IR in cells stimulated by androgen or by a combination of androgen and IGFI, as evaluated by flow cytometric analysis and immunoblotting. Furthermore, upon transfection of β(1A) siRNA and consequent downregulation of IGF-IR, neither activation of AKT, an effector of IGF-IR, nor AR levels are affected. We conclude that β(1A) integrin expression is critical for maintaining the regulatory crosstalk between IGF-IR and AR.     

Epidermal growth factor, transforming growth factor-α, and epidermal growth factor receptor expression and localization in the canine endometrium during the estrous cycle and in bitches with pyometra

Gene expression and immunohistochemical localization of epidermal growth factor (EGF), transforming growth factor-α (TGF-α), and epidermal growth factor receptor (EGF-R) were compared between the endometrium of bitches (Canis familiaris) with pyometra accompanied by cystic endometrial hyperplasia (CEH) and that of healthy bitches at similar stages of the estrous cycle. In normal bitches, endometrial TGF-α mRNA levels were highest at proestrus and gradually decreased as the cycle progressed to anestrus. Epidermal growth factor receptor mRNA levels were not significantly affected by the stage of the estrous cycle. Epidermal growth factor mRNA levels were higher at Day 35 of diestrus than at other stages of the estrous cycle (P < 0.05). In bitches with pyometra, endometrial TGF-α and EGF-R mRNA levels did not differ significantly from those at diestrus in normal bitches, but EGF mRNA levels were lower than those at Day 35 of diestrus in normal bitches (P < 0.05). In normal bitches, positive immunohistochemical staining for TGF-α, EGF, and EGF-R was mainly present in the glandular and luminal epithelial cells of the endometrium. In contrast, in bitches with pyometra, immunoreactivity for EGF was clearly present in endometrial stromal cells. Inflammatory cells that had infiltrated the endometrial stroma stained strongly for TGF-α and EGF-R. Luminal and glandular epithelial cells also stained positive for EGF-R. In conclusion, expression of TGF-α by inflammatory cells and a low level of expression and differential localization of EGF may be involved in aberrant growth of endometrial glands and development of CEH.     

Clustering heart rate dynamics is associated with β-adrenergic receptor polymorphisms: analysis by information-based similarity index

Background

Genetic polymorphisms in the gene encoding the β-adrenergic receptors (β-AR) have a pivotal role in the functions of the autonomic nervous system. Using heart rate variability (HRV) as an indicator of autonomic function, we present a bottom-up genotype–phenotype analysis to investigate the association between β-AR gene polymorphisms and heart rate dynamics.

Methods

A total of 221 healthy Han Chinese adults (59 males and 162 females, aged 33.6±10.8 years, range 19 to 63 years) were recruited and genotyped for three common β-AR polymorphisms: β₁-AR Ser49Gly, β₂-AR Arg16Gly and β₂-AR Gln27Glu. Each subject underwent two hours of electrocardiogram monitoring at rest. We applied an information-based similarity (IBS) index to measure the pairwise dissimilarity of heart rate dynamics among study subjects.

Results

With the aid of agglomerative hierarchical cluster analysis, we categorized subjects into major clusters, which were found to have significantly different distributions of β₂-AR Arg16Gly genotype. Furthermore, the non-randomness index, a nonlinear HRV measure derived from the IBS method, was significantly lower in Arg16 homozygotes than in Gly16 carriers. The non-randomness index was negatively correlated with parasympathetic-related HRV variables and positively correlated with those HRV indices reflecting a sympathovagal shift toward sympathetic activity.

Conclusions

We demonstrate a bottom-up categorization approach combining the IBS method and hierarchical cluster analysis to detect subgroups of subjects with HRV phenotypes associated with β-AR polymorphisms. Our results provide evidence that β₂-AR polymorphisms are significantly associated with the acceleration/deceleration pattern of heart rate oscillation, reflecting the underlying mode of autonomic nervous system control.