Accumulation of alpha- and beta-globin messenger RNAs in mouse erythroleukemia cells

The accumulation of alpha- and beta-globin mRNA sequences in murine erythroleukemia cells (MELC) treated with various inducers has been studied using specific alpha- and beta-globin complementary DNAs (cDNAs). In cells cultured with dimethylsulfoxide (Me2SO), hexamethylene bisacetamide (HMBA) or butyric acid, accumulation of alpha-globin mRNA is detectable after 16, 12 and 8 hr of culture, respectively. An increase in beta-globin mRNA sequences is not detected until 20-24 hr after culture. In cells exposed to hemin, both alpha- and beta-globin mRNAs are detectable by 6 hr of culture, and a constant ratio of alpha/beta-mRNA is maintained during induction. In maximally induced cells, the alpha/beta-globin mRNA ratios are approximately 1 in cells induced by Me2SO and HMBA, and 0.66 and 0.3-0.50 in cells induced by butyric acid and hemin, respectively. Thus different inducers of erythroid differentiation in MELC lead to different times of onset of the expression of alpha- and beta-like genes. In addition, the relative accumulation of alpha- and beta-globulin mRNAs in induced cells differs with various types of inducers.     

Reconstitution of receptors and GTP-binding regulatory proteins (G proteins) in Sf9 cells. A direct evaluation of selectivity in receptor.G protein coupling

The selectivity in coupling of various receptors to GTP-binding regulatory proteins (G proteins) was examined directly by a novel assay entailing the use of proteins overexpressed in Spodoptera frugiperda (Sf9) cells. Activation of G proteins was monitored in membranes prepared from Sf9 cells co-expressing selected pairs of receptors and G proteins (i.e. alpha, beta1, and gamma2 subunits). Membranes were incubated with [35S]guanosine 5'-(3-O-thio)triphosphate (GTPgammaS) +/- an agonist, and the amount of radiolabel bound to the alpha subunit was quantitated following immunoprecipitation. When expressed without receptor (but with beta1gamma2), the G protein subunits alphaz, alpha12, and alpha13 did not bind appreciable levels of [35S]GTPgammaS, consistent with a minimal level of GDP/[35S]GTPgammaS exchange. In contrast, the subunits alphas and alphaq bound measurable levels of the nucleotide. Co-expression of the 5-hydroxytryptamine1A (5-HT1A) receptor promoted binding of [35S]GTPgammaS to alphaz but not to alpha12, alpha13, or alphas. Binding to alphaz was enhanced by inclusion of serotonin in the assay. Agonist activation of both thrombin and neurokinin-1 receptors promoted a modest increase in [35S]GTPgammaS binding to alphaz and more robust increases in binding to alphaq, alpha12, and alpha13. Binding of [35S]GTPgammaS to alphas was strongly enhanced only by the activated beta1-adrenergic receptor. Our data identify interactions of receptors and G proteins directly, without resort to measurements of effector activity, confirm the coupling of the 5-HT1A receptor to Gz and extend the list of receptors that interact with this unique G protein to the receptors for thrombin and substance P, imply constitutive activity for the 5-HT1A receptor, and demonstrate for the first time that the cloned receptors for thrombin and substance P activate G12 and G13.     

Expression of the protein tyrosine phosphatase beta2 gene in mouse erythroleukemia cells induces terminal erythroid differentiation

We have cloned cDNA for protein tyrosine phosphatase beta2, which had been implicated in erythroid differentiation of mouse erythroleukemia cells. Expression of cDNA constructs, in which beta2 cDNA is placed under the control of mouse metallothionein-I promoter, by ZnCl2 converted a significant portion (20 to 38%) of the cells to erythroid-like cells, which is 25-50% of the erythroid differentiation efficiency observed by conventional erythroid-inducing agents. Furthermore, introduction and expression of altered protein tyrosine phosphatase beta2 cDNA constructs designed to produce the enzyme lacking the phosphatase activity inhibited erythroid differentiation by 100-20%, depending upon the concentration of erythroid-inducing agents employed. These results strongly suggest that protein tyrosine phosphatase beta2 is involved in triggering erythroid differentiation in mouse erythroleukemia cells.     

Successful replication of parvovirus B19 in the human megakaryocytic leukemia cell line MB-02

The pathogenic human parvovirus B19 has been shown to undergo productive replication in the erythroid lineage in primary normal human hematopoietic progenitor cells. However, none of the established erythroleukemia cell lines has allowed B19 virus replication in vitro. The remarkable erythroid tissue tropism of B19 virus was evaluated with a human megakaryocytic leukemia cell line, MB-02, which is dependent on the growth factor granulocyte-macrophage colony-stimulating factor but can be induced to undergo erythroid differentiation following treatment with erythropoietin (Epo). Whereas these cells did not support B19 virus DNA replication in the presence of granulocyte-macrophage colony-stimulating factor alone, active viral DNA replication was observed if the cells were exposed to Epo for 5 to 10 days prior to B19 virus infection, as detected by the presence of the characteristic B19 virus DNA replicative intermediates on Southern blots. No replication occurred if the cells were treated with Epo for 3 days or less. In addition, complete expression of the B19 virus genome also occurred in Epo-treated MB-02 cells, as detected by Northern blot analysis. B19 progeny virions were released into culture supernatants that were biologically active in secondary infection of normal human bone marrow cells. The availability of the only homogeneous permanent cell line in which induction of erythroid differentiation leads to a permissive state for B19 virus replication in vitro promises to yield new and useful information on the molecular basis of the erythroid tissue tropism as well as parvovirus B19-induced pathogenesis.     

alpha-Thrombin upregulates G alpha i3 in human vascular endothelial cells

BACKGROUND AND PURPOSE: During thrombosis, alpha-thrombin becomes sequestered by fibrin and the subendothelial basement membrane, and it is available to interact with the vasculature over prolonged periods. In this study, we investigated the long-term effect of alpha-thrombin on G alpha i3 and G alpha s levels in human vascular endothelial cells (EC). Because obesity is associated with changes in receptor signaling in many animal models, we also explored the influence of this clinical risk factor. METHODS: Primary cultures of human EC were exposed to alpha-thrombin for 16 hours, and immunologically detectable G alpha i3 and G alpha s levels were measured. RESULTS: alpha-Thrombin (100 nmol/L) increased G alpha i3 levels in EC derived from the cerebral microvasculature and superficial temporal artery (4.2 +/- 1.2-fold and 2.8 +/- 0.32-fold, respectively) but had no significant effect on EC derived from omental artery (P > .6) or from the superficial temporal artery of obese (body mass index > or = 28 kg/m2) patients (P > .4). The expression of G alpha s was unchanged in all cell types (P > or = .1). Two other circulating peptides, vasoactive intestinal peptide and endothelin-1, failed to alter the expression of either G protein in EC from the cerebral microvasculature, further demonstrating the specificity of the alpha-thrombin effect. However, thrombin receptor activating protein-14 mimicked the alpha-thrombin response and increased G alpha i3 in EC derived from the cerebral microvasculature and superficial temporal artery. CONCLUSIONS: We conclude that alpha-thrombin increases G alpha i3 expression in some EC through activation of its tethered liganded receptor. Obesity appears to suppress this action of alpha-thrombin.     

Anti-S-100 antibody recognizes ellipsoid-associated cells and other dendritic cells in the chicken spleen

We examined the activation of genes induced by erythropoietin (Epo) in erythroid progenitor cells that were isolated from the spleens of mice infected with anemia-inducing strain of the Friend virus. These erythroid progenitor cells, termed FVA cells, undergo in vitro differentiation to erythrocytes under the influence of Epo within 2 to 3 days. We used a differential hybridization procedure to screen a cDNA library constructed from FVA cells that were treated with Epo 2U/mL in the absence of serum for 2 hours. Of 20,000 recombinant phages, 47 plaques hybridized preferentially to cDNA probe prepared from Epo-stimulated cells. We found at least three different Epo-responsive genes (ERGs) and one of them corresponds to the mouse virus-like (VL30) element, similar to already reported BVL-1. The induction of VL30, which was evident within 30 minutes after Epo exposure, reached a maximum by 1 hour and remained stable for up to 4 hours. The treatment of FVA cells with cycloheximide (CHX) 10 micrograms/mL, which in itself activates the expression of VL30 caused a superinduction of the Epo signal. Changes in intracellular Ca2+ concentrations, either raised by ionomycin or depleted by EGTA, had no effect on the Epo-induced VL30 expression. In addition, protein kinase C (PKC) inhibitors such as staurosporine (3 mumol/L) or H7 (20 mumol/L) and a tyrosine kinase inhibitor, genistein (200 mumol/L), did not inhibit the Epo-induced expression of VL30. TPA (100 ng/mL), a PKC agonist, did not induce VL30 expression. Although the physiologic role of VL30 in the differentiation of erythroid progenitor cells is not known, our findings demonstrate that VL30 is an early ERG, and may be a useful indicator of the initial molecular actions of Epo.     

Difference of (Ca2+)i movements in platelets stimulated by thrombin and TRAP: the involvement of alpha(IIb)beta3-mediated TXA2 synthesis

This study investigated the difference of [Ca2+]i movement in platelets in response to thrombin and TRAP. The involvement of alpha(IIb)beta3 in this signaling was also studied. Stimulation of platelets with thrombin at 0.03 U/ml caused platelet aggregation and a two-peak increase in [Ca2+]i. The second peak of [Ca2+]i, but not the first peak was abolished by the inhibition of platelet aggregation with alpha(IIb)beta3 antagonists or by scavenging endogenous ADP with apyrase. A cyclooxygenase inhibitor, aspirin, and a TXA2 receptor antagonist, BM 13505, also abolished the second peak of [Ca2+]i but not the first peak, although these regents did not inhibit aggregation. Under the same assay conditions, measurement of TXB2 demonstrated that alpha(IIb)beta3 antagonists and aspirin almost completely inhibited the production of TXB2. In contrast to thrombin-stimulation, TRAP caused only a single peak of [Ca2+]i even in the presence of platelet aggregation, and a high level of [Ca2+]i increase was needed for the induction of platelet aggregation. The inhibition of aggregation with alpha(IIb)beta3 antagonists had no effect on [Ca2+]i change and TXB2 production induced by TRAP. Inhibition studies using anti-GPIb antibodies suggested that GPIb may be involved in the thrombin response, but not in the TRAP. Our findings suggest that low dose thrombin causes a different [Ca2+]i response and TXA2 producing signal from TRAP. Endogenous ADP release and fibrinogen binding to alpha(IIb)beta3 are responsible for the synthesis of TXA2 which results in the induction of the second peak of [Ca2+]i in low thrombin- but not TRAP-stimulated platelets.     

Injuries to the phrenic nerve resulting in diaphragmatic paralysis with special reference to stretch trauma

The effectiveness of purines and purine analogues as inducers of erythroid differentiation in cultured murine erythroleukemia cells has been investigated. These cell lines have previously been shown to differentiate in vitro in response to dimethylsulfoxide (DMSO) and a number of other polar solvents. Two purine analogues, 6-thioguanine and 6-mercaptopurine, as well as the naturally occuring purine, purine, hypoxanthine, are shown to be extremely potent inducers. 6-Thioguanine is effective at a concentration of 0.06 mM, 750 fold lower than the DMSO concentration required for equivalent induction. 6-Mercaptopurine and hypoxanthine are effective inducers at a concentration of approximately 2 mM. Accumulation of globin mRNA was monitored during induction with purine inducers and shown to be similar in amount to globin mRNA levels reached in DMSO-induced cultures. Induction of differentiation by all three compounds follows a similar time course to induction with DMSO. All three compounds are potent inducers of HGPRT (hypoxanthine-guanine phosphoribosyltransferase)-negative cell lines; hence incorporation of purines into DNA is not required for induction of differentiation. Comparison of these compounds with other purines and purine analogues suggests a high degree of specificity in their interaction with a cellular target.     

Effect of thyroid hormones on G proteins in synaptosomes of chick embryo

We have described a thyroid hormone receptor in synaptosomes of the chick embryo brain. To understand how the hormones exert their actions at this level, we performed a series of studies to demonstrate that this receptor could be linked to G proteins. Guanosine 5'-[gamma-thio]triphosphate (GTP gamma S)(100 muM) lowered the binding capacity of the receptor high affinity site from 8.9 +/- 1.3 to 3.4 +/- 1.3 ng T3/mg protein, a finding consistent with the coupling of receptor to G proteins. Furthermore, ADP ribosylation with pertussis toxin showed that thyroid hormones induced a dose-dependent increase in the inactive alpha 0-subunit of the G0 protein. This effect was detected at 10 pM, with a maximal increase (mean +/- SEM, 50 +/- 3.6%) at 100 nM, and T4 was as effective as T3. Both hormones also decreased the intrinsic guanine triphosphatase activity of G proteins by lowering the binding of GTP to the alpha-subunit and their rate of hydrolysis. This inhibition was greater with T4 (25 +/- 5%) than with T3 (14 +/- 2%), suggesting that the former could be the more active hormone at the synaptosomal level. The effect on guanine triphosphatase activity confirms that the synaptosomal thyroid hormone receptor is coupled to a G(zero) protein. These results demonstrate that thyroid hormones increase or favor the ADP ribosylation of G alpha(zero) by pertussis toxin. Thus, they enhance the alpha(zero)-GDP form of the G(zero) protein, namely its inactive conformation. By decreasing the activity of this protein, these hormones may modulate the formation of second messengers in synaptosomes and intervene in the regulation of neuronal proliferation and differentiation induced by several factors. Therefore, thyroid hormones may exert their action on brain maturation at least in part by modulating G alpha(zero) through their synaptosomal receptor.     

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.     

Erythroid potentiating activity of tissue inhibitor of metalloproteinases on the differentiation of erythropoietin-responsive mouse erythroleukemia cell line, ELM-I-1-3, is closely related to its cell growth potentiating activity

The erythroid-potentiating activity (EPA) of the tissue inhibitor of metalloproteinase-1 (TIMP-1) was re-examined using ELM-I-1-3, a mouse erythroleukemia cell line, which responded well to erythropoietin. Depletion of pre-existing TIMP-1 from fetal calf serum in culture medium using monoclonal antibody suppressed erythropoietin-induced differentiation as measured by the induction of hemoglobin, commitment assay and globin mRNAs. The removal of TIMP-1 also suppressed the proliferation of ELM-I-1-3 as measured by cell number and de novo DNA synthesis. These changes were reversed by the addition of purified TIMP-1 to the culture medium. Anti-TIMP-1 antibody also blocked both hexamethylene bisacetamide (HMBA)-induced erythroid differentiation and the proliferation of both ELM-I-1-3 and Friend erythroleukemia cells. Considering previous reports analyzing the chemical induction of Friend mouse erythroleukemia cell differentiation, our results suggest that erythropoietin- or HMBA-induced erythroid differentiation might also be coupled with cell proliferation. Our 3H thymidine-uptake experiment shows that TIMP-1 removal was also effective in the inhibition of cell growth of various other cell lines in addition to erythroleukemia cell lines. These results suggest that EPA action of TIMP-1 on erythroid leukemia cell lines is closely related to its activity to promote the cell growth of various cell lines and cells including erythroleukemia cell lines.