Receptor-mediated Signal Transduction Research Articles

Limiting angiotensin II signaling with a cell‐penetrating peptide mimicking the second intracellular loop of the angiotensin II type‐I receptor

A cell-penetrating peptide consisting of the second intracellular loop (IC2) of the angiotensin II (AngII) type-I receptor (AT1) linked to the HIV-transactivating regulatory protein (TAT) domain was used to identify the role of this motif In intracellular signal transduction. HEK-293 cells stably transfected with AT1R cDNA and primary cultures of human pulmonary artery smooth muscle cells expressing endogenous AT1 receptor were exposed to the cell-penetrating peptide construct, and the effect on angiotensin II signaling was determined. The AT1 IC2 peptide effectively inhibited AngII-stimulated phosphatidylinositol turnover and calcium influx. It also limited the activation of Akt/PKB as determined by an inhibition of phosphorylation of Akt at Ser473, and completely abolished the AngII-dependent activation of the transcriptional factor NFkappaB. In contrast, the AT1 IC2 peptide had no effect on AngII/AT1 receptor activation of ERK. These results illustrate the potential of using cell-penetrating peptides to both delineate receptor-mediated signal transduction and to selectively regulate G protein-coupled receptor signaling.

Suppression mechanisms of flavonoids on aryl hydrocarbon receptor-mediated signal transduction

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates biological and toxicological effects by binding to its agonists such as 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD). Previously we demonstrated that flavonoids suppressed the TCDD-induced DNA-binding activity of the AhR in a structure-dependent manner. In this study, we investigated the mechanisms by which flavonoids suppressed the AhR-mediated signal transduction in mouse hepatoma Hepa-1c1c7 cells. Flavones and flavonols suppressed the TCDD-induced nuclear translocation of the AhR and dissociation of its partner proteins, heat shock protein 90 and X-associated protein 2, whereas flavanones and catechins did not. Flavonoids of all these four subclasses suppressed the phosphorylation of both AhR and Arnt and the formation of a heterodimer consisting of these proteins. Since certain flavonoids are known to inhibit mitogen-activated protein kinases (MAPKs), we confirmed the contribution of MAPK/ERK kinase (MEK) to the AhR-mediated signal transduction by using U0126, an inhibitor of MEK1/2. U0126 suppressed TCDD-induced phosphorylation of the AhR and Arnt followed by the DNA-binding activity of the AhR. Flavanones and catechins suppressed the TCDD-induced phosphorylation of ERK1/2. The inhibition of MEK/ERK phosphorylation is one of the mechanisms by which flavanones and catechins suppress the AhR-mediated signal transduction in Hepa-1c1c7 cells.

On the expanding terminology in the GPCR field: The meaning of receptor mosaics and receptor heteromers

The oligomerization of G protein-coupled receptors (GPCRs) is a fact that deserves further attention as increases both the complexity and diversity of the receptor-mediated signal transduction, thus enriching the cell signaling. Consequently, in the present review we tackle among others the problems concerning the terminology used to describe aspects surrounding the GPCRs oligomerization phenomenon. Therefore, the theoretical implications of the GPCR oligomerization will be briefly discussed together with possible implications of this phenomenon especially for new strategies in drug development.

Effects of desipramine on the cell cycle and apoptosis in Ca3/7 mouse skin squamous carcinoma cells

Desipramine (DMI) has been reported to induce glucocorticoid receptor-mediated signal transduction in recent studies. It has been suggested that a non-glucocorticoid receptor signaling pathway might play an important role in skin squamous carcinoma Ca3/7 cells. The aim of this study was to investigate the growth inhibitory effects of DMI on Ca3/7 cells by evaluating the mRNA expression of genes related to apoptosis and cell cycle progression. Hoechst nuclear staining and DNA fragmentation assays were used to detect apoptosis, and the cell cycle was analyzed by flow cytometry. Apoptotic bodies in the nuclei of cells and DNA fragmentation were observed when the Ca3/7 cells were treated with 20 microM DMI for 24 h. Quantitative RT-PCR (reverse transcriptional-polymerase chain reaction) showed that DMI caused a decrease in Bcl-2 and survivin but not Bcl-xL gene expression and an increase in the expression of Bax, Apaf-1, caspase-3 and caspase-7 in a dose- and time-dependent manner. DMI also caused translocation of the apoptosis-inducing factor from the cytoplasm to the nucleus as well as cell cycle arrest in the Ca3/7 cells. Quantitative RT-PCR revealed that DMI decreased the expression of the PCNA gene and caused an increase in the expression of the p21 and p27 genes in the Ca3/7 cells. Our results showed that DMI inhibited the growth of Ca3/7 cells by inducing both apoptosis and cell cycle arrest.

Abstract 4024: Single-allele K-ras mutation mediates CXCR4 signaling in PanIN and pancreatic cancer cells

Abstract Single-allele K-ras mutation mediates CXCR4 signaling in PanIN and pancreatic cancer cells Xiaoming Shen, Jianming Lu, Stefanie Christopher, Andrew M. Lowy, Joseph Kim Department of Oncologic Surgery, City of Hope Division of Surgical Oncology, Moores Comprehensive Cancer Center, UCSD Introduction: We previously demonstrated that the activated chemokine receptor CXCR4 signals through MAPK and PI-3K/AKT pathways in pancreatic intraepithelial neoplasia (PanIN) and invasive pancreatic cancer to enhance cell proliferation. It remains unclear whether mutant-derived constitutive K-Ras activity regulates CXCR4 signaling. Our aim was to characterize the role of mutant K-ras in CXCR4 signal transduction. Methods: Human pancreatic cancer cell lines with wild-type K-ras (BxPC-3 and Hs766T), single-allele mutant K-ras (PANC-1 and FG), and double-allele mutant K-ras (AsPC-1 and MIAPaCa-2) were evaluated. An established murine PanIN cell line with single allele mutant K-ras was also assessed. First, CXCR4 and K-Ras expression in all the cell lines were measured by Western blot assay. Then, changes in K-Ras activity induced by CXCL12, the selective CXCR4 ligand, were measured by a Raf-Ras binding domain ELISA assay. To further characterize the interaction between K-Ras and CXCR4, a K-ras knockdown assay was used to examine its effects on CXCL12-mediated activation of ERK1/2 and cell proliferation. Results: PCR and DNA sequencing verified K-ras mutation status in all the cell lines. Western blot assay showed that all cells expressed K-Ras and all cells expressed CXCR4, except MIAPaCa-2 (double-allele mutant K-ras). When CXCR4-expressing cells were exposed to CXCL12, we observed significantly increased K-Ras activity in single-allele mutant K-ras cells (what is the magnitude of change?111% for panIN and 18.6% for panc1), but not wild-type and double-allele mutant K-ras cells. Furthermore, the increased K-Ras activity corresponded with CXCL12-induced proliferation, which was only observed in the single-allele mutant K-ras cells. K-ras knockdown in the single allele mutant cells (PANC-1 and PanIN) blocked CXCL12-induced phosphorylation of ERK1/2 and abrogated cell proliferation. Because of concern that knockdown of dominant K-Ras signaling may block receptor-mediated signal transduction, we also exposed cells to epidermal growth factor, which induced phosphorylation of ERK1/2 despite K-ras knockdown. Conclusion: Our results verify that the activated chemokine receptor CXCR4 promotes the proliferation of PanIN and pancreatic cancer cells. We also show that while K-Ras conducts CXCR4 signals, only single-allele mutant K-ras mediates CXCR4-induced increases in K-Ras activity and the subsequent downstream enhancement in cell proliferation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4024.

Abstract 2526: CP690550 inhibits interleukin-2 (IL-2), IL-9 and IL-15 mediated JAK3 activation and proliferation of T cells obtained from patients with HTLV-1-associated adult T cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP)

Abstract The retrovirus, human T cell lymphotrophic virus-1 (HTLV-1) is the etiologic agent of ATL and the neurological disorder, HAM/TSP. Infection with HTLV-1 results in viral tax protein mediated constitutive activation of the JAK3/STAT5 pathway in patients' T-lymphocytes that is believed to result in malignant transformation or autoimmunity. JAK3 plays a key role in cytokine receptor-mediated signal transduction including the receptors for IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21 that use the common γ- chain. This suggests that inhibitors of JAK3 may be of benefit in HTLV-1-associated diseases. We evaluated the effect of a novel small molecule JAK3 inhibitor, CP690550 on the cytokine-dependent cell line, NK92. CP690550 at 50 or 100 nM effectively inhibited the proliferation of IL-2-stimulated NK92 cells, but not NK92 that were stimulated with IL-12 or the combination of IL-6 and its receptor (IL-6R) that do not utilize the common γ-chain and JAK3 for signaling and proliferation. We also demonstrated the relative specificity of this inhibitor on another cytokine-dependent cell line, 32Dβ where at 50 or 100 nM CP690550 inhibited IL-2 and JAK3 mediated proliferation but did not affect murine IL-3-mediated cell proliferation. The activation mediated phosphorylation of STAT5 in NK92 cells in response to IL-2 stimulation was inhibited by CP690550. We evaluated the action of CP690550 on the ex vivo proliferation of peripheral blood mononuclear cells (PBMC) from selected patients with the smoldering or chronic subtypes of ATL, or HAM/TSP whose PBMC are associated with the production of cytokines, in particular, IL-2, IL-9 and IL-15 that use the common γ-chain and JAK3. CP690550 at 50 nM inhibited the ex vivo spontaneous proliferation of PBMC from 11 patients with ATL and 9 patients with HAM/TSP at 6-day by means of 66% and 86.4%, respectively. CP690550 also inhibited the proliferation of NK92 cells stimulated with the 6-day ex vivo PBMC culture supernatants from ATL and HAM/TSP patients by a mean of 83.9% and 82%, respectively. We also demonstrated that CP690550 effectively inhibited the activation of the JAK3/STAT5 pathway in isolated T-cells from ATL patients. On the basis of this study, the JAK3 inhibitor CP690550 deserve further study as a potential therapeutic agent for select patients with HTLV-1-associated ATL and HAM/TSP. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2526.

The SCHOOL of nature: II. Protein order, disorder and oligomericity in transmembrane signaling.

Recent reports have revealed that many proteins that do not adopt globular structures under native conditions, thus termed intrinsically disordered proteins (IDPs), are involved in cell signaling. Intriguingly, physiologically relevant oligomerization of IDPs has been recently observed and shown to exhibit unique biophysical characteristics, including the lack of significant changes in chemical shift and peak intensity upon binding. In this work, I summarize several distinct features of protein disorder that are especially important as related to receptor-mediated transmembrane signal transduction. I also hypothesize that interactions of IDPs with their protein or lipid partners represent a general biphasic process with the "no disorder-to-order" fast interaction which, depending on the interacting partner, may or may not be accompanied by the slow formation of a secondary structure. Further, I suggest signaling-related functional connections between protein order, disorder, and oligomericity and hypothesize that receptor oligomerization induced or tuned upon ligand binding outside the cell is translated across the membrane into protein oligomerization inside the cell, thus providing a general platform, the Signaling Chain HOmoOLigomerization (SCHOOL) platform, for receptor-mediated signaling. This structures our current multidisciplinary knowledge and views of the mechanisms governing the coupling of recognition to signal transduction and cell response. Importantly, this approach not only reveals previously unrecognized striking similarities in the basic mechanistic principles of function of numerous functionally diverse and unrelated surface membrane receptors, but also suggests the similarity between therapeutic targets, thus opening new horizons for both fundamental and clinically relevant studies.

2065 TOLL-LIKE RECEPTOR 3 (TLR3) PRETREATMENT PROTECTS IN RENAL ISCHEMIC INJURY

You have accessJournal of UrologyTransplantation & Vascular Surgery: Renal Transplantation, Vascular Surgery I1 Apr 20102065 TOLL-LIKE RECEPTOR 3 (TLR3) PRETREATMENT PROTECTS IN RENAL ISCHEMIC INJURY Jason Hedges and Michael Conlin Jason HedgesJason Hedges Tigard, OR More articles by this author and Michael ConlinMichael Conlin Portland, OR More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.2125AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Ischemic preconditioning, a phenomenon induced by periods of brief ischemia followed by reperfusion, renders an organ more tolerant to future ischemia/reperfusion injury. Pretreatment with other agonists such as lipopolysaccharide (LPS) that activate pro-inflammatory and inflammatory mediators have been shown to protect in renal ischemic injury. Toll-like receptors mediate signal transduction pathways of the innate immune system that govern inflammatory cytokines. Using polyinosine-polycytidylic acid (Poly I:C), a ligand for Toll-like receptor 3 (TLR3), we determined the role of TLR3 preconditioning on renal function and histology in a murine renal ischemia model. METHODS A total of 24 C57BL/6J mice (Jackson Laboratories, Maine) were divided into 4 groups. Group 1 was vehicle control (normal saline); Group 2 was our positive control (LPS 16 mg/kg); Group 3 was Poly I:C (1.6 mg/kg, a synthetic analog of dsRNA and ligand for TLR3); and Group 4 was sham operation. Groups 1-3 underwent intraperitoneal injection 48 hours prior to the ischemic event. On day 0, mice were anesthetized and underwent bilateral renal pedicle clamping for 45 minutes. After unclamping, Doppler ultrasound was used to verify renal pedicle blood flow. Midline incisions were then closed and the mice were allowed to recover. Blood samples were taken on days 0 and 2 for serum creatinine analysis. Kidneys were harvested on day 2 for histological study. RESULTS All mice survived the 45 minutes of bilateral renal pedicle clamping. Group 1 (normal saline) had 4-fold greater increase (p<0.05) in serum creatinine levels after 2 days compared to Group 4 (sham operation). Group 2 (LPS) had significantly less creatinine rise than group 1. Group 3 (Poly I:C) serum creatinine levels after 2 days were the same as Group 4 (sham operation). On histological study, Group 1 (normal saline) had severe fibrosis and cellular damage, Group 2 (LPS) had minor inflammation, while Group 3 (Poly I:C) and Group 4 (sham operation) had normal histology. CONCLUSIONS Bilateral renal pedicle clamping for 45 minutes results in elevated serum creatinine levels and histological renal damage after 2 days. Pretreatment of the mice with LPS and Poly I:C (a ligand for TLR3) protected the mice from both a rise in serum creatinine levels and histological evidence of renal damage. These results taken together indicate that TLR3 may play an important novel role in protection of renal ischemic injury. © 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e803 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jason Hedges Tigard, OR More articles by this author Michael Conlin Portland, OR More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Altered glial–neuronal crosstalk: Cornerstone in the pathogenesis of hepatic encephalopathy

Hepatic encephalopathy (HE) is a serious neuropsychiatric complication of liver failure, characterized neuropathologically by astrocyte swelling, microglial activation and Alzheimer Type II astrocytosis. Molecular studies in HE brain reveal altered expression of genes coding for key astroglial proteins including early losses of expression of GFAP and the glutamate transporter EEAT-2 with concomitant increases of the astrocytic/microglial mitochondrial benzodiazepine receptor (MBR). Decreased expression of EAAT-2 results in decreased glutamate transport and impaired cycling of glutamate–glutamine between astrocytes and neurons, as well as increased extracellular glutamate, activation of the NMDA receptor-mediated cGMP-NO signal transduction pathway, and nitration of tyrosine residues on key astroglial proteins such as glutamine synthetase (GS) and the MBR. GS is uniquely responsible for the removal of excess ammonia in brain. Ammonia-induced activation of MBR in astrocytes and/or microglia results in stimulation of the synthesis of neurosteroids such as allopregnanolone with positive allosteric GABA-A receptor neuromodulatory properties. Allopregnanolone concentrations are increased up to 7-fold in HE brain. Attenuation of microglial activation by minocycline results in a delay in onset of HE and prevents brain edema in liver failure. Mild hypothermia is likewise beneficial in acute liver failure resulting in normalization of extracellular brain glutamate and prevention of oxidative/nitrosative stress in experimental animals with HE resulting from either ischemic or toxic liver injuries.

FoxO1 – the key for the pathogenesis and therapy of acne?

Five main factors play a pivotal role in the pathogenesis of acne: androgen dependence, follicular retention hyperkeratosis, increased sebaceous lipogenesis, increased colonization with P. acnes, and inflammatory events. This paper offers a solution for the pathogenesis of acne and explains all major pathogenic factors at the genomic level by a relative deficiency of the nuclear transcription factor FoxO1. Nuclear FoxO1 suppresses androgen receptor, other important nuclear receptors and key genes of cell proliferation, lipid biosynthesis and inflammatory cytokines. Elevated growth factors during puberty and persistent growth factor signals due to Western life style stimulate the export of FoxO1 out of the nucleus into the cytoplasm via activation of the phos-phoinositide-3-kinase (PI3K)/Akt pathway. By this mechanism, genes and nuclear receptors involved in acne are derepressed leading to increased androgen receptor-mediated signal transduction, increased cell proliferation of androgen-dependent cells, induction of sebaceous lipogenesis and upregulation of Toll-like-receptor-2-dependent inflammatory cytokines. All known acne-inducing factors exert their action by reduction of nuclear FoxO1 levels. In contrast, retinoids, antibiotics and dietary intervention will increase the nuclear content of FoxO1, thereby normalizing increased transcription of genes involved in acne. Various receptor-mediated growth factor signals are integrated at the level of PI3K/Akt activation which finally results in nuclear FoxO1 deficiency.

Plasma Membrane Heterogeneity and Receptor Mediated Signaling

The complex role of plasma membrane structure in orchestrating receptor-mediated signal transduction is addressed in collaborative studies investigating how antigen crosslinking of IgE-receptors on mast cells initiates signaling pathways leading to multiple cellular responses. Segregation of liquid ordered regions from disordered regions of the plasma membrane provides protection from transmembrane phosphatases and thereby a mechanism for crosslinking-dependent phosphorylation of IgE-receptors by active Lyn kinase in the first signaling event. Defined clustering of IgE-receptors with patterned lipid bilayers enables fluorescence visualization of co-redistributing signaling components with spatial resolution on the micron scale. Nanoscale resolution of clustering components is visualized with scanning electron microscopy and super-resolution fluorescence microscopy. Single molecule dynamics can be characterized in nanofabricated devices. These integrated approaches for examining membrane structural heterogeneity and functional consequences will be discussed.

Abstract A183: Ex vivo imaging of EGF receptor-mediated signal transduction for prediction of anti-EGFR treatment efficacy

Abstract Objectives: EGFR is a promising treatment target in solid tumors. Two major types of anti-EGFR agents have entered the clinical setting: antibodies and small-molecule EGFR TKIs. EGFR-TKI inhibits autophosphorylation of EGFR by binding to ATP binding site of tyrosine kinase domain of EGFR and interrupts the signal transduction. On the other hand, anti-EGER therapeutic antibodies interrupt EGFR signaling by blocking ligand binding. In addition, as for antibodies, the effects of the ADCC are also expected. In this study, we aimed at developing a method to estimate the effectiveness of anti-EGFR agents by analyzing these mechanisms using ex vivo imaging technique. Methods: To establish a method that confirms the inhibition effect of the EGFR-signal transduction, we prepared fluorescent-labeled ligand and developed the imaging strategy to visualize membrane binding and subsequent internalization of labeled EGF. In addition, fluorescent-labeled NK-based effector cells that have been established by introducing fcgr3a gene into a NK leukemia cell line were used for characterization of ADCC susceptibility of individual tumor cells. In our strategy, a continuous confocal laser scanning microscopy was used for tracing fluorescent-labeled EGF or effector cells as well as tumor cell death. Results: Fluorescent probe bearing EGF was highly specific and potent in the binding and activation of the EGF receptor, being rapidly internalized into endosomes. Pretreatment with therapeutic anti-EGFR antibody completely inhibited the ligand-binding and subsequent events. Meanwhile, EGFR TKIs partially prevented endocytotic step. K-ras mutation status did not significantly affect the ligand binding and internalization. The imaging analysis of the ADCC using primary colorectal cancer specimens revealed that cetuximab-coated tumor cells were gradually damaged by direct interaction of effector cells. ADCC susceptibility of individual tumor cells has varied to some extent. Conclusion: We have established imaging methods of the early events of EGFR signaling pathway and ADCC reaction. This imaging method soffer the rapid reliable means that analyze the effect of the EGFR targeted agent to the individual tumor cells. By combining with already established prognostic factors such as K-ras mutation status or Fc receptor polymorphism, our imaging analysis will propose novel strategy to predict an individual treatment prognosis more precisely. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A183.

Desensitization of vascular endothelin receptors by G protein-coupled receptor kinase 2

At the heart of receptor-mediated signal transduction are two intertwined processes: translation of signals carried by stimulating agents to cells through intracellular messenger molecules and termination/restriction of signalling by cells. The latter is a critical mechanism that protects cells from overreacting to an extended or appropriate stimulus and makes cells ready to react to forthcoming stimulation. G protein-coupled receptors (GPCRs) are a family of receptors composed of seven transmembrane domains that respond to various stimuli. GPCRs usually undergo rapid desensitization after activation, in which GPCR kinases (GRKs), a family consisting of seven members (GRK 1–7), play a critical role.1 Endothelins (ETs) are a family of peptides comprising ET1, ET2, and ET3.2 ETs exert their actions through two types of receptors, ETA and ETB, which belong to the GPCR family.3 Activation of ETA receptors results in vasoconstriction through the generation of 1,4,5-inositol trisphosphate and diacylglycerol as intracellular messengers, which, in turn, trigger intracellular Ca2+ release and protein kinase C activation. Among the ET family members, ET1 is a potent vasoconstrictor that induces contraction at nanomolar concentrations in several vascular beds.3 Significant desensitization of ET1-induced cellular response was soon reported after ET was discovered in 1988,4 but the underlying mechanisms were not fully understood. Morris … *Corresponding author. Tel: +1 905 525 9140 Extn 22477; Fax: +1 905 523 1224. E-mail address : gaoyu{at}mcmaster.ca

Tyrosine Phosphorylation of 3BP2 Regulates B Cell Receptor-mediated Activation of NFAT

Adaptor protein c-Abl SH3 domain-binding protein-2 (3BP2, also referred to SH3BP2) regulates immune receptor-mediated signal transduction. In this report we focused on the molecular mechanism of 3BP2 function in B cell receptor (BCR) signaling. Engagement of BCR induces tyrosine phosphorylation of 3BP2. Genetic analysis demonstrated that Syk is critical for BCR-mediated tyrosine phosphorylation of 3BP2. Mutational analysis of 3BP2 revealed that both Tyr(183) and Src homology 2 (SH2) domain are necessary for 3BP2-mediated BCR-induced activation of nuclear factor of activated T cells (NFAT). Point mutation of Tyr(183) or Arg(486) in the SH2 domain of 3BP2 diminished BCR-mediated tyrosine phosphorylation of 3BP2. Endogenous 3BP2 forms a complex with tyrosine-phosphorylated cellular signaling molecules. Peptide binding experiments demonstrated that only phosphorylated Tyr(183) in 3BP2 could form a complex with the SH2 domain(s) of phospholipase Cgamma2 and Vav1 from B cell lysates. These interactions were represented by using bacterial glutathione S-transferase-phospholipase Cgamma2 or -Vav1 SH2 domain. Furthermore, pulldown and Far Western experiments showed that the 3BP2-SH2 domain directly binds to B cell linker protein (BLNK) after BCR stimulation. These results demonstrated that 3BP2 induces the protein complex with cellular signaling molecules through phosphorylation of Tyr(183) and SH2 domain leading to the activation of NFAT in B cells.

Prorenin induces ERK activation in endothelial cells to enhance neovascularization independently of the renin–angiotensin system

Prorenin is an enzymatically inactive precursor of renin, and its biological function in endothelial cells (ECs) is unknown despite its relevance with the incidence of diabetic microvascular complications. Recently, (pro)renin receptor was identified, and the receptor-associated prorenin system has been discovered, whereas its expression as well as function in ECs remain unclear. In the present study, we found that ECs express the (pro)renin receptor, and that prorenin provoked ERK activation through (pro)renin receptor independently of the renin–angiotensin system (RAS). Prorenin stimulated the proliferation, migration and tube-formation of ECs, while it inhibited endothelial apoptosis induced by serum and growth factor depletion. MEK inhibitor abrogated these proangiogenic effects of prorenin, while AT1 receptor antagonist or angiotensin-converting enzyme inhibitor failed to block them. In vivo neovascularization in the Matrigel-plugs implanted into mouse flanks was significantly enhanced by prorenin, in which significant ERK activation was detected in ECs. Furthermore, tumor xenografts stably transfected with prorenin demonstrated the significantly accelerated growth rate concomitantly with enhanced intratumoral neovascularization. Our data demonstrated that the RAS-independent (pro)renin receptor-mediated signal transduction plays a pivotal role in the regulation of ECs function as well as in the neovascularization, and thus prorenin is potentially involved in the pathophysiology of diabetic microvascular complications as well as cancers.

Subcellular compartmentalization of FADD as a new level of regulation in death receptor signaling

Fas-associated protein with death domain (FADD) is an essential adaptor protein in death receptor-mediated signal transduction. During apoptotic signaling, FADD functions in the cytoplasm, where it couples activated receptors with initiator caspase-8. However, in resting cells, FADD is predominantly stored in the nucleus. In this study, we examined the modalities of FADD intracellular trafficking. We demonstrate that, upon CD95 activation, FADD redistributes from the nucleus to the cytoplasm. This inducible nuclear-cytoplasmic translocation of FADD is independent of CD95 internalization, formation of the death-inducing signaling complex, and caspase-8 activation. In contrast to nuclear export of FADD, its subsequent recruitment and accumulation at endosomes containing internalized CD95 requires a caspase-8-dependent feedback loop. These data indicate the existence of differential pathways directing FADD nuclear export and cytoplasmic trafficking, and identify subcellular compartmentalization of FADD as a novel regulatory mechanism in death receptor signaling.

Antineoplastic effects of melatonin on a rare malignancy of mesenchymal origin: melatonin receptor‐mediated inhibition of signal transduction, linoleic acid metabolism and growth in tissue‐isolated human leiomyosarcoma xenografts

Melatonin provides a circadian signal that regulates linoleic acid (LA)-dependent tumor growth. In rodent and human cancer xenografts of epithelial origin in vivo, melatonin suppresses the growth-stimulatory effects of linoleic acid (LA) by blocking its uptake and metabolism to the mitogenic agent, 13-hydroxyoctadecadienoic acid (13-HODE). This study tested the hypothesis that both acute and long-term inhibitory effects of melatonin are exerted on LA transport and metabolism, and growth activity in tissue-isolated human leiomyosarcoma (LMS), a rare, mesenchymally-derived smooth muscle tissue sarcoma, via melatonin receptor-mediated inhibition of signal transduction activity. Melatonin added to the drinking water of female nude rats bearing tissue-isolated LMS xenografts and fed a 5% corn oil (CO) diet caused the rapid regression of these tumors (0.17 +/- 0.02 g/day) versus control xenografts that continued to grow at 0.22 +/- 0.03 g/day over a 10-day period. LMS perfused in situ for 150 min with arterial donor blood augmented with physiological nocturnal levels of melatonin showed a dose-dependent suppression of tumor cAMP production, LA uptake, 13-HODE release, extracellular signal-regulated kinase (ERK 1/2), mitogen activated protein kinase (MEK), Akt activation, and [(3)H]thymidine incorporation into DNA and DNA content. The inhibitory effects of melatonin were reversible and preventable with either melatonin receptor antagonist S20928, pertussis toxin, forskolin, or 8-Br-cAMP. These results demonstrate that, as observed in epithelially-derived cancers, a nocturnal physiological melatonin concentration acutely suppress the proliferative activity of mesenchymal human LMS xenografts while long-term treatment of established tumors with a pharmacological dose of melatonin induced tumor regression via a melatonin receptor-mediated signal transduction mechanism involving the inhibition of tumor LA uptake and metabolism.

TRAIL receptor mediates inflammatory cytokine release in an NF-κB-dependent manner

In the present article, we report that DR4 or DR5 overexpression dramatically activates the release of the inflammatory cytokines IL-8, TNF-alpha, CCL20, MIP-2 and MIP-1beta in an NF-kappaB-dependent manner in 293T, MDA-MB-231 and HCT-116 cells. We showed that death receptor-mediated signals were extracellular domain-independent, whereas the effect of overexpression of the DR4 intracellular domain was much less potent. The TRADD-TRAF2-NIK-IKKalpha/beta signaling cascade, which plays an essential role in TNF-induced NF-kappaB activation, was found to be involved in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor-mediated signal transduction. The FADD-caspase signaling pathway, which has been reported to be mostly related to apoptosis, was identified as being essential for DR4 or DR5 overexpression-mediated NF-kappaB activation and cytokine secretion and crosstalks with the TRADD-TRAF2-NIK-IKKalpha/beta signaling cascade. Furthermore, a DR5 agonistic antibody (AD5-10) triggered the inflammatory cytokine release. These data, together with previous reports, provide strong evidence that TRAIL and TRAIL receptors play an important role in inflammation.

(Pro)renin Receptor–Mediated Signal Transduction and Tissue Renin-Angiotensin System Contribute to Diabetes-Induced Retinal Inflammation

OBJECTIVEThe term “receptor-associated prorenin system” (RAPS) refers to the pathogenic mechanisms whereby prorenin binding to its receptor dually activates the tissue renin-angiotensin system (RAS) and RAS-independent intracellular signaling via the receptor. The aim of the present study was to define the association of the RAPS with diabetes-induced retinal inflammation.RESEARCH DESIGN AND METHODSLong-Evans rats, C57BL/6 mice, and angiotensin II type 1 receptor (AT1-R)-deficient mice with streptozotocin-induced diabetes were treated with (pro)renin receptor blocker (PRRB). Retinal mRNA expression of prorenin and the (pro)renin receptor was examined by quantitative RT-PCR. Leukocyte adhesion to the retinal vasculature was evaluated with a concanavalin A lectin perfusion–labeling technique. Retinal protein levels of vascular endothelial growth factor (VEGF) and intercellular adhesion molecule (ICAM)-1 were examined by ELISA. Retinal extracellular signal–regulated kinase (ERK) activation was analyzed by Western blotting.RESULTSInduction of diabetes led to significant increase in retinal expression of prorenin but not the (pro)renin receptor. Retinal adherent leukocytes were significantly suppressed with PRRB. Administration of PRRB inhibited diabetes-induced retinal expression of VEGF and ICAM-1. To clarify the role of signal transduction via the (pro)renin receptor in the diabetic retina, we used AT1-R–deficient mice in which the RAS was deactivated. Retinal adherent leukocytes in AT1-R–deficient diabetic mice were significantly suppressed with PRRB. PRRB suppressed the activation of ERK and the production of VEGF, but not ICAM-1, in AT1-R–deficient diabetic mice.CONCLUSIONSThese results indicate a significant contribution of the RAPS to the pathogenesis of diabetes-induced retinal inflammation, suggesting the possibility of the (pro)renin receptor as a novel molecular target for the treatment of diabetic retinopathy.

Critical and Multiple Roles for the CD3ε Intracytoplasmic Tail in Double Negative to Double Positive Thymocyte Differentiation

The preTCR is associated with signal-transducing CD3gamma, delta, epsilon, and zeta polypeptides. It is generally agreed that CD3 chains play redundant roles in the receptor-mediated signal transduction. In the present study, we show that the intracytoplasmic (IC) domain of CD3epsilon is essential for early thymocyte maturation. We demonstrate that the IC domain-deleted CD3epsilon fails to restore the double negative (DN) to double positive (DP) thymocyte development in CD3epsilon-deficient mice. Additional experiments show that the membrane proximal basic amino acid rich sequence in the IC domain of CD3epsilon is sufficient for the DN to DP differentiation, whereas the proline rich sequence is required for efficient proliferation. This is probably due to impaired ligand independent recruitment of Nck to the proline rich sequence motif of CD3epsilon within the context of the preTCR. The data presented in this study elucidates mechanistic basis for the preTCR-induced proliferation of the DN thymocytes and have identified distinct roles for individual motifs of CD3epsilon in the preTCR-mediated differentiation and proliferation. These data provide the first genetic and phenotypic evidence for requirement of the IC domain of a CD3 chain in thymocyte development.