Fenofibrate induces PPARα and BMP2 expression to stimulate osteoblast differentiation.

The peroxisome proliferator-activated receptor (PPAR)-α agonist fenofibrate is used as a lipid-lowering agent to reduce cholesterol and triglyceride in blood. In this study, we investigated whether fenofibrate affects osteoblast differentiation of osteogenic precursor cells. Quantitative real-time PCR and alkaline phosphatase (ALP) staining assays revealed that fenofibrate can enhance the osteoblast differentiation of C3H10T1/2 and MC3T3-E1 cells. In contrast with fenofibrate, the PPARγ agonist rosiglitazone decreased or did not affect the expression of osteogenic genes in these cells. Fenofibrate dose- and time-dependently increased PPARα expression, and concomitantly increased the expression of bone morphogenetic protein 2 (BMP2). Knockdown of PPARα abolished fenofibrate-induced BMP2 expression, activity of the BMP2 promoter gene, and calcium deposition. The chromatin immunoprecipitation assay demonstrated that fenofibrate increased BMP2 expression by inducing direct binding of PPARα to the BMP2 promoter region. Taken together, we suggest that fenofibrate has a stimulatory effect on osteoblast differentiation via the elevation of PPARα levels and the PPARα-mediated BMP2 expression. Our findings provide fenofibrate as a useful agent for controlling hypercholesterolemic patients with osteoporosis.

Aminoacyl tRNA Synthetase--Interacting Multifunctional Protein 1 Activates NK Cells via Macrophages In Vitro and In Vivo.

Aminoacyl tRNA synthetase-interacting multifunctional protein 1 (AIMP1) has been reported to have antitumor effects in various tumor models. However, mechanisms by which AIMP1 ameliorates tumorigenesis are not well understood. As NK cells are a major cell type involved in antitumor activities and AIMP1 is known to activate professional APCs, we determined whether AIMP1 induced NK cell activation directly or via these APCs. AIMP1 induced the expression of surface activation markers on murine NK cells in total splenocytes, although AIMP1 did not directly induce these activation markers of NK cells. The inductive effect of AIMP1 on NK cell activation disappeared in macrophage-depleted splenocytes, indicating that macrophages were required for the AIMP1-induced activation of NK cells. Furthermore, coculture experiments showed that AIMP1 activated NK cells in the presence of isolated macrophages, but failed to activate NK cells when cultured alone or with dendritic cells or B cells. Although AIMP1 significantly promoted TNF-α production by macrophages, the secreted TNF-α partially affected the NK cell activation. Transwell cocultivation analysis revealed that direct contact between macrophages and NK cells was required for the AIMP1-induced NK cell activation. In addition, AIMP1 significantly enhanced cytotoxicity of NK cells against Yac-1 cells. Furthermore, the in vivo administration of AIMP1 also induced NK cell activation systemically with a macrophage-dependent manner. Importantly, AIMP1 dramatically reduced the lung metastasis of melanoma cells, which was mediated by NK cells. Taken together, our results show that AIMP1 induces antitumor responses by NK cell activation mainly via macrophages.

l-Asparaginase-mediated downregulation of c-Myc promotes 1,25(OH)2 D3 -induced myeloid differentiation in acute myeloid leukemia cells.

Treatment of acute myeloid leukemia (AML) largely depends on chemotherapy, but current regimens have been unsatisfactory for long-term remission. Although differentiation induction therapy utilizing 1,25(OH)2 D3 (VD3) has shown great promise for the improvement of AML treatment efficacy, severe side effects caused by its supraphysiological dose limit its clinical application. Here we investigated the combinatorial effect of l-asparaginase (ASNase)-mediated amino acid depletion and the latent alternation of VD3 activity on the induction of myeloid differentiation. ASNase treatment enhanced VD3-driven phenotypic and functional differentiation of three-different AML cell lines into monocyte/macrophages, along with c-Myc downregulation. Using gene silencing with shRNA and a chemical blocker, we found that reduced c-Myc is a critical factor for improving VD3 efficacy. c-Myc-dependent inhibition of mTORC1 signaling and induction of autophagy were involved in the enhanced AML cell differentiation. In addition, in a postculture of AML cells after each treatment, ASNase supports the antileukemic effect of VD3 by inhibiting cell growth and inducing apoptosis. Finally, we confirmed that the administration of ASNase significantly improved VD3 efficacy in the prolongation of survival time in mice bearing tumor xenograft. Our results are the first to demonstrate the extended application of ASNase, which is currently used for acute lymphoid leukemia, in VD3-mediated differentiation induction therapy for AML, and suggest that this drug combination may be a promising novel strategy for curing AML.

MST1 deficiency promotes B cell responses by CD4+ T cell-derived IL-4, resulting in hypergammaglobulinemia.

MST1 deficiency causes T and B cell lymphopenia, resulting in combined immunodeficiency. However, MST1-deficient patients also exhibit autoimmune-like symptoms such as hypergammaglobulinemia and autoantibody production. Recent studies have shown that the autoimmune responses observed in MST1-deficient patients were most likely attributable to defective regulatory T (Treg) cells instead of intrinsic signals in MST1-lacking B cells. Nevertheless, it is not determined how MST1 deficiency in T cells breaks B cell tolerance and causes systemic autoimmune-like phenotypes. In this study, we confirmed that Mst1-/- mice developed hypergammaglobulinemia associated with increased levels of IgG, IgA, and IgE. We also showed that uncontrolled B cell responses were resulted from the IL-4-rich environment created by CD4+ T cells. Defective MST1-FOXO1 signaling down-regulated Treg cells, resulting in the collapse of immune tolerance where the populations of Th2 and T follicular helper cells expanded. In conclusion, we suggest that MST1 acts as a molecular brake to maintain immune tolerance by regulating T cell-mediated B cell activation.

Aminoacyl-tRNA synthetase-interacting multifunctional protein 1 suppresses tumor growth in breast cancer-bearing mice by negatively regulating myeloid-derived suppressor cell functions.

Myeloid-derived suppressor cells (MDSCs) are one of the most important cell types that contribute to negative regulation of immune responses in the tumor microenvironment. Recently, aminoacyl-tRNA synthetase-interacting multifunctional protein 1 (AIMP1), a novel pleiotropic cytokine, was identified as an antitumor protein that inhibits angiogenesis and induces antitumor responses. However, the effect of AIMP1 on MDSCs in the tumor environment remains unclear. In the present study, we demonstrated that AIMP1 significantly inhibited tumor growth in 4T1 breast cancer-bearing mice and reduced MDSCs population of tumor sites and spleens of tumor-bearing mice. AIMP1 reduced expansion of MDSCs from bone marrow-derived cells in the tumor-conditioned media. AIMP1 also negatively regulated suppressive activities of MDSCs by inhibiting IL-6 and NO production, and Arg-1 expression. Furthermore, treatment of breast cancer-bearing mice with AIMP1 decreased the capacity of MDSCs to suppress T cell proliferation and Treg cell induction. Western blot and inhibition experiments showed that downregulation of MDSCs functions by AIMP1 may result from attenuated activation of STATs, Akt, and ERK. These findings indicate that AIMP1 plays an essential role in negative regulation of suppressive functions of MDSCs. Therefore, it has a significant potential as a therapeutic agent for cancer treatment.

5-diphenylacetamido-indirubin-3'-oxime as a novel mitochondria-targeting agent with anti-leukemic activities.

Current treatment for leukemia largely depends on chemotherapy. Despite the progress in treatment efficacy of chemotherapy, a poor outcome consequent upon chemoresistance against conventional anti-cancer drugs still remains to be solved. In this study, we report 5-diphenylacetamido-indirubin-3'-oxime (LDD398) as a novel mitochondria-targeting anti-leukemic agent, which is a derivative of indirubin used in traditional medicine. Treatment with LDD398 resulted in caspase activation, cell death, and growth arrest at G2/M phases in leukemia cells. Interestingly, LDD398 quickly collapsed mitochondrial membrane potential (MMP) within 1 h, accompanied by cytochrome c release into cytosol and severe depletion of cellular ATP. However, the LDD398-induced cellular events was significantly mitigated by blockage of mitochondrial permeability transition pore (MPTP) opening with chemical and genetic modifications, strongly supporting that LDD398 executes its anti-leukemic activity via an inappropriate opening of MPTP and a consequent depletion of ATP. The most meaningful finding was the prominent effectiveness of LDD398 on primary leukemia cells and also on malignant leukemia cells resistant to anticancer drugs. Our results demonstrate that, among a series of indirubin derivatives, LDD398 induces leukemia cell death via a different mode from indirubin or conventional chemotherapeutics, and can be employed as a potent anti-cancer agent in the treatment for newly diagnosed and relapsed leukemia.

Boeravinone B, a natural rotenoid, inhibits osteoclast differentiation through modulating NF-κB, MAPK and PI3K/Akt signaling pathways.

Osteoporosis is a major public health concern, which requires novel therapeutic strategies to prevent or mitigate bone loss. Natural compounds have attracted attention as potential therapeutic agents due to their safety and efficacy. In this study, we investigated the regulatory activities of boeravinone B (BOB), a natural rotenoid isolated from the medicinal plant Boerhavia diffusa, on the differentiation of osteoclasts and mesenchymal stem cells (MSCs), the two main cell components responsible for bone remodeling. We found that BOB inhibited osteoclast differentiation and function, as determined by TRAP staining and pit formation assay, with no significant cytotoxicity. Furthermore, our results showing that BOB ameliorates ovariectomyinduced bone loss demonstrated that BOB is also effective in vivo. BOB exerted its inhibitory effects on osteoclastogenesis by downregulating the RANKL/RANK signaling pathways, including NF-κB, MAPK, and PI3K/Akt, resulting in the suppression of osteoclast-specific gene expression. Further experiments revealed that, at least phenomenologically, BOB promotes osteoblast differentiation of bone marrow-derived MSCs but inhibits their differentiation into adipocytes. In conclusion, our study demonstrates that BOB inhibits osteoclastogenesis and promotes osteoblastogenesis in vitro by regulating various signaling pathways. These findings suggest that BOB has potential value as a novel therapeutic agent for the prevention and treatment of osteoporosis. [BMB Reports 2023; 56(10): 545-550].

Structure and activation of the RING E3 ubiquitin ligase TRIM72 on the membrane.

Defects in plasma membrane repair can lead to muscle and heart diseases in humans. Tripartite motif-containing protein (TRIM)72 (mitsugumin 53; MG53) has been determined to rapidly nucleate vesicles at the site of membrane damage, but the underlying molecular mechanisms remain poorly understood. Here we present the structure of Mus musculus TRIM72, a complete model of a TRIM E3 ubiquitin ligase. We demonstrated that the interaction between TRIM72 and phosphatidylserine-enriched membranes is necessary for its oligomeric assembly and ubiquitination activity. Using cryogenic electron tomography and subtomogram averaging, we elucidated a higher-order model of TRIM72 assembly on the phospholipid bilayer. Combining structural and biochemical techniques, we developed a working molecular model of TRIM72, providing insights into the regulation of RING-type E3 ligases through the cooperation of multiple domains in higher-order assemblies. Our findings establish a fundamental basis for the study of TRIM E3 ligases and have therapeutic implications for diseases associated with membrane repair.

Isolation of High Purity Mouse Mesenchymal Stem Cells through Depleting Macrophages Using Liposomal Clodronate.

BACKGROUND: The use of mouse bone marrow mesenchymal stem cells (mBMSCs) represents a promising strategy for performing preclinical studies in the field of cell-based regenerative medicine; however, mBMSCs obtained via conventional isolation methods have two drawbacks, i.e., (i) they are heterogeneous due to frequent macrophage contamination, and (ii) they require long-term culturing for expansion. METHODS: In the present study, we report a novel strategy to generate highly pure mBMSCs using liposomal clodronate. This approach is based on the properties of the two cell populations, i.e., BMSCs (to adhere to the plasticware in culture dishes) and macrophages (to phagocytose liposomes). RESULTS: Liposomal clodronate added during the first passage of whole bone marrow culture was selectively engulfed by macrophages in the heterogeneous cell population, resulting in their effective elimination without affecting the MSCs. This method allowed the generation of numerous high-purity Sca-1+CD44+F4/80- mBMSCs (> 95%) with just one passaging. Comparative studies with mBMSCs obtained using conventional methods revealed that the mBMSCs obtained in the present study had remarkably improved experimental utilities, as demonstrated by in vitro multilineage differentiation and in vivo ectopic bone formation assays. CONCLUSION: Our newly developed method, which enables the isolation of mBMSCs using simple and convenient protocol, will aid preclinical studies based on the use of MSCs.

Evaluation of cellular response and drug delivery efficacy of nanoporous stainless steel material.

OBJECTIVE: Various surface modification techniques that can further improve the function and usability of stainless steel as a medical device have been reported. In the present study, the physical and biological properties of nanoporous stainless steel as well as its usefulness for drug delivery were assessed. METHODS: The specimen was prepared with a circular disk shape (15 mm in diameter and 1 mm in thickness). The disk was subjected to electropolishing at a constant voltage of 20 V and 10 A for 10 min in an acidic environment (50% H2SO4). Everolimus (EVL) was used as a testing drug for drug-loading capacity of the material surface and release kinetics. The physiobiological properties of the material were assessed using platelet adhesion, and smooth muscle cell (SMC) adhesion, migration, and proliferation assays. RESULTS: The surface roughness of the postpolishing group was greater than that of the nonpolishing group. Platelet adhesion and SMC adhesion and migration were inhibited in the postpolishing group compared to those in the prepolishing group. In the postpolishing group, the total amount of EVL on the surface (i.e., drug storage rate) was higher and the drug release rate was lower, with half the amount of the EVL released within 4 days compared with only 1 day for that of the prepolishing group. CONCLUSION: Taken together, this stainless steel with a nanoporous surface could be used as a medical device for controlling cellular responses and carrying drugs.

Resveratrol-mediated reversal of doxorubicin resistance in acute myeloid leukemia cells via downregulation of MRP1 expression.

Chemo-resistance to anti-cancer drugs is a major obstacle in efforts to develop a successful treatment of acute myeloid leukemia (AML). In this study, we investigate whether resveratrol, a common ingredient in a broad variety of fruits and vegetables, can reverse drug resistance in AML cells. Three doxorubicin-resistant AML cell lines (AML-2/DX30, AML-2/DX100, AML-2/DX300) were prepared via long-term exposure to doxorubicin for more than 3 months. DNA microarray analysis demonstrated that many genes were differentially expressed in the resistant cells, as compared with the wild type AML-2/WT cells. In particular, the expression level of the MRP1 gene was significantly increased in the AML-2/DX300 cells, as compared to that detected in AML-2 cells. Importantly, the resveratrol was shown not only to induce cell growth arrest and apoptotic death in doxorubicin-resistant AML cells, but was also shown to downregulate the expression of an MRP1 gene. Furthermore, resveratrol treatment induced a significant increase in the uptake of 5(6)-carboxyfluorescein diacetate, a MRP1 substrate, into the doxorubicin-resistant AML-2/DX300 cells. The results of this study show that resveratrol may facilitate the cellular uptake of doxorubicin via an induced downregulation of MRP1 expression, and also suggest that it may prove useful in overcoming doxorubicin resistance, or in sensitizing doxorubicin-resistant AML cells to anti-leukemic agents.

CUEDC2 controls osteoblast differentiation and bone formation via SOCS3-STAT3 pathway.

The CUE domain-containing 2 (CUEDC2) protein plays critical roles in many biological processes, such as the cell cycle, inflammation, and tumorigenesis. However, whether CUEDC2 is involved in osteoblast differentiation and plays a role in bone regeneration remains unknown. This study investigated the role of CUEDC2 in osteogenesis and its underlying molecular mechanisms. We found that CUEDC2 is expressed in bone tissues. The expression of CUEDC2 decreased during bone development and BMP2-induced osteoblast differentiation. The overexpression of CUEDC2 suppressed the osteogenic differentiation of precursor cells, while the knockdown of CUEDC2 showed the opposite effect. In vivo studies showed that the overexpression of CUEDC2 decreased bone parameters (bone volume, bone area, and bone mineral density) during ectopic bone formation, whereas its knockdown increased bone volume and the reconstruction percentage of critical-size calvarial defects. We found that CUEDC2 affects STAT3 activation by regulating SOCS3 protein stability. Treatment with a chemical inhibitor of STAT3 abolished the promoting effect of CUEDC2 silencing on osteoblast differentiation. Together, we suggest that CUEDC2 functions as a key regulator of osteoblast differentiation and bone formation by targeting the SOCS3-STAT3 pathway. CUEDC2 manipulation could serve as a therapeutic strategy for controlling bone disease and regeneration.

The primary cilium directs osteopontin-induced migration of mesenchymal stem cells by regulating CD44 signaling and Cdc42 activation.

The primary cilium acts as a sensory organelle with diverse receptors and ion channels to detect extracellular cues and regulate cellular functions, including cell migration. The migration of mesenchymal stem cells (MSCs) to bone remodeling sites is important for bone homeostasis. Recently, we have suggested that osteopontin (OPN) is a significant chemoattractant in MSC migration to bone remodeling sites. The objective of this study was to determine whether the primary cilium acts as a chemoattractant sensory unit to detect OPN cues and control MSC migration. We found that the loss of primary cilium induced by silencing of IFT88 reduced OPN-induced migration of MSCs. The effect of IFT88 silencing on cellular attachment, spreading, and proliferation was negligible. The loss of primary cilium did not affect the level of integrinß1 or CD44, two known receptors for OPN. Interestingly, CD44 was localized to the primary cilium by OPN stimulus. Knockdown of IFT88 or CD44 dysregulated OPN-induced signaling activation and abolished OPN-induced Cdc42 activation. Our findings suggest that the primary cilium acts as a chemoattractant sensor for OPN to regulate MSC migration by controlling not only CD44-mediated OPN signaling, but also Cdc42-mediated actin cytoskeleton rearrangement.

Enhanced invasiveness of drug-resistant acute myeloid leukemia cells through increased expression of matrix metalloproteinase-2.

The acquired drug resistance as well as extramedullary tissue infiltration of leukemic cells is a major obstacle in leukemia treatment. Excessive egress of leukemia cell blasts results in invasion into various organs or tissues, which is facilitated by the catalytic activities of matrix metalloproteinases (MMPs). However, the migration of chemoresistant leukemia cells remains unclear. Here, we generated drug-resistant variants of the human acute myeloid leukemia cell line (AML-2/WT) by stepwise exposure to anticancer drugs and evaluated the level of MMP-2 in the drug-resistant variants, along with their invasiveness. Each of the drug-resistant cell variants demonstrated predominant increases in the expression and gelatinolytic activity of MMP-2 as well as in invasiveness, which were significantly suppressed by both a MMP-2 inhibitor and a blocking antibody. Knockdown experiments using MMP-2 short hairpin RNA also indicated that its upregulation was strongly associated with the cells' increased invasive properties. Importantly, elevated levels of MMP-2 activity and invasiveness were observed in ex vivo mononuclear cell of bone marrow from patients with poor responses to chemotherapy. These findings suggest that advanced malignancy due to acquired drug resistance is responsible for the progressive invasiveness of leukemia cells via MMP-2.

Defective expression of deoxycytidine kinase in cytarabine-resistant acute myeloid leukemia cells.

Resistance to cytarabine (Ara-C) incapacitates the therapeutic effort during the treatment of acute myeloid leukemia (AML). To elucidate mechanism responsible for the development of resistance to Ara-C, we established the Ara-C resistant AML-2/WT cell sublines, AML-2/IDAC and AML-2/ARC. We then conducted DNA microarray analysis to compare the AML-2/IDAC cells with parental AML-2/WT cells. The results of the microarray analysis revealed a severe defect in the expression of deoxycytidine kinase (dCK), which plays a key role in the transformation of Ara-C to the active form in AML-2/IDAC cells. A similar event was observed in AML-2/ARC cells, but not in Ara-C sensitive AML-2/IDA cells that were resistant to idarubicin. The decreased expression of dCK also resulted in lower activity in both Ara-C resistant variants. However, no significant difference in the intracellular concentration of Ara-C was observed among the cells tested, which indicates that the Ara-C resistant phenotype in our models occurred due to the lower expression and activity of dCK rather than a change in the ability to take up Ara-C. Additionally, in vitro assays using BM cells from AML patients revealed that the expression of dCK and the sensitivity to Ara-C were correlated. Taken together, these findings demonstrate that dCK can regulate the in vitro cellular response to Ara-C in AML cells.

Alleviation of the drug-resistant phenotype in idarubicin and cytosine arabinoside double-resistant acute myeloid leukemia cells by indomethacin.

Chemoresistance to anticancer drugs is a major issue in the successful treatment of acute myeloid leukemia (AML). In this study, we developed an AML cell line (AML-2/IDAC) that is resistant to treatment with a combination of idarubicin and cytosine arabinoside (Id/AraC) by chronic exposure for more than 3 months. We then investigated the ability of indomethacin to alleviate the chemoresistance of AML-2/IDAC cells. Treatment with indomethacin alone induced growth arrest, but not the death of AML-2/IDAC cells. However, when AML-2/IDAC cells were treated with combinations of indomethacin and Id/AraC, the cell death and apoptosis rate of AML-2/IDAC cells were significantly increased in a dose- and time-dependent manner. The combined treatment with indomethacin and Id/AraC caused the collapse of the mitochondrial membrane potential and was also demonstrated to enhance the activities of caspase-3 and -8 in AML-2/IDAC cells. Furthermore, indomethacin down-regulated expression of the ABCA3 and MRP1 genes, which were over-expressed in AML-2/IDAC cells. Taken together, the results of this study suggest that indomethacin can be used to increase the therapeutic potential against drug-resistant AML when combined with anti-leukemic drugs.

Indeno[1,2-c]isoquinolines as enhancing agents on all-trans retinoic acid-mediated differentiation of human myeloid leukemia cells.

Induction of differentiation is a new and promising approach to cancer therapy, well illustrated by the treatment of acute myeloid leukemia with all-trans retinoic acid (ATRA). Using combination of ATRA and chemotherapy, adverse effects such as retinoic acid syndrome have decreased, and long-term survival has improved. In this study, we demonstrated that the indeno[1,2-c]isoquinolines markedly enhanced differentiation of human myeloid leukemia HL-60 and NB4 cells when simultaneously combined with a low dose of ATRA. Of the tested compounds, 6-(4-methoxybenzyl)-2,11-dimethyl-6H,11H-indeno[1,2-c]isoquinolin-5-one (IIQ-16), an indeno[1,2-c]isoquinoline derivative, showed the highest differentiation-enhancing activity via a pathway involved with protein kinase C, extracellular signal-regulated kinase, and c-Jun N-terminal kinase. The ability to enhance the differentiation potential of ATRA by IIQ-16 may improve outcomes in the therapy of acute promyelocytic leukemia.

Elevated extracellular calcium ions promote proliferation and migration of mesenchymal stem cells via increasing osteopontin expression.

Supplementation of mesenchymal stem cells (MSCs) at sites of bone resorption is required for bone homeostasis because of the non-proliferation and short lifespan properties of the osteoblasts. Calcium ions (Ca2+) are released from the bone surfaces during osteoclast-mediated bone resorption. However, how elevated extracellular Ca2+ concentrations would alter MSCs behavior in the proximal sites of bone resorption is largely unknown. In this study, we investigated the effect of extracellular Ca2+ on MSCs phenotype depending on Ca2+ concentrations. We found that the elevated extracellular Ca2+ promoted cell proliferation and matrix mineralization of MSCs. In addition, MSCs induced the expression and secretion of osteopontin (OPN), which enhanced MSCs migration under the elevated extracellular Ca2+ conditions. We developed in vitro osteoclast-mediated bone resorption conditions using mouse calvaria bone slices and demonstrated Ca2+ is released from bone resorption surfaces. We also showed that the MSCs phenotype, including cell proliferation and migration, changed when the cells were treated with a bone resorption-conditioned medium. These findings suggest that the dynamic changes in Ca2+ concentrations in the microenvironments of bone remodeling surfaces modulate MSCs phenotype and thereby contribute to bone regeneration.

Enhancing effects of ceramide derivatives on 1,25-dihydroxyvitamin D(3)-induced differentiation of human HL-60 leukemia cells.

Differentiation-inducing therapy by agents such as 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] represents a useful approach for the treatment for cancer, including acute myeloid leukemia (AML). Recent studies demonstrated that the combined administration of 1,25-(OH)(2)D(3) and differentiation-enhancing agents could alleviate the side effects of 1,25-(OH)(2)D(3) and improve the rate of long term survival. In this study, we determined the enhancing activities of ceramide derivatives on 1,25-(OH)(2)D(3)-induced differentiation of human myeloid leukemia HL-60 cells. Importantly, some of these derivatives -- namely, A2, B3, and H9 -- enhanced the 1,25-(OH)(2)D(3)-induced differentiation of HL-60 cells in a concentration-dependent manner. In addition, the morphologic studies using Giemsa staining and flow cytometric analysis demonstrated that the combined treatment of 1,25-(OH)(2)D(3) with one of the three analogues, A2, B3, and H9, directed the HL-60 cells into monocytic lineage, but not into granulocytic lineage. The inhibition studies demonstrated that A2, B3, and H9, enhanced 1,25-(OH)(2)D(3)-induced differentiation of HL-60 cells via the PI3-K/PKC/JNK/ERK pathways. The ability of ceramide derivatives to enhance the differentiation-inducing potential of 1,25-(OH)(2)D(3) may contribute to an effective therapy for AML.

The 1:2 complex between RavZ and LC3 reveals a mechanism for deconjugation of LC3 on the phagophore membrane.

Hosts utilize macroautophagy/autophagy to clear invading bacteria; however, bacteria have also developed a specific mechanism to survive by manipulating the host cell autophagy mechanism. One pathogen, Legionella pneumophila, can hinder host cell autophagy by using the specific effector protein RavZ that cleaves phosphatidylethanolamine-conjugated LC3 on the phagophore membrane. However, the detailed molecular mechanisms associated with the function of RavZ have hitherto remained unclear. Here, we report on the biochemical characteristics of the RavZ-LC3 interaction, the solution structure of the 1:2 complex between RavZ and LC3, and crystal structures of RavZ showing different conformations of the active site loop without LC3. Based on our biochemical, structural, and cell-based analyses of RavZ and LC3, both distant flexible N- and C-terminal regions containing LC3-interacting region (LIR) motifs are important for substrate recognition. These results suggest a novel mechanism of RavZ action on the phagophore membrane and lay the groundwork for understanding how bacterial pathogens can survive autophagy.