The thrombopoietin/MPL axis is activated in the Gata1low mouse model of myelofibrosis and is associated with a defective RPS14 signature.

Myelofibrosis (MF) is characterized by hyperactivation of thrombopoietin (TPO) signaling, which induces a RPS14 deficiency that de-regulates GATA1 in megakaryocytes by hampering its mRNA translation. As mice carrying the hypomorphic Gata1low mutation, which reduces the levels of Gata1 mRNA in megakaryocytes, develop MF, we investigated whether the TPO axis is hyperactive in this model. Gata1low mice contained two times more Tpo mRNA in liver and TPO in plasma than wild-type littermates. Furthermore, Gata1low LSKs expressed levels of Mpl mRNA (five times greater than normal) and protein (two times lower than normal) similar to those expressed by LSKs from TPO-treated wild-type mice. Gata1low marrow and spleen contained more JAK2/STAT5 than wild-type tissues, an indication that these organs were reach of TPO-responsive cells. Moreover, treatment of Gata1low mice with the JAK inhibitor ruxolitinib reduced their splenomegaly. Also in Gata1low mice activation of the TPO/MPL axis was associated with a RSP14 deficiency and a discordant microarray ribosome signature (reduced RPS24, RPS26 and SBDS expression). Finally, electron microscopy revealed that Gata1low megakaryocytes contained poorly developed endoplasmic reticulum with rare polysomes. In summary, Gata1low mice are a bona fide model of MF, which recapitulates the hyperactivation of the TPO/MPL/JAK2 axis observed in megakaryocytes from myelofibrotic patients.

TRAIL promotes a pro-survival signal in erythropoietin-deprived human erythroblasts through the activation of an NF-kB/IkBalpha pathway.

The biological activity of TNF-related apoptosis inducing ligand (TRAIL) was analyzed in primary human erythroblasts derived from mononuclear cells of blood donors, kept in culture in the presence of 20 percent foetal calf serum, growth factors (EPO, SCF, IL-3) and glucocorticoids (10-6 M dexamethasone, 10-6 M oestradiol) or under growth factor and serum starvation. In the presence of growth factors and serum, primary erythroblasts showed a differential expression of TRAIL-Receptors (Rs) at various degrees of maturation and responded to TRAIL treatment with a mild cytotoxicity. On the other hand, in the absence of serum and growth factors, TRAIL treatment unexpectedly up-regulated TRAIL-R4 decoy receptor and promoted erythroblast survival. The concomitant activation of NF-kB/IkB survival pathway was detected with Western blotting and immunofluorescence procedures and confirmed by experiments performed with SN50, a pharmacological inhibitor of the NF-kB/IkB pathway. Our study indicates that TRAIL has a twofold activity on erythroid lineages: it induces a mild erythroid cell cytotoxicity in the presence of serum and growth factors, while it promotes erythroid cell survival through the activation of the NF-kB/IkB pathway under starvation conditions.

Differential localization of P-selectin and von Willebrand factor during megakaryocyte maturation.

An important step in megakaryocyte maturation is the appropriate assembly of at least two distinct subsets of alpha-granules. The mechanism that sorts the alpha-granule components into distinct structures and mediates their release in response to specific stimuli is now emerging. P-selectin and von Willebrand factor are two proteins present in the alpha-granules that recognize P-selectin glycoprotein ligand on neutrophils and collagen in the subendothelial matrix. These proteins may play an important role in determining the differential release of the alpha-granule contents in response to external stimuli. If P-selectin and von Willebrand factor are localized in the same or different alpha-granules is not known. To clarify this question, we analyzed by immunoelectron microscopy the localization of von Willebrand factor and P-selectin during the maturation of wild-type and Gata1(low) megakaryocytes induced in vivo by treating animals with thrombopoietin. Gata1(low) is a hypomorphic mutation that blocks megakaryocyte maturation, reduces the levels of von Willebrand factor expression and displaces P-selectin on the demarcation membrane system. The maturation block induced by this mutation is partially rescued by treatment in vivo with thrombopoietin. In immature megakaryocytes, both wild-type and Gata1(low), the two receptors were co-localized in the same cytoplasmic structures. By contrast, the two proteins were segregated to separate alpha-granule subsets as the megakaryocytes matured. These observations support the hypothesis that P-selectin and von Willebrand factor may ensure differential release of the alpha-granule content in response to external stimuli.

Regulation of CREB activation by p38 mitogen activated protein kinase during human primary erythroblast differentiation.

Among the molecular events underlying erythroid differentiation, we analyzed the signalling pathway leading to cAMP response element binding (CREB) nuclear transcription factor activation. Normal donor blood light density cells differentiated to pro-erythroblasts during the proliferative phase (10 days) of the human erythroblast massive amplification (HEMA) culture, and to orthochromatic erythroblasts, during the differentiation phase (4 additional days) of the culture. Since erythropoietin was present all over the culture, also pro-erythroblasts left in proliferative medium for 14 days continued their maturation without reaching the final steps of differentiation. p38 mitogen activated protein kinase (p38 MAPK) and CREB maximal activation occurred upon 4 days of differentiation induction, whereas a lower activation was detectable in the cells maintained in parallel in proliferative medium (14 days). Interestingly, when SB203580, a specific p38 MAPK inhibitor, was added to the culture the percentage of differentiated cells decreased along with p38 MAPK and CREB phosphorylation. All in all, our results evidence a role for p38 MAPK in activating CREB metabolic pathway in the events leading to erythroid differentiation.

Peroxiredoxin genes are not induced in myeloid leukemia cells exposed to ionizing radiation.

Peroxiredoxins (Prx) comprise an extended family of small antioxidant proteins which conserve a thioredoxin-dependent catalytic function that can contribute to cell protection from reactive oxygen species (ROS). ROS generation is one of the deleterious intracellular effects of ionizing radiation, but the role of Prx during radiation treatment has not been extensively explored. Present experiments measure effects of ionizing radiation on expression of human Prx types I (PAGA), II (NKEF-B) and IV (AOE372) in human myeloid leukemia cells (K562). Prx gene transcription was analyzed by amplifying with RT-PCR cDNAs complementary to each Prx-specific coding sequence and by identifying the derived products with Southern blotting procedure. Transcripts of GAPDH were used as the endogenous standard for semi-quantitative comparisons. No consistent increase in Prx gene expression was detected at time intervals up to 72 h after gamma radiation doses that caused cell cycle arrest and nuclear damage (maximum 20 Gy). Immunoblots also were consistent with a prolonged expression or stability of the Prx I/II proteins. Similarly, a cytotoxic concentration of the oxidant hemin, which stimulates rapid hemoglobinization of K562 cells, caused no induction of Prx gene expression. Our results indicate a high Prx stability in human radio-resistant leukemia cells.

Inefficient phospholipase C activation and reduced Lck expression characterize the signaling defect of umbilical cord T lymphocytes.

Adult and neonatal immunocompetent cells exhibit important functional distinctions, including differences in cytokine production and susceptibility to tolerance induction. We have investigated the molecular features that characterize the immune response of cord blood-derived T lymphocytes compared with that of adult T lymphocytes. Our findings demonstrate that phospholipase C (PLC) isozymes, which play a pivotal role in the control of protein kinase C activation and Ca2+ mobilization, are differently expressed in cord and adult T lymphocytes. PLCbeta1 and delta1 are expressed at higher levels in cord T cells, while PLCbeta2 and gamma1 expression is higher in adult T lymphocytes. PLCdelta2 and gamma2 appear to be equally expressed in both cell types. In addition, a functional defect in PLC activation via CD3 ligation or pervanadate treatment, stimuli that activate tyrosine kinases, was observed in cord blood T cells, whereas treatment with aluminum tetrafluoride (AlF4-), a G protein activator, demonstrated a similar degree of PLC activation in cord and adult T cells. The impaired PLC activation of cord blood-derived T cells was associated with a a very low expression of the Src kinase, Lck, along with a reduced level of ZAP70. No mitogenic response to CD3 ligation was observed in cord T cells. However, no signaling defect was apparent downstream of PLC activation, as demonstrated by the mitogenic response of cord T cells to the pharmacologic activation of protein kinase C and Ca2+ by treatment with PMA and ionomycin. Thus, neonatal cord blood-derived T cells show a signaling immaturity associated with inadequate PLCgamma activation and decreased Lck expression.

Immunocytochemical localization of phospholipase C isozymes in cord blood and adult T-lymphocytes.

The response of T-cells to peptide antigen plus major histocompatibility complex (MHC) consists of a series of cellular events collectively called T-cell activation. An essential component of this pathway is phospholipase C (PLC)gamma1, whose hydrolytic activity increases rapidly after binding of ligands to the T-cell receptor (TCR) and consequent activation of tyrosine kinases. Recent studies also suggest a GTP binding protein-dependent activation of PLCbeta during the early steps of T-cell activation. On the basis of these findings, we first checked the expression of PLC isoforms by Western blotting and by confocal and electron microscopy techniques, and then we looked for the phosphoinositide breakdown induced by CD3 engagement in cord and adult T-lymphocytes. Our results indicated that PLCbeta1 was almost exclusively expressed in cord T-cells, whereas PLCbeta2 was more strongly represented in the adult. The amount of PLCgamma1 was found to be larger in the adult than in cord cells. No significant differences were found in PLCgamma2 and delta2 expression. PLCdelta1 was scarcely detectable. On CD3 stimulation, adult lymphocytes gave rise, as expected, to a dramatic increase in phosphoinositide breakdown, whereas in cord cells this response was scarcely detected. These results indicate that a shift in PLC expression occurs in the postnatal period and that this change is associated with induction of the capability to respond to CD3 engagement with phosphoinositide hydrolysis.

Immunocytochemical detection of phosphatidylinositol 3 kinase in Burkitt lymphoma cells.

PI 3-kinase, an enzyme responsible for the phosphorylation of the D3 position of the inositol ring of phosphatidylinositol (PI), is recognized to be involved in the regulation of many cellular processes such as mitogenic signalling, inhibition of apoptosis, intracellular vesicle trafficking/secretion, regulation of actin and integrin functions and regulation of protein kinases induced by tumour necrosis factor, oncoproteins and ultraviolet light. Here we report the subcellular distribution and the phosphorylative pattern of p85 alpha subunit of PI 3-kinase in Burkitt lymphoma cells exposed to R interferon alpha treatment. Immunocytochemical analysis of this enzyme, performed by confocal microscopy, revealed an increased expression of this protein at cytoplasmic level after 90 min of interferon alpha treatment. Western blotting analyses performed on nuclear and cytoplasmic fractions confirmed the overexpression found by confocal microscopy at cytoplasmic level in the 90 min interferon alpha treated cells still persisting in the 24 hr treated samples. Such an overexpression was paralleled by an increase of tyrosine phosphorylation both at cytoplasmic and nuclear level suggesting that an enhanced requirement for cytoplasmic expression and phosphorylation of PI 3-kinase might be necessary to the cell for regulating some cytoplasmic-nuclear cross talk involved in the control of Burkitt lymphoma cell metabolism following interferon alpha treatment.

Novel evidence of expression and activity of ecto-phospholipase C gamma1 in human T lymphocytes.

Although much is known about the intracellular phospholipase C (PLC) specific for inositol phospholipids, few data are available about the presence of a less common PLC at the external side of the membrane bilayer of some cell types. This ectoenzyme seems to play particular roles in cellular function by hydrolyzing inositol lipids located on the outer leaflet of the plasma membrane. Here, we provide the first evidence that peripheral T lymphocytes express a discrete level of a PLCgamma1 at the outer leaflet of the plasma membrane. Flow cytometry showed that the PLCgamma1-positive (PLCgamma1(+)) cells (approximately 37%) were CD8(+) and CD45RA+. Biochemical evidence indicated that (1) this ectoenzyme displays a mass similar to the cytoplasmic form, (2) it is phosphorylated on tyrosine residues, and (3) its activity is Ca2+-dependent. In addition, this enzyme appeared to be correlated with the proliferative state of the cell, since stimulation with phytohemagglutinin (PHA) downregulated both its expression and activity, which were restored by treatment with an antiproliferative agent like natural interferon beta. Moreover, the different kinetics of formation of its hydrolytic products, inositol 1 phosphate and inositol 1:2 cyclic phosphate (Ins(1)P and Ins(1:2 cycl)P), formed upon incubation of the lymphocytes with [3H]-lyso-phosphatidylinositol (PI), allow the hypothesis of a selective involvement of the two inositol phosphates in the mechanisms regulating the metabolism of particular T-lymphocyte subsets.

Phospholipase C gamma 1 overexpression and activation induced by interferon beta in human T lymphocytes: an ISGF3-independent response.

Interferons exert their antiviral, antiproliferative and immunoregulatory activities by stimulating the expression of several genes. Such genes disclose a common element within their promoters, defined Interferon Stimulated Response Element (ISRE), which binds a nuclear factor(s) translocated from the cytoplasm to the nucleus (ISGF3) after the binding of interferon (IFN) to the specific receptor. Here we report the induction of the synthesis and of the hydrolytic activity of phospholipase C gamma 1 (PLC gamma 1) in human T lymphocytes by IFN-beta. The increased level of PLC gamma 1 becomes evident after 90 min of IFN-beta treatment and is still detectable after 24 h. Neither the PLC gamma 1 overexpression induced by IFN nor the increased hydrolytic activity of the enzyme appear to be affected by pretreatment of the cells with the protein tyrosine kinase inhibitor genistein, which is known to prevent the association of ISGF3 components. These results suggest that in human T lymphocytes IFN-beta can activate other transcription factor(s) distinct from ISGF3 to regulate PLC gamma 1 expression. In addition, the ability of this enzyme to hydrolyse PIP2, also in the presence of genistein, implies the possibility that this enzyme can exert its hydrolytic activity independently of protein tyrosine kinase activation.

Synergistic regulatory effects of TNF alpha, IL-1 alpha and IFN alpha on the growth and differentiation of Daudi lymphoma cells.

The regulatory effects of the combined treatment of tumour necrosis factor alpha (TNF alpha), interleukin-1 alpha (IL-1 alpha) and interferon alpha (IFN alpha) on the growth and differentiation of Daudi lymphoma cells were investigated. By means of anti-BrdU monoclonal antibodies and [3H-thymidine] incorporation a reduced proliferation rate was shown both through a combination of TNF alpha with either IL-1 alpha or IFN alpha and, above all, through simultaneous treatment with the three cytokines. In parallel, the degree of differentiation was evaluated via morphological criteria and detection of Fc receptors (FcR) and appeared higher after treatment with the three cytokines. Our results provide evidence of the increased sensitivity of this cell line to this combined treatment supporting the existence of a synergistic interaction in inducing the antiproliferative and differentiative effects.

Nuclear translocation of beta II PKC isoenzyme in phorbol ester-stimulated KM-3 pre-B human leukemic cells.

Members of the protein kinase C (PKC) family play a key role in regulating cell growth and differentiation in response to several stimuli, including hormones, neurotransmitters, and growth factors. The different properties and substrate specificity of the PKC isoforms are not fully understood, and they are assumed to have specific functions in intracellular signaling. In lymphoid cells, the effects of PMA and Ca2+ ionophore, singly or in combination, on activation and expression of Ca(2+)-dependent PKC at the level of protein and messenger RNA have been examined. Starting from these observations and the possibility that differential isoenzyme expression might contribute to the differences in phorbol ester sensitivity of lymphoid cells, it seemed worthwhile to investigate the expression and the modulation of PKC isoforms in KM-3 cells, a human pre-B cell line, upon treatment with phorbol 12-myristate 13-acetate (PMA). Using multiparametric analysis we detected three PKC isoforms in the KM-3 cell line: alpha, beta II, and zeta. PMA treatment causes an intranuclear translocation of the beta II isoform, via the nuclear pore complex, associated with the interchromatinic regions. These data suggest that the beta II isoenzyme may play a strategic role in signal transduction and regulation of specific gene expression in B lymphocytes.

Phosphoinositide signalling enzymes in human T lymphocytes: modulation of phosphoinositidase C isoform gamma 1 upon interferon treatment.

In human T lymphocytes immunoblotting analysis of phosphoinositidase C (PIC) isoforms has shown that gamma 1 is the most represented isoform both at the cytoplasmic and nuclear level, and that interferon beta produces, within 90 min of treatment, an increase of its expression. These results, also supported by immunoelectronmicroscopic investigation, strongly suggest the involvement of PIC gamma 1 in the cascade of molecular events generated by the interaction between interferon and its cell surface receptors.

Evidence for an early and transient involvement of nuclear inositol lipids in subcellular signalling events related to DNA repair processes.

The involvement of nuclear inositol lipids in the processes related to DNA repair upon ionizing radiation has been investigated in Murine Erythroleukaemia cells. Early changes in the in vitro phosphatidylinositol-bisphosphate phosphorylation in isolated nuclei were found to precede transiently the marked increase in DNA synthesis occurring after irradiation. Such an increase detected by anti-BrdU monoclonal antibodies has been found to be related mainly to DNA polymerase beta activity as revealed by the kinetic analysis of in vitro DNA synthesis. The results here presented allow us to speculate on a possible involvement of nuclear inositol lipids in the cascade of the early events leading to the regulation of DNA repair in the nucleus.

Age-related events in human active T lymphocytes: changes in the phosphoinositidase C activity.

Since PHA-stimulated active T lymphocytes from aged humans showed changes in the metabolic pattern of inositol lipids in comparison with young subjects, we studied the possible role of phosphoinositidase C (PIC) in the generation of this phenomenon. The breakdown of exogenous [3H]phosphatidylinositol 4,5-bisphosphate was found to be optimal at neutral pH and Ca++ concentrations close to millimolar levels. Under these conditions PIC activity of resting lymphocytes did not differ in aged and young subjects, while, after short periods of PHA stimulation (up to 4 hr) the substrate hydrolysis was lower and delayed in the elderly group in comparison with that of controls. Our findings support the hypothesis that the age-related default of this enzyme, responsible for the age-related changes in the inositol lipid pathway of this peculiar subpopulation, could be involved, as a primary event, in the mechanisms leading to the reduced proliferative response of aged active T lymphocytes.

Inositol lipid-mediated intranuclear signalling: a comparative analysis of in vivo labelling in interferon alpha-sensitive and -resistant Daudi lymphoma cells.

Changes in inositol lipid and diacylglycerol metabolism have been analysed in Daudi lymphoma cells treated up to 24 h with human DNA recombinant interferon alpha. Results showing a different response of nuclear phosphoinositides and diacylglycerol, compared to whole cells, suggest that the intranuclear signalling system activated by interferon in Daudi cells involves nuclear inositol lipid metabolism. A well-characterized clone of Daudi cells selected for resistance to the antiproliferative action of interferon provided controls for the specificity of results.

Further considerations on the thermal stabilization of the nuclear matrix in mouse erythroleukemia cells.

The morphology and the polypeptide composition of the nuclear matrix obtained from 37 degrees C incubated nuclei has been studied in mouse erythroleukemia cells. From a structural point of view, in the absence of heat treatment, the matrix lacked identifiable nucleolar remnants and the internal fibrogranular meshwork whereas a peripheral lamina was seen. On the contrary, the matrix obtained from heat exposed nuclei displayed very electrondense nucleolar remnants and an abundant inner network. These results were obtained irrespective of the type of extracting agent (2M NaCl or 0.2 M (NH4)2SO4) used to remove histones and other soluble proteins. The heat stabilization of the matrix could not be prevented by sulfhydryl blocking chemicals such as iodoacetamide and n-ethylmaleimide, thus suggesting that heat does not stabilize the matrix by inducing the formation of disulfide bonds. Only limited differences in the polypeptide pattern of matrix isolated under different conditions were seen using one-dimensional pore gradient polyacrylamide gels stained with both Coomassie Brilliant Blue and silver despite the fact that the matrix fraction from heat treated nuclei retained about three fold more protein in comparison with controls. The same results were obtained also by means of two-dimensional non-equilibrium gel electrophoresis.

Changes in inositol lipid metabolism and protein kinase C translocation in nuclei of mitogen stimulated Swiss 3T3 cells.

The correlation between changes in nuclear polyphosphoinositide levels preceding PKC translocation to the nucleus and the onset of DNA synthesis has been discussed. Using two different clones of Swiss 3T3 fibroblasts belonging to the same original cell line, one of which is unresponsive to mitogenic stimulation with IGF-I on its own or in combination with bombesin, it has been observed that a rapid and transient breakdown of nuclear PIP and PIP2 occurs only in responsive cells and this precedes the translocation of PKC to the nucleus, as evidenced by immunochemical analysis as well as by enzymatic activity. Therefore, it seems that a direct link exists between nuclear polyphosphoinositide metabolism, PKC translocation to the nucleus and cell division. Since IGF-I acts at the plasma membrane through a tyrosine kinase receptor it seems that the mitogenic stimulation induced by this factor utilizes different signalling pathways at the plasma membrane and at the nucleus. Because of the evidence that type I IGF receptor is expressed in both responsive and unresponsive cells and that the receptor machinery at the plasma membrane is active the lack of the transient changes in nuclear inositol lipids and of PKC translocation in unresponsive cells further suggests that the cell nucleus is capable of an autonomous signalling system based on polyphosphoinositide metabolism.