Expression of the Receptor Tyrosine Kinase EphB2 on Dendritic Cells Is Modulated by Toll-Like Receptor Ligation but Is Not Required for T Cell Activation

The Eph receptor tyrosine kinases interact with their ephrin ligands on adjacent cells to facilitate contact-dependent cell communication. Ephrin B ligands are expressed on T cells and have been suggested to act as co-stimulatory molecules during T cell activation. There are no detailed reports of the expression and modulation of EphB receptors on dendritic cells, the main antigen presenting cells that interact with T cells. Here we show that mouse splenic dendritic cells (DC) and bone-marrow derived DCs (BMDC) express EphB2, a member of the EphB family. EphB2 expression is modulated by ligation of TLR4 and TLR9 and also by interaction with ephrin B ligands. Co-localization of EphB2 with MHC-II is also consistent with a potential role in T cell activation. However, BMDCs derived from EphB2 deficient mice were able to present antigen in the context of MHC-II and produce T cell activating cytokines to the same extent as intact DCs. Collectively our data suggest that EphB2 may contribute to DC responses, but that EphB2 is not required for T cell activation. This result may have arisen because DCs express other members of the EphB receptor family, EphB3, EphB4 and EphB6, all of which can interact with ephrin B ligands, or because EphB2 may be playing a role in another aspect of DC biology such as migration.     

Role of Cx43 Phosphorylation and MAP Kinase Activation in EGF Induced Enhancement of Cell Communication in Human Kidney Epithelial Cells

Epidermal growth factor (EGF) has been found to induce enhanced gap junctional intercellular communication (GJIC) in the human kidney epithelial cell line K7. This is in contrast to what is reported for other cell types, which all show decreased GJIC in response to EGF. In the present study it is shown that 12-O-tetradecanoylphorbol-13-acetate (TPA) and EGF induce similar phosphorylation pattern of the gap junction protein connexin43 (Cx43) in K7 cells, although their effects on GJIC are opposite. Tyrosine phosphorylation of a 42 kD protein was observed to be induced concomitantly with phosphorylation of Cx43. EGF was however found to induce only serine phosphorylation of Cx43, indicating that the tyrosine kinase activity of the EGF receptor was not directly affecting the gap junction protein. The 42 kD protein phosphorylated on tyrosine was identified to be a mitogen activated protein (MAP) kinase. Both EGF and TPA was found to activate MAP kinase in these cells. Phosphorylation of Cx43 and enhancement of GJIC in response to EGF occurred with difference in time course. Phosphorylation of Cx43 was completed within 15 min, while the enhanced GJIC appeared 2-3 h later. It is therefore possible that regulation of synthesis or transport of Cx43 is responsible for the increase in GJIC, rather than direct involvement of Cx43 phosphorylation. This is in support of our previous finding that protein synthesis is necessary for EGF induced upregulation of GJIC in K7 cells.     

The Tyrosine Kinase c-Met Contributes to the Pro-tumorigenic Function of the p38 Kinase in Human Bile Duct Cholangiocarcinoma Cells

Pro-tumorigenic function of the p38 kinase plays a critical role in human cholangiocarcinogenesis. However, the underlying mechanism remains incompletely understood. Here, we report that c-Met, the tyrosine kinase receptor for hepatocyte growth factor (HGF), contributes to the pro-tumorigenic ability of p38 in human cholangiocarcinoma cells. Both p38 and c-Met promote the proliferation and invasion of human cholangiocarcinoma cells. Importantly, inhibition or knockdown of p38 decreased the basal activation of c-Met. Tyrosine phosphatase inhibitor studies revealed that p38 promotes the activity of c-Met, at least in part, by inhibiting dephosphorylation of the receptor. Moreover, density enhanced phosphatase-1 (DEP-1) is involved in p38-mediated inhibiting dephosphorylation of c-Met. Furthermore, p38 inhibits the degradation of c-Met. Taken together, these data provide a potential mechanism to explain how p38 promotes human cholangiocarcinoma cell proliferation and invasion. We propose that the link between p38 and c-Met is implicated in the progression of human cholangiocarcinoma.     

Activation of Extracellular Signal-Regulated Kinase but Not of p38 Mitogen-Activated Protein Kinase Pathways in Lymphocytes Requires Allosteric Activation of SOS

Thymocytes convert graded T cell receptor (TCR) signals into positive selection or deletion, and activation of extracellular signal-related kinase (ERK), p38, and Jun N-terminal protein kinase (JNK) mitogen-activated protein kinases (MAPKs) has been postulated to play a discriminatory role. Two families of Ras guanine nucleotide exchange factors (RasGEFs), SOS and RasGRP, activate Ras and the downstream RAF-MEK-ERK pathway. The pathways leading to lymphocyte p38 and JNK activation are less well defined. We previously described how RasGRP alone induces analog Ras-ERK activation while SOS and RasGRP cooperate to establish bimodal ERK activation. Here we employed computational modeling and biochemical experiments with model cell lines and thymocytes to show that TCR-induced ERK activation grows exponentially in thymocytes and that a W729E allosteric pocket mutant, SOS1, can only reconstitute analog ERK signaling. In agreement with RasGRP allosterically priming SOS, exponential ERK activation is severely decreased by pharmacological or genetic perturbation of the phospholipase Cγ (PLCγ)-diacylglycerol-RasGRP1 pathway. In contrast, p38 activation is not sharply thresholded and requires high-level TCR signal input. Rac and p38 activation depends on SOS1 expression but not allosteric activation. Based on computational predictions and experiments exploring whether SOS functions as a RacGEF or adaptor in Rac-p38 activation, we established that the presence of SOS1, but not its enzymatic activity, is critical for p38 activation.     

A Novel Role of Protein Tyrosine Kinase2 in Mediating Chloride Secretion in Human Airway Epithelial Cells

Ca²⁺ activated Cl⁻ channels (CaCC) are up-regulated in cystic fibrosis (CF) airway surface epithelia. The presence and functional properties of CaCC make it a possible therapeutic target to compensate for the deficiency of Cl⁻ secretion in CF epithelia. CaCC is activated by an increase in cytosolic Ca²⁺, which not only activates epithelial CaCCs, but also inhibits epithelial Na⁺ hyperabsorption, which may also be beneficial in CF. Our previous study has shown that spiperone, a known antipsychotic drug, activates CaCCs and stimulates Cl⁻ secretion in polarized human non-CF and CF airway epithelial cell monolayers in vitro, and in Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) knockout mice in vivo. Spiperone activates CaCC not by acting in its well-known role as an antagonist of either 5-HT2 or D2 receptors, but through a protein tyrosine kinase-coupled phospholipase C-dependent pathway. Moreover, spiperone independently activates CFTR through a novel mechanism. Herein, we performed a mass spectrometry analysis and identified the signaling molecule that mediates the spiperone effect in activating chloride secretion through CaCC and CFTR. Proline-rich tyrosine kinase 2 (PYK2) is a non-receptor protein tyrosine kinase, which belongs to the focal adhesion kinase family. The inhibition of PYK2 notably reduced the ability of spiperone to increase intracellular Ca²⁺ and Cl⁻ secretion. In conclusion, we have identified the tyrosine kinase, PYK2, as the modulator, which plays a crucial role in the activation of CaCC and CFTR by spiperone. The identification of this novel role of PYK2 reveals a new signaling pathway in human airway epithelial cells.     

Protein Kinase Cδ Mediates Neurogenic but Not Mitogenic Activation of Mitogen-Activated Protein Kinase in Neuronal Cells

In several neuronal cell systems, fibroblast-derived growth factor (FGF) and nerve growth factor (NGF) act as neurogenic agents, whereas epidermal growth factor (EGF) acts as a mitogen. The mechanisms responsible for these different cellular fates are unclear. We report here that although FGF, NGF, and EGF all activate mitogen-activated protein (MAP) kinase (extracellular signal-related kinase [ERK]) in rat hippocampal (H19-7) and pheochromocytoma (PC12) cells, the activation of ERK by the neurogenic agents FGF and NGF is dependent upon protein kinase Cdelta (PKCdelta), whereas ERK activation in response to the mitogenic EGF is independent of PKCdelta. Antisense PKCdelta oligonucleotides or the PKCdelta-specific inhibitor rottlerin inhibited FGF- and NGF-induced, but not EGF-induced, ERK activation. In contrast, EGF-induced ERK activation was inhibited by the phosphatidylinositol-3-kinase inhibitor wortmannin, which had no effect upon FGF-induced ERK activation. Rottlerin also inhibited the activation of MAP kinase kinase (MEK) in response to activated Raf, but had no effect upon c-Raf activity or ERK activation by activated MEK. These results indicate that PKCdelta functions either downstream from or in parallel with c-Raf, but upstream of MEK. Inhibition of PKCdelta also blocked neurite outgrowth induced by FGF and NGF in PC12 cells and by activated Raf in H19-7 cells, indicating a role for PKCdelta in the neurogenic effects of FGF, NGF, and Raf. Interestingly, the PKCdelta requirement is apparently cell type specific, since FGF-induced ERK activation was independent of PKCdelta in NIH 3T3 murine fibroblasts, in which FGF is a mitogen. These data demonstrate that PKCdelta contributes to growth factor specificity and response in neuronal cells and may also promote cell-type-specific differences in growth factor signaling.     

Obesity-Induced Insulin Resistance in Human Skeletal Muscle Is Characterised by Defective Activation of p42/p44 MAP Kinase

Insulin resistance (IR), an impaired cellular, tissue and whole body response to insulin, is a major pathophysiological defect of type 2 diabetes mellitus. Although IR is closely associated with obesity, the identity of the molecular defect(s) underlying obesity-induced IR in skeletal muscle remains controversial; reduced post-receptor signalling of the insulin receptor substrate 1 (IRS1) adaptor protein and downstream effectors such as protein kinase B (PKB) have previously been implicated. We examined expression and/or activation of a number of components of the insulin-signalling cascade in skeletal muscle of 22 healthy young men (with body mass index (BMI) range, 20–37 kg/m²). Whole body insulin sensitivity (M value) and body composition was determined by the hyperinsulinaemic (40 mU. min⁻¹.m⁻².), euglycaemic clamp and by dual energy X-ray absorptiometry (DEXA) respectively. Skeletal muscle (vastus lateralis) biopsies were taken before and after one hour of hyperinsulinaemia and the muscle insulin signalling proteins examined by western blot and immunoprecipitation assay. There was a strong inverse relationship between M-value and BMI. The most striking abnormality was significantly reduced insulin-induced activation of p42/44 MAP kinase, measured by specific assay, in the volunteers with poor insulin sensitivity. However, there was no relationship between individuals'' BMI or M-value and protein expression/phosphorylation of IRS1, PKB, or p42/44 MAP kinase protein, under basal or hyperinsulinaemic conditions. In the few individuals with poor insulin sensitivity but preserved p42/44 MAP kinase activation, other signalling defects were evident. These findings implicate defective p42/44 MAP kinase signalling as a potential contributor to obesity-related IR in a non-diabetic population, although clearly multiple signalling defects underlie obesity associated IR.     

Barrier-Protective Effects of Activated Protein C in Human Alveolar Epithelial Cells

Acute lung injury (ALI) is a clinical manifestation of respiratory failure, caused by lung inflammation and the disruption of the alveolar-capillary barrier. Preservation of the physical integrity of the alveolar epithelial monolayer is of critical importance to prevent alveolar edema. Barrier integrity depends largely on the balance between physical forces on cell-cell and cell-matrix contacts, and this balance might be affected by alterations in the coagulation cascade in patients with ALI. We aimed to study the effects of activated protein C (APC) on mechanical tension and barrier integrity in human alveolar epithelial cells (A549) exposed to thrombin. Cells were pretreated for 3 h with APC (50 µg/ml) or vehicle (control). Subsequently, thrombin (50 nM) or medium was added to the cell culture. APC significantly reduced thrombin-induced cell monolayer permeability, cell stiffening, and cell contraction, measured by electrical impedance, optical magnetic twisting cytometry, and traction microscopy, respectively, suggesting a barrier-protective response. The dynamics of the barrier integrity was also assessed by western blotting and immunofluorescence analysis of the tight junction ZO-1. Thrombin resulted in more elongated ZO-1 aggregates at cell-cell interface areas and induced an increase in ZO-1 membrane protein content. APC attenuated the length of these ZO-1 aggregates and reduced the ZO-1 membrane protein levels induced by thrombin. In conclusion, pretreatment with APC reduced the disruption of barrier integrity induced by thrombin, thus contributing to alveolar epithelial barrier protection.     

Src Tyrosine Kinase Activation by 4-Hydroxynonenal Upregulates p38, ERK/AP-1 Signaling and COX-2 Expression in YPEN-1 Cells

4-Hydroxynonenal (4-HNE), a major end product of lipid peroxidation, is highly reactive and involved in various cellular processes, such as inflammatory signaling. However, to date, the mechanistic roles of 4-HNE in inflammatory signaling related to protein tyrosine kinases have not been elucidated. In the present study, we investigated the interaction between 4-HNE and Src (a non-receptor tyrosine kinase) for its involvement in the molecular modulation of the inflammatory signaling pathway utilizing the YPEN-1 cell system. Immunoprecipitation experiments showed that 4-HNE phosphorylates (activates) Src at Tyr416 via adduct formation. In addition, LC-MS/MS and a docking simulation model revealed an addiction site at the Cys248 residue of Src, resulting in the stimulation of downstream p38, ERK/AP-1 and cyclooxygenase-2 (COX-2) signaling in YPEN-1 cells. The role of 4-HNE-activated Src in downstream inflammatory signaling was further investigated using dasatinib (a Src inhibitor) and by siRNA knockdown of Src. p38 and ERK were directly regulated by Src, as revealed by immunoblotting of the phosphorylated forms of mitogen-activated protein kinases (MAPKs), which are key elements in the signaling transduction pathway initiated by Src. The study also shows that Src modulates the HNE-enhanced activation of AP-1 and the expression of COX-2 (a target gene of AP-1). Together, the results of this study show that 4-HNE stimulates Src tyrosine kinase in activation of the inflammation process.     

MAPK Activation Is Essential for Waddlia chondrophila Induced CXCL8 Expression in Human Epithelial Cells

Background

Waddlia chondrophila (W. chondrophila) is an emerging agent of respiratory and reproductive disease in humans and cattle. The organism is a member of the order Chlamydiales, and shares many similarities at the genome level and in growth studies with other well-characterised zoonotic chlamydial agents, such as Chlamydia abortus (C. abortus). The current study investigated the growth characteristics and innate immune responses of human and ruminant epithelial cells in response to infection with W. chondrophila.

Methods

Human epithelial cells (HEp2) were infected with W. chondrophila for 24h. CXCL8 release was significantly elevated in each of the cell lines by active-infection with live W. chondrophila, but not by exposure to UV-killed organisms. Inhibition of either p38 or p42/44 MAPK significantly inhibited the stimulation of CXCL8 release in each of the cell lines. To determine the pattern recognition receptor through which CXCL8 release was stimulated, wild-type HEK293 cells which express no TLR2, TLR4, NOD2 and only negligible NOD1 were infected with live organisms. A significant increase in CXCL8 was observed.

Conclusions/Significance

W. chondrophila actively infects and replicates within both human and ruminant epithelial cells stimulating CXCL8 release. Release of CXCL8 is significantly inhibited by inhibition of either p38 or p42/44 MAPK indicating a role for this pathway in the innate immune response to W. chondrophila infection. W. chondrophila stimulation of CXCL8 secretion in HEK293 cells indicates that TLR2, TLR4, NOD2 and NOD1 receptors are not essential to the innate immune response to infection.     

Role of Protein Kinase C,PI3-kinase and Tyrosine Kinase in Activation of MAP Kinase by Glucose and Agonists of G-protein Coupled Receptors in INS-1 Cells

MAP (mitogen-activated protein) kinase (also called Erk 1/2) plays a crucial role in cell proliferation and differentiation. Its impact on secretory events is less well established. The interplay of protein kinase C (PKC), PI3-kinase nd cellular tyrosine kinase with MAP kinase activity using inhibitors and compounds such as glucose, phorbol 12-myristate 13-acetate (PMA) and agonists of G-protein coupled receptors like gastrin releasing peptide (GRP), oxytocin (OT) and glucose-dependent insulinotropic peptide (GIP) was investigated in INS-1 cells, an insulin secreting cell line. MAP kinase activity was determined by using a peptide derived from the EGF receptor as a MAP kinase substrate and [P32]ATP. Glucose as well as GRP, OT and GIP exhibited a time-dependent increase in MAP kinase activity with a maximum at time point 2.5 min. All further experiments were performed using 2.5 min incubations. The flavone PD 098059 is known to bind to the inactive forms of MEK1 (MAPK/ERK-Kinase) thus preventing activation by upstream activators. 20 μM PD 098059 (IC₅₀=51 μM) inhibited MAP kinase stimulated by either glucose, GRP, OT, GIP or PMA. Inhibiton (“downregulation”) of PKC by a long term (22h) pretreatment with 1 μM PMA did not influence MAP kinase activity when augmented by either of the above mentioned compound. To investigate whether PI3-kinase and cellular tyrosine kinase are involved in G-protein mediated effects on MAP kinase, inhibitors were used: 100 nM wortmannin (PI3-kinase inhibitor) reduced the effects of GRP, OT and GIP but not that of PMA; 100 μM genistein (tyrosine kinase inhibitor) inhibited the stimulatory effect of either above mentioned compound on MAP kinase activation. Inhibition of MAP kinase by 20 μM PD 098059 did not influence insulin secretion modulated by either compound (glucose, GRP, OT or GIP). [H3]Thymidine incorporation, however, was severely inhibited by PD 098059. Thus MAP kinase is important for INS-1 cell proliferation but not for its insulin secretory response with respect to major initiators and modulators of insulin release. The data indicate that MAP kinase is active and under the control of MAP kinase. PKC is upstream of a genisteinsensitive tyrosine kinase and probably downstream of a PI3-kinase in INS-1 cells.     

The Tyrosine Kinase p56lck Mediates Activation of Swelling-induced Chloride Channels in Lymphocytes

Osmotic cell swelling activates Cl⁻ channels to achieve anion efflux. In this study, we find that both the tyrosine kinase inhibitor herbimycin A and genetic knockout of p56^lck, a src-like tyrosine kinase, block regulatory volume decrease (RVD) in a human T cell line. Activation of a swelling-activated chloride current (I_Cl−swell) by osmotic swelling in whole-cell patch-clamp experiments is blocked by herbimycin A and lavendustin. Osmotic activation of I_Cl−swell is defective in p56^lck-deficient cells. Retransfection of p56^lck restores osmotic current activation. Furthermore, tyrosine kinase activity is sufficient for activation of I_Cl−swell. Addition of purified p56^lck to excised patches activates an outwardly rectifying chloride channel with 31 pS unitary conductance. Purified p56^lck washed into the cytoplasm activates I_Cl−swell in native and p56^lck-deficient cells even when hypotonic intracellular solutions lead to cell shrinkage. When whole-cell currents are activated either by swelling or by p56^lck, slow single-channel gating events can be observed revealing a unitary conductance of 25–28 pS. In accordance with our patch-clamp data, osmotic swelling increases activity of immunoprecipitated p56^lck. We conclude that osmotic swelling activates I_Cl−swell in lymphocytes via the tyrosine kinase p56^lck.     

Overexpression of MERTK Receptor Tyrosine Kinase in Epithelial Cancer Cells Drives Efferocytosis in a Gain-of-Function Capacity

MERTK, a member of the TAM (TYRO3, AXL, and MERTK) receptor tyrosine kinases, has complex and diverse roles in cell biology. On the one hand, knock-out of MERTK results in age-dependent autoimmunity characterized by failure of apoptotic cell clearance, while on the other, MERTK overexpression in cancer drives classical oncogene pathways leading to cell transformation. To better understand the interplay between cell transformation and efferocytosis, we stably expressed MERTK in human MCF10A cells, a non-tumorigenic breast epithelial cell line devoid of endogenous MERTK. While stable expression of MERTK in MCF10A resulted in enhanced motility and AKT-mediated chemoprotection, MERTK-10A cells did not form stable colonies in soft agar, or enhance proliferation compared with parental MCF10A cells. Concomitant to chemoresistance, MERTK also stimulated efferocytosis in a gain-of-function capacity. However, unlike AXL, MERTK activation was highly dependent on apoptotic cells, suggesting MERTK may preferentially interface with phosphatidylserine. Consistent with this idea, knockdown of MERTK in breast cancer cells MDA-MB 231 reduced efferocytosis, while transient or stable expression of MERTK stimulated apoptotic cell clearance in all cell lines tested. Moreover, human breast cancer cells with higher endogenous MERTK showed higher levels of efferocytosis that could be blocked by soluble TAM receptors. Finally, through MERTK, apoptotic cells induced PD-L1 expression, an immune checkpoint blockade, suggesting that cancer cells may adopt MERTK-driven efferocytosis as an immune suppression mechanism for their advantage. These data collectively identify MERTK as a significant link between cancer progression and efferocytosis, and a potentially unrealized tumor-promoting event when MERTK is overexpressed in epithelial cells.     

Low-Dose,Long-Wave UV Light Does Not Affect Gene Expression of Human Mesenchymal Stem Cells

Light is a non-invasive tool that is widely used in a range of biomedical applications. Techniques such as photopolymerization, photodegradation, and photouncaging can be used to alter the chemical and physical properties of biomaterials in the presence of live cells. Long-wave UV light (315 nm–400 nm) is an easily accessible and commonly used energy source for triggering biomaterial changes. Although exposure to low doses of long-wave UV light is generally accepted as biocompatible, most studies employing this wavelength only establish cell viability, ignoring other possible (non-toxic) effects. Since light exposure of wavelengths longer than 315 nm may potentially induce changes in cell behavior, we examined changes in gene expression of human mesenchymal stem cells exposed to light under both 2D and 3D culture conditions, including two different hydrogel fabrication techniques, decoupling UV exposure and radical generation. While exposure to long-wave UV light did not induce significant changes in gene expression regardless of culture conditions, significant changes were observed due to scaffold fabrication chemistry and between cells plated in 2D versus encapsulated in 3D scaffolds. In order to facilitate others in searching for more specific changes between the many conditions, the full data set is available on Gene Expression Omnibus for querying.     

Identification of p42 Mitogen-Activated Protein Kinase as a Tyrosine Kinase Substrate Activated by Maximal Electroconvulsive Shock in Hippocampus

Abstract: Recent studies have demonstrated that administration of an electroconvulsive shock produces a rapid and transient increase in tyrosyl phosphorylation of a ∼40-kDa protein in rat brain. Initial characterization of this protein's chromatographic properties indicated that it might be a member of a recently identified family of kinases, referred to as mitogen-activated protein (MAP) kinases, that are activated by tyrosyl phosphorylation. In the present study, we have used MAP kinase antisera to assess the identity of this protein. We have found that the ∼40-kDa phosphotyrosine-containing protein comigrates with p42 MAP kinase (p42^mapk) and not with two other 44-kDa MAP kinase family members detected by these antisera. Western blots of proteins immunoprecipitated with MAP kinase antibodies confirm that p42^mapk displays increased tyrosyl phosphorylation after an electroconvulsive stimulus. Chromatographic separation of hippocampal extracts indicates that MAP kinase activity elutes in parallel with p42^mapk. Accordingly, these studies identify p42^mapk as a tyrosyl kinase substrate that is activated by this stimulus and suggest that this form of MAP kinase may be selectively regulated by neuronal stimulation.     

Slik and the Receptor Tyrosine Kinase Breathless Mediate Localized Activation of Moesin in Terminal Tracheal Cells

A key element in the regulation of subcellular branching and tube morphogenesis of the Drosophila tracheal system is the organization of the actin cytoskeleton by the ERM protein Moesin. Activation of Moesin within specific subdomains of cells, critical for its interaction with actin, is a tightly controlled process and involves regulatory inputs from membrane proteins, kinases and phosphatases. The kinases that activate Moesin in tracheal cells are not known. Here we show that the Sterile-20 like kinase Slik, enriched at the luminal membrane, is necessary for the activation of Moesin at the luminal membrane and regulates branching and subcellular tube morphogenesis of terminal cells. Our results reveal the FGF-receptor Breathless as an additional necessary cue for the activation of Moesin in terminal cells. Breathless-mediated activation of Moesin is independent of the canonical MAP kinase pathway.