1,25-Dihydroxyvitamin D3 and fetal lung maturation: immunogold detection of VDR expression in pneumocytes type II cells and effect on fructose 1,6 bisphosphatase.

Lung maturation before birth includes type II pneumocyte differentiation with progressive disappearance of glycogen content and onset of surfactant synthesis. We have shown previously that 1,25-(OH)2D3 increases surfactant synthesis and secretion by type II cells and decreases their glycogen content in fetal rat lung explants. Recently, the gene coding fructose 1,6 bisphosphatase (F1,6BP), a regulatory enzyme of gluconeogenesis, has been identified in type II cells and its promoter bears a Vitamin D response element. Present results show:The coexistence of type II cells at different stages of maturation. in rat fetal lung on day 21 of gestation (electron microscopy), and the association between maturation of type II cells and disappearance of their glycogen content. The immunogold labeling of all type II cells when using the 9A7g VDR-antibody, with significantly more abundant gold particles in cells exhibiting an intermediate glycogen content. The expression of F1,6BP mRNA in a human type II cell line (NCI-H441) and the increase of this expression after 18h incubation with 1,25-(OH)2D3 (10(-8)M). These results bring further evidence for a physiological role of 1,25-(OH)2D3 during type II pneumocyte maturation. Activation of F1,6BP may participate to the 1,25-(OH)2D3 action on surfactant synthesis via the gluconeogenesis pathway.

Signaling revisited in acute promyelocytic leukemia.

Although transcription factors are still the main focus to understanding leukemogenesis, recent results strongly suggest that alteration of a receptor and/or subsequent signaling plays a critical and co-operative role in the pathogenesis of acute myeloid leukemia (AML). The t(15;17) translocation, found in 95% of APL, encodes a PML-RARalpha fusion protein. A main model proposed for acute promyelocytic leukemia (APL) is that PML-RARalpha exerts its oncogenic effects by repressing retinoic acid-inducible genes critical to myeloid differentiation. Dysregulation of these genes may result in abnormal signaling, thereby freeing pre-leukemic cells from controls which normally induce the onset of differentiation. It is also likely that treatment of APL cells by retinoic acid induces de novo up-regulation of the same genes which are dominantly repressed by PML-RARalpha and whose expression is required for reactivation of the differentiation program. Identification of such genes together with the signaling pathways interrupted at the early stages of leukemia transformation and reactivated during retinoic acid-induced differentiation in APL cells will contribute to the development of new molecular targets for treatment of leukemia.

Myeloblastin is an Myb target gene: mechanisms of regulation in myeloid leukemia cells growth-arrested by retinoic acid.

A pivotal role has been assigned to Myb in the control of myeloid cell growth. Although Myb is a target of retinoic acid, little is known about the mechanisms by which it may contribute to induced growth arrest in leukemia cells. Indeed, few Myb target genes are known to be linked to proliferation. Myeloblastin is involved in the control of proliferation in myeloid leukemia cells. It is expressed early during hematopoiesis and is a granulocyte colony-stimulating factor-responsive gene. Myeloblastin can confer factor-independent growth to hematopoietic cells, an early step in leukemia transformation. The myeloblastin promoter contains PU.1, C/EBP, and Myb binding sites, each of which are critical for constitutive expression in myeloid cells. Inhibition of myeloblastin expression in leukemia cells growth-arrested by retinoic acid is demonstrated to depend on Myb down-regulation. Myb is shown to induce myeloblastin expression and abolish its down-regulation by retinoic acid. Altogether, the data offer a clue as to how a myeloid-specific transcriptional machinery can be accessible to regulation by retinoic acid and point to myeloblastin as a novel target of Myb. This link between Myb and myeloblastin suggests a previously nonidentified Myb pathway through which growth arrest is induced by retinoic acid in myeloid leukemia cells.

PRAM-1 is a novel adaptor protein regulated by retinoic acid (RA) and promyelocytic leukemia (PML)-RA receptor alpha in acute promyelocytic leukemia cells.

The t(15;17) translocation, found in 95% of acute promyelocytic leukemia, encodes a promyelocytic leukemia (PML)-retinoic acid receptor alpha (RARalpha) fusion protein. Complete remission of acute promyelocytic leukemia can be obtained by treating patients with all-trans retinoic acid, and PML-RARalpha plays a major role in mediating retinoic acid effects in leukemia cells. A main model proposed for acute promyelocytic leukemia is that PML-RARalpha exerts its oncogenic effects by repressing the expression of retinoic acid-inducible genes critical to myeloid differentiation. By applying subtraction cloning to acute promyelocytic leukemia cells, we identified a retinoic acid-induced gene, PRAM-1 (PML-RARalpha target gene encoding an Adaptor Molecule-1), which encodes a novel adaptor protein sharing structural homologies with the SLAP-130/fyb adaptor. PRAM-1 is expressed and regulated during normal human myelopoiesis. In U937 myeloid precursor cells, PRAM-1 expression is inhibited by expression of PML-RARalpha in the absence of ligand and de novo superinduced by retinoic acid. PRAM-1 associates with other adaptors, SLP-76 and SKAP-55HOM, in myeloid cell lines and with protein tyrosine kinase lyn. By providing the first evidence that PML-RARalpha dysregulates expression of an adaptor protein, our data open new insights into signaling events that are disrupted during transformation by PML-RARalpha and induced by retinoic acid during de novo differentiation of acute promyelocytic leukemia cells.

Myeloblastin is a granulocyte colony-stimulating factor-responsive gene conferring factor-independent growth to hematopoietic cells.

Hematopoiesis depends on a pool of quiescent hematopoietic stem/progenitor cells. When exposed to specific cytokines, a portion of these cells enters the cell cycle to generate an amplified progeny. Myeloblastin (MBN) initially was described as involved in proliferation of human leukemia cells. The granulocyte colony-stimulating factor (G-CSF), which stimulates the proliferation of granulocytic precursors, up-regulates MBN expression. Here we show that constitutive overexpression of MBN confers factor-independent growth to murine bone marrow-derived Ba/F3/G-CSFR cells. Our results point to MBN as a G-CSF responsive gene critical to factor-independent growth and indicate that expression of the G-CSF receptor is a prerequisite to this process. A 91-bp MBN promoter region containing PU.1, C/EBP, and c-Myb binding sites is responsive to G-CSF treatment. Although PU.1, C/EBP, and c-Myb transcription factors all were critical for expression of MBN, its up-regulation by G-CSF was associated mainly with PU.1. These findings suggest that MBN is an important target of PU.1 and a key protease for factor-independent growth of hematopoietic cells.

A protein kinase C-independent pathway leading to c-Jun-dependent expression of 100-kDa Ras GTPase-activating protein in JEG-3 human choriocarcinoma cells.

Although the 100-kDa Ras GTPase-activating protein (p100 RasGAP) has been reported to exist specifically in human placental trophoblasts, the molecular mechanisms responsible for regulating its expression remain unclear. In this study we used okadaic acid, an inhibitor of serine/threonine phosphatase 1 and 2 A, as a probe to explore the signaling pathway regulating the expression of p100 RasGAP in JEG-3 human placental choriocarcinoma cells. Treatment of JEG-3 cells with okadaic acid provoked dose- and time-dependent stimulation of p100 RasGAP expression without marked modification of expression of p120 RasGAP, another isoform of RasGAP. Co-treatment of cells with okadaic acid and the protein kinase C activator, phorbol 12-myristate 13-acetate, exerted an additive effect on p100 RasGAP induction. Moreover, the response of the p100 RasGAP de novo synthesis to okadaic acid was not affected by the selective inhibitor of protein kinase C, GF 109203X. Thus this study identified a novel signaling pathway regulating p100 RasGAP expression, which is independent of protein kinase C. In addition, okadaic acid treatment resulted in the activation of ERK2 (p42 MAP kinase) and the induction of both c-Jun and c-Fos proteins without activating JNK (c-Jun NH2-terminal kinase). Significantly, blockade of c-Jun expression with antisense c-jun oligonucleotides suppressed p100 RasGAP expression. Taken together, it is concluded that okadaic acid induces the expression of p100 RasGAP protein in JEG-3 cells preceded by activation of ERK and AP-1 cascade, and that this okadaic acid-induced p100 RasGAP expression is independent of protein kinase C-mediated pathway but requires c-Jun/AP-1 function.

Evidence for a novel RasGAP-associated protein of 105 kDa in both mature trophoblasts and differentiating choriocarcinoma cells.

A novel tyrosine-phosphorylated, RasGAP-associated protein of 105 kDa (p105) is found in normal human term placental trophoblasts, as well as in JEG-3 human choriocarcinoma cells induced to differentiate by okadaic acid (OA). This p105 RasGAP-associated protein is distinct from other RasGAP-associated proteins described so far, none of which has either a molecular size close to p105 or a trophoblastic cell origin. The p105 appears, accompanied by p120 and p100 RasGAP expression, after OA treatment of JEG-3 cells but is almost undetectable in the absence of stimulation. Moreover, the p105 is the first discovered RasGAP-associated protein bound to p100 RasGAP. The natural occurrence of the p105 in normal mature trophoblasts isolated from human term placenta suggests that it may be linked to the differentiation state of human trophoblasts. Hence, this p105 RasGAP-associated protein might be considered a marker of human trophoblast differentiation.

Proteinase 3 mRNA expression is induced in monocytes but not in neutrophils of patients with cystic fibrosis.

Proteinase 3 (PR3), a serine proteinase which can degrade lung tissue, is present in the cystic fibrosis (CF) sputum. In the present study, PR3 protein and mRNA expression was determined in circulating neutrophils and monocytes. CF neutrophils contained similar PR3 concentrations as healthy controls and poorly expressed PR3 mRNA. In contrast, CF monocytes showed significantly higher PR3 concentrations than controls, together with an upregulation of PR3 mRNA expression especially during pulmonary exacerbation. Interestingly, antibiotic treatment fully abrogated PR3 mRNA expression and decreased PR3 protein in monocytes. Our findings highlight a potential role of monocyte-derived PR3 in CF-associated airway inflammation.

Evidence for a role of NF-kappaB in the survival of hematopoietic cells mediated by interleukin 3 and the oncogenic TEL/platelet-derived growth factor receptor beta fusion protein.

Interleukin 3 (IL-3) and other hematopoietic cytokines transduce signals that stimulate DNA synthesis and cell survival. In certain chronic myelomonocytic leukemias, a TEL/platelet-derived growth factor receptor beta (PDGFRbeta) fusion protein is produced as a consequence of the t(5;12) translocation. It contains the amino terminus of the transcription factor TEL fused to the transmembranous and cytoplasmic domains of the PDGFRbeta. It is oncogenic as it substitutes for IL-3, thus promoting cell growth and preventing apoptotic cell death. The mechanism by which TEL/PDGFRbeta generates survival signals remains undefined. Here, we report that both IL-3 and TEL/PDGFRbeta initiate a signaling cascade that leads to the activation of the transcriptional factor NF-kappaB. In fact, either cytokine deprivation of IL-3-dependent Ba/F3 cells or exposure of TEL/PDGFRbeta-expressing cells to the specific inhibitor of the PDGFR tyrosine kinase, CGP53716, caused a strong decrease in NF-kappaB activity followed by extensive cell death. Further, treatment with the proteasome inhibitor Z-IE(O-t-Bu)A-leucinal suppressed IL-3 and TEL/PDGFRbeta-dependent survival. The same result was seen upon overexpression of an unphosphorylable form of IkappaBalpha. Because both conditions inactivate NF-kappaB by preventing its translocation into the nucleus, that process seems to be essential for cell survival in response to IL-3 and TEL/PDGFRbeta. Moreover, overexpression of a dominant-negative mutant of the protooncogene c-Myc, a downstream target of NF-kappaB, had a similar effect. We conclude that NF-kappaB plays an important role in maintaining cell survival in response to IL-3 and TEL/PDGFRbeta and that c-Myc may be a downstream effector mediating this effect.

Myc is essential for transformation by TEL/platelet-derived growth factor receptor beta (PDGFRbeta).

The t(5;12) translocation identified in patients with chronic myelomonocytic leukemia (CMML) encodes a TEL/platelet-derived growth factor receptor beta (PDGFRbeta) fusion protein. A key hypothesis for how the TEL/PDGFRbeta fusion protein would function as an oncogene is that it represents a constitutively active version of the normal PDGFRbeta. A link between the function of the t(5;12)-encoded TEL/PDGFRbeta fusion protein and Myc expression is suggested by the fact that Myc is induced by PDGF and is essential for entry of cells into the S phase of the cell cycle. We here show that the kinase activity of TEL/PDGFRbeta is necessary for Ba/F3 cells to acquire interleukin-3 (IL-3) independence and that, in contrast to their untransfected counterpart, Ba/F3 cells stably transfected with TEL/PDGFRbeta maintain a high level of Myc expression after removal of IL-3. Using dominant negative mutants of Myc, we show that a threshold of active Myc is essential for TEL/PDGFRbeta to transform Ba/F3 and Rat-1 cells. The findings that the kinase activity of TEL/PDGFRbeta and a threshold of active Myc are involved in TEL/PDGFRbeta transformation may allow for the development of therapeutic strategies in patients with t(5;12)+ CMML using specific inhibitors of the PDGFRbeta kinase as well as compounds designed to interfere specifically with Myc.

The majority of myeloid-antigen-positive (My+) childhood B-cell precursor acute lymphoblastic leukaemias express TEL-AML1 fusion transcripts.

The t(12:21) translocation fuses the TEL and AML1 genes and has been found in up to 28% of paediatric B-cell precursor acute lymphoblastic leukaemias (BCP-ALL). The AML1 gene is a transcription factor which regulates expression of several myeloid differentiation associated genes. A molecular analysis of TEL-AML1, E2A-PBX1, MLL-AF4, BCR-ABL expression and an immunophenotypic study of CD13/CD33 myeloid antigen expression have been performed prospectively on tumour cells from 96 paediatric BCP-ALL patients. Percentages of CD13 or CD33 expressing leukaemic cells were found to be higher in TEL-AML1 positive cases (n = 22) than in TEL-AML1 negative (n = 74) cases (P<0.001). In 22/96 cases (23%) >10% of neoplastic cells were found to express at least one of the two markers. In 14 of these cases (63%), TEL-AML1 expression was detected, whereas t(4;11), t(11;19) and t(9;22) translocations were found by molecular methods in only three cases (14%). In four cases (18%) no molecular marker was found. These data show that TEL-AML1 expression is significantly associated with myeloid antigen expression by leukaemic cells and suggests that the prognostic significance of myeloid antigen expression in paediatric ALLs should be re-evaluated in the light of molecular cytogenetic markers.

HIV-1 p17 and IFN-gamma both induce fructose 1,6-bisphosphatase.

The p17 matrix protein of the human immunodeficiency virus type 1 (HIV-1) plays a crucial role in AIDS pathogenesis. It orchestrates viral assembly and directs the preintegration complex to the nucleus of infected cells. Recently, the three-dimensional structure of p17 was shown to resemble that of interferon-gamma (IFN-gamma), suggesting that both proteins might share analogous functions. We demonstrate that in monocytes, p17 shares with IFN-gamma the ability to induce 1alpha-hydroxylase activity and to activate fructose 1,6-bisphosphatase gene expression in the presence of 25-hydroxyvitamin D3. However, p17 does not bind to the IFN-gamma cell membrane receptor and fails to increase expression of IFN-gamma-induced proteins, such as tryptophanyl-tRNA synthetase, Fc gammaRI, and HLA DR or B7/BB1 antigens. Altogether, our results raise the possibility that the structural resemblance between p17 and IFN-gamma causes the selective activation of a common pathway resulting in the production of 1,25-dihydroxyvitamin D3. We also found that unlike IFN-gamma, p17 increases the intracellular ATP content. Since transport of the HIV-1 preintegration complex through the nuclear membrane is an ATP-dependent process, our observation suggests that p17 plays a double role in this active transport, not only by acting as a chaperone molecule but also by recruiting the necessary energy for this process.

[Effect of translocation t(15;17) on the gene expression regulation of myeloblastin during all trans retinoic acid induced myeloid differentiation in human leukemic cells]. / Effets de la translocation t(15;17) sur la régulation de l'expression du gène myéloblastine--mbn--au cours de la différenciation myéloïde induite par l'acide rétinoïque tout-trans des cellules leucémiques humaines.

Myeloblastin (mbn) is a serine protease involved in the control of growth and differentiation of human leukemic cells. In the promyelocytic-like human leukemia cell line HL-60 this protease is inhibited during retinoic acid (RA) induced differentiation. The t(15;17) translocation, specifically associated with the human acute promyelocytic leukemia (APL), fuses the retinoic acid receptor alpha (RAR alpha) to a novel gene PML generating the hybrid protein PML-RAR. We have shown that while mbn was early down-regulated in HL60 cells treated with all trans RA, the inhibition of this gene was considerably delayed in NB4 cells, which carry the t(15;17) translocation, upon treatment with the same inducer. This observation suggested that the changes in the myeloblastin regulation by RA found in NB4 cells could be ascribed to the presence of the fusion protein PML-RAR. To verify this hypothesis we have cloned the putative promoter region of mbn gene. Transactivation properties of endogenous retinoic acid receptors on this region have been tested in transfection experiments of HL60 and NB4 cell lines before and after treatment with all trans RA. We found that RA induced a significant inhibition of the luciferase reporter gene in HL60 cells. In contrast, a strong stimulation of luciferase activity was observed in NB4 cells treated with RA. The analysis of the promoter region allowed us to identify a new response element for retinoic acid receptors, named mREpal, which is probably affected by the product of t(15;17) translocation.

Retinoic acid downmodulates erythroid differentiation and GATA1 expression in purified adult-progenitor culture.

All-trans retinoic acid (RA) is an important morphogen in vertebrate development, a normal constituent in human adult blood and is also involved in the control of cell growth and differentiation in acute promyelocytic leukemia. We have examined the effects of RA on normal hematopoiesis by using early hematopoietic progenitor cells (HPC) stringently purified from adult peripheral blood. In clonogenetic fetal calf serum-supplemented (FCS+) or -nonsupplemented (FCS-) culture treated with saturating levels of interleukin-3 (IL-3) granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (Ep) (combined with c-kit ligand in FCS(-)-culture conditions), RA induces a dramatic dose-dependent shift from erythroid to granulomonocytic colony formation, the latter colonies being essentially represented by granulocytic clones. This shift is apparently not caused by a recruitment phenomenon, because in FCS+ culture, the total number of colonies is not significantly modified by RA addition. In FCS- liquid-suspension culture supplemented with saturating Ep level and low-dose IL-3/GM-CSF, adult HPC undergo unilineage erythropoietic differentiation: Here again, treatment with high-dose RA induces a shift from the erythroid to granulocytic differentiation pathway. Studies on RA time-response or pulse treatment in semisolid or liquid culture show that early RA addition is most effective, thus indicating that early but not late HPC are sensitive to its action. We then analyzed the expression of the master GATA1 gene, which encodes a finger transcription factor required for normal erythroid development; addition of RA to HPC stimulated into unilineage erythropoietic differentiation in liquid culture caused a virtually complete inhibition of GATA1 mRNA induction. These results indicate that RA directly inhibits the erythroid differentiation program at the level of early adult HPC, and may lead to a shift from the erythroid to granulocytic differentiation pathway. This phenomenon is correlated with inhibition of GATA1 induction in the early stages of erythropoietic differentiation.

Regulation of myeloblastin messenger RNA expression in myeloid leukemia cells treated with all-trans retinoic acid.

Retinoic acid is known to induce differentiation of human myeloid leukemia cells in vitro. Recently, all-trans retinoic acid has been used to induce remissions in patients with acute promyelocytic leukemia, probably through differentiation of the leukemia cells. Myeloblastin (mbn) is a protease that has been identified in the human leukemia cell line HL-60. Downregulation of this protease can inhibit proliferation and induce differentiation of HL-60-derived leukemia cells. Here we have investigated the regulation of mbn messenger RNA (mRNA) expression in two human leukemia cell lines, HL-60 and NB4, treated with all-trans retinoic acid. Under this treatment, downregulation of mbn mRNA was observed in both cell lines, but was considerably delayed in NB4 cells that carry the t(15;17) translocation characteristic of acute promyelocytic leukemia. We have found that multiple mechanisms were involved in the control of mbn mRNA expression. These mechanisms were different in HL-60 and NB4 cells. Our results show that in HL-60 cells, all-trans retinoic acid rapidly decreased transcription of mbn. In contrast, in the t(15;17)-positive NB4 cells treated with all-trans retinoic acid, upregulation of mbn mRNA expression was followed by a late downregulation, both achieved via posttranscriptional mechanisms.

Wegener autoantigen and myeloblastin are encoded by a single mRNA.

Myeloblastin is a serine protease that has been identified in the human leukemia cell line HL-60. Down-regulation of this protease can inhibit proliferation and induce differentiation of promyelocyte-like human leukemic cells. Proteinase 3, a serine protease of human neutrophils, has been identified as the Wegener autoantigen. A high level of homology between myeloblastin and proteinase 3 has suggested that they may be a single serine protease. We have recently completed the 5'-terminal nucleotide sequence of proteinase 3 and shown that its mRNA was also expressed in HL-60 cells and in cells from patients with acute myeloid leukemia. Here we demonstrate that myeloblastin and proteinase 3 are encoded by a single mRNA.

1 alpha,25-dihydroxyvitamin D3-induced regulation of protein kinase C gene expression during HL-60 cell differentiation.

The human promyelocytic leukemia cell line HL-60 differentiates in vitro when treated with various inducers. It has previously been shown that protein kinase C (PKC) isozymes are modulated during granulocytic differentiation of HL-60 cells induced by dimethyl sulfoxide or retinoic acid (M. Makowske, R. Ballester, Y. Cayre, and O.M. Rosen, J. Biol. Chem., 263: 3402-3410, 1988; K. Hashimoto, A. Kishimoto, H. Aihara, I. Yasuda, K. Mikawa, and Y. Nishizuka, FEBS Left., 263: 31-34, 1990). HL-60 responds to 1 alpha, 25-dihydroxyvitamin D3 (1,25-(OH)2D3) or to 12-O-tetradecanoylphorbol-13-acetate by giving rise to monocytic cells. In the present study, we demonstrate that treatment of HL-60 cells with 1,25-(OH)2D3 causes dramatic increases in PKC-alpha and PKC-beta protein levels detected by immunoblotting with PKC isoform-specific antibodies and in Ca(2+)- and phospholipid-dependent protein kinase activity. We also observed a transient increase in the steady-state levels of PKC-alpha and PKC-beta mRNA species in Northern blotting experiments, with maximal induction occurring 48 h after addition of 1,25-(OH)2D3. Analyses of 1,25-(OH)2D3-induced PKC mRNA expression by nuclear run-on transcription experiments suggest that the observed increases in PKC mRNA levels may occur by a posttranscriptional mechanism(s). In contrast to the transient increases in PKC mRNA levels, the increases in PKC Mr 80,000 protein species and in PKC enzyme activity were progressive in HL-60 cells treated with 1,25-(OH)2D3 between 1 and 5 days, thus implying the existence of a further up-regulation of PKC proteins occurring at the translational and/or posttranslational levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Down-regulation of a serine protease, myeloblastin, causes growth arrest and differentiation of promyelocytic leukemia cells.

Cells from the human leukemia cell line HL-60 undergo terminal differentiation when exposed to inducing agents. Differentiation of these cells is always accompanied by withdrawal from the cell cycle. Here we describe the isolation of a cDNA encoding a novel serine protease that is present in HL-60 cells and is down-regulated during induced differentiation of these cells. We have named this protease myeloblastin. Down-regulation of myeloblastin mRNA occurs with both monocytic and granulocytic inducers. Myeloblastin mRNA is undetectable in fully differentiated HL-60 cells as well as in human peripheral blood monocytes. We found that regulation of myeloblastin mRNA in HL-60 cells is serum dependent. Inhibition of myeloblastin expression by an antisense oligodeoxynucleotide inhibits proliferation and induces differentiation of promyelocyte-like leukemia cells.