HIV service provision and training needs in outpatient mental health settings

The erythropoietin receptor (EpoR) has been previously shown to contain a cytoplasmic C-terminal negative regulatory domain, experimental deletion or mutation of which leads to increased sensitivity of expressing cells to the effects erythropoietin (Epo). We have studied a naturally occurring C-terminal truncation mutant of the human EpoR by stably transfecting the growth factor-dependent hematopoietic tissue culture cell line 32D with expression plasmids containing either the wildtype or mutant human EpoR cDNA, thus rendering the cells dependent on Epo for viability and proliferation. In Epo dose-response assays, cells expressing the mutant EpoR displayed hyperresponsiveness to Epo compared with cells expressing comparable numbers of the wild-type EpoR cultured in the presence of fetal bovine serum. We investigated whether enhanced Epo sensitivity of cells expressing the truncated EpoR is associated with alteration in Epo receptor-mediated activation of Stat5, which could have a role in Epo-induced proliferation. Although maximal Stat5 activation in response to a given concentration of Epo was comparable in 32D cells expressing the wild-type or truncated EpoRs, the time course of Epo-induced Stat5 activation was very different. Gel-mobility shift studies revealed the presence of Stat5 DNA-binding activity in nuclear and cytoplasmic extracts of cells expressing the truncated EpoR for a significantly longer time than that observed in similar extracts of cells expressing the wild-type EpoR consistent with decreased rate of inactivation of Stat5 in cells expressing the mutant EpoR. Epo-dependent tyrosine phosphorylation of both Stat5 and Jak2 was also substantially prolonged in cells expressing the truncated EpoR. These results suggest a role for Stat5 in regulation of Epo-mediated cell growth and implicate altered kinetics of Epo-induced Jak2 and Stat5 activation in the pathogenesis of familial erythrocytosis associated with this naturally occurring EpoR gene mutation.     

The prolactin receptor rescues EpoR-/- erythroid progenitors and replaces EpoR in a synergistic interaction with c-kit

We recently showed that a retrovirally transduced prolactin receptor (PrlR) efficiently supports the differentiation of wild-type burst-forming unit erythroid (BFU-e) and colony-forming unit erythroid (CFU-e) progenitors in response to prolactin and in the absence of erythropoietin (Epo). To examine directly whether the Epo receptor (EpoR) expressed by wild-type erythroid progenitors was essential for their terminal differentiation, we infected EpoR-/- progenitors with retroviral constructs encoding either the PrlR or a chimeric receptor containing the extracellular domain of the PrlR and intracellular domain of EpoR. In response to prolactin, both receptors were equally efficient in supporting full differentiation of the EpoR-/- progenitors into erythroid colonies in vitro. Therefore, there is no requirement for an EpoR-unique signal in erythroid differentiation; EpoR signaling has no instructive role in red blood cell differentiation. A synergistic interaction between EpoR and c-kit is essential for the production of normal numbers of red blood cells, as demonstrated by the severe anemia of mice mutant for either c-kit or its ligand, stem cell factor. We show that the addition of stem cell factor potentiates the ability of the PrlR to support differentiation of both EpoR-/- and wild-type CFU-e progenitors. This synergism is quantitatively equivalent to that observed between c-kit and EpoR. Therefore, there is no requirement for an EpoR-unique signal in the synergistic interaction between c-kit and EpoR.     

Notch1 and Notch2 inhibit myeloid differentiation in response to different cytokines

We have compared the ability of two mammalian Notch homologs, mouse Notchl and Notch2, to inhibit the granulocytic differentiation of 32D myeloid progenitor cells. 32D cells undergo granulocytic differentiation when stimulated with either granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF). Expression of the activated intracellular domain of Notch1 inhibits the differentiation induced by G-CSF but not by GM-CSF; conversely, the corresponding domain of Notch2 inhibits differentiation in response to GM-CSF but not to G-CSF. The region immediately C-terminal to the cdc10 domain of Notch confers cytokine specificity on the cdc10 domain. The cytokine response patterns of Notch1 and Notch2 are transferred with this region, which we have termed the Notch cytokine response (NCR) region. The NCR region is also associated with differences in posttranslational modification and subcellular localization of the different Notch molecules. These findings suggest that the multiple forms of Notch found in mammals have structural differences that allow their function to be modulated by specific differentiation signals.     

STAT5 activation correlates with erythropoietin receptor-mediated erythroid differentiation of an erythroleukemia cell line

Interaction between erythropoietin (EPO) and its membrane receptor induces the proliferation and differentiation of erythroid progenitors. EPO has been shown to activate the JAK2-STAT5 pathway in various hematopoietic cell lines, although the physiological role of this pathway is unclear. We have previously shown that epidermal growth factor activates a chimeric receptor bearing the extracellular domain of the epidermal growth factor receptor linked to the cytoplasmic domain of the EPO receptor, resulting in proliferation of interleukin-3-dependent hematopoietic cells and erythroid differentiation (globin synthesis) of EPO-responsive erythroleukemia cells. In the present study, we introduced various deletion and tyrosine to phenylalanine substitution in the cytoplasmic domain of the chimeric receptor and expressed these mutant chimeras in an EPO-responsive erythroleukemia cell line, ELM-I-1. Mutant chimeric receptors retaining either Tyr343 or Tyr401 could activate STAT5, judged by tyrosine-phosphorylation of STAT5 and induction of CIS, a target gene of STAT5. These mutants were able to induce erythroid differentiation. However, a chimeric receptor containing both Y343F and Y401F mutations could not activate STAT5 nor induce erythroid differentiation. Thus, Tyr343 or Tyr401 of the EPO receptor are independently necessary for erythroid differentiation as well as STAT5 activation. Moreover, exogenous expression of dominant-negative STAT5 suppressed EPO-dependent erythroid differentiation. These findings suggest that STAT5 plays an important role in erythroid differentiation through the EPO receptor cytoplasmic domain.     

Physical and functional interaction between p72(syk) and erythropoietin receptor

Erythropoietin (Epo) regulates the proliferation and differentiation of erythroid cells through interaction with a cell surface receptor (EpoR) that belongs to the cytokine receptor family. The Jak2 tyrosine kinase was previously shown to bind to the EpoR, to be activated upon Epo stimulation, and to play a critical role in Epo-induced proliferation. However, little is known about the role of other tyrosine kinases in Epo signaling. In this paper, we examined whether Syk was involved in EpoR activation. Coimmunoprecipitation experiments showed that the phosphorylated EpoR was associated with the Syk kinase in activated UT7 cells. The interaction of Epo with its receptor led to an increased kinase activity. The use of recombinant Syk Src homology 2 (SH2) domains expressed in tandem or individually revealed that both N- and C-SH2 domains of Syk participated in EpoR binding with a major contribution of the C-terminal SH2 domain. Far Western blotting further indicated that Syk directly binds to the EpoR and that the interaction of Syk with EpoR only occurred after Epo activation. These data suggest that phosphorylation of EpoR on tyrosine residues may mediate Syk binding to the receptor through interaction between the two SH2 domains of Syk and tyrosines of the receptor. We propose that in addition to Jak2, Syk protein kinase may be a component of EpoR signaling.     

The response of IL-3 dependent B6SUtA bone marrow cells to both erythropoietin and chemical inducers of differentiation

To develop cell lines which respond to both a physiological cytokine and chemical agents by the induction of differentiation pathway, factor dependent B6SUtA murine bone marrow cells were transfected with the erythropoietin receptor (EpoR). Clones were obtained that exhibited different sensitivities to erythopoietin (Epo), with one clone exhibiting erythroid differentiation in response to Epo, while in another Epo acted as a proliferation stimulus. Moreover, parental B6SUtA cells were sensitive to the initiation of differentiation by butyrate, diazepam and hemin. Thus, B6SUtA cells appear to represent a unique model to dissect the signaling molecules involved in the growth and differentiation pathways employed by Epo and non-physiological chemicals.     

Distinct roles for leukemia inhibitory factor receptor alpha-chain and gp130 in cell type-specific signal transduction

Leukemia inhibitory factor (LIF) induces a variety of disparate biological responses in different cell types. These responses are thought to be mediated through the functional LIF receptor (LIFR), consisting of a heterodimeric complex of LIFR alpha-chain (LIFRalpha) and gp130. The present study investigated the relative capacity of the cytoplasmic domains of each receptor subunit to signal particular responses in several cell types. To monitor the signaling potential of LIFRalpha and gp130 individually, we constructed chimeric receptors by linking the extracellular domain of granulocyte colony-stimulating factor receptor (GCSFR) to the transmembrane and cytoplasmic regions of either LIFRalpha or gp130. Both chimeric receptors and the full-length GCSFR in expressed in M1 myeloid leukemic cells to measure differentiation induction, in embryonic stem cells to measure differentiation inhibition, and in Ba/F3 cells to measure cell proliferation. Our results demonstrated that whereas GCSFR-gp130 receptor homodimer mediated a GCSF-induced signal in all three cell types, the GCSFR-LIFRalpha receptor homodimer was only functional in embryonic stem cells. These findings suggest that the signaling potential of gp130 and LIFRalpha cytoplasmic domains may differ depending upon the tissue and cellular response initiated.     

A substance P (neurokinin-1) receptor mutant carboxyl-terminally truncated to resemble a naturally occurring receptor isoform displays enhanced responsiveness and resistance to desensitization

Two isoforms of the substance P (SP) receptor, differing in the length of the cytoplasmic carboxyl-terminus by approximately 8 kDa, have been detected previously in rat salivary glands and other tissues. The binding and functional properties of these two isoforms have been investigated using full-length (407 amino acids) and carboxyl-terminally truncated (324 amino acids) rat SP receptors transfected stably into Chinese hamster ovary cells. Both the full-length and the truncated receptor bound radiolabeled SP with a similar Kd ( approximately 0.1 nM). The average number of high affinity SP binding sites per cell was 1.0 x 10(5) and 0.3 x 10(5) for the full-length and the truncated SP receptor, respectively. In both cell lines, SP induced a rapid but transient increase in cytosolic calcium concentration ([Ca2+]i), which consisted of the release of Ca2+ from intracellular stores and the influx of extracellular Ca2+. Both components are dependent on phospholipase C activation. Although the full-length and the truncated receptor utilize the same calcium pathways, they differ in their EC50 values (0.28 nM for the full-length; 0.07 nM for the truncated). These differences in responsiveness may be related to the observed differences in receptor desensitization. The truncated receptor, in contrast to the full-length receptor, does not undergo rapid and long-lasting desensitization. Cells possessing the short isoform of the SP receptor would thus be expected to exhibit a prolonged responsiveness.     

Early events in erythropoietin-induced signaling

The cloning of the erythropoietin receptor (EpoR) in 1989 has allowed very rapid progress in our understanding of the early intracellular events that may be triggered by erythropoietin (Epo) in erythroid progenitor cells. From studies carried out primarily with cell lines expressing exogenous wild-type and mutant EpoRs, it appears that the activated EpoR is capable of triggering many of the same cascades that are utilized by receptors possessing endogenous tyrosine kinase domains. The major challenge over the next decade lies in seeing if these same signaling pathways are also utilized by normal Epo-responsive erythroid progenitors, discriminating between proliferation and differentiation inducing events in these cells, and determining whether various hematologic disorders can be attributed to aberrations in these signaling pathways.     

Biased suppression of hematopoiesis and multiple developmental defects in chimeric mice containing Shp-2 mutant cells

Shp-2 is a cytoplasmic tyrosine phosphatase that contains two Src homology 2 (SH2) domains at the N terminus. Biochemical data suggests that Shp-2 acts downstream of a variety of receptor and cytoplasmic tyrosine kinases. A targeted deletion mutation in the N-terminal SH2 (SH2-N) domain results in embryonic lethality of homozygous mutant mice at midgestation. In vitro embryonic stem (ES) cell differentiation assays suggest that Shp-2 might play an important role in hematopoiesis. By aggregating homozygous mutant (Shp-2(-/-)) ES cells and wild-type (WT) embryos, we created Shp-2(-/-)-WT chimeric animals. We report here an essential role of Shp-2 in the control of blood cell development. Despite the widespread contribution of mutant cells to various tissues, no Shp-2(-/-) progenitors for erythroid or myeloid cells were detected in the fetal liver and bone marrow of chimeric animals by using the in vitro CFU assay. Furthermore, hematopoiesis was defective in Shp-2(-/-) yolk sacs. In addition, the Shp-2 mutation caused multiple developmental defects in chimeric mice, characterized by short hind legs, aberrant limb features, split lumbar vertebrae, abnormal rib patterning, and pathological changes in the lungs, intestines, and skin. These results demonstrate a functional involvement of Shp-2 in the differentiation of multiple tissue-specific cells and in body organization. More importantly, the requirement for Shp-2 is more stringent in hematopoiesis than in other systems.     

Negative regulation of granulocytic differentiation in the myeloid precursor cell line 32Dcl3 by ear-2, a mammalian homolog of Drosophila seven-up, and a chimeric leukemogenic gene, AML1/ETO

The polyomavirus enhancer binding protein 2alphaB (AML1/PEBP2alphaB/Cbfa2) plays a pivotal role in granulocyte colony-stimulating factor (G-CSF)-mediated differentiation of a myeloid progenitor cell line, 32Dc13. In this article, we report the identification of a PEBP2alphaB interacting protein, Ear-2, an orphan member of the nuclear hormone receptor superfamily that directly binds to and can inhibit the function of PEBP2alphaB. Ear-2 is expressed in proliferating 32Dc13 cells in presence of interleukin 3 but is down-regulated during differentiation induced by G-CSF. Interestingly, AML1/ETO(MTG8), a leukemogenic chimeric protein can block the differentiation of 32Dc13 cells, which is accompanied by the sustained expression of ear-2. Overexpression of Ear-2 can prevent G-CSF-induced differentiation, strongly suggesting that ear-2 is a key negative regulator of granulocytic differentiation. Our results indicate that a dynamic balance existing between PEBP2alphaB and Ear-2 appears to determine the choice between growth or differentiation for myeloid cells.     

TLS/FUS, a pro-oncogene involved in multiple chromosomal translocations, is a novel regulator of BCR/ABL-mediated leukemogenesis

The leukemogenic potential of BCR/ABL oncoproteins depends on their tyrosine kinase activity and involves the activation of several downstream effectors, some of which are essential for cell transformation. Using electrophoretic mobility shift assays and Southwestern blot analyses with a double-stranded oligonucleotide containing a zinc finger consensus sequence, we identified a 68 kDa DNA-binding protein specifically induced by BCR/ABL. The peptide sequence of the affinity-purified protein was identical to that of the RNA-binding protein FUS (also called TLS). Binding activity of FUS required a functional BCR/ABL tyrosine kinase necessary to induce PKCbetaII-dependent FUS phosphorylation. Moreover, suppression of PKCbetaII activity in BCR/ABL-expressing cells by treatment with the PKCbetaII inhibitor CGP53353, or by expression of a dominant-negative PKCbetaII, markedly impaired the ability of FUS to bind DNA. Suppression of FUS expression in myeloid precursor 32Dcl3 cells transfected with a FUS antisense construct was associated with upregulation of the granulocyte-colony stimulating factor receptor (G-CSFR) and downregulation of interleukin-3 receptor (IL-3R) beta-chain expression, and accelerated G-CSF-stimulated differentiation. Downregulation of FUS expression in BCR/ABL-expressing 32Dcl3 cells was associated with suppression of growth factor-independent colony formation, restoration of G-CSF-induced granulocytic differentiation and reduced tumorigenic potential in vivo. Together, these results suggest that FUS might function as a regulator of BCR/ABL leukemogenesis, promoting growth factor independence and preventing differentiation via modulation of cytokine receptor expression.     

Characterization of the domains of the erythropoietin receptor necessary for induction of cell growth and differentiation

To define the cytoplasmic region(s) of the erythropoietin receptor (EpoR) necessary for promotion of growth and induction of differentiation, mutated EpoR cDNAs containing truncations and conversions of tyrosine residues to phenylalanines were generated. Constructs were introduced into IL-3 dependent Ba/F3 cells by electroporation, and individual transfectants were propagated in methylcellulose-containing medium in the presence of erythropoietin (Epo). Truncated receptor at amino acid 350 was partially deficient in promoting cellular growth and the receptor lacking the box 2 region was not capable of inducing cellular growth. When a large number of cells were screened in Epo-containing liquid medium, clones arose in which Epo-dependent growth was due to activation of the endogenous EpoR gene. Analysis of the degree of Epo-dependent differentiation of the transfectants, based on the steady-state levels of beta major-globin mRNA, showed that the carboxyl terminal 133 amino acids and tyrosyl residues located at positions 429/431 and 460/464 were not necessary for the induction of differentiation. Examination of clones from diverse origins demonstrated that an inverse relationship existed between the rate of Epo-induced cellular replication and the degree of Epo-induced differentiation.     

Differentiation and growth arrest signals are generated through the cytoplasmic region of gp130 that is essential for Stat3 activation

Interleukin-6 (IL-6) induces growth arrest and macrophage differentiation through its receptor in a murine myeloid leukaemic cell line, M1, although it is largely unknown how the IL-6 receptor generates these signals. By using chimeric receptors consisting of the extracellular domain of growth hormone receptor and the transmembrane and cytoplasmic domain of gp130 with progressive C-terminal truncations, we showed that the membrane-proximal 133, but not 108, amino acids of gp130 could generate the signals for growth arrest, macrophage differentiation, down-regulation of c-myc and c-myb, induction of junB and IRF1 and Stat3 activation. Mutational analysis of this region showed that the tyrosine residue with the YXXQ motif was critical not only for Stat3 activation but also for growth arrest and differentiation, accompanied by down-regulation of c-myc and c-myb and immediate early induction of junB and IRF1. The tight correlation between Stat3 activation and other IL-6 functions was further observed in the context of the full-length cytoplasmic region of gp130. The result suggest that Stat3 plays an essential role in the signals for growth arrest and differentiation.     

Granulocytic sarcoma of the jejunum: a rare cause of small bowel obstruction

We report the case of a 40-yr-old man presenting with symptoms of small bowel obstruction. Small bowel x-rays revealed a stricture of the mid-jejunum. Push enteroscopy found a polypoid mass at 1 meter of the ligament of Treitz. Histopathological examination of the biopsy and surgical specimens showed a diffuse infiltrate of the mucosa made of medium to large cells, which were stained on immunohistochemistry by the leucocyte marker CD45 and the histiocyte/monocyte marker CD68 but were negative for the B and T cell markers. Cytological examination of the ascitic fluid revealed many myelobasts with cytoplasmic Auer rods and positive myeloperoxidase staining. There was no evidence of blood or bone marrow involvement suggestive of acute leukemia or myeloproliferative disorders. These findings were consistent with the diagnosis of preleukemic granulocytic sarcoma (or chloroma). Chemotherapy led to complete remission, but 21 months later the patient developed an acute myeloid leukemia. He died from aspergillus pneumonitis, 10 months after bone marrow allograft. Preleukemic granulocytic sarcoma of the small bowel is a rare condition and its diagnosis is usually not easy, requiring histochemical or immunohistochemical studies. Most cases have progressed to acute myeloid leukemia.