Dexamethasone modulates ErbB tyrosine kinase expression and signaling through multiple and redundant mechanisms in cultured rat hepatocytes

Glucocorticoids paradoxically exert both stimulatory and inhibitory effects on the proliferation of cultured rat hepatocytes. We studied the effects of dexamethasone, a synthetic glucocorticoid, on the proliferation of cultured rat hepatocytes. The timing of growth factor addition modified the action of high-dose dexamethasone (10(-6) M) on DNA synthesis. When we added transforming growth factor-alpha at the time of plating, 10(-6) M dexamethasone weakly stimulated DNA synthesis by 26% relative to cells cultured in dexamethasone-free media. When we delayed growth factor addition until 24-48 h after plating, 10(-6) M dexamethasone inhibited DNA synthesis by 50%. Using immunological methods, we analyzed the expression and signaling patterns of the ErbB kinases in dexamethasone-treated cells. High-dose dexamethasone stabilized the expression of epidermal growth factor receptor (EGFr) and ErbB3, and it suppressed the de novo expression of ErbB2 that occurs during the third and fourth day of culture in 10(-8) M dexamethasone. High-dose dexamethasone by 72 h suppressed basal and EGF-associated phosphorylation of ERK and Akt. The reduction in ERK1/2 phosphorylation correlated with suppression of a culture-dependent increase in Son-of sevenless 1 (Sos1) and ERK1/2 expression. High-dose dexamethasone in hepatocytes stabilized or upregulated several inhibitory effectors of EGFr/ErbB2 and ERK, including receptor-associated late transducer (RALT) and MKP-1, respectively. Thus 10(-6) M dexamethasone exerts a time-dependent and redundant inhibitory effect on EGFr-mediated proliferative signaling in hepatocytes, targeting not only the ErbB proteins but also their various positive and negative effectors.     

ErbB4 signaling during breast and neural development: novel genetic models reveal unique ErbB4 activities

The erbB4 gene encodes one of the four members of the mammalian ErbB family of transmembrane tyrosine kinases. The ErbB4 protein plays a role as a receptor for the neuregulins, a large group of structurally related molecules and a few other epidermal growth factor (EGF)-related polypeptides, such as heparin-binding EGF, betacellulin and epiregulin. The importance of this receptor tyrosine kinase in development has been demonstrated by the generation of mice with a targeted inactivation of the erbB4 gene. Such mice die by embryonic day eleven due to defective trabeculation in the heart, precluding analysis of phenotypes at later stages in development and in the adult. Now, using two unique genetic approaches our laboratories succeeded in overcoming this obstacle. In the first approach, the heart defects of ErbB4 null mutant mice were rescued by transgenic expression of an ErbB4 cDNA under a cardiac-specific myosin promoter. This allowed the generation of ErbB4 mutants that develop into adulthood and are fertile. In the second approach, the role of ErbB4 during mammary gland development was specifically addressed by Cre-mediated deletion of both erbB4 alleles within the mammary epithelium. Below we discuss the progress made studying these genetic models in understanding the physiological roles of ErbB4 with a focus on the mammary gland and the nervous system.     

Ligand discrimination in signaling through an ErbB4 receptor homodimer

The epidermal growth factor (EGF)-like family of growth factors elicits cellular responses by stimulating the dimerization, autophosphorylation, and tyrosine kinase activities of the ErbB family of receptor tyrosine kinases. Although several different EGF-like ligands are capable of binding to a single ErbB family member, it is generally thought that the biological and biochemical responses of a single receptor dimer to different ligands are indistinguishable. To test whether an ErbB receptor dimer is capable of discriminating among ligands we have examined the effect of four EGF-like growth factors on signaling through the ErbB4 receptor homodimer in CEM/HER4 cells, a transfected human T cell line ectopically expressing ErbB4 in an ErbB-null background. Despite stimulating similar levels of gross receptor tyrosine phosphorylation, the EGF-like growth factors betacellulin, neuregulin-1beta, neuregulin-2beta, and neuregulin-3 exhibited different biological potencies in a cellular growth assay. Moreover, the different ligands induced different patterns of recruitment of intracellular signaling proteins to the activated receptor and induced differential usage of intracellular kinase signaling cascades. Finally, two-dimensional phosphopeptide mapping of ligand-stimulated ErbB4 revealed that the different growth factors induce different patterns of receptor tyrosine phosphorylation. These results indicate that ErbB4 activation by growth factors is not generic and suggest that individual ErbB receptors can discriminate between different EGF-like ligands within the context of a single receptor dimer. More generally, our observations significantly modify our understanding of signaling through receptor tyrosine kinases and point to a number of possible models for ligand-mediated signal diversification.     

Neurexin-neuroligin signaling in synapse development

Neurexins and neuroligins are emerging as central organizing molecules for excitatory glutamatergic and inhibitory GABAergic synapses in mammalian brain. They function as cell adhesion molecules, bridging the synaptic cleft. Remarkably, each partner can trigger formation of a hemisynapse: neuroligins trigger presynaptic differentiation and neurexins trigger postsynaptic differentiation. Recent protein interaction assays and cell culture studies indicate a selectivity of function conferred by alternative splicing in both partners. An insert at site 4 of beta-neurexins selectively promotes GABAergic synaptic function, whereas an insert at site B of neuroligin 1 selectively promotes glutamatergic synaptic function. Initial knockdown and knockout studies indicate that neurexins and neuroligins have an essential role in synaptic transmission, particularly at GABAergic synapses, but further studies are needed to assess the in vivo functions of these complex protein families.     

ErbB signaling in cardiac development and disease

The ErbB family of receptor tyrosine kinases (RTKs) is a family of receptors that allow cells to interact with the extracellular environment and transduce signals to the nucleus that promote differentiation, migration and proliferation necessary for proper heart morphogenesis and function. This review focuses on the role of the ErbB family of receptor tyrosine kinases, and their importance in proper heart morphogenesis, as well as their role in maintenance and function of the adult heart. Studies from transgenic mouse models have shown the importance of ErbB receptors in heart development, and provide insight into potential future therapeutic targets to help reduce congenital heart defect (CHD) mortality rates and prevent disease in adults. Cancer therapeutics have also shed light to the ErbB receptors and signaling network, as undesired side effects have demonstrated their importance in adult cardiomyocytes and prevention of cardiomyopathies. This review will discuss ErbB receptor tyrosine kinases (RTK) in heart development and disease including valve formation and partitioning of a four-chambered heart as well as cardiotoxicity when ErbB signaling is attenuated in adults.     

p120 catenin regulates dendritic spine and synapse development through Rho-family GTPases and cadherins

Both the cadherin-catenin complex and Rho-family GTPases have been shown to regulate dendrite development. We show here a role for p120 catenin (p120ctn) in regulating spine and synapse formation in the developing mouse brain. p120catenin gene deletion in hippocampal pyramidal neurons in vivo resulted in reduced spine and synapse densities along dendrites. In addition, p120 catenin loss resulted in reduced cadherin levels and misregulation of Rho-family GTPases, with decreased Rac1 and increased RhoA activity. Analyses in vitro indicate that the reduced spine density reflects aberrant Rho-family GTPase signaling, whereas the effects on spine maturation appear to result from reduced cadherin levels and possibly aberrant Rho-family GTPase signaling. Thus, p120ctn acts as a signal coordinator between cadherins and Rho-family GTPases to regulate cytoskeletal changes required during spine and synapse development.     

Constitutively active ErbB4 and ErbB2 mutants exhibit distinct biological activities.

ErbB4 is a member of the epidermal growth factor receptor(EGFR) family of tyrosine kinases, which includes EGFR/ErbB1, ErbB2/HER2/Neu, and ErbB3/HER3. These receptors play important roles both in normal development and in neoplasia. For example, deregulated signaling by ErbB1 and ErbB2 is observed in many human malignancies. In contrast, the roles that ErbB4 plays in tumorigenesis and normal biological processes have not been clearly defined. To identify the biological responses that are coupled to ErbB4, we have constructed three constitutively active ErbB4 mutants. Unlike a constitutively active ErbB2 mutant, the ErbB4 mutants are not coupled to increased cell proliferation, loss of contact inhibition, or anchorage independence in a rodent fibroblast cell line. This suggests that ErbB2 and ErbB4 may play distinct roles in tumorigenesis in vivo.     

Mechanical stimuli and nutrients regulate rapamycin-sensitive signaling through distinct mechanisms in skeletal muscle

The mammalian target of rapamycin (mTOR) has been identified as a growth factor and nutrient-sensitive molecule that controls the translational machinery and cell growth. Rapamycin-sensitive (RS) signaling events have also been shown to be necessary for mechanical load-induced growth of skeletal muscle, but the mechanisms involved in the mechanical activation of RS signaling are not known. The finding that mechanical stimuli induce nutrient uptake in skeletal muscle raises the possibility that mechanically induced RS signaling is mediated via a nutrient-dependent mechanism. To investigate this hypothesis, skeletal muscles (ex vivo) were stimulated with nutrients or intermittent mechanical stretch and the phosphorylation of p70S6k [P-p70(389)], PKB [P-PKB], mTOR [P-mTOR(2481)], and p38 [P-p38] was assessed. In comparison to vehicle-treated controls, both nutrient and mechanical stimuli induced P-p70(389), neither stimulus altered P-PKB or P-mTOR(2481), and only mechanical stimuli induced P-p38. The nutrient and mechanically induced increase in P-p70(389) was blocked by rapamycin, but only nutrient-induced signaling to P-p70(389) was blocked by wortmannin. Furthermore, the mechanically induced increase in P-p70(389) was not impaired by the removal of exogenous nutrients. Taken together, these results indicate that exogenous nutrients are not required for mechanically induced RS signaling and that nutrient and mechanical stimuli activate RS signaling through distinct upstream mechanisms.     

Pak1 and Pak2 mediate tumor cell invasion through distinct signaling mechanisms

Pak kinases are thought to play critical roles in cell migration and invasion. Here, we analyze the roles of Pak1 and Pak2 in breast carcinoma cell invasion using the transient transfection of small interfering RNA. We find that although both Pak1 and Pak2 contribute to breast carcinoma invasion stimulated by heregulin, these roles are mediated by distinct signaling mechanisms. Thus, whereas the depletion of Pak1 interferes with the heregulin-mediated dephosphorylation of cofilin, the depletion of Pak2 does not. The depletion of Pak1 also has a stronger inhibitory effect on lamellipodial protrusion than does the depletion of Pak2. Interestingly, Pak1 and Pak2 play opposite roles in regulating the phosphorylation of the myosin light chain (MLC). Whereas the depletion of Pak1 decreases phospho-MLC levels in heregulin-stimulated cells, the depletion of Pak2 enhances MLC phosphorylation. Consistent with their opposite effects on MLC phosphorylation, Pak1 and Pak2 differentially modulate focal adhesions. Pak2-depleted cells display an increase in focal adhesion size, whereas in Pak1-depleted cells, focal adhesions fail to mature. We also found that the depletion of Pak2, but not Pak1, enhances RhoA activity and that the inhibition of RhoA signaling in Pak2-depleted cells decreases MLC phosphorylation and restores cell invasion. In summary, this work presents the first comprehensive analysis of functional differences between the Pak1 and Pak2 isoforms.     

Orchestration of ErbB3 signaling through heterointeractions and homointeractions

Members of the ErbB family of receptor tyrosine kinases are capable of both homointeractions and heterointeractions. Because each receptor has a unique set of binding sites for downstream signaling partners and differential catalytic activity, subtle shifts in their combinatorial interplay may have a large effect on signaling outcomes. The overexpression and mutation of ErbB family members are common in numerous human cancers and shift the balance of activation within the signaling network. Here we report the development of a spatial stochastic model that addresses the dynamics of ErbB3 homodimerization and heterodimerization with ErbB2. The model is based on experimental measures for diffusion, dimer off-rates, kinase activity, and dephosphorylation. We also report computational analysis of ErbB3 mutations, generating the prediction that activating mutations in the intracellular and extracellular domains may be subdivided into classes with distinct underlying mechanisms. We show experimental evidence for an ErbB3 gain-of-function point mutation located in the C-lobe asymmetric dimerization interface, which shows enhanced phosphorylation at low ligand dose associated with increased kinase activity.     

ErbB and HB-EGF signaling in heart development and function

The epidermal growth factor (EGF)-ErbB signaling network is composed of multiple ligands of the EGF family and four tyrosine kinase receptors of the ErbB family. In higher vertebrates, these four receptors bind a multitude of ligands. Ligand binding induces the formation of various homo- and heterodimers of ErbB, potentially providing for a high degree of signal diversity. ErbB receptors and their ligands are expressed in a variety of tissues throughout development. Recent advances in gene targeting strategies in mice have revealed that the EGF-ErbB signaling network has fundamental roles in development, proliferation, differentiation, and homeostasis in mammals. The heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family of growth factors that binds to and activates the EGF receptor (EGFR/ErbB1) and ErbB4. Recent studies using several mutant mice lacking HB-EGF expression have revealed that HB-EGF has a critical role in normal heart function and in normal cardiac valve formation in conjunction with ErbB receptors. HB-EGF signaling through ErbB2 is essential for the maintenance of homeostasis in the adult heart, whereas HB-EGF signaling through EGFR is required during cardiac valve development. In this review, we introduce and discuss the role of ErbB receptors in heart function and development, focusing on the physiological function of HB-EGF in these processes.     

Small noncoding vault RNA modulates synapse formation by amplifying MAPK signaling

The small noncoding vault RNA (vtRNA) is a component of the vault complex, a ribonucleoprotein complex found in most eukaryotes. Emerging evidence suggests that vtRNAs may be involved in the regulation of a variety of cellular functions when unassociated with the vault complex. Here, we demonstrate a novel role for vtRNA in synaptogenesis. Using an in vitro synapse formation model, we show that murine vtRNA (mvtRNA) promotes synapse formation by modulating the MAPK signaling pathway. mvtRNA is transported to the distal region of neurites as part of the vault complex. Interestingly, mvtRNA is released from the vault complex in the neurite by a mitotic kinase Aurora-A–dependent phosphorylation of MVP, a major protein component of the vault complex. mvtRNA binds to and activates MEK1 and thereby enhances MEK1-mediated ERK activation in neurites. These results suggest the existence of a regulatory mechanism of the MAPK signaling pathway by vtRNAs as a new molecular basis for synapse formation.     

Interleukin 7 and interleukin 4 stimulate human thymocyte growth through distinct mechanisms

One of the major functions of cytokines is their ability to regulate cell growth and differentiation. The complexity of this process has been highlighted by recent studies on murine thymocytes; it has been shown that a number of cytokines interact to regulate thymocyte growth. We have investigated the effects of interleukin 4 (IL-4) and interleukin 7 (IL-7) on human thymocyte proliferation. Although maximal proliferation was dependent upon the presence of the mitogen phytohaemagglutinin (PHA), IL-7 alone stimulated thymocyte growth. In order to determine if this proliferation was due to the induction of IL-2, this pathway was inhibited by the addition of blocking antibody to the IL-2 receptor. Proliferation induced with IL-7 plus PHA, but not that induced by IL-7 alone, could be blocked by this treatment. In contrast, IL-4 stimulated thymocyte proliferation only in the presence of PHA; this proliferation was not inhibited by antibodies to the IL-2 receptor. Our findings show that both IL-7 and IL-4 can act as growth factors for human thymocytes, and that these cytokines stimulate proliferation through distinct mechanisms.     

The GTPase Rem2 regulates synapse development and dendritic morphology

Rem2 is a member of the Rad/Rem/Rem2/Gem/Kir subfamily of small Ras-like GTPases that was identified as an important mediator of synapse development. We performed a comprehensive, loss- of-function analysis of Rem2 function in cultured hippocampal neurons using RNAi to substantially decrease Rem2 protein levels. We found that knockdown of Rem2 decreases the density and maturity of dendritic spines, the primary site of excitatory synapses onto pyramidal neurons in the hippocampus. Knockdown of Rem2 also alters the gross morphology of dendritic arborizations, increasing the number of dendritic branches without altering total neurite length. Thus, Rem2 functions to inhibit dendritic branching and promote the development of dendritic spines and excitatory synapses. Interestingly, binding to the calcium-binding protein calmodulin is required for the Rem2 regulation of dendritic branching. However, this interaction is completely dispensable for synapse development. Overall, our results suggest that Rem2 regulates dendritic branching and synapse development via distinct and overlapping signal transduction pathways.     

The protein dendrite arborization and synapse maturation 1 (Dasm-1) is dispensable for dendrite arborization

The development of a highly branched dendritic tree is essential for the establishment of functional neuronal connections. The evolutionarily conserved immunoglobulin superfamily member, the protein dendrite arborization and synapse maturation 1 (Dasm-1) is thought to play a critical role in dendrite formation of dissociated hippocampal neurons. RNA interference-mediated Dasm-1 knockdown was previously shown to impair dendrite, but not axonal, outgrowth and branching (S. H. Shi, D. N. Cox, D. Wang, L. Y. Jan, and Y. N. Jan, Proc. Natl. Acad. Sci. USA 101:13341-13345, 2004). Here, we report the generation and analysis of Dasm-1 null mice. We find that genetic ablation of Dasm-1 does not interfere with hippocampal dendrite growth and branching in vitro and in vivo. Moreover, the absence of Dasm-1 does not affect the modulation of dendritic outgrowth induced by brain-derived neurotrophic factor. Importantly, the previously observed impairment in dendrite growth after Dasm-1 knockdown is also observed when the Dasm-1 knockdown is performed in cultured hippocampal neurons from Dasm-1 null mice. These findings indicate that the dendrite arborization phenotype was caused by off-target effects and that Dasm-1 is dispensable for hippocampal dendrite arborization.     

TNF receptor 1 mediates dendritic cell maturation and CD8 T cell response through two distinct mechanisms

TNF-α and its two receptors (TNFR1 and 2) are known to stimulate dendritic cell (DC) maturation and T cell response. However, the specific receptor and mechanisms involved in vivo are still controversial. In this study, we show that in response to an attenuated mouse hepatitis virus infection, DCs fail to mobilize and up-regulate CD40, CD80, CD86, and MHC class I in TNFR1(-/-) mice as compared with the wild-type and TNFR2(-/-) mice. Correspondingly, virus-specific CD8 T cell response was dramatically diminished in TNFR1(-/-) mice. Adoptive transfer of TNFR1-expressing DCs into TNFR1(-/-) mice rescues CD8 T cell response. Interestingly, adoptive transfer of TNFR1-expressing naive T cells also restores DC mobilization and maturation and endogenous CD8 T cell response. These results show that TNFR1, not TNFR2, mediates TNF-α stimulation of DC maturation and T cell response to mouse hepatitis virus in vivo. They also suggest two mechanisms by which TNFR1 mediates TNF-α-driven DC maturation, as follows: a direct effect through TNFR1 expressed on immature DCs and an indirect effect through TNFR1 expressed on naive T cells.     

Agomelatine modulates calcium signaling through protein kinase C and phospholipase C-mediated mechanisms in rat sensory neurons

Agomelatine, a novel antidepressant exerting its effects through melatonergic and serotonergic systems, implicated to be effective against pain including neuropathic pain but without any knowledge of mechanism of action. To explore the possible role of agomelatine on nociceptive transmission at the peripheral level, the effects of agomelatine on intracellular calcium ([Ca²⁺]_i) signaling in peripheral neurons were investigated in cultured rat dorsal root ganglion (DRG) neurons. Using the fura-2-based calcium imaging technique, the effects of agomelatine on [Ca²⁺]_i and roles of the second messenger-mediated pathways were assessed. Agomelatine caused [Ca²⁺]_i signaling in a dose-dependent manner when tested at 10 and 100 μM concentration. Luzindole, a selective melatonin receptor antagonist, almost completely blocked the agomelatine-induced calcium signals. The agomelatine-induced calcium transients were also nearly abolished following pretreatment with the 100 ng/ml pertussis toxin, a Gi/o protein inhibitor. The stimulatory effects of agomelatine on [Ca²⁺]_i transients were significantly reduced by applications of phospholipase C (PLC) and protein kinase C (PKC) blockers, 10 μM U73122, and 10 μM chelerythrine chloride, respectively. The obtained results of agomelatine-induced [Ca²⁺]_i signals indicates that peripheral mechanisms are involved in analgesic effects of agomelatine. These mechanisms seems to involve G-protein-coupled receptor activation and PLC and PKC mediated mechanisms.     

Wip1 phosphatase positively modulates dendritic spine morphology and memory processes through the p38MAPK signaling pathway

Dendritic spine morphology is modulated by protein kinase p38, a mitogen-activated protein (MAPK), in the hippocampus. Protein p38MAPK is a substrate of wip1, a protein phosphatase. The role of wip1 in the central nervous system (CNS) has never been explored. Here, we report a novel function of wip1 in dendritic spine morphology and memory processes. Wip1 deficiency decreases dendritic spine size and density in pyramidal neurons of the hippocampal CA1 region. Simultaneously, impairments in object recognition tasks and contextual memory occur in wip1 deficient mice, but are reversed in wip1/p38 double mutant mice. Thus, our findings demonstrate that wip1 modulates dendritic morphology and memory processes through the p38MAPK signaling pathway. In addition to the well-characterized role of the wip1/p38MAPK in cell death and differentiation, we revealed the novel contribution of wip1 to cognition and dendritic spine morphology, which may suggest new approaches to treating neurodegenerative disorders.     

Wip1 phosphatase positively modulates dendritic spine morphology and memory processes through the p38MAPK signaling pathway

Dendritic spine morphology is modulated by protein kinase p38, a mitogen-activated protein (MAPK), in the hippocampus. Protein p38MAPK is a substrate of wip1, a protein phosphatase. The role of wip1 in the central nervous system (CNS) has never been explored. Here, we report a novel function of wip1 in dendritic spine morphology and memory processes. Wip1 deficiency decreases dendritic spine size and density in pyramidal neurons of the hippocampal CA1 region. Simultaneously, impairments in object recognition tasks and contextual memory occur in wip1 deficient mice, but are reversed in wip1/p38 double mutant mice. Thus, our findings demonstrate that wip1 modulates dendritic morphology and memory processes through the p38MAPK signaling pathway. In addition to the well-characterized role of the wip1/p38MAPK in cell death and differentiation, we revealed the novel contribution of wip1 to cognition and dendritic spine morphology, which may suggest new approaches to treating neurodegenerative disorders.     

Biased signaling of the angiotensin II type 1 receptor can be mediated through distinct mechanisms

Background

Seven transmembrane receptors (7TMRs) can adopt different active conformations facilitating a selective activation of either G protein or β-arrestin-dependent signaling pathways. This represents an opportunity for development of novel therapeutics targeting selective biological effects of a given receptor. Several studies on pathway separation have been performed, many of these on the Angiotensin II type 1 receptor (AT1R). It has been shown that certain ligands or mutations facilitate internalization and/or recruitment of β-arrestins without activation of G proteins. However, the underlying molecular mechanisms remain largely unresolved. For instance, it is unclear whether such selective G protein-uncoupling is caused by a lack of ability to interact with G proteins or rather by an increased ability of the receptor to recruit β-arrestins. Since uncoupling of G proteins by increased ability to recruit β-arrestins could lead to different cellular or in vivo outcomes than lack of ability to interact with G proteins, it is essential to distinguish between these two mechanisms.

Methodology/Principal Findings

We studied five AT1R mutants previously published to display pathway separation: D74N, DRY/AAY, Y292F, N298A, and Y302F (Ballesteros-Weinstein numbering: 2.50, 3.49–3.51, 7.43, 7.49, and 7.53). We find that D74N, DRY/AAY, and N298A mutants are more prone to β-arrestin recruitment than WT. In contrast, receptor mutants Y292F and Y302F showed impaired ability to recruit β-arrestin in response to Sar¹-Ile⁴-Ile⁸ (SII) Ang II, a ligand solely activating the β-arrestin pathway.

Conclusions/Significance

Our analysis reveals that the underlying conformations induced by these AT1R mutants most likely represent principally different mechanisms of uncoupling the G protein, which for some mutants may be due to their increased ability to recruit β-arrestin2. Hereby, these findings have important implications for drug discovery and 7TMR biology and illustrate the necessity of uncovering the exact molecular determinants for G protein-coupling and β-arrestin recruitment, respectively.