Growth control or terminal differentiation: endogenous production and differential activities of vitamin A metabolites in HL-60 cells.

Dakir E., Mollinedo F.

MicroRNAs (miRNAs, miRs) are short non-coding post-transcriptional regulators of gene expression in normal physiology and disease. Acute myeloid leukemia is characterized by accumulation of malignantly transformed immature myeloid precursors, and differentiation therapy, used to overcome this differentiation blockage, has become a successful therapeutic option. The human HL-60 acute leukemia cell line serves as a cell culture model for granulocytic maturation, and dimethyl sulfoxide (DMSO) incubation leads to its differentiation towards neutrophil-like cells, as assessed by biochemical, functional and morphological parameters. DMSO-induced HL-60 cell differentiation constitutes an excellent model to examine molecular processes that turn a proliferating immortal leukemic cell line into mature non-proliferating and apoptosis-prone neutrophil-like end cells. By performing genome-wide miRNA profiling and functional assays, we have identified a signature of 86 differentially expressed canonical miRNAs (51 upregulated; 35 downregulated) during DMSO-induced granulocytic differentiation of HL-60 cells. Quantitative real-time PCR was used to validate miRNA expression. Among these differentially expressed canonical miRNAs, we found miR-125a-5p upregulation and miR-17-92 cluster downregulation acted as major regulators of granulocytic differentiation in HL-60 cells. Enforced expression of miR-125a-5p promoted granulocytic differentiation in HL-60 cells, whereas miR-17-92 ectopic expression inhibited DMSO-induced HL-60 granulocytic differentiation. Ectopic expression of miR-125a-5p also promoted granulocytic differentiation in human acute promyelocytic leukemia NB4 cells, as well as in naïve human primary CD34+-hematopoietic progenitor/stem cells. These findings provide novel molecular insights into the identification of miRNAs regulating granulocytic differentiation of human leukemia cells and normal CD34+-hematopoietic progenitor/stem cells, and may assist in the development of novel miRNA-targeted therapies for leukemia.

Tan L., Xu H., Chen G., Wei X., Yu B., Ye J., Xu L., Tan H.

High-mobility group AT-hook 2 (HMGA2) may serve as an architectural transcription factor, and it can regulate a range of normal biological processes including proliferation and differentiation. Upregulation of HMGA2 expression is correlated to the undifferentiated phenotype of immature leukaemic cells. However, the underlying mechanism of HMGA2-dependent myeloid differentiation blockage in leukaemia is unknown. To reveal the role and mechanism of HMGA2 in differentiation arrest of myeloid leukaemia cells, the quantitative expression of HMGA2 and homeobox A9 (HOXA9) was analysed by real-time PCR (qRT-PCR). The regulatory function of HMGA2 in blockage of differentiation in human myeloid leukaemia was investigated through in vitro assays (XTT assay, May–Grünwald–Giemsa, flow cytometry analysis and western blot). We found that the expression of HMGA2 and HOXA9 was reduced during the process of granulo-monocytic maturation of acute myeloid leukaemia (AML) cells, knockdown of HMGA2 promotes terminal (granulocytic and monocytic) differentiation of myeloid leukaemia primary blasts and cell lines, and HOXA9 was significantly downregulated in leukaemic cells with knockdown of HMGA2. Downregulation of HOXA9 in myeloid leukaemia cells led to increased differentiation capacity in vitro. Our data suggest that increased expression of HMGA2 represents a possible new mechanism of myeloid differentiation blockage of leukaemia. Aberrant expression of HMGA2 may enhance HOXA9-dependent leukaemogenesis and myeloid leukaemia phenotype. Disturbance of the HMGA2–HOXA9 pathway is probably a therapeutic strategy in myeloid leukaemia.

Hammerling U.

For the past century, vitamin A has been considered to serve as a precursor for retinoids that facilitate vision or as a precursor for retinoic acid (RA), a signaling molecule that modulates gene expression. However, vitamin A circulates in plasma at levels that far exceed the amount needed for vision or the synthesis of nanomolar levels of RA, and this suggests that vitamin A alcohol (i.e. retinol) may possess additional biological activity. We have pursued this question for the last 20 years, and in this chapter, we unfold the story of our quest and the data that support a novel and distinct role for vitamin A (alcohol) action. Our current model supports direct binding of vitamin A to the activation domains of serine/threonine kinases, such as protein kinase C (PKC) and Raf isoforms, where it is involved in redox activation of these proteins. Redox activation of PKCs was first described by the founders of the PKC field, but several hurdles needed to be overcome before a detailed understanding of the biochemistry could be provided. Two discoveries moved the field forward. First, was the discovery that the PKCδ isoform was activated by cytochrome c, a protein with oxidoreduction activity in mitochondria. Second, was the revelation that both PKCδ and cytochrome c are tethered to p66Shc, an adapter protein that brings the PKC zinc-finger substrate into close proximity with its oxidizing partner. Detailed characterization of the PKCδ signalosome complex was made possible by the work of many investigators. Our contribution was determining that vitamin A is a vital co-factor required to support an unprecedented redox-activation mechanism. This unique function of vitamin A is the first example of a general system that connects the one-electron redox chemistry of a heme protein (cytochrome c) with the two-electron chemistry of a classical phosphoprotein (PKCδ). Furthermore, contributions to the regulation of mitochondrial energetics attest to biological significance of vitamin A alcohol action.

Ruiz F. ., Porté S., Gallego O., Moro A., Ardèvol A., Del Río-Espínola A., Rovira C., Farrés J., Parés X.

Human AKR (aldo–keto reductase) 1C proteins (AKR1C1–AKR1C4) exhibit relevant activity with steroids, regulating hormone signalling at the pre-receptor level. In the present study, investigate the activity of the four human AKR1C enzymes with retinol and retinaldehyde. All of the enzymes except AKR1C2 showed retinaldehyde reductase activity with low Km values (~1 μM). The kcat values were also low (0.18–0.6 min−1), except for AKR1C3 reduction of 9-cis-retinaldehyde whose kcat was remarkably higher (13 min−1). Structural modelling of the AKR1C complexes with 9-cis-retinaldehyde indicated a distinct conformation of Trp227, caused by changes in residue 226 that may contribute to the activity differences observed. This was partially supported by the kinetics of the AKR1C3 R226P mutant. Retinol/retinaldehyde conversion, combined with the use of the inhibitor flufenamic acid, indicated a relevant role for endogenous AKR1Cs in retinaldehyde reduction in MCF-7 breast cancer cells. Overexpression of AKR1C proteins depleted RA (retinoic acid) transactivation in HeLa cells treated with retinol. Thus AKR1Cs may decrease RA levels in vivo. Finally, by using lithocholic acid as an AKR1C3 inhibitor and UVI2024 as an RA receptor antagonist, we provide evidence that the pro-proliferative action of AKR1C3 in HL-60 cells involves the RA signalling pathway and that this is in part due to the retinaldehyde reductase activity of AKR1C3.

Li Z., Yao S., Alini M., Stoddart M.J.

The present study aimed to investigate the role of a retinoic acid receptor-β (RARβ) inhibitor LE135 on TGF-β induced chondrogenesis of human bone marrow mesenchymal stem cells (hMSCs). Pellet culture with exogenous transforming growth factor-β (TGF-β), and a mechanically loaded scaffold system were used to provide two culture models. All samples were cultured for 8 days and changes in early gene expression were determined. Glycosaminoglycan and mRNA expression data showed that LE135 itself did not induce any chondrogenic response in either pellet culture or scaffold culture of hMSCs. LE135 actually inhibited the chondrogenic response caused by exogenous TGF-β, or endogenous TGF-β induced by mechanical load, while the expression of genes normally associated with osteogenesis was not affected. This suggests that the inhibitor LE135 affects the osteochondral differentiation pathway at a different stage, inhibiting chondrogenic gene expression while having no effect on genes normally associated with the osteogenic phenotype. Alternatively, it might be that different cells were proceeding down different lineages. Some cells were undergoing chondrogenesis and this was affected by LE135, while other cells underwent osteogenic differentiation and were not affected by LE135.

Acin‐Perez R., Hoyos B., Zhao F., Vinogradov V., Fischman D.A., Harris R.A., Leitges M., Wongsiriroj N., Blaner W.S., Manfredi G., Hammerling U.

Semba R.D.

Bousquet M., Quelen C., Rosati R., Mansat-De Mas V., La Starza R., Bastard C., Lippert E., Talmant P., Lafage-Pochitaloff M., Leroux D., Gervais C., Viguié F., Lai J., Terre C., Beverlo B., et. al.

Most chromosomal translocations in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) involve oncogenes that are either up-regulated or form part of new chimeric genes. The t(2;11)(p21;q23) translocation has been cloned in 19 cases of MDS and AML. In addition to this, we have shown that this translocation is associated with a strong up-regulation of miR-125b (from 6- to 90-fold). In vitro experiments revealed that miR-125b was able to interfere with primary human CD34+ cell differentiation, and also inhibited terminal (monocytic and granulocytic) differentiation in HL60 and NB4 leukemic cell lines. Therefore, miR-125b up-regulation may represent a new mechanism of myeloid cell transformation, and myeloid neoplasms carrying the t(2;11) translocation define a new clinicopathological entity.

Chiu H., Fischman D.A., Hammerling U.

Dawson H.D., Collins G., Pyle R., Key M., Weeraratna A., Deep-Dixit V., Nadal C.N., Taub D.D.

Vitamin A (VA) deficiency induces a type 1 cytokine response and exogenously provided retinoids can induce a type 2 cytokine response both in vitro and in vivo. The precise mechanism(s) involved in this phenotypic switch are inconsistent and have been poorly characterized in humans. In an effort to determine if retinoids are capable of inducing Th2 cytokine responses in human T cell cultures, we stimulated human PBMCs with immobilized anti-CD3 mAb in the presence or absence of all-trans retinoic acid (ATRA) or 9-cis-RA. Stimulation of human PBMCs and purified T cells with ATRA and 9-cis-RA increased mRNA and protein levels of IL-4, IL-5, and IL-13 and decreased levels of IFN-γ, IL-2, IL-12p70 and TNF-α upon activation with anti-CD3 and/or anti-CD28 mAbs. These effects were dose-dependent and evident as early as 12 hr post stimulation. Real time RT-PCR analysis revealed a dampened expression of the Th1-associated gene, T-bet, and a time-dependent increase in the mRNA for the Th2-associated genes, GATA-3, c-MAF and STAT6, upon treatment with ATRA. Besides Th1 and Th2 cytokines, a number of additional proinflammatory and regulatory cytokines including several chemokines were also differentially regulated by ATRA treatment. These data provide strong evidence for multiple inductive roles for retinoids in the development of human type-2 cytokine responses.

Hoyos B., Hammerling U.

Hoyos B., Jiang S., Hammerling U.

Redox activations of serine/threonine kinases represent alternate pathways in which vitamin A plays a crucial co-factor role. Vitamin A binds the zinc finger domain of c-Raf with nanomolar affinity. The retinoid-binding site has been mapped within this structure by scanning mutagenesis. The deduced contact sites were found anchored on Phe-8, counting from the 1st conserved histidine of the zinc finger. These sites agreed with contact amino acids identified by computational docking. The boundaries of a related binding pocket were identified by mutagenesis and partially confirmed by docking trials in the protein kinase C-α C1A zinc finger. They comprised Phe-7, Phe-8, and Trp-22. This trio was absent from the αC1B domain, explaining why the latter did not bind retinol. Reconfiguring at a minimum the two corresponding amino acids of αC1B, Thr-7 and Tyr-22, to conform to αC1A converted this domain to a binder. Deletion of the predicted retinoid-binding site in the full-length molecule created a mutant c-Raf that was deficient in retinol-dependent redox activation but fully responsive to epidermal growth factor. Our findings indicate that ligation of retinol to a specific site embedded in the regulatory domain is an important feature of c-Raf regulation in the redox pathway.

Semba R.D.

Key Points

Grubbs C.J., Hill D.L., Bland K.I., Beenken S.W., Lin T., Eto I., Atigadda V.R., Vines K.K., Brouillette W.J., Muccio D.D.

Studies were performed in female Sprague-Dawley rats to determine the efficacy of a new RXR specific retinoid (9cUAB30) when combined with tamoxifen in the prevention of mammary cancers and to determine various pharmacokinetic parameters of the retinoid. When administered by gavage, 9cUAB30 was rapidly absorbed and had a serum t(1/2) of 13.5 h. Since the retinoid was administered in the diet for the chemoprevention study, a 28-day study in which 9cUAB30 was given at dose levels of 200, 400, and 600 mg/kg diet revealed fairly constant serum levels regardless of dose or length of treatment; possibly accounting for the observed low toxicity of this compound. When suboptimal doses of 9cUAB30 were given in the methylnitrosourea (MNU)-induced mammary cancer model, the following average number of mammary cancers were observed: 9cUAB30 (150 mg/kg diet), 4.3; tamoxifen (0.4 mg/kg diet), 4.6; 9cUAB30 (150 mg/kg diet)+tamoxifen (0.4 mg/kg diet), 2.6; and controls, 6.0. Thus, the combination of the agents resulted in an increased effect in preventing mammary cancers; suggesting that cancer cell proliferation was inhibited by the compounds blocking different pathways.

Korichneva I., Waka J., Hammerling U.

Cardiomyocytes suffering irreversible damage under oxidative stress during ischemia activate their suicide program. Mitochondria play a key role in this process, while they themselves are subject to regulation by a number of signaling pathways. We demonstrate here that retinoids influence mitochondrial function in cardiomyocytes. Depending on their chemical nature, retinoids can either ameliorate or exacerbate stress-related damage. Thus, vitamin A, retinol, was protective because retinol deprivation enhanced oxidative damage, as indicated by rapid loss of mitochondrial membrane potential. Supplementation with a physiological concentration of retinol reversed this effect. Anhydroretinol (AR), a known antagonist, which works by displacing retinol from the common binding sites on serine/threonine kinases, also caused mitochondrial membrane depolarization. The AR effect was both Ca(2+)-dependent and cyclosporin-sensitive, suggesting an upstream signaling mechanism rather than direct membrane effect. Our results agree with a model where retinol supports mitochondrial integrity by enabling upstream signaling processes. The consequences of disrupting these processes by AR are opening of the permeability transition pore, release of cytochrome c, and activation of the suicide program.