Protein-tyrosine phosphatase DEP-1 controls receptor tyrosine kinase FLT3 signaling

Fms-like tyrosine kinase 3 (FLT3) plays an important role in hematopoietic differentiation, and constitutively active FLT3 mutant proteins contribute to the development of acute myeloid leukemia. Little is known about the protein-tyrosine phosphatases (PTP) affecting the signaling activity of FLT3. To identify such PTP, myeloid cells expressing wild type FLT3 were infected with a panel of lentiviral pseudotypes carrying shRNA expression cassettes targeting different PTP. Out of 20 PTP tested, expressed in hematopoietic cells, or presumed to be involved in oncogenesis or tumor suppression, DEP-1 (PTPRJ) was identified as a PTP negatively regulating FLT3 phosphorylation and signaling. Stable 32D myeloid cell lines with strongly reduced DEP-1 levels showed site-selective hyperphosphorylation of FLT3. In particular, the sites pTyr-589, pTyr-591, and pTyr-842 involved in the FLT3 ligand (FL)-mediated activation of FLT3 were hyperphosphorylated the most. Similarly, acute depletion of DEP-1 in the human AML cell line THP-1 caused elevated FLT3 phosphorylation. Direct interaction of DEP-1 and FLT3 was demonstrated by "substrate trapping" experiments showing association of DEP-1 D1205A or C1239S mutant proteins with FLT3 by co-immunoprecipitation. Moreover, activated FLT3 could be dephosphorylated by recombinant DEP-1 in vitro. Enhanced FLT3 phosphorylation in DEP-1-depleted cells was accompanied by enhanced FLT3-dependent activation of ERK and cell proliferation. Stable overexpression of DEP-1 in 32D cells and transient overexpression with FLT3 in HEK293 cells resulted in reduction of FL-mediated FLT3 signaling activity. Furthermore, FL-stimulated colony formation of 32D cells expressing FLT3 in methylcellulose was induced in response to shRNA-mediated DEP-1 knockdown. This transforming effect of DEP-1 knockdown was consistent with a moderately increased activation of STAT5 upon FL stimulation but did not translate into myeloproliferative disease formation in the 32D-C3H/HeJ mouse model. The data indicate that DEP-1 is negatively regulating FLT3 signaling activity and that its loss may contribute to but is not sufficient for leukemogenic cell transformation.     

p38 Regulates Pigmentation via Proteasomal Degradation of Tyrosinase

The synthesis of melanin pigments, or melanogenesis, is regulated by the balance of a variety of signal transduction pathways. Among these pathways, p38 MAPK signaling was found to be involved in stress-induced melanogenesis and to be activated by α-melanocyte-stimulating hormone (α-MSH) and ultraviolet irradiation. Previous studies have shown that α-MSH-stimulated melanogenesis can be inhibited by blocking p38 MAPK activity with SB203580, a pyridinyl imidazole compound. Consistent with this, we observed that pyridinyl imidazoles (SB203580 and SB202190) inhibited both basal and α-MSH-induced melanogenesis in B16 melanoma cells. However, SB202474, which has no ability to inhibit p38 MAPK activity and is usually used as a negative control compound in p38 MAPK studies, also suppressed melanin synthesis induction. Furthermore, the independence of the p38 kinase pathway from the repression of melanogenesis by pyridinyl imidazole compounds was also confirmed by small interfering RNA experiments. Interfering with p38 MAPK expression surprisingly stimulated melanogenesis and tyrosinase family protein expression. Although the molecular mechanism(s) by which p38 promotes the degradation of melanogenic enzymes remain to be determined, the involvement of the ubiquitin-proteasome pathway was demonstrated by co-treatment with the proteasome-specific inhibitor MG132 and the relative decrease in the ubiquitination of tyrosinase in cells transfected with p38-specific small interfering RNA.     

A novel form of melanoma apoptosis resistance: melanogenesis up-regulation in apoptotic B16-F0 cells delays ursolic acid-triggered cell death

Melanoma is one of the most aggressive forms of cancer with a continuously growing incidence worldwide and is usually resistant to chemotherapy agents, which is due in part to a strong resistance to apoptosis. The resistance mechanisms are complex and melanoma cells may have diverse possibilities for regulating apoptosis to generate apoptotic deficiencies. In this study, we investigated the relationship between melanogenesis and resistance to apoptosis induced by ursolic acid, a natural chemopreventive agent, in B16-F0 melanoma cells. We demonstrated that cells undergoing apoptosis are able to delay their own death. It appeared that tyrosinase and TRP-1 up-regulation in apoptotic cells and the subsequent production of melanin were clearly implicated in an apoptosis resistance mechanism; while TRP-2, a well known mediator of melanoma resistance to cell death, was repressed. Our results confirm the difficulty of treating melanomas, since, even undergoing apoptosis, cells are nevertheless able to trigger a resistance mechanism to delay death.     

Hyaluronan synthase 2 (HAS2) promotes breast cancer cell invasion by suppression of tissue metalloproteinase inhibitor 1 (TIMP-1)

Invasion and metastasis are the primary causes of breast cancer mortality, and increased knowledge about the molecular mechanisms involved in these processes is highly desirable. High levels of hyaluronan in breast tumors have been correlated with poor patient survival. The involvement of hyaluronan in the early invasive phase of a clone of breast cancer cell line MDA-MB-231 that forms bone metastases was studied using an in vivo-like basement membrane model. The metastatic to bone tumor cells exhibited a 7-fold higher hyaluronan-synthesizing capacity compared with MDA-MB-231 cells predominately due to an increased expression of hyaluronan synthase 2 (HAS2). We found that knockdown of HAS2 completely suppressed the invasive capability of these cells by the induction of tissue metalloproteinase inhibitor 1 (TIMP-1) and dephosphorylation of focal adhesion kinase. HAS2 knockdown-mediated inhibition of basement membrane remodeling was rescued by HAS2 overexpression, transfection with TIMP-1 siRNA, or addition of TIMP-1-blocking antibodies. Moreover, knockdown of HAS2 suppressed the EGF-mediated induction of the focal adhesion kinase/PI3K/Akt signaling pathway. Thus, this study provides new insights into a possible mechanism whereby HAS2 enhances breast cancer invasion.     

N-formyl-methionyl-leucyl-phenylalanine (fMLP) promotes osteoblast differentiation via the N-formyl peptide receptor 1-mediated signaling pathway in human mesenchymal stem cells from bone marrow

Binding of N-formyl-methionyl-leucyl-phenylalanine (fMLP) to its specific cell surface receptor, N-formyl peptide receptor (FPR), triggers different cascades of biochemical events, eventually leading to cellular activation. However, the physiological role of fMLP and FPR during differentiation of mesenchymal stem cells is unknown. In this study, we attempted to determine whether fMLP regulates differentiation of mesenchymal stem cells derived from bone marrow. Analysis by quantitative-PCR and flow cytometry showed significantly increased expression of FPR1, but not FPR2 and FPR3, during osteoblastic differentiation. fMLP, a specific ligand of FPR1, promotes osteoblastic commitment and suppresses adipogenic commitment under differentiation conditions. Remarkably, fMLP-stimulated osteogenesis is associated with increased expression of osteogenic markers and mineralization, which were blocked by cyclosporine H, a selective FPR1 antagonist. In addition, fMLP inhibited expression of peroxisome proliferator-activated receptor-γ1, a major regulator of adipocytic differentiation. fMLP-stimulated osteogenic differentiation was mediated via FPR1-phospholipase C/phospholipase D-Ca(2+)-calmodulin-dependent kinase II-ERK-CREB signaling pathways. Finally, fMLP promoted bone formation in zebrafish and rabbits, suggesting its physiological relevance in vivo. Collectively, our findings provide novel insight into the functional role of fMLP in bone biology, with important implications for its potential use as a therapeutic agent for treatment of bone-related disorders.     

The SH2B1 adaptor protein associates with a proximal region of the erythropoietin receptor

Gene targeting experiments have shown that the cytokine erythropoietin (EPO), its cognate erythropoietin receptor (EPO-R), and associated Janus tyrosine kinase, JAK2, are all essential for erythropoiesis. Structural-functional and murine knock-in experiments have suggested that EPO-R Tyr-343 is important in EPO-mediated mitogenesis. Although Stat5 binds to EPO-R phosphotyrosine 343, the initial Stat5-deficient mice did not have profound erythroid abnormalities suggesting that additional Src homology 2 (SH2) domain-containing effectors may bind to EPO-R Tyr-343 and couple to downstream signaling pathways. We have utilized cloning of ligand target (COLT) screening to demonstrate that EPO-R Tyr(P)-343 and Tyr(P)-401 bind to the SH2 domain-containing adaptor protein SH2B1β. Immunoprecipitation and in vitro mixing experiments reveal that EPO-R binds to SH2B1 in an SH2 domain-dependent manner and that the sequence that confers SH2B1 binding to the EPO-R is pYXXL. Previous studies have shown that SH2B1 binds directly to JAK2, but we show that in hematopoietic cells, SH2B1β preferentially associates with the EPO-R. SH2B1 is capable of constitutive association with EPO-R, which is necessary for its optimal SH2-dependent recruitment to EPO-R-Tyr(P)-343/Tyr(P)-401. We also demonstrate that SH2B1 is responsive to EPO stimulation and becomes phosphorylated, most likely on serines/threonines, in an EPO dose- and time-dependent manner. In the absence of SH2B1, we observe enhanced activation of signaling pathways downstream of the EPO-R, indicating that SH2B1 is a negative regulator of EPO signaling.     

MicroRNA-21 Is a Downstream Effector of AKT That Mediates Its Antiapoptotic Effects via Suppression of Fas Ligand

MicroRNA-21 (miR-21) is highly up-regulated during hypertrophic and cancerous cell growth. In contrast, we found that it declines in cardiac myocytes upon exposure to hypoxia. Thus, the objective was to explore its role during hypoxia. We show that miR-21 not only regulates phosphatase and tensin homologue deleted on chromosome 10 (PTEN), but also targets Fas ligand (FasL). During prolonged hypoxia, down-regulation of miR-21 proved necessary and sufficient for enhancing expression of both proteins. We demonstrate here for the first time that miR-21 is positively regulated via an AKT-dependent pathway, which is depressed during prolonged hypoxia. Accordingly, hypoxia-induced down-regulation of miR-21 and up-regulation of FasL and PTEN were reversed by activated AKT and reproduced by a dominant negative mutant, wortmannin, or PTEN. Moreover, the antiapoptotic function of AKT partly required miR-21, which was sufficient for inhibition of caspase-8 activity and mitochondrial damage. In consensus, overexpression of miR-21 in a transgenic mouse heart resulted in suppression of ischemia-induced up-regulation of PTEN and FasL expression, an increase in phospho-AKT, a smaller infarct size, and ameliorated heart failure. Thus, we have identified a unique aspect of the function of AKT by which it inhibits apoptosis through miR-21-dependent suppression of FasL.     

G protein-coupled receptor signaling via Src kinase induces endogenous human transient receptor potential vanilloid type 6 (TRPV6) channel activation

Ca(2+) homeostasis plays a critical role in a variety of cellular processes. We showed previously that stimulation of the prostate-specific G protein-coupled receptor (PSGR) enhances cytosolic Ca(2+) and inhibits proliferation of prostate cells. Here, we analyzed the signaling mechanisms underlying the PSGR-mediated Ca(2+) increase. Using complementary molecular, biochemical, electrophysiological, and live-cell imaging techniques, we found that endogenous Ca(2+)-selective transient receptor potential vanilloid type 6 (TRPV6) channels are critically involved in the PSGR-induced Ca(2+) signal. Biophysical characterization of the current activated by PSGR stimulation revealed characteristic properties of TRPV6. The molecular identity of the involved channel was confirmed using RNA interference targeting TrpV6. TRPV6-mediated Ca(2+) influx depended on Src kinase activity. Src kinase activation occurred independently of G protein activation, presumably by direct interaction with PSGR. Taken together, we report that endogenous TRPV6 channels are activated downstream of a G protein-coupled receptor and present the first physiological characterization of these channels in situ.     

Signaling via macrophage G2A enhances efferocytosis of dying neutrophils by augmentation of Rac activity

Phosphatidylserine (PS) and oxidized PS species have been identified as key ligands on apoptotic cells important for their recognition and removal (efferocytosis) by phagocytes, a requisite step for resolution of inflammation. We have recently demonstrated that lysophosphatidylserine (lyso-PS) generated and retained on neutrophils following short term activation of the NADPH oxidase in vitro and in vivo enhanced their clearance via signaling through the macrophage G-protein-coupled receptor G2A. Here, we investigated the signaling pathway downstream of G2A. Lyso-PS, either made endogenously in apoptosing neutrophils or supplied exogenously in liposomes along with lyso-PS(neg) apoptotic cells, signaled to macrophages in a G2A-dependent manner for their enhanced production of prostaglandin E2 (PGE2) via a calcium-dependent cytosolic phospholipase A2/cyclooxygenase-mediated mechanism. Subsequent signaling by PGE2 via EP2 receptors activated macrophage adenylyl cyclase and protein kinase A. These events, in turn, culminated in enhanced activity of Rac1, resulting in an increase in both the numbers of macrophages efferocytosing apoptotic cells and the numbers of cells ingested per macrophage. These data were surprising in light of previous reports demonstrating that signaling by PGE2 and adenylyl cyclase activation are associated with macrophage deactivation and inhibition of apoptotic cell uptake. Further investigation revealed that the impact of this pathway, either the enhancement or inhibition of efferocytosis, was exquisitely sensitive to concentration effects of these intermediaries. Together, these data support the hypothesis that lyso-PS presented on the surface of activated and dying neutrophils provides a tightly controlled, proresolution signal for high capacity clearance of neutrophils in acute inflammation.     

Functional characterization of putative cholesterol binding sequence (CRAC) in human type-1 cannabinoid receptor

Endocannabinoid signaling modulates a variety of neuroinflammatory and neurodegenerative diseases, mainly through the activation of type-1 and type-2 (CB(1)R and CB(2)R) cannabinoid receptors. CB(1)R is negatively regulated by membrane cholesterol, while CB(2)R is unaffected. Here, we identified in the transmembrane helix 7 of human CBRs a consensus sequence already known in other proteins as cholesterol recognition/interaction amino acid sequence and consensus pattern. As this motif is different in the two CBR subtypes, we mutated lysine 402 of CB(1)R into glycine, to obtain a cholesterol recognition/interaction amino acid sequence and consensus similar to that of CB(2)R. Both mutated and wild-type receptors were transiently expressed in human neuronal SH-SY5Y cells, and their localization and functioning were investigated using biochemical assays and immunofluorescence labelling. We found a reduced propensity of the mutant CB(1)R to reside in cholesterol-rich microdomains and, by means of fluorescence recovery after photobleaching analysis, we documented its loss of sensitivity to increased membrane cholesterol content. These results seem to uncover the existence of a new structural determinant in cannabinoid receptors, that is likely implicated in directing their interaction with cholesterol-rich microdomains of cell membranes.     

Second extracellular loop of human glucagon-like peptide-1 receptor (GLP-1R) has a critical role in GLP-1 peptide binding and receptor activation

The glucagon-like peptide-1 receptor (GLP-1R) is a therapeutically important family B G protein-coupled receptor (GPCR) that is pleiotropically coupled to multiple signaling effectors and, with actions including regulation of insulin biosynthesis and secretion, is one of the key targets in the management of type II diabetes mellitus. However, there is limited understanding of the role of the receptor core in orthosteric ligand binding and biological activity. To assess involvement of the extracellular loop (ECL) 2 in ligand-receptor interactions and receptor activation, we performed alanine scanning mutagenesis of loop residues and assessed the impact on receptor expression and GLP-1(1-36)-NH(2) or GLP-1(7-36)-NH(2) binding and activation of three physiologically relevant signaling pathways as follows: cAMP formation, intracellular Ca(2+) (Ca(2+)(i)) mobilization, and phosphorylation of extracellular signal-regulated kinases 1 and 2 (pERK1/2). Although antagonist peptide binding was unaltered, almost all mutations affected GLP-1 peptide agonist binding and/or coupling efficacy, indicating an important role in receptor activation. However, mutation of several residues displayed distinct pathway responses with respect to wild type receptor, including Arg-299 and Tyr-305, where mutation significantly enhanced both GLP-1(1-36)-NH(2)- and GLP-1(7-36)-NH(2)-mediated signaling bias for pERK1/2. In addition, mutation of Cys-296, Trp-297, Asn-300, Asn-302, and Leu-307 significantly increased GLP-1(7-36)-NH(2)-mediated signaling bias toward pERK1/2. Of all mutants studied, only mutation of Trp-306 to alanine abolished all biological activity. These data suggest a critical role of ECL2 of the GLP-1R in the activation transition(s) of the receptor and the importance of this region in the determination of both GLP-1 peptide- and pathway-specific effects.     

Critical role for antiapoptotic Bcl-xL and Mcl-1 in human macrophage survival and cellular IAP1/2 (cIAP1/2) in resistance to HIV-Vpr-induced apoptosis

Macrophages are resistant to HIV cytopathic effects, which contributes to viral persistence and reservoir formation. HIV viral protein R (Vpr) is a potent apoptosis-inducing agent for primary monocytes. Because the biologically active Vpr is found in serum and cerebrospinal fluid of HIV-infected patients, we investigated the apoptotic effect of Vpr on monocyte-derived macrophages and phorbol 12-myristate 13-acetate-activated THP1 macrophages. Our results show that primary monocytes and THP1 cells develop resistance to Vpr-induced apoptosis following differentiation into macrophages. To determine the effect of Vpr on the expression of antiapoptotic proteins, we show that in contrast to the undifferentiated cells, Vpr did not down-regulate the expression of antiapoptotic inhibitors of apoptosis (IAPs) and Bcl2 family members in macrophages, suggesting their involvement in resistance to Vpr-induced apoptosis. However, knocking down Bcl-xL and Mcl-1 proteins induced spontaneous apoptosis with no impact on susceptibility to Vpr-induced apoptosis. In contrast, down-regulation of cellular IAP1 (cIAP1) and cIAP2 by using siRNAs and SMAC (second mitochondria-derived activator of caspases) mimetic sensitized macrophages to Vpr-induced apoptosis. Overall, our results suggest that resistance to Vpr-induced apoptosis is specifically mediated by cIAP1/2 genes independent of Bcl-xL and Mcl-1, which play a key role in maintaining cell viability. Moreover, IAP modulation may be a potential strategy to eliminate HIV persistence in macrophages.     

High cell surface death receptor expression determines type I versus type II signaling

Previous studies have suggested that there are two signaling pathways leading from ligation of the Fas receptor to induction of apoptosis. Type I signaling involves Fas ligand-induced recruitment of large amounts of FADD (FAS-associated death domain protein) and procaspase 8, leading to direct activation of caspase 3, whereas type II signaling involves Bid-mediated mitochondrial perturbation to amplify a more modest death receptor-initiated signal. The biochemical basis for this dichotomy has previously been unclear. Here we show that type I cells have a longer half-life for Fas message and express higher amounts of cell surface Fas, explaining the increased recruitment of FADD and subsequent signaling. Moreover, we demonstrate that cells with type II Fas signaling (Jurkat or HCT-15) can signal through a type I pathway upon forced receptor overexpression and that shRNA-mediated Fas down-regulation converts cells with type I signaling (A498) to type II signaling. Importantly, the same cells can exhibit type I signaling for Fas and type II signaling for TRAIL (TNF-α-related apoptosis-inducing ligand), indicating that the choice of signaling pathway is related to the specific receptor, not some other cellular feature. Additional experiments revealed that up-regulation of cell surface death receptor 5 levels by treatment with 7-ethyl-10-hydroxy-camptothecin converted TRAIL signaling in HCT116 cells from type II to type I. Collectively, these results suggest that the type I/type II dichotomy reflects differences in cell surface death receptor expression.