All-trans Retinoic Acid Stimulation Research Articles

Exogenous CD38 upregulation enables high-efficacy dually cascade targeted molecular therapy of leukemia

Acute myeloid leukemia (AML) patients face poor prognosis with high mortality rate, mainly due to the lack of suitable antigens and shortage of effective targeted therapeutics. Here, we utilize exogenous all-trans retinoic acid (ATRA) to upregulate CD38 level on AML cells and therefore boost the AML-targeting of daratumumab-directed polymersomal volasertib molecular targeted drugs (DPVol), for potent and safe AML depletion via cascade targeting. DPVol with a small size of ∼ 30 nm had tailored daratumumab density, efficient and robust Vol loading, as well as reduction-triggered intracellular Vol release. ATRA stimulation substantially increased CD38 level on AML cell lines and primary cells isolated from AML patients as much as 20-fold, and accordingly enhanced the cellular uptake and anti-AML activity of DPVol, displaying a low IC50 of 16.3 nM in MV-4-11 cells. Intriguingly, ATRA + DPVol combination treatment dramatically reduced the leukemia burden in orthotopic CD38low MV-4-11 and Molm-13-Luc AML bearing mice without causing toxicity, resulting in 4.3–5.8-fold prolonged survival with 20–40 % of mice cured, superior to DPVol and ATRA + PVol. The exogenous stimulation mediated CD38 upregulation in combination with DPVol may provide a safe and cascade targeting strategy for potent treatment of leukemia.

Sel1l May Contributes to the Determinants of Neuronal Lineage and Neuronal Maturation Regardless of Hrd1 via Atf6-Sel1l Signaling.

The endoplasmic reticulum (ER) is the primary site of intracellular quality control involved in the recognition and degradation of unfolded proteins. A variety of stresses, including hypoxia and glucose starvation, can lead to accumulation of unfolded proteins triggering the ER-associated degradation (ERAD) pathway. Suppressor Enhancer Lin12/Notch1 Like (Sel1l) acts as a "gate keeper" in the quality control of de novo synthesized proteins and complexes with the ubiquitin ligase Hrd1 in the ER membrane. We previously demonstrated that ER stress-induced aberrant neural stem cell (NSC) differentiation and inhibited neurite outgrowth. Inhibition of neurite outgrowth was associated with increased Hrd1 expression; however, the contribution of Sel1l remained unclear. To investigate whether ER stress is induced during normal neuronal differentiation, we semi-quantitatively evaluated mRNA expression levels of unfolded protein response (UPR)-related genes in P19 embryonic carcinoma cells undergoing neuronal differentiation in vitro. Stimulation with all-trans retinoic acid (ATRA) for 4days induced the upregulation of Nestin and several UPR-related genes (Atf6, Xbp1, Chop, Hrd1, and Sel1l), whereas Atf4 and Grp78/Bip were unchanged. Small-interfering RNA (siRNA)-mediated knockdown of Sel1l uncovered that mRNA levels of the neural progenitor marker Math1 (also known as Atoh1) and the neuronal marker Math3 (also known as Atoh3 and NeuroD4) were significantly suppressed at 4days after ATRA stimulation. Consistent with this result, Sel1l silencing significantly reduced protein levels of immature neuronal marker βIII-tubulin (also known as Tuj-1) at 8days after induction of neuronal differentiation, whereas synaptogenic factors, such as cell adhesion molecule 1 (CADM1) and SH3 and multiple ankyrin repeat domain protein 3 (Shank3) were accumulated in Sel1l silenced cells. These results indicate that neuronal differentiation triggers ER stress and suggest that Sel1l may facilitate neuronal lineage through the regulation of Math1 and Math3 expression.

The role of ZFP580 in the regulation of rat VSMCs migration by ATRA and its mechanism

To investigate the probable roles of the novel C2H2 zinc finger transcription factor ZFP580 on all-transretinoic acid (ATRA)-regulated VSMCs migration and underlying mechanisms. Rat aortic VSMCs were isolated, cultured and identified. VSMCs were treated with ATRA at the concentrations of 0, 5, 10 or 20 μmol/L for 24 hours. The migration ability of VSMCs was observed in each group and compared with control group which was treated by 0 μmol/L ATRA. The mRNA and protein expression levels of ZFP580 were detected by QPCR and Western blot. ZFP580 protein expression in VSMCs was detected under ATRA stimulation when ERK inhibitor PD98059 was used to inhibit the protein expression of ERK. Adenovirus transfection technology was used to obtain VSMCs with overexpression or low expression of ZFP580, and QPCR and Western blot were used to detect the mRNA and protein levels of MMP-2, MMP-9 and ZFP580. On the 10th day of VSMCs culture, immunofluorescence showed that SM22 alpha antibody, as a specific marker of smooth muscle cells, was positive. Compared to the control group, VSMCs migration was reduced by 32%, 43%, and 59% in the group of 5, 10, and 20 μmol/L ATRA pretreatment. Compared with the control group, VSMCs treated by 20 μmol/L ATRA reduced the cell migration by 49%, 36% and 22% at 24, 48 and 72 h. The mRNA and protein expression levels of ZFP580 were increased with the increase of ATRA stimulation solubility and the extension of stimulation time. ERK was increased significantly after 15 min of ATRA stimulation. Pretreatment with ERK inhibitor PD98059 (20 μmol/L) inhibited the expression of ERK protein and reduced the expression of ATRA-induced ZFP580 protein. Overexpression of ZFP580 inhibited the expressions of MMP-2 and MMP-9, whereas down-expression of ZFP580 promoted the expressions of MMP-2 and MMP-9. ATRA increased the expression of ZFP580 through the ERK signaling pathway, while ZFP580 was involved in ATRA's inhibition of VSMCs migration by affecting the expression of downstream MMP-2 and MMP-9.

Differentiation of stem cells upon deprivation of exogenous FGF2: a general approach to study spontaneous differentiation of hESCs in vitro

Establishing a model for in vitro differentiation of human embryonic stem cells (hESCs) towards the germ cell lineage could be used to identify molecular mechanisms behind germ cell differentiation that may help in understanding human infertility. Here, we evaluate whether a lack of exogenous fibroblast growth factor 2 (FGF2) is supporting spontaneous differentiation of hESCs cultured on human foreskin fibroblast (hFF) monolayers towards germ cell lineage. Additionally to depriving the hESCs of exogenous FGF2, cells were stimulated with all-trans retinoic acid (ATRA). To get a more comprehensive impression on effects of removal of FGF2 and stimulation with ATRA, we combined the results of three cell lines for each experimental setting. When combining gene expression profiles of three cell lines for 96 genes, only 6 genes showed a significant up-regulation in all cell lines, when no FGF2 was added to the media for 12 weeks. None of these genes are related to the germ lineage, whereas genes for neuronal cells (PAX6 and NR6A1) and endothelial cells (FLT-1 and PTF1A) were up-regulated. To induce and support the differentiation towards the germ lineage we stimulated hESCs with different concentrations of ATRA for 7 and 14 days. We observed no significant difference in gene expression on RNA level when combining all cell lines. Whereas, the overall outcome was negative, one of these cell lines demonstrated an up-regulation of DDX4 on RNA and protein level after 7 days of ATRA stimulation. In summary, our data showed that the lack of exogenous FGF2 results in up-regulation of genes crucial for neuronal and endothelial cell differentiation of hESCs, but not in the up-regulation of genes related to germ cell differentiation when cultured on hFFs. Additionally, we demonstrated that ATRA supplementation did not result in a general specific direction of hESCs towards the germ lineage.

Retinoic Acid Receptor α Mediates All-trans-retinoic Acid-induced Klf4 Gene Expression by Regulating Klf4 Promoter Activity in Vascular Smooth Muscle Cells

The transcription factor Krüppel-like factor 4 (KLF4) plays a critical role in vascular smooth muscle cell (VSMC) differentiation induced by all-trans-retinoic acid (ATRA). Although it has been demonstrated that ATRA stimulation augments both KLF4 protein and mRNA levels in VSMCs, the molecular mechanisms by which ATRA regulates Klf4 transcription are unknown. In this study, we examined the roles of ATRA-selective nuclear retinoic acid receptors (RARs) in the transcriptional regulation of Klf4. The introduction of small interfering RNA and an RAR antagonist demonstrated that RARα, but not RARβ or RARγ, mediated ATRA-induced Klf4 expression. A luciferase assay for the Klf4 promoter showed that three GC boxes in the proximal Klf4 promoter were indispensible for ATRA-induced Klf4 transcription and that RARα enhanced Klf4 promoter activity in a GC box-dependent manner. Furthermore, chromatin immunoprecipitation and oligonucleotide pulldown assays demonstrated that the transcription factors KLF4, Sp1, and YB1 directly bound to the GC boxes of the proximal Klf4 promoter. Upon RARα agonist stimulation, RARα was recruited to the Klf4 promoter through its interaction with KLF4, Sp1, and YB1 to form a transcriptional activation complex on the three GC boxes of the Klf4 promoter. These results suggest that RARα serves as an essential co-activator for ATRA signaling and that the recruitment of RARα to the KLF4-Sp1-YB1 complex, which leads to Klf4 expression in VSMCs, is independent of a retinoic acid response element.

Neutral Sphingomyelinase-2 Mediates Growth Arrest by Retinoic Acid through Modulation of Ribosomal S6 Kinase

All-trans-retinoic acid (ATRA) induces growth arrest of many cell types. Previous studies have reported that ATRA can modulate cellular sphingolipids, but the role of sphingolipids in the ATRA response is not clear. Using MCF-7 cells as a model system, we show that ATRA stimulates an increase in ceramide levels followed by G(0)/G(1) growth arrest. Notably, induction of nSMase2 was the primary effect of ATRA on the sphingolipid network and was both time- and dose-dependent. Importantly, pretreatment with nSMase2 siRNA significantly inhibited ATRA effects on ceramide levels and growth arrest. In contrast, nSMase2 overexpression was sufficient to increase ceramide levels and induce G(0)/G(1) growth arrest of asynchronous MCF-7 cells. Surprisingly, neither ATRA stimulation nor nSMase2 overexpression had significant effects on classical cell cycle regulators such as p21/WAF1 or retinoblastoma. In contrast, ATRA suppressed phosphorylation of ribosomal S6 kinase (S6K) and its downstream targets S6 and eIF4B. Importantly, these effects were significantly inhibited by nSMase2 siRNA. Reciprocally, nSMase2 overexpression was sufficient to suppress S6K phosphorylation and signaling. Notably, neither ATRA effects nor nSMase2 effects on S6K phosphorylation required the ceramide-activated protein phosphatase PP2A, previously identified as important for S6K regulation. Finally, nSMase2 overexpression was sufficient to decrease translation as measured by methionine incorporation and analysis of polyribosome profiles. Taken together, these results implicate nSMase2 as a major component of ATRA-induced growth arrest of MCF-7 cells and identify S6K as a novel downstream target of nSMase2.

Chemokine induction by all-trans retinoic acid and arsenic trioxide in acute promyelocytic leukemia: triggering the differentiation syndrome

AbstractIn acute promyelocytic leukemia (APL), differentiation therapy with all-trans retinoic acid (ATRA) and/or arsenic trioxide can induce a differentiation syndrome (DS) with massive pulmonary infiltration of differentiating leukemic cells. Because chemokines are implicated in migration and extravasation of leukemic cells, chemokines might play a role in DS. ATRA stimulation of the APL cell line NB4 induced expression of multiple CC-chemokines (CCLs) and their receptors (> 19-fold), resulting in increased chemokine levels and chemotaxis. Induction of CCL2 and CCL24 was directly mediated by ligand-activated retinoic acid receptors. In primary leukemia cells derived from APL patients at diagnosis, ATRA induced chemokine production as well. Furthermore, in plasma of an APL patient with DS, we observed chemokine induction, suggesting that chemokines might be important in DS. Dexamethasone, which efficiently reduces pulmonary chemokine production, did not inhibit chemokine induction in APL cells. Finally, chemokine production was also induced by arsenic trioxide as single agent or in combination with ATRA. We propose that differentiation therapy may induce chemokine production in the lung and in APL cells, which both trigger migration of leukemic cells. Because dexamethasone does not efficiently reduce leukemic chemokine production, pulmonary infiltration of leukemic cells may induce an uncontrollable hyperinflammatory reaction in the lung.

MiR-132 and MiR-212 as Regulators of WT1 and GATA2 in Acute Myeloid Leukemia.

Abstract 1283Poster Board I-305Wilms' tumor 1 (WT1) and GATA binding protein 2 (GATA2) transcription factors are highly expressed in hematopoietic stem cells and progenitors. Differentiation of precursor blood cells towards mature blood cells is accompanied by rapid downregulation of both transcription factors. Overexpression of WT1 has been observed in the majority of acute myeloid leukemia (AML) cases. Furthermore, in 10-15% of the AML cases mutations in the WT1 gene occur, which have been correlated with poor prognosis. Aberrant expression of GATA2 in AML has been described as well, but no mutations in this gene have been reported in AML so far. How the (aberrant) expression of WT1 and GATA2 is controlled is not completely clear. A regulatory role for microRNAs (miRNAs) has been described for several transcription factors which regulate hematopoiesis. MiRNAs negatively regulate gene expression by translational repression or degradation of target messenger RNAs (mRNAs). In the present study we investigated the interplay between miRNAs and transcription factors that are involved in myeloid development and malignant transformation towards AML. We studied the expression of 158 miRNAs in the APL cell line NB4 during induction of granulocytic differentiation with all-trans retinoic acid (ATRA). Quantitative PCR specific for mature miRNAs was performed (Applied Biosystems). Twenty out of 158 miRNAs were more than 10-fold upregulated upon differentiation induction with ATRA. MiR-132 and miR212, which are derived from the same pri-miRNA transcript, were most strongly upregulated during ATRA-induced granulocytic differentiation (1200- and 350-fold respectively at 96 hours after ATRA-stimulation). In vitro ATRA-induction of primary APL cells also resulted in upregulation of miR-132 and miR-212. Computational target prediction algorithms were used to identify transcription factors which may be targeted by miR-132 and miR-212. Subsequently, the expression pattern of the predicted targets was determined experimentally in NB4 cells before and after differentiation induction with ATRA using microarray-based mRNA profiling (Affymetrix). In addition, further verification of target gene expression during ATRA-induced differentiation was performed using quantitative PCR. The transcription factors WT1 and GATA2 were predicted as targets of miR-132 and miR-212 by two out of four different prediction programs that were used. Both transcription factors contained putative binding sites for miR-132 and miR-212 in their 3'UTR. When tested on microarray and by quantitative PCR, the expression of WT1 and GATA2 was indeed strongly downregulated during ATRA-induced granulocytic differentiation of NB4 cells (65- and 165-fold respectively at 96 hours after ATRA stimulation) as well as in primary leukemia cells derived from APL patients (30- and 10-fold respectively at 48 hours after ATRA-stimulation). During ATRA-induced differentiation the expression levels of WT1 were positively correlated with the expression levels of GATA2. In addition, WT1 expression was also strongly correlated with GATA2 expression in a cohort of 27 pre-treatment AML cases as well as in 7 healthy controls, suggesting that these genes might be co-regulated to a large extent. To directly prove that WT1 and GATA2 are indeed targeted by miR-132 and miR-212, we are currently performing lentiviral-based overexpression studies of both miRNAs to determine the effect on endogenous WT1 and GATA2 mRNA expression. MicroRNAs which target WT1 and GATA2 may be valuable tools in controlling the aberrant expression of WT1 and GATA2 observed in AML. DisclosuresNo relevant conflicts of interest to declare.

All- trans retinoic acid-induced ADAM28 degrades proteoglycans in human chondrocytes

In order to elucidate the mechanism of cartilage degradation in osteoarthritis (OA), we established a cell assay system. Under the stimulation of all- trans retinoic acid (ATRA), the human chondrosarcoma cell line HCS-2/8 increased proteoglycan release from inactivated bovine nasal cartilage (BNC) and the results suggested the involvement of membrane-bound metalloproteinase(s). Therefore, we focused on the induction of a disintegrin and metalloproteinase (ADAM) superfamily upon ATRA stimulation. Of all ADAMs tested, only ADAM28 was induced by ATRA in HCS-2/8 cells and also in human primary chondrocytes. We found that transfection of ADAM28 or its alternatively spliced soluble form augmented proteoglycan release in the cell assay; however, a mutant soluble form in which a portion of the disintegrin domain was deleted did not have proteoglycan-releasing activity, implying the importance of the domain for enzyme localization and substrate recognition for cartilage degradation in OA.

Dysregulation of elastin expression by fibroblasts in pulmonary emphysema: role of cellular retinoic acid binding protein 2

Background:All-trans retinoic acid (ATRA) stimulates elastin synthesis by lung fibroblasts and induces alveolar regeneration in animal models of pulmonary emphysema. However, ATRA treatment has had disappointing results in human emphysema....

Arsenic Trioxide Augments Chk2/p53-mediated Apoptosis by Inhibiting Oncogenic Wip1 Phosphatase

The oncogenic Wip1 phosphatase (PPM1D) is induced upon DNA damage in a p53-dependent manner and is required for inactivation or suppression of DNA damage-induced cell cycle checkpoint arrest and of apoptosis by dephosphorylating and inactivating phosphorylated Chk2, Chk1, and ATM kinases. It has been reported that arsenic trioxide (ATO), a potent cancer chemotherapeutic agent, in particular for acute promyelocytic leukemia, activates the Chk2/p53 pathway, leading to apoptosis. ATO is also known to activate the p38 MAPK/p53 pathway. Here we show that phosphatase activities of purified Wip1 toward phosphorylated Chk2 and p38 in vitro are inhibited by ATO in a dose-dependent manner. Furthermore, DNA damage-induced phosphorylation of Chk2 and p38 in cultured cells is suppressed by ectopic expression of Wip1, and this Wip1-mediated suppression can be restored by the presence of ATO. We also show that treatment of acute promyelocytic leukemia cells with ATO resulted in induction of phosphorylation and activation of Chk2 and p38 MAPK, which are required for ATO-induced apoptosis. Importantly, this ATO-induced activation of Chk2/p53 and p38 MAPK/p53 apoptotic pathways can be enhanced by siRNA-mediated suppression of Wip1 expression, further indicating that ATO inhibits Wip1 phosphatase in vivo. These results exemplify that Wip1 is a direct molecular target of ATO.

Lentiviral PU.1 overexpression restores differentiation in myeloid leukemic blasts

PU.1, a transcription factor of the ETS family, plays a pivotal role in normal hematopoiesis, and particularly in myeloid differentiation. Altered PU.1 function is possibly implicated in leukemogenesis, as PU.1 gene mutations were identified in some patients with acute myeloid leukemia (AML) and as several oncogenic products (AML1-ETO, promyelocytic leukemia-retinoic acid receptor alpha, FMS-like receptor tyrosine kinase 3 internal tandem duplication) are associated with PU.1 downregulation. To demonstrate directly a role of PU.1 in the blocked differentiation of leukemic blasts, we transduced cells from myeloid cell lines and primary blasts from AML patients with a lentivector encoding PU.1. In NB4 cells we obtained increases in PU.1 mRNA and protein, comparable to increases obtained with all-trans retinoic acid-stimulation. Transduced cells showed increased myelomonocytic surface antigen expression, decreased proliferation rates and increased apoptosis. Similar results were obtained in primary AML blasts from 12 patients. These phenotypic changes are characteristic of restored blast differentiation. PU.1 should therefore constitute an interesting target for therapeutic intervention in AML.

Id1 and Id2 Are Retinoic Acid Responsive Genes and Induce a G0/G1 Arrest in Acute Promyelocytic Leukemia Cells.

Acute promyelocytic leukemia (APL) is uniquely sensitive to treatment with all-trans retinoic acid (ATRA), which overcomes the differentiation arrest and induces terminal granulocytic differentiation of the leukemic blasts. In 98% of the cases of APL, the leukemic cells express a promyelocytic leukemia (PML)- retinoic acid receptor a (RARa) fusion protein as a result of a t(15;17) chromosome translocation. Previously, we have identified Id1 and Id2 as direct retinoic acid target genes being upregulated after ATRA stimulation in the APL cell line NB4 as well as in primary leukemic cells from APL patients. Id (inhibitor of DNA-binding) proteins act as antagonists of basic helix-loop-helix (bHLH) transcription factors by trapping them in heterodimeric complexes, thereby inhibiting DNA-binding and gene transactivation. Various bHLH proteins are pivotal in the control of differentiation and proliferation in various tissues (like muscle and nerve). We have studied the expression pattern of E2A, an ubiquitously expressed bHLH protein, which is generally considered as a promiscuous heterodimerization partner of other, more tissue restricted bHLH proteins. The expression of E2A was high in untreated APL cells and strongly downregulated upon ATRA stimulation. The simultaneous upregulation of Id1 and Id2, and the downregulation of E2A suggest a role for bHLH proteins in the induction of differentiation of APL cells upon treatment with ATRA. To assess the importance of Id1 and Id2 induction for the neutrophilic differentiation of the cells, we have overexpressed both proteins in the APL cell line NB4 using amphotropic retroviral transduction. Ectopic expression of Id1 and Id2 resulted in respectively 27% (n=3, SD= 9%) and 48% (n=3, SD=25%) inhibition of clonogenic growth in semi-solid medium, compared to vector-transduced control cells. Apart from the reduction in the number of colonies, overexpression of Id1 and Id2 did not alter the ATRA sensitivity of APL cells. NB4 liquid cultures revealed that Id1 and Id2 overexpression resulted in inhibition of proliferation and an increase of the percentage of cells in G0/G1, without having an effect on differentiation or apoptosis. These results indicate that Id1 and Id2 are important retinoic acid responsive genes in APL, and suggest that the inhibition of specific bHLH transcription factor complexes may play a role in the therapeutic effect of ATRA in APL.

All- trans retinoic acid directly up-regulates thrombopoietin transcription in human bone marrow stromal cells

Objective All- trans retinoic acid (ATRA) has been used as the first-line therapy for patients with acute promyelocytic leukemia (APL). We previously reported that ATRA increased serum thrombopoietin (TPO) levels accompanied by thrombocytosis during ATRA therapy for APL. In this study, we investigated the mechanism of transcriptional regulation of TPO gene by ATRA using human bone marrow stromal cells. Materials and methods Real time reverse transcriptase polymerase chain reaction and Western blotting were performed to quantify TPO mRNA and protein levels in cells from the human bone marrow stromal cell line KM101. Luciferase-based reporter assay, electrophoretic mobility shift assay (EMSA), and chromatin immunoprecipitation (ChIP) assay were performed to identify a retinoic acid responsive element in the promoter region of TPO gene (TPO-RARE). Results TPO mRNA expression was up-regulated by approximately 2.9 times 8 hours after stimulation with 10 −6 M ATRA in KM101 cells. In contrast, ATRA did not alter TPO mRNA expression in cells from the human hepatoma cell line HepG2. Protein level of KM101 cells also was increased with 10 −6 M ATRA for 48 hours in KM101 cells. We found the synthesized RARα protein bound to [γ- 32P]-labeled TPO-RARE probe and its binding was competed by adding 200× amount of cold TPO-RARE probe by EMSA. In addition, [γ- 32P]-labeled TPO-RARE probe bound to KM101 nuclear protein extract was supershifted by anti-RARα antibody and modified by treatment with ATRA. The relative luciferase activity of TPO gene was increased by 2.2× and the histone H4 was acetylated through TPO-RARE after ATRA stimulation in KM101 cells by ChIP assay. Conclusions These data support the direct up-regulation of TPO transcription by ATRA stimulation in human bone marrow stromal cells and propose one of the mechanisms of thrombocytosis during ATRA therapy for APL.

C-fms expression correlates with monocytic differentiation in PML-RAR alpha+ acute promyelocytic leukemia.

We have investigated the expression of the M-CSF receptor (c-fms) in 16 freshly isolated acute promyelocytic leukemias (APL) expressing the PML/RAR alpha fusion protein. In parallel, we evaluated the capacity of these cells to differentiate along the granulocytic and monocytic pathways. c-fms was constitutively and constantly expressed in all cases sensitive in vivo to all-trans retinoic acid (ATRA) and its expression was further potentiated following in vitro induction with ATRA. Furthermore, gel-shift analysis of APL cells showed elevated levels of PU.1 binding activity to the M-CSF receptor promoter, particularly after ATRA stimulation. Interestingly, the rise of PU.1 binding activity as well as of PU.1 levels after ATRA treatment was significantly higher in APL patients exhibiting monocytic maturation, as compared to those that did not undergo monocytic differentiation. A variable proportion of ATRA-induced APL cells exhibited monocyte-like morphology and immunophenotype: the proportion of monocytic cells was consistently increased by combined treatment with ATRA and diverse hematopoietic growth factors cocktails, which always comprised M-CSF. Monocytic cells originating from in vitro ATRA-induced maturation of APL cells derive from the leukemic clone as suggested by two lines of evidence: (1) monocytic cells harbor the 15;17 translocation; (2) monocytic cells possess Auer bodies. The c-fms(bright) leukemic blasts preferentially showed the capacity for monocytic differentiation as compared to the c-fms(dim/-) subset: indeed, enforced expression of c-fms into NB4, a PML/RAR alpha+ cell line, favored the onset of monocytic maturation. Finally, low c-fms expression was observed in an APL relapsing patient resistant to ATRA, as well as in an APL case with t(11;17), PLZF/RAR alpha+. These observations indicate that PML/RAR alpha+ APL blasts are bipotent for differentiation through both neutrophilic and monocytic lineages, whereby monocytic differentiation is linked to c-fms expression and stimulation.

Up-regulation of Acid Sphingomyelinase during Retinoic Acid-induced Myeloid Differentiation of NB4, a Human Acute Promyelocytic Leukemia Cell Line

All-trans-retinoic acid (ATRA) induces myeloid differentiation of a human promyelocytic leukemia cell line, NB4, but does not affect its subclone NB4/RA harboring a point-mutated ligand-binding domain (AF2) in retinoic acid receptor alpha (RARalpha) gene. We found that ATRA induced the 4-fold elevation of acid sphingomyelinase (ASMase) activity 24 h after treatment in NB4 cells, but not in NB4/RA cells. ATRA did not affect neutral sphingomyelinase activity in either NB4 or NB4/RA. Upon treatment with ATRA, ceramide, the product of an ASMase reaction, accumulated in NB4 cells. Northern blot analysis showed a marked elevation of the ASMase mRNA 8 h after ATRA treatment, reaching a plateau at 24 h. Regulation of ASMase gene expression was studied by a promoter analysis using luciferase reporter assay. The 5'-upstream flanking region of human ASMase gene (-519/+300) conjugated with the luciferase gene was introduced into COS-7 cells. Luciferase activity in transformed cells markedly increased in response to ATRA stimulation when the wild type RARalpha or the PML/RARalpha hybrid protein was co-expressed. Deletion experiments revealed that a short sequence at the 5'-end (-519/-485) was indispensable for the ATRA response. Within this short region, two retinoic acid-responsive element-like motifs (TGCCCG and TCTCCT) and one AP2-like motif (CCCTTCCC) were identified. Deletion and base-substitution experiments showed that all three motifs are required for the full expression induced by ATRA. Electrophoresis mobility shift assays with the nuclear extract of ATRA-treated NB4 cells showed that proteins were bound specifically to the probe being mediated by all three motifs in the promoter sequence.

Retinoic acid stimulates plasminogen activator inhibitor 2 production by blood mononuclear cells and inhibits urokinase-induced extracellular proteolysis.

Retinoids have been shown to modulate several functions of mononuclear phagocytes. We investigated the in vitro effect of all-trans-retinoic acid (ATRA) on the production of two major fibrinolytic components, urokinase-type plasminogen activator (u-PA) and PA inhibitor 2 (PAI-2), by human blood mononuclear cells (MNC). ATRA caused a dose-dependent (range 0.01-10 microM) accumulation of PAI-2 antigen and activity into the cell culture medium, with a maximal increase (about 5-fold over control) at a concentration of 1-10 microM. Similarly, a dose-dependent increase in PAI-2 antigen was observed in cell extracts upon ATRA stimulation. Northern blot analysis showed a parallel increase in the amount of PAI-2 mRNA in ATRA-treated cells. Time-course experiments with 1 microM ATRA showed enhanced PAI-2 mRNA expression as early as 2 h, reaching a maximum at 4-6 h and then declining at 18-24 h, and a time-dependent increase in PAI-2 antigen in the cell culture medium. At variance with PAI-2, u-PA was not influenced by the drug. To establish whether ATRA-induced changes influenced the fibrinolytic process, we evaluated the effect of MNC stimulated with ATRA on u-PA-induced degradation of diluted plasma clots. ATRA-treated cells markedly inhibited clot lysis induced by low concentrations of u-PA. The effect was due to enhanced extracellular PAI-2 accumulation since it was observed with conditioned medium from ATRA-treated cells; it was abolished by the addition of neutralizing anti-PAI-2 antibodies and was negligible when single-chain t-PA was used instead of u-PA. Since monocyte/macrophage-mediated, plasminogen-dependent extracellular proteolysis has been proposed as an important mechanism of tissue damage in several inflammatory states, our findings might contribute to better explain the anti-inflammatory properties of retinoids.