Active conformation of the erythropoietin receptor: random and cysteine-scanning mutagenesis of the extracellular juxtamembrane and transmembrane domains

In the absence of erythropoietin (Epo) cell surface Epo receptors (EpoR) are dimeric; dimerization is mediated mainly by the transmembrane domain. Binding of Epo changes the orientation of the two receptor subunits. This conformational change is transmitted through the juxtamembrane and transmembrane domains, leading to activation of JAK2 kinase and induction of proliferation and survival signals. To define the active EpoR conformation(s) we screened libraries of EpoRs with random mutations in the transmembrane domain and identified several point mutations that activate the EpoR in the absence of ligand, including changes of either of the first two transmembrane domain residues (Leu(226) and Ile(227)) to cysteine. Following this discovery, we performed cysteine-scanning mutagenesis in the EpoR juxtamembrane and transmembrane domains. Many mutants formed disulfide-linked receptor dimers, but only EpoR dimers linked by cysteines at positions 223, 226, or 227 activated EpoR signal transduction pathways and supported proliferation of Ba/F3 cells in the absence of cytokines. These data suggest that activation of dimeric EpoR by Epo binding is achieved by reorienting the EpoR transmembrane and the connected cytosolic domains and that certain disulfide-bonded dimers represent the activated dimeric conformation of the EpoR, constitutively activating downstream signaling. Based on our data and the previously determined structure of Epo bound to a dimer of the EpoR extracellular domain, we present a model of the active and inactive conformations of the Epo receptor.     

A "classical" homodimeric erythropoietin receptor is essential for the antiapoptotic effects of erythropoietin on differentiated neuroblastoma SH-SY5Y and pheochromocytoma PC-12 cells

The hematopoietic cytokine erythropoietin (Epo) exerts cytoprotective effects on several types of neuronal cells both in vivo and in culture. Detailed molecular mechanisms underlying this phenomenon have not been elucidated and even the identity of the cytoprotective Epo receptors in neuronal cells is controversial. Here we show that Epo prevents staurosporine-induced apoptosis of differentiated human neuroblastoma SH-SY5Y cells, and activates the STAT5, AKT and MAPK signaling pathways. Differentiated SH-SY5Y cells have fewer than 50 high affinity Epo surface binding sites per cell, which could not be detected by standard assays measuring binding of 125I-labeled Epo. However, by measuring endocytosis of 125I-Epo, we could reliably quantify very small numbers of high-affinity Epo surface binding sites. Using SH-SY5Y cells stably expressing an Epo receptor (EpoR) shRNA and thus lacking detectable EpoR expression, we show that high affinity binding of Epo to these neuronal cells is mediated by the hematopoietic EpoR, and that this EpoR is also essential for the antiapoptotic activity of Epo. In contrast, a mutant Epo that has an intact binding site 1 but a non-functional binding site 2 and hence binds only to one cell surface EpoR molecule ("site 2" Epo mutant) displays significantly lower antiapoptotic activity than wild-type Epo. Furthermore, expression of the GM-CSF/IL-3/IL-5 receptor common beta chain, which was proposed to be responsible for the cytoprotective activity of Epo on certain types of neuronal cells, was undetectable in differentiated SH-SY5Y cells. Epo also alleviated staurosporine-induced apoptosis of rat PC-12 pheochromocytoma cells while the R103A "site 2" Epo mutant did not, and we could not detect expression of the common beta chain in PC-12 cells. Together our results indicate that Epo exerts its antiapoptotic effects on differentiated SH-SY5Y and PC-12 cells through the standard stoichiometry of one molecule of Epo binding to two EpoR subunits, comprising the "classical" Epo receptor signaling complex.     

Antiviral Activity of the Proteasome on Incoming Human Immunodeficiency Virus Type 1

Following cell surface receptor binding and membrane fusion, human immunodeficiency virus (HIV) virion cores are released in the cytoplasm. Incoming viral proteins represent potential targets for cytosolic proteases. We show that treatment of target cells with the proteasome inhibitors MG132 and lactacystin increased the efficiency of HIV infection. Proteasome inhibitors were active at the early steps of the viral cycle. Incoming p24^Gag proteins accumulated in the cytosol, and larger amounts of proviral DNA were synthesized. In vitro, purified 20S proteasome degraded HIV virion components. Thus, degradation of incoming viral proteins by the proteasome represents an early intracellular defense against infection.     

The distinct erythropoietin functions that promote cell survival and proliferation are affected by aluminum exposure through mechanisms involving erythropoietin receptor

Erythropoietin (Epo) promotes the development of erythroid progenitors by triggering intracellular signals through the binding to its specific receptor (EpoR). Previous results related to the action of aluminum (Al) on erythropoiesis let us suggest that the metal affects Epo interaction with its target cells. In order to investigate this effect on cell activation by the Epo-EpoR complex, two human cell lines with different dependence on Epo were subjected to Al exposure. In the Epo-independent K562 cells, Al inhibited Epo antiapoptotic action and triggered a simultaneous decrease in protein and mRNA EpoR levels. On the other hand, proliferation of the strongly Epo-dependent UT-7 cells was enhanced by long-term Al treatment, in agreement with the upregulation of EpoR expression during Epo starvation. Results provide some clues to the way by which Epo supports cell survival and growth, and demonstrate that not all the intracellular factors needed to guarantee the different signaling pathways of Epo-cell activation are available or activated in cells expressing EpoR. This study then suggests that at least one of the mechanisms by which Al interfere with erythropoiesis might involve EpoR modulation.     

Activation of erythropoietin signaling by receptor dimerization

The hormone erythropoietin (Epo) is essential for red blood cell development. Epo binds a high affinity receptor on the surface of erythroid progenitor cells, stimulating receptor dimerization and activation of the intracellular signal transduction pathways that support erythroid cell survival, proliferation and differentiation. Biochemical and structural analysis of the erythropoietin receptor (EpoR) is revealing the molecular mechanisms of EpoR function, leading the way to the development of small molecule Epo mimetics. This review focuses on the role EpoR dimerization plays in receptor function.     

Delayed and sustained activation of p42/p44 mitogen-activated protein kinase induced by proteasome inhibitors through p21(ras) in PC12 cells

Proteolysis by the ubiquitin/proteasome pathway regulates the intracellular level of several proteins, some of which control cell proliferation and cell cycle progression. To determine what kinds of signaling cascades are activated or inhibited by proteasome inhibition, we treated PC12 cells with specific proteasome inhibitors and subsequently performed in-gel kinase assays. N-Acetyl-Leu-Leu-norleucinal and lactacystin, which inhibit the activity of the proteasome, induced the activation of p42/p44 mitogen-activated protein (MAP) kinases [extracellular signal-regulated kinases (ERKs) 1 and 2]. In contrast, N-acetyl-Leu-Leu-methional, which inhibits the activity of calpains, but not of the proteasome, failed to induce ERK activation. Uniquely, the kinetics of MAP kinase activation induced by proteasome inhibitors are very slow compared with those resulting from activation by nerve growth factor; ERK activation is detectable only after a 5-h treatment with the inhibitors, and its activity remained unchanged for at least until 27 h. Proteasome inhibitor-initiated ERK activation is inhibited by pretreatment with the ERK kinase inhibitor PD 98059, as well as by overexpression of a dominant-negative form of Ras. Thus, proteasome inhibitors induce sustained ERK activation in a Ras-dependent manner. Proteasome inhibitor-induced neurite outgrowth, however, is not inhibited by PD 98059, indicating that sustained activation of ERKs is not the factor responsible for proteasome inhibitor-induced morphological differentiation. Our data suggest the presence of a novel mechanism for activation of the MAP kinase cascade that involves proteasome activity.     

Cellular trafficking and degradation of erythropoietin and novel erythropoiesis stimulating protein (NESP)

Erythropoietin (Epo) is essential for the production of mature red blood cells, and recombinant Epo is commonly used to treat anemia, but how Epo is degraded and cleared from the body is not understood. Glycosylation of Epo is required for its in vivo bioactivity, although not for in vitro receptor binding or stimulation of Epo-dependent cell lines; Epo glycosylation actually reduces the affinity of Epo for the Epo receptor (EpoR). Interestingly, a hyperglycosylated analog of Epo, called novel erythropoiesis-stimulating protein (NESP), has a lower affinity than Epo for the EpoR but has greater in vivo activity and a longer serum half-life than Epo. We hypothesize that a major mechanism for degradation of Epo in the body occurs in cells expressing the Epo receptor, through receptor-mediated endocytosis of Epo followed by degradation in lysosomes, and therefore investigated the trafficking and degradation of Epo and NESP by EpoR-expressing cells. We show that Epo and NESP are degraded only by cultured cells that express the EpoR, and their receptor binding, dissociation, and trafficking properties determine their rates of intracellular degradation. Epo binds surface EpoR faster than NESP (k(on) = 5.0 x 10(8) m(-1) min(-1) versus 1.1 x 10(8) m(-1) min(-1)) but dissociates slower (k(off) = 0.029 min(-1) versus 0.042 min(-1)). Surface-bound Epo and NESP are internalized at the same rate (k(in) = 0.06 min(-1)), and after internalization 60% of each ligand is resecreted intact and 40% degraded. Our kinetic model of Epo and NESP receptor binding, intracellular trafficking, and degradation explains why Epo is degraded faster than NESP at the cellular level.     

Protein kinase C alpha controls erythropoietin receptor signaling

Protein kinase C (PKC) is implied in the activation of multiple targets of erythropoietin (Epo) signaling, but its exact role in Epo receptor (EpoR) signal transduction and in the regulation of erythroid proliferation and differentiation remained elusive. We analyzed the effect of PKC inhibitors with distinct modes of action on EpoR signaling in primary human erythroblasts and in a recently established murine erythroid cell line. Active PKC appeared essential for Epo-induced phosphorylation of the Epo receptor itself, STAT5, Gab1, Erk1/2, AKT, and other downstream targets. Under the same conditions, stem cell factor-induced signal transduction was not impaired. LY294002, a specific inhibitor of phosphoinositol 3-kinase, also suppressed Epo-induced signal transduction, which could be partially relieved by activators of PKC. PKC inhibitors or LY294002 did not affect membrane expression of the EpoR, the association of JAK2 with the EpoR, or the in vitro kinase activity of JAK2. The data suggest that PKC controls EpoR signaling instead of being a downstream effector. PKC and phosphoinositol 3-kinase may act in concert to regulate association of the EpoR complex such that it is responsive to ligand stimulation. Reduced PKC-activity inhibited Epo-dependent differentiation, although it did not effect Epo-dependent "renewal divisions" induced in the presence of Epo, stem cell factor, and dexamethasone.     

Erythropoietin and IL-3 Induce Tyrosine Phosphorylation of CrkL and Its Association with Shc,SHP-2, and Cbl in Hematopoietic Cells

The present study demonstrates that erythropoietin (Epo) and IL-3 induce tyrosine phosphorylation of the SH2/SH3-containing adapter protein CrkL and its transient association with tyrosine-phosphorylated SHP-2, Shc, and Cbl in a murine IL-3-dependent cell line, 32D, expressing the Epo receptor (EpoR). In these cells, CrkL was constitutively complexed with the guanine nucleotide exchange factor C3G, which was found to coimmunoprecipitate with Shc from Epo- or IL-3-stimulated cells. Studies using cells expressing mutant EpoRs showed that the Epo-induced tyrosine phosphorylation of CrkL is dependent on the membrane-proximal EpoR cytoplasmic region involved in the activation of Jak2 as well as the C-terminal 145 amino acid region which is required for tyrosine phosphorylation of SHP-2 and Shc. It was further revealed that CrkL is recruited to the tyrosine-phosphorylated EpoR, most likely through its interaction with tyrosine-phosphorylated Shc and SHP-2. These results suggest that CrkL is involved in the signaling pathways from the receptors for Epo and IL-3, most likely by modulating the activity of the Ras family GTPases through its interaction with C3G.     

Role of the cytokine-induced SH2 domain-containing protein CIS in growth hormone receptor internalization

The cytokine-inducible SH2 domain-containing protein CIS inhibits signaling from the growth hormone (GH) receptor (GHR) to STAT5b by a proteasome-dependent mechanism. Here, we used the GH-responsive rat liver cell line CWSV-1 to investigate the role of CIS and the proteasome in GH-induced GHR internalization. Cell-surface GHR localization and internalization were monitored in GH-stimulated cells by confocal immunofluorescence microscopy using an antibody directed against the GHR extracellular domain. In GH na?ve cells, GHR was detected in small, randomly distributed granules on the cell surface and in the cytoplasm, with accumulation in the perinuclear area. GH treatment induced a rapid (within 5 min) internalization of GH.GHR complexes, which coincided with the onset of GHR tyrosine phosphorylation and the appearance in the cytosol of distinct granular structures containing internalized GH. GHR signaling to STAT5b continued for approximately 30-40 min, however, indicating that GHR signaling and deactivation of the GH.GHR complex both proceed from an intracellular compartment. The internalization of GH and GHR was inhibited by CIS-R107K, a dominant-negative SH2 domain mutant of CIS, and by the proteasome inhibitors MG132 and epoxomicin, which prolong GHR signaling to STAT5b. GH pulse-chase studies established that the internalized GH.GHR complexes did not recycle back to the cell surface in significant amounts under these conditions. Given the established specificity of CIS-R107K for blocking the GHR signaling inhibitory actions of CIS, but not those of other SOCS/CIS family members, these findings implicate CIS and the proteasome in the control of GHR internalization following receptor activation and suggest that CIS-dependent receptor internalization is a prerequisite for efficient termination of GHR signaling.     

Low doses of EPO activate MAP kinases but not JAK2-STAT5 in rat vascular smooth muscle cells.

Previous reports have shown a direct effect of erythropoietin (Epo) on vascular smooth muscle cells (VSMCs). Our aim was to assess expression of the Epo receptor (EpoR) on VSMCs and to study the activation of two major signaling cascades activated by Epo, namely JAK2/STAT5 and MAPK pathways. All experiments were performed in parallel using the Epo-responsive UT7 cell line. From semiquantitative RT-PCR experiments, VSMCs were estimated to express approximately 30-fold less EpoR mRNA than UT7 cells. Epo-induced phosphorylation of proteins involved in the EpoR/JAK2/STAT5 cascade could not be detected in VSMCs, even using pharmacological doses of Epo (250 IU/ml). In contrast, a strong activation of MAP kinase pathway was detected with as low as 10 IU/ml Epo. We suggest that MAPK activation reflects a physiologically relevant effect of Epo on VSMCs that may be correlated to cell proliferation.     

Therapeutic targeting of cancer cell cycle using proteasome inhibitors

Proteasomes are multicatalytic protease complexes in the cell, involved in the non-lysosomal recycling of intra-cellular proteins. Proteasomes play a critical role in regulation of cell division in both normal as well as cancer cells. In cancer cells this homeostatic function is deregulated leading to the hyperactivation of the proteasomes. Proteasome inhibitors (PIs) are a class of compounds, which either reversibly or irreversibly block the activity of proteasomes and induce cancer cell death. Interference of PIs with the ubiquitin proteasome pathway (UPP) involved in protein turnover in the cell leads to the accumulation of proteins engaged in cell cycle progression, which ultimately put a halt to cancer cell division and induce apoptosis. Upregulation of many tumor suppressor proteins involved in cell cycle arrest are known to play a role in PI induced cell cycle arrest in a variety of cancer cells. Although many PIs target the proteasomes, not all of them are effective in cancer therapy. Some cancers develop resistance against proteasome inhibition by possibly activating compensatory signaling pathways. However, the details of the activation of these pathways and their contribution to resistance to PI therapy remain obscure. Delineation of these pathways may help in checking resistance against PIs and deducing effective combinational approaches for improved treatment strategies. This review will discuss some of the signaling pathways related to proteasome inhibition and cell division that may help explain the basis of resistance of some cancers to proteasome inhibitors and underline the need for usage of PIs in combination with traditional chemotherapy.     

Antiapoptotic effects of erythropoietin in differentiated neuroblastoma SH-SY5Y cells require activation of both the STAT5 and AKT signaling pathways

The hematopoietic cytokine erythropoietin (Epo) prevents neuronal death during ischemic events in the brain and in neurodegenerative diseases, presumably through its antiapoptotic effects. To explore the role of different signaling pathways in Epo-mediated antiapoptotic effects in differentiated human neuroblastoma SH-SY5Y cells, we employed a prolactin receptor (PrlR)/erythropoietin receptor (EpoR) chimera system, in which binding of prolactin (Prl) to the extracellular domain activates EpoR signaling in the cytosol. On induction of apoptosis by staurosporine, Prl supports survival of the SH-SY5Y cells expressing the wild-type PrlR/EpoR chimera. In these cells Prl treatment strongly activates the STAT5, AKT, and MAPK signaling pathways and induces weak activation of the p65 NF-kappaB factor. Selective mutation of the eight tyrosine residues of the EpoR cytoplasmic domain results in impaired or absent activation of either STAT5 (mutation of Tyr(343)) or AKT (mutation of Tyr(479)) or both (mutation of all eight tyrosine residues). Most interestingly, Prl treatment does not prevent apoptosis in cells expressing mutant PrlR/EpoR chimeras in which either the STAT5 or the AKT signaling pathways are not activated. In contrast, ERK 1/2 is fully activated by all mutant PrlR/EpoR chimeras, comparable with the level seen with the wild-type PrlR/EpoR chimera, implying that activation of the MAPK signaling pathway per se is not sufficient for antiapoptotic activity. Therefore, the antiapoptotic effects of Epo in neuronal cells require the combinatorial activation of multiple signaling pathways, including STAT5, AKT, and potentially MAPK as well, in a manner similar to that observed in hematopoietic cells.     

The Erythropoietin/Erythropoietin Receptor Signaling Pathway Promotes Growth and Invasion Abilities in Human Renal Carcinoma Cells

Co-expression of erythropoietin (Epo) and erythropoietin receptor (EpoR) has been found in various non-hematopoietic cancers including hereditary and sporadic renal cell carcinomas (RCC), but the Epo/EpoR autocrine and paracrine mechanisms in tumor progression have not yet been identified. In this study, we used RNA interference method to down-regulate EpoR to investigate the function of Epo/EpoR pathway in human RCC cells. Epo and EpoR co-expressed in primary renal cancer cells and 6 human RCC cell lines. EpoR signaling was constitutionally phosphorylated in primary renal cancer cells, 786-0 and Caki-1 cells, and recombinant human Epo (rhEpo) stimulation had no significant effects on further phosphorylation of EpoR pathway, proliferation, and invasiveness of the cells. Down-regulation of EpoR expression in 786-0 cells by lentivirus-introduced siRNA resulted in inhibition of growth and invasiveness in vitro and in vivo, and promotion of cell apoptosis. In addition, rhEpo stimulation slightly antagonized the anti-tumor effect of Sunitinib on 786-0 cells. Sunitinib could induce more apoptotic cells in 786-0 cells with knockdown EpoR expression. Our results suggested that Epo/EpoR pathway was involved in cell growth, invasion, survival, and sensitivity to the multi-kinases inhibitor Sunitinib in RCC cells.     

Implications of proteasome inhibition: an enhanced macrophage phenotype

The objective of this study was to elucidate the role of the cellular proteasome on endotoxin-mediated activation of the macrophage. To study this role, THP-1 cells were stimulated with lipopolysaccharide (LPS) with selective cells being pretreated with the proteasome inhibitor, lactacystin or MG-132. LPS stimulation led to the phosphorylation and degradation of IRAK, followed by activation of JNK/SAPK, ERK 1/2, and p38. Subsequently, LPS induced the degradation of IkappaB, and the nuclear activation of NF-kappaB and AP-1. Activation of these pathways was associated with the production of IL-6, IL-8, IL-10, and TNF-alpha. Proteasome inhibition with either lactacystin or MG-132 attenuated LPS-induced IRAK degradation, and enhanced activation of JNK/SAPK, ERK 1/2, and p38. Proteasome inhibition, also, led to increased LPS-induced AP-1 activation, and attenuated LPS-induced IkappaB degradation resulting in abolished NF-kappaB activation. Proteasome inhibition led to significant modulation of LPS-induced cytokine production; increased IL-10, no change in IL-6, and decreased IL-8, and TNF-alpha. Thus, this study demonstrates that cellular proteasome is critical to regulation of LPS-induced signaling within the macrophage, and inhibition of the proteasome results in a conversion to an anti-inflammatory phenotype.     

Proteasome inhibition induces neurite outgrowth through posttranslational modification of TrkA receptor

The ubiquitin-proteasome pathway regulates many biological processes, including protein degradation, receptor endocytosis, protein sorting, subnuclear trafficking and neuronal differentiation. While proteasome inhibition is known to induce neurite outgrowth, the signaling mechanisms that mediate these effects have not been defined. In this study, we investigated the underlying mechanisms that link proteasome inhibition with neurite generation. We found that the proteasome inhibitors, MG132 and lactacystin, induced neurite outgrowth and also activated extracellular signal-regulated kinase/mitogen activated protein kinase and phosphatidylinositol-3-kinase/AKT pathways. These proteasome inhibitors also induced phosphorylation and ubiquitination of TrkA receptors, indicating that proteasome inhibition activates the major pathways of TrkA signaling. However, in contrast to nerve growth factor stimulation, which induces internalization of surface TrkA receptors, proteasome inhibitor-induced neurite outgrowth did not require TrkA receptor internalization. These results indicate that the ubiquitin-proteasome system regulates neurite formation through posttranslational modification of TrkA receptors.     

Treatment of COS-7 cells with proteasome inhibitors or gamma-interferon reduces the increase in caspase 3 activity associated with staurosporine-induced apoptosis.

Proteasomes play a major role in intracellular protein degradation and have been implicated in apoptosis. In this study we have investigated proteasome activity and the effects of inhibition of proteasomes or modulation of proteasome complexes on staurosporine-induced apoptosis in COS-7 cells. Staurosporine treatment of COS-7 cells had little direct effect on proteasome activity and did not cause dissociation of 26S proteasomes. There was also no major redistribution of proteasomes accompanying apoptosis in COS-7 cells. However, when the cells were pretreated with proteasome inhibitors, both the caspase 3 activity of the cells and the percentage of apoptotic cells measured by the TUNEL assay were reduced compared to staurosporine-treated cells, which had no inhibitor added. Proteasome inhibitors were also found to reduce the activation of caspase 3 in living cells which was assayed using a FRET-based method. However, proteasome inhibitors did not prevent some of the morphological changes associated with staurosporine-induced apoptosis. Pretreatment of cells with gamma-interferon, which increases immunoproteasomes and PA28 complexes and reduces 26S proteasome levels, had an antiapoptotic effect. These results are consistent with a role for 26S proteasomes in regulating the activation of caspase 3 through the degradation of key regulatory proteins.     

Calpain is involved in the HIV replication from the latently infected OM10.1 cells

Treatment of OM10.1 cells latently infected with human immunodeficiency virus type 1 (HIV-1) with phorbol ester and calcium ionophore (A23187) induced virus replication which was blocked by N-Ac-Leu-Leu-norleucinal (ALLnL), a calpain inhibitor I, and not by lactacystin, a specific proteasome inhibitor. When the purified NF-kappa B/I kappa B complex was treated with mu-calpain, the specific DNA-binding activity was demonstrated by using electrophoretic mobility shift assay in vitro. This effect of mu-calpain was inhibited by ALLnL and calpastatin and not by lactacystin. In fact, we found that mu-calpain efficiently degraded I kappa B alpha. Furthermore, our Western blotting analysis has revealed that mu-calpain cleaves I kappa B alpha at its N-terminal and C-terminal regions that were previously reported to be involved in the interaction with NF-kappa B p65. These observations indicate that in monocyte/macrophage cells calcium signaling is involved in NF-kappa B activation through activation of calpain and thus calpain inhibitors may be effective in inhibiting the activation of latently infected HIV.