Epidermal Growth Factor and Granulocyte Colony Stimulating Factor Signaling Are Synergistic for Hematopoietic Regeneration.

Hematopoietic regeneration following chemotherapy may be distinct from regeneration following radiation. While we have shown that epidermal growth factor (EGF) accelerates regeneration following radiation, its role following chemotherapy is currently unknown. We sought to identify EGF as a hematopoietic growth factor for chemotherapy-induced myelosuppression. Following 5-fluorouracil (5-FU), EGF accelerated hematopoietic stem cell regeneration and prolonged survival compared with saline-treated mice. To mitigate chemotherapy-induced injury to endothelial cells in vivo, we deleted Bax in VEcadherin+ cells (VEcadherinCre;BaxFL/FL mice). Following 5-FU, VEcadherinCre;BaxFL/FL mice displayed preserved hematopoietic stem/progenitor content compared with littermate controls. 5-FU and EGF treatment resulted in increased cellular proliferation, decreased apoptosis, and increased DNA double-strand break repair by non-homologous end-joining recombination compared with saline-treated control mice. When granulocyte colony stimulating factor (G-CSF) is given with EGF, this combination was synergistic for regeneration compared with either G-CSF or EGF alone. EGF increased G-CSF receptor (G-CSFR) expression following 5-FU. Conversely, G-CSF treatment increased both EGF receptor (EGFR) and phosphorylation of EGFR in hematopoietic stem/progenitor cells. In humans, the expression of EGFR is increased in patients with colorectal cancer treated with 5-FU compared with cancer patients not on 5-FU. Similarly, EGFR signaling is responsive to G-CSF in humans in vivo with both increased EGFR and phospho-EGFR in healthy human donors following G-CSF treatment compared with donors who did not receive G-CSF. These data identify EGF as a hematopoietic growth factor following myelosuppressive chemotherapy and that dual therapy with EGF and G-CSF may be an effective method to accelerate hematopoietic regeneration. Stem Cells 2018;36:252-264.

Assembly of a beta2-adrenergic receptor--GluR1 signalling complex for localized cAMP signalling.

Central noradrenergic signalling mediates arousal and facilitates learning through unknown molecular mechanisms. Here, we show that the beta(2)-adrenergic receptor (beta(2)AR), the trimeric G(s) protein, adenylyl cyclase, and PKA form a signalling complex with the AMPA-type glutamate receptor subunit GluR1, which is linked to the beta(2)AR through stargazin and PSD-95 and their homologues. Only GluR1 associated with the beta(2)AR is phosphorylated by PKA on beta(2)AR stimulation. Peptides that interfere with the beta(2)AR-GluR1 association prevent this phosphorylation of GluR1. This phosphorylation increases GluR1 surface expression at postsynaptic sites and amplitudes of EPSCs and mEPSCs in prefrontal cortex slices. Assembly of all proteins involved in the classic beta(2)AR-cAMP cascade into a supramolecular signalling complex and thus allows highly localized and selective regulation of one of its major target proteins.

Morphine induces AMPA receptor internalization in primary hippocampal neurons via calcineurin-dependent dephosphorylation of GluR1 subunits.

Chronic morphine treatment resulting in the alteration of postsynaptic levels of AMPA receptors, thereby modulating synaptic strength, has been reported. However, the mechanism underlying such drug-induced synaptic modification has not been resolved. By monitoring the GluR1 trafficking in primary hippocampal neurons using the pHluorin-GluR1 imaging and biotinylation studies, we observed that prolonged morphine exposure significantly induced loss of synaptic and extrasynaptic GluR1 by internalization. The morphine-induced GluR1 endocytosis was independent of neural network activities or NMDA receptor activities, as neither blocking the sodium channels with tetrodotoxin nor NMDA receptors with dl-APV altered the effects of morphine. Instead, morphine-induced GluR1 endocytosis is attributed to a change in the phosphorylation state of the GluR1 at Ser(845) as morphine significantly decreased the dephosphorylation of GluR1 at this site. Such changes in Ser(845) phosphorylation required morphine-induced activation of calcineurin, based on the observations that a calcineurin inhibitor, FK506, completely abrogated the dephosphorylation, and morphine treatment led to an increase in calcineurin enzymatic activity, even in the presence of dl-APV. Importantly, pretreatment with FK506 and overexpression of the GluR1 mutants, S845D (phospho-mimic) or S845A (phospho-blocking) attenuated the morphine-induced GluR1 endocytosis. Therefore, the calcineurin-mediated GluR1-S845 dephosphorylation is critical for the morphine-induced changes in the postsynaptic AMPA receptor level. Together, these findings reveal a novel molecular mechanism for opioid-induced neuronal adaptation and/or synaptic impairment.

Selective ERBB2 and BCL2 Inhibition Is Synergistic for Mitochondrial-Mediated Apoptosis in MDS and AML Cells.

The ERBB2 proto-oncogene is associated with an aggressive phenotype in breast cancer. Its role in hematologic malignancies is incompletely defined, in part because ERBB2 is not readily detected on the surface of cancer cells. We demonstrate that truncated ERBB2, which lacks the extracellular domain, is overexpressed on primary CD34+ myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) cells compared with healthy hematopoietic cells. This overexpression of ERBB2 is associated with aberrant, oncogenic signaling with autophosphorylation of multiple tyrosine sites. Like in breast cancers, ERBB2 can exist as truncated isoforms p95ERBB2 and p110ERBB2 in MDS and AML. Neutralization of ERBB2 signaling with ERBB2 tyrosine kinase inhibitors (i.e., lapatinib, afatinib, and neratinib) increases apoptotic cell death and reduces human engraftment of MDS cells in mice at 21 weeks posttransplantation. Inhibition of ERBB2 modulates the expression of multiple pro- and anti-apoptotic mitochondrial proteins, including B-cell lymphoma 2 (BCL2). Dual blockade with ERBB2 and BCL2 inhibitors triggers additional reductions of BCL2 phosphorylation and myeloid cell leukemia-1 (MCL1) expression compared with single drug treatment. Dual therapy was synergistic at all tested doses, with a dose reduction index of up to 29 for lapatinib + venetoclax compared with venetoclax alone. Notably, these agents operated together and shifted cancer cells to a pro-apoptotic phenotype, resulting in increased mitochondrial cytochrome c release and activated caspase-3-mediated cell death. IMPLICATIONS: These findings warrant study of ERBB2 and BCL2 combination therapy in patients with MDS and AML. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/19/5/886/F1.large.jpg.

Targeting High Mobility Group Box-1 (HMGB1) Promotes Cell Death in Myelodysplastic Syndrome.

PURPOSE: Myelodysplastic syndrome (MDS) is associated with a dysregulated innate immune system. The purpose of this study was to determine whether modulation of the innate immune system via high mobility group box-1 (HMGB1) could reduce cell viability in MDS. EXPERIMENTAL DESIGN: We quantified HMGB1 in an MDS cell line MDS-L and in primary MDS cells compared with nonmalignant hematopoietic cells. We performed loss-of-function studies of HMGB1 using pooled siRNAs and a small-molecule inhibitor sivelestat compared with standard chemotherapy. We measured levels of engraftment of MDS-L cells in NOD-scidIL2Rgnull (NSG) mice following treatment with sivelestat. Mechanistically, we interrogated cell survival pathways and 45 targets within the NFκB pathway using both protein analysis and a proteome profiler array. RESULTS: We discovered that HMGB1 had increased expression in both MDS-L cells and in primary CD34+ MDS cells compared with healthy CD34+ hematopoietic cells. Sivelestat impaired MDS cell expansion, increased cellular death, and spared healthy hematopoietic cells. MDS-L marrow engraftment is reduced significantly at 17 weeks following treatment with sivelestat compared with control mice. Treatment of CD34+ MDS cells with sivelestat and azacitidine or decitabine was additive to increase apoptotic cell death compared with chemotherapy alone. Sivelestat promoted apoptosis with increased expression of PUMA, activated caspase 3, and increased DNA double-strand breaks. Inhibition of HMGB1 reduced levels of Toll-like receptors (TLR) and suppressed activation of NFκB in MDS-L cells. CONCLUSIONS: Inhibition of HMGB1 could promote MDS cell death and alter innate immune responses via suppression of NFκB pathways.

CCR5 Signaling Promotes Murine and Human Hematopoietic Regeneration following Ionizing Radiation.

Hematopoietic stem and progenitor cells (HSPCs) depend on regulatory cytokines from the marrow microenvironment. From an unbiased cytokine screen of murine marrow supernatants, we identified C-C motif chemokine ligand 5 (CCL5) as an endothelial cell-secreted hematopoietic growth factor. Following treatment with CCL5, hematopoietic regeneration is accelerated and survival is prolonged after radiation. In mice with deletion of Ccr5, hematopoietic regeneration is delayed compared to control mice. Deletion of Ccr5 specifically in hematopoietic cells was sufficient to delay regeneration, while the deletion of Ccr5 in stromal/endothelial cells was not. Mechanistically, CCL5 promotes hematopoietic cell cycling and cell survival. Like murine hematopoietic cells, human hematopoietic cells (cord blood, healthy marrow, and peripheral blood) increase CCR5 expression after radiation exposure to promote cell survival. These data establish that CCL5 and CCR5 signaling play critical roles in hematopoietic regeneration and could serve as therapeutic targets to shorten the duration of myelosuppression.

Endothelial Cell-Derived Extracellular Vesicles Mitigate Radiation-Induced Hematopoietic Injury.

PURPOSE: Extracellular vesicles (EVs) are shed vesicles that bear a combination of nucleic acids and proteins. EVs are becoming recognized as a mode of cell-to-cell communication. Because hematopoietic stem cells reside in proximity to endothelial cells (ECs), we investigated whether EC-derived EVs could regulate hematopoietic stem cell regeneration after ionizing radiation. METHODS AND MATERIALS: We generated EVs derived from primary murine marrow ECs. We sought to determine the response of irradiated hematopoietic stem and progenitor cells to syngeneic or allogeneic EVs in culture assays. Starting 24 hours after either sublethal or lethal irradiation, mice were treated with EVs or saline or cultured primary marrow endothelial cells to determine the hematopoietic response in vivo. RESULTS: We demonstrate that EVs bear nuclear material and express EC-specific markers. Treatment with EVs promoted cell expansion and increased the number of colony-forming units compared to irradiated, hematopoietic cell cultures treated with cytokines alone. After total body irradiation, EV-treated mice displayed preserved marrow cellularity, marrow vessel integrity, and prolonged overall survival compared with controls treated with saline. Treatment of irradiated hematopoietic stem/progenitor cells (HSPCs) with EVs from different genetic strains showed results similar to treatment of HSPCs from syngeneic EVs. Mechanistically, treatment of irradiated HSPCs with EVs resulted in decreased levels of annexin V+ apoptotic cell death, which is mediated in part by tissue inhibitor of metalloproteinase-1. CONCLUSIONS: Our findings show that syngeneic or allogeneic EVs could serve as cell-derived therapy to deliver physiologic doses of nucleic acids and growth factors to hematopoietic cells to accelerate hematopoietic regeneration.

Formyl peptide receptor like 1 differentially requires mitogen-activated protein kinases for the induction of glial fibrillary acidic protein and interleukin-1alpha in human U87 astrocytoma cells.

Mitogen-activated protein kinases (MAPKs) are not only pivotal mediators of signal transduction but they also regulate diverse biological processes ranging from survival, proliferation and differentiation to apoptosis. By using human U87 astrocytoma and transfected FPRL1/CHO cells, we have demonstrated that activation of FPRL1 with WKYMVM effectively phosphorylated JNK and ERK. Interestingly, p38 MAPK activation was only seen with FPRL1/CHO cells. The MAPK phosphorylations in response to WKYMVM were blocked by WRW(4) (a selective FPRL1 antagonist), but not cyclosporine H (a well-known FPR antagonist). The key signaling intermediates in the MAPK pathways were also delineated. G(i)/G(o) proteins, Src family tyrosine kinases, but not phosphatidylinositol-3 kinase, protein kinase C and calmodulin-dependent kinase II, were required to transmit signals from FPRL1 toward JNK, ERK and p38 MAPK. Furthermore, phospholipase Cbeta was distinctively involved in the regulation of JNK but not the other MAPKs. Importantly, WKYMVM-stimulated U87 cells triggered noticeable increases in glial fibrillary acidic protein (GFAP) and interleukin-1alpha (IL-1alpha), which are correlated with reactive astrocytosis. In contrast, GFAP expression was not altered following stimulation with N-formyl-methionyl-leucyl-phenylalanine. Moreover, inhibitions of G(i)/G(o) proteins and JNK completely abolished both GFAP and IL-1alpha upregulations by FPRL1, while blockade of the MEK/ERK cascade exclusively suppressed the GFAP production. Consistently, overexpression of MEK1 and constitutively active JNKK in U87 cells led to ERK and JNK activation, respectively, which was accompanied with markedly increased GFAP production. We have thus identified a possible linkage among FPRL1, MAPKs, astrocytic activation and the inflammatory response.

Formyl peptide-receptor like-1 requires lipid raft and extracellular signal-regulated protein kinase to activate inhibitor-kappa B kinase in human U87 astrocytoma cells.

Formyl peptide-receptor like-1 (FPRL-1) may possess critical roles in Alzheimer's diseases, chemotaxis and release of neurotoxins, possibly through its regulation of nuclear factor-kappaB (NFkappaB). Here we illustrate that activation of FPRL-1 in human U87 astrocytoma or Chinese hamster ovary cells stably expressing the receptor resulted in the phosphorylations of inhibitor-kappaB kinase (IKK), an onset kinase for NFkappaB signaling cascade. FPRL-1 selective hexapeptide Trp-Lys-Tyr-Met-Val-Met (WKYMVM) promoted IKK phosphorylations in time- and dose-dependent manners while pre-treatment of pertussis toxin abrogated the Galpha(i/o)-dependent stimulations. The FPRL-1-mediated IKK phosphorylation required extracellular signal-regulated protein kinase (ERK), phosphatidylinositol 3-kinase and cellular Src (c-Src), but not c-Jun N-terminal kinase and p38 mitogen-activated protein kinase. Despite its ability to mobilize Ca(2+), WKYMVM did not require Ca(2+) for the modulation of IKK phosphorylation. Activation of FPRL-1 also induced NFkappaB-driven luciferase expression. Interestingly, cholesterol depletion from plasma membrane by methyl-beta-cyclodextrin abolished the FPRL-1-stimulated IKK phosphorylation, denoting the important role of lipid raft integrity in the FPRL-1 to IKK signaling. Furthermore, we demonstrated that in U87 cells, several signaling intermediates in the FPRL-1-IKK pathway including Galpha(i2), c-Src and ERK were constitutively localized at the raft microdomains. WKYMVM administration not only resulted in higher amount of ERK recruitment to the raft region, but also specifically stimulated raft-associated c-Src and ERK phosphorylations. Taken together, these results demonstrate that FPRL-1 is capable of activating NFkappaB signaling through IKK phosphorylation and this may serve as a useful therapeutical target for FPRL-1-related diseases.

Naltrexone Facilitates Learning and Delays Extinction by Increasing AMPA Receptor Phosphorylation and Membrane Insertion.

BACKGROUND: The opioid antagonists naloxone/naltrexone are involved in improving learning and memory, but their cellular and molecular mechanisms remain unknown. We investigated the effect of naloxone/naltrexone on hippocampal α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) trafficking, a molecular substrate of learning and memory, as a probable mechanism for the antagonists activity. METHODS: To measure naloxone/naltrexone-regulated AMPAR trafficking, pHluorin-GluA1 imaging and biochemical analyses were performed on primary hippocampal neurons. To establish the in vivo role of GluA1-Serine 845 (S845) phosphorylation on the behavioral effect induced by inhibition of the endogenous µ-opioid receptor (MOR) by naltrexone, MOR knockout, and GluA1-S845A mutant (in which Ser(845) was mutated to Ala) mice were tested in a water maze after chronic naltrexone administration. Behavioral responses and GluA1 levels in the hippocampal postsynaptic density in wild-type and GluA1-S845A mutant mice were compared using western blot analysis. RESULTS: In vitro prolonged naloxone/naltrexone exposure significantly increased synaptic and extrasynaptic GluA1 membrane expression as well as GluA1-S845 phosphorylation. In the MOR knockout and GluA1-S845A mutant mice, naltrexone did not improve learning, which suggests that naltrexone acts via inhibition of endogenous MOR action and alteration of GluA1 phosphorylation. Naltrexone-treated wild-type mice had significantly increased phosphorylated GluA1-S845 and GluA1 levels in their hippocampal postsynaptic density on the third day of acquisition, which is the time when naltrexone significantly improved learning. CONCLUSIONS: The beneficial effect of naltrexone on spatial learning and memory under normal conditions appears to be the result of increasing GluA1-S845 phosphorylation-dependent AMPAR trafficking. These results can be further explored in a mouse model of memory loss.

Regulation of STAT3 by mu-opioid receptors in human neuroblastoma SH-SY5Y cells.

Heptahelical opioid receptors are implicated in the transcriptional regulation of neuronal development. Here we demonstrated that activation of mu-opioid receptors in human neuroblastoma SH-SY5Y cells led to the activation of signal transducer and activator of transcription 3 (STAT3), a transcription factor central to the regulation of numerous biological processes. The mu-opioid-induced activation of STAT3 is sensitive to receptor was further shown to pertussis toxin treatment and required JAK and Src tyrosine kinases, but not phosphatidylinositol 3-kinase. This mu-opioid-induced response was mediated via the extracellular signal-regulated protein kinase in a Raf-1-independent manner. The present study provides a foundation to explore the importance of STAT3 signaling in the regulation of neuronal growth and differentiation by the mu-opioid receptor.

Activation of the human FPRL-1 receptor promotes Ca2+ mobilization in U87 astrocytoma cells.

The human formyl peptide receptor like 1 (FPRL-1) is a variant of the Gi-coupled formyl-peptide receptor. Functional FPRL-1 is endogenously expressed in the U87 astrocytoma cell line and there is accumulating evidence to suggest that FPRL-1 may be involved in neuroinflammation associated with the pathogenesis of Alzheimer's disease. In this study, we examined the ability of FPRL-1 to mobilize intracellular Ca2+ in U87 astrocytoma cells, as well as in Chinese hamster ovary (CHO) cells stably expressing FPRL-1. We showed that Trp-Lys-Tyr-Met-Val-Met-NH2 (WKYMVM), a specific agonist for FPRL-1, stimulated Ca2+ influx in both U87 and FPRL-1/CHO cells. These effects can be inhibited by the FPRL-1 selective antagonist, WRW4. Involvement of Gi proteins was demonstrated with the use of pertussis toxin, while inhibitors of store-operated channels (SOC) including 1-[2-(4-methoxyphenyl)]-2-[3-(4-methpxyphenyl)propoxy]ethyl-1H-imidazole hydrochloride (SKF96365) and 2-aminoethoxydiphenyl borate (2-APB) were found to abolish the WKYMVM-induced Ca2+ increase. However, intracellular Ca2+ mobilization in both cell lines were unaffected by the phospholipase Cbeta inhibitor U73122 or selective ryanodine receptor inhibitors. Our data demonstrated that activation of Gi-coupled FPRL-1 can lead to Ca2+ influx possibly via SOCs in U87 and FPRL-1/CHO cells.

Rac and Cdc42-dependent regulation of c-Jun N-terminal kinases by the delta-opioid receptor.

Heptahelical opioid receptors utilize Gi proteins to regulate a multitude of effectors including the classical adenylyl cyclases and the more recently discovered mitogen-activated protein kinases (MAPKs). The c-Jun NH2-terminal kinases (JNKs) belong to one of three subgroups of MAPKs. In NG108-15 neuroblastoma x glioma hybrid cells that endogenously express delta-opioid receptors, delta-agonist dose-dependently stimulated JNK activity in a pertussis toxin-sensitive manner. By using COS-7 cells transiently transfected with the cDNAs of delta-opioid receptor and hemagglutinin (HA)-tagged JNK, we delineated the signaling components involved in this pathway. Sequestration of Gbetagamma subunits by transducin suppressed the opioid-induced JNK activity. The possible involvement of the small GTPases was also examined. Expression of dominant negative mutants of Rac and Cdc42 blocked the opioid-induced JNK activation, and a partial inhibition was observed in the presence of the dominant negative mutant of Ras. In contrast, the dominant negative mutant of Rho did not affect the opioid-induced JNK activation. In addition, the receptor-mediated JNK activation was dependent on Src family tyrosine kinases, but independent of phosphatidylinositol-3 kinase and EGF receptor tyrosine kinases. Collectively, these results demonstrate functional regulation of JNK by the delta-opioid receptor, and this pathway requires Gbetagamma, Src kinases and the small GTPases Rac and Cdc42.

Phosphatidylinositol-3 kinase is distinctively required for mu-, but not kappa-opioid receptor-induced activation of c-Jun N-terminal kinase.

Opioid receptors are the therapeutic targets of narcotic analgesics. All three types of opioid receptors (mu, delta and kappa) are prototypical G(i)-coupled receptors with common signaling characteristics in their regulation of intracellular events. Nevertheless, numerous signaling processes are differentially regulated by the three receptors. We have recently demonstrated that stimulation of delta-opioid receptor can up-regulate the activity of the c-Jun N-terminal kinase (JNK) in a pertussis toxin-sensitive manner (Kam et al. 2003; J. Neurochem. 84, 503-513). The present study revealed that the mu-opioid receptor could stimulate JNK in both SH-SY5Y cells and transfected COS-7 cells. The mechanism by which the mu-opioid receptor stimulated JNK was delineated with the use of specific inhibitors and dominant-negative mutants of signaling intermediates. Activation of JNK by the mu-opioid receptor was mediated through G beta gamma, Src kinase, son-of-sevenless (Sos), Rac and Cdc42. Interestingly, unlike the delta-opioid receptors, the mu-opioid receptor required phosphatidylinositol-3 kinase (PI3K) to activate JNK. The mu-opioid receptor-induced JNK activation was effectively inhibited by wortmannin or the coexpression of a dominant negative mutant of PI3K gamma. Like the delta-opioid receptor, activation of JNK by the kappa-opioid receptor occurred in a PI3K-independent manner. These studies revealed that the mu-opioid receptor utilize a distinct mechanism to regulate JNK.

Kappa-opioid receptor signals through Src and focal adhesion kinase to stimulate c-Jun N-terminal kinases in transfected COS-7 cells and human monocytic THP-1 cells.

Opioid peptides exert diverse physiological functions through their cognate receptors. One subtype of the opioid receptors, kappa-opioid receptor, is endogenously expressed in human monocytic THP-1 cells. Stimulation of the THP-1 cells with a kappa-opioid receptor-selective agonist exerted a Gi-dependent activation of c-Jun N-terminal kinase (JNK). To further investigate the signaling mechanism by which the kappa-opioid receptor regulates JNK activity, heterologous expression assays in COS-7 cells were utilized. Overexpression of Galphat in COS-7 cells clearly suppressed kappa-opioid receptor-stimulated JNK activity, indicating that the pathway is primarily regulated by Gbetagamma. In both THP-1 and transfected COS-7 cells, pretreatment of the selective Src family kinase inhibitor pyrazolopyrimidine PP1 abolished the JNK activation, whereas the epidermal growth factor receptor inhibitor AG1478 [N-(3-chlorophenyl)-6,7-dimethoxy-4-quinazolinanine] failed to do that. Furthermore, the JNK activation in response to kappa-opioid receptor was suppressed by an autophosphorylation-resistant mutant of focal adhesion kinase (FAK). Consistently, activated kappa-opioid receptor induced Src stimulation and FAK autophosphorylation and promoted the formation of Src-FAK complex. The participation of small GTPases as well as a guanine nucleotide exchange factor was also implicated because dominant-negative mutants of Rac, Cdc42, and Son-of-sevenless (Sos) attenuated the agonist-induced activation of JNK. These studies demonstrate that the activation of JNK by kappa-opioid receptors is routed via Gbetagamma, Src, FAK, Sos, Rac, and Cdc42.