A Crosstalk between the Receptor Tyrosine Kinase-Like Orphan Receptors ROR1/2 and S1P Signaling Pathways in Lung Cancer.

OBJECTIVE: Availability of multimodal treatment strategies, including targeted therapies and immunotherapies, have improved the survival of non-small cell lung carcinoma (NSCLC). However, some patients still progress or respond poorly due to inherent resistance, acquired resistance, or lack of druggable driver mutations. Sphingosine-1-phosphate (S1P) and receptor tyrosine kinase-like orphan receptor (ROR1/2) signaling pathways are activated during lung carcinogenesis. METHODS: In this study, we have evaluated the crosstalk of S1P and ROR1/2 signaling pathways in lung cancer cells. RESULTS: S1P treatment of lung cancer cells decreases ROR1 and ROR2 transcript levels. While treatment with PF-543, a pharmacological SphK1 inhibitor or genetic knockdown of SPHK1 by shRNA, raises ROR1 and ROR2. Furthermore, simultaneous inhibition of SphK1 along with ROR1 reduced the migration of lung cancer cells. CONCLUSION: These findings demonstrate the reciprocal regulation of both pathways, suggesting that both pathways have an inverse relation i.e, in the absence of one pathway, another pathway may take charge of the other pathway. Therefore, simultaneously targeting both pathways could serve as a potential therapeutic target for lung cancer treatment.

Plasma S1P Orchestrates the Reverse Transendothelial Migration of Aortic Intimal Myeloid Cells in Mice.

BACKGROUND: Myeloid cells (MCs) reside in the aortic intima at regions predisposed to atherosclerosis. Systemic inflammation triggers reverse transendothelial migration (RTM) of intimal MCs into the arterial blood, which orchestrates a protective immune response that clears intracellular pathogens from the arterial intima. Molecular pathways that regulate RTM remain poorly understood. S1P (sphingosine-1-phosphate) is a lipid mediator that regulates immune cell trafficking by signaling via 5 G-protein-coupled receptors (S1PRs [S1P receptors]). We investigated the role of S1P in the RTM of aortic intimal MCs. METHODS: Intravenous injection of lipopolysaccharide was used to model a systemic inflammatory stimulus that triggers RTM. CD11c+ intimal MCs in the lesser curvature of the ascending aortic arch were enumerated by en face confocal microscopy. Local gene expression was evaluated by transcriptomic analysis of microdissected intimal cells. RESULTS: In wild-type C57BL/6 mice, lipopolysaccharide induced intimal cell expression of S1pr1, S1pr3, and Sphk1 (a kinase responsible for S1P production). Pharmacological modulation of multiple S1PRs blocked lipopolysaccharide-induced RTM and modulation of S1PR1 and S1PR3 reduced RTM in an additive manner. Cre-mediated deletion of S1pr1 in MCs blocked lipopolysaccharide-induced RTM, confirming a role for myeloid-specific S1PR1 signaling. Global or hematopoietic deficiency of Sphk1 reduced plasma S1P levels, the abundance of CD11c+ MCs in the aortic intima, and blunted lipopolysaccharide-induced RTM. In contrast, plasma S1P levels, the abundance of intimal MCs, and lipopolysaccharide-induced RTM were rescued in Sphk1-/- mice transplanted with Sphk1+/+ or mixed Sphk1+/+ and Sphk1-/- bone marrow. Stimulation with lipopolysaccharide increased endothelial permeability and intimal MC exposure to circulating factors such as S1P. CONCLUSIONS: Functional and expression studies support a novel role for S1P signaling in the regulation of lipopolysaccharide-induced RTM and the homeostatic maintenance of aortic intimal MCs. Our data provide insight into how circulating plasma mediators help orchestrate intimal MC dynamics.

Substrate promiscuity of inositol 1,4,5-trisphosphate kinase driven by structurally-modified ligands and active site plasticity.

D-myo-inositol 1,4,5-trisphosphate (InsP3) is a fundamental second messenger in cellular Ca2+ mobilization. InsP3 3-kinase, a highly specific enzyme binding InsP3 in just one mode, phosphorylates InsP3 specifically at its secondary 3-hydroxyl group to generate a tetrakisphosphate. Using a chemical biology approach with both synthetised and established ligands, combining synthesis, crystallography, computational docking, HPLC and fluorescence polarization binding assays using fluorescently-tagged InsP3, we have surveyed the limits of InsP3 3-kinase ligand specificity and uncovered surprisingly unforeseen biosynthetic capacity. Structurally-modified ligands exploit active site plasticity generating a helix-tilt. These facilitated uncovering of unexpected substrates phosphorylated at a surrogate extended primary hydroxyl at the inositol pseudo 3-position, applicable even to carbohydrate-based substrates. Crystallization experiments designed to allow reactions to proceed in situ facilitated unequivocal characterization of the atypical tetrakisphosphate products. In summary, we define features of InsP3 3-kinase plasticity and substrate tolerance that may be more widely exploitable.

Sphingolipid metabolism controls mammalian heart regeneration.

Utilization of lipids as energy substrates after birth causes cardiomyocyte (CM) cell-cycle arrest and loss of regenerative capacity in mammalian hearts. Beyond energy provision, proper management of lipid composition is crucial for cellular and organismal health, but its role in heart regeneration remains unclear. Here, we demonstrate widespread sphingolipid metabolism remodeling in neonatal hearts after injury and find that SphK1 and SphK2, isoenzymes producing the same sphingolipid metabolite sphingosine-1-phosphate (S1P), differently regulate cardiac regeneration. SphK2 is downregulated during heart development and determines CM proliferation via nuclear S1P-dependent modulation of histone acetylation. Reactivation of SphK2 induces adult CM cell-cycle re-entry and cytokinesis, thereby enhancing regeneration. Conversely, SphK1 is upregulated during development and promotes fibrosis through an S1P autocrine mechanism in cardiac fibroblasts. By fine-tuning the activity of each SphK isoform, we develop a therapy that simultaneously promotes myocardial repair and restricts fibrotic scarring to regenerate the infarcted adult hearts.

Loss of Pip4k2c confers liver-metastatic organotropism through insulin-dependent PI3K-AKT pathway activation.

Liver metastasis (LM) confers poor survival and therapy resistance across cancer types, but the mechanisms of liver-metastatic organotropism remain unknown. Here, through in vivo CRISPR-Cas9 screens, we found that Pip4k2c loss conferred LM but had no impact on lung metastasis or primary tumor growth. Pip4k2c-deficient cells were hypersensitized to insulin-mediated PI3K/AKT signaling and exploited the insulin-rich liver milieu for organ-specific metastasis. We observed concordant changes in PIP4K2C expression and distinct metabolic changes in 3,511 patient melanomas, including primary tumors, LMs and lung metastases. We found that systemic PI3K inhibition exacerbated LM burden in mice injected with Pip4k2c-deficient cancer cells through host-mediated increase in hepatic insulin levels; however, this circuit could be broken by concurrent administration of an SGLT2 inhibitor or feeding of a ketogenic diet. Thus, this work demonstrates a rare example of metastatic organotropism through co-optation of physiological metabolic cues and proposes therapeutic avenues to counteract these mechanisms.

N-terminal acetyltransferase 6 facilitates enterovirus 71 replication by regulating PI4KB expression and replication organelle biogenesis.

Enterovirus 71 (EV71) is one of the major pathogens causing hand, foot, and mouth disease in children under 5 years old, which can result in severe neurological complications and even death. Due to limited treatments for EV71 infection, the identification of novel host factors and elucidation of mechanisms involved will help to counter this viral infection. N-terminal acetyltransferase 6 (NAT6) was identified as an essential host factor for EV71 infection with genome-wide CRISPR/Cas9 screening. NAT6 facilitates EV71 viral replication depending on its acetyltransferase activity but has little effect on viral release. In addition, NAT6 is also required for Echovirus 7 and coxsackievirus B5 infection, suggesting it might be a pan-enterovirus host factor. We further demonstrated that NAT6 is required for Golgi integrity and viral replication organelle (RO) biogenesis. NAT6 knockout significantly inhibited phosphatidylinositol 4-kinase IIIß (PI4KB) expression and PI4P production, both of which are key host factors for enterovirus infection and RO biogenesis. Further mechanism studies confirmed that NAT6 formed a complex with its substrate actin and one of the PI4KB recruiters-acyl-coenzyme A binding domain containing 3 (ACBD3). Through modulating actin dynamics, NAT6 maintained the integrity of the Golgi and the stability of ACBD3, thereby enhancing EV71 infection. Collectively, these results uncovered a novel mechanism of N-acetyltransferase supporting EV71 infection.IMPORTANCEEnterovirus 71 (EV71) is an important pathogen for children under the age of five, and currently, no effective treatment is available. Elucidating the mechanism of novel host factors supporting viral infection will reveal potential antiviral targets and aid antiviral development. Here, we demonstrated that a novel N-acetyltransferase, NAT6, is an essential host factor for EV71 replication. NAT6 could promote viral replication organelle (RO) formation to enhance viral replication. The formation of enterovirus ROs requires numerous host factors, including acyl-coenzyme A binding domain containing 3 (ACBD3) and phosphatidylinositol 4-kinase IIIß (PI4KB). NAT6 could stabilize the PI4KB recruiter, ACBD3, by inhibiting the autophagy degradation pathway. This study provides a fresh insight into the relationship between N-acetyltransferase and viral infection.

Novel PNKP mutations associated with reduced DNA single-strand break repair and severe microcephaly, seizures, and developmental delay.

BACKGROUND: Microcephaly with early-onset seizures (MCSZ) is a neurodevelopmental disorder caused by pathogenic variants in the DNA strand break repair protein, polynucleotide kinase 3'-phosphatase (PNKP). METHODS: We have used whole genome sequencing and Sanger sequencing to identify disease-causing variants, followed by a minigene assay, Western blotting, alkaline comet assay, γH2AX, and ADP-ribose immunofluorescence. RESULTS: Here, we describe a patient with compound heterozygous variants in PNKP, including a missense variant in the DNA phosphatase domain (T323M) and a novel splice acceptor site variant within the DNA kinase domain that we show leads to exon skipping. We show that primary fibroblasts derived from the patient exhibit greatly reduced levels of PNKP protein and reduced rates of DNA single-strand break repair, confirming that the mutated PNKP alleles are dysfunctional. CONCLUSION: The data presented show that the detected compound heterozygous variants result in reduced levels of PNKP protein, which affect the repair of both oxidative and TOP1-induced single-strand breaks, and most likely causes MCSZ in this patient.

The accumulation of sphingosine kinase 2 disrupts the DNA damage response and promotes resistance to genotoxic agents.

There is a known correlation between cancer and changes in sphingolipids, specifically the production of sphingosine-1-phosphate (S1P) by sphingosine kinases 1 and 2 (SPHK1/SPHK2). However, a potential relationship between bioactive lipid molecules, SPHK, and the response to DNA damage is unknown. We aimed to evaluate the response of oral keratinocytes and head and neck cancer cell lines with SPHK2/S1P alterations to DNA damage. This assessment is intended to establish a link between these alterations and tumorigenesis as well as resistance to therapy. SPHK2 overexpression in oral squamous cells promoted evasion of apoptosis and cell cycle control, which increased the resistance to genotoxic agents (chemical and physical). Cells that have SPHK2 overexpression are more prone to DNA damage, which allows those damaged cells to survive and multiply. This is associated with a decrease in overall DNA methylation. These discoveries help to clarify the connection between SPHK2 and the response to DNA damage, as well as its capacity to aid in the resistance against genotoxic agents, including those used in cancer treatment.

Interaction of ROMK2 channel with lipid kinases DGKE and AGK: Potential channel activation by localized anionic lipid synthesis.

In this study, we utilized enzyme-catalyzed proximity labeling with the engineered promiscuous biotin ligase Turbo-ID to identify the proxisome of the ROMK2 channel. This channel resides in various cellular membrane compartments of the cell including the plasma membrane, endoplasmic reticulum and mitochondria. Within mitochondria, ROMK2 has been suggested as a pore-forming subunit of mitochondrial ATP-regulated potassium channel (mitoKATP). We found that ROMK2 proxisome in addition to previously known protein partners included two lipid kinases: acylglycerol kinase (AGK) and diacylglycerol kinase ε (DGKE), which are localized in mitochondria and the endoplasmic reticulum, respectively. Through co-immunoprecipitation, we confirmed that these two kinases are present in complexes with ROMK2 channels. Additionally, we found that the products of AGK and DGKE, lysophosphatidic acid (LPA) and phosphatidic acid (PA), stimulated the activity of ROMK2 channels in artificial lipid bilayers. Our molecular docking studies revealed the presence of acidic lipid binding sites in the ROMK2 channel, similar to those previously identified in Kir2 channels. Based on these findings, we propose a model wherein localized lipid synthesis, mediated by channel-bound lipid kinases, contributes to the regulation of ROMK2 activity within distinct intracellular compartments, such as mitochondria and the endoplasmic reticulum.

A novel homozygous variant in PMVK is associated with enhanced IL1ß secretion and a hyper-IgD syndrome-like phenotype.

The evolutionarily conserved mevalonate pathway plays an important role in the synthesis of cholesterol and isoprenoid compounds. Mevalonate kinase (MVK) and phosphomevalonate kinase (PMVK) enzymes regulate key rate-limiting steps in this pathway by sequentially phosphorylating mevalonic acid to yield downstream metabolites that regulate protein prenylation and cell signaling. Biallelic pathogenic variants in MVK cause a spectrum of rare autoinflammatory disorders that encompass milder forms of hyper-IgD syndrome (HIDS) at one end and the more severe mevalonic aciduria on the other. In contrast, pathogenic variants reported in PMVK are heterozygous and associated with porokeratosis, a skin disorder with no systemic manifestations. Recently, biallelic variants in PMVK were reported as a cause for an autoinflammatory disorder for the first time in two unrelated patients. In this study, we describe a child with recurrent arthritis and a HIDS-like phenotype harboring a novel homozygous variant c.398 C>T (p.Ala133Val) in PMVK. Mononuclear cells isolated from the patient showed significantly elevated production of interleukin 1ß, a key cytokine that shapes the inflammatory response in HIDS. Protein modeling studies suggested potential defects in PMVK enzyme activity. These results posit a further expanding of the genotypic spectrum of autoinflammatory disease to include biallelic PMVK variants.

Screening fructosamine-3-kinase (FN3K) inhibitors, a deglycating enzyme of oncogenic Nrf2: Human FN3K homology modelling, docking and molecular dynamics simulations.

Fructosamine-3-kinase (FN3K) is involved in the deglycation of Nrf2, a significant regulator of oxidative stress in cancer cells. However, the intricate functional aspects of FN3K and Nrf2 in breast cancers have not been explored vividly. The objectives of this study are to design the human FN3K protein using homology modeling followed by the screening of several anticancer molecules and examining their efficacy to modulate FN3K activity, Nrf2-mediated antioxidant signalling. Methods pertinent to homology modeling, virtual screening, molecular docking, molecular dynamics simulations, assessment of ADME properties, cytotoxicity assays for anticancer molecules of natural/synthetic origin in breast cancer cells (BT-474, T-47D), and Western blotting were used in this study. The screened anticancer molecules including kinase inhibitors of natural and synthetic origin interacted with the 3-dimensional structure of the catalytic domain in human FN3K protein designed through homology modeling by significant CDOCKER interaction energies. Subsequently, gefitinib, sorafenib, neratinib, tamoxifen citrate, and cyclosporine A enhanced the expression of FN3K in BT-474 cell lines with simultaneous alteration in Nrf2-driven antioxidant signalling. Oxaliplatin significantly downregulated FN3K expression and modulated Nrf2-driven antioxidant signalling when compared to cisplatin and other anticancer drugs. Hence, the study concluded the potential implications of existing anticancer drugs to modulate FN3K activity in breast cancers.

Structural basis for the conserved roles of PI4KA and its regulatory partners and their misregulation in disease.

The type III Phosphatidylinositol 4-kinase alpha (PI4KA) is an essential lipid kinase that is a master regulator of phosphoinositide signalling at the plasma membrane (PM). It produces the predominant pool of phosphatidylinositol 4-phosphate (PI4P) at the PM, with this being essential in lipid transport and in regulating the PLC and PI3K signalling pathways. PI4KA is essential and is highly conserved in all eukaryotes. In yeast, the PI4KA ortholog stt4 predominantly exists as a heterodimer with its regulatory partner ypp1. In higher eukaryotes, PI4KA instead primarily forms a heterotrimer with a TTC7 subunit (ortholog of ypp1) and a FAM126 subunit. In all eukaryotes PI4KA is recruited to the plasma membrane by the protein EFR3, which does not directly bind PI4KA, but instead binds to the TTC7/ypp1 regulatory partner. Misregulation in PI4KA or its regulatory partners is involved in myriad human diseases, including loss of function mutations in neurodevelopmental and inflammatory intestinal disorders and gain of function in human cancers. This review describes an in-depth analysis of the structure function of PI4KA and its regulatory partners, with a major focus on comparing and contrasting the differences in regulation of PI4KA throughout evolution.

Investigating potential of cholic acid, syringic acid, and mangiferin as cancer therapeutics through sphingosine kinase 1 inhibition.

The signaling of sphingosine kinase 1 (SphK1) and sphingosine-1-phosphate (S1P) regulates various diseases, including multiple sclerosis, atherosclerosis, rheumatoid arthritis, inflammation-related ailments, diabetes, and cancer. SphK1 is considered an attractive potential drug target and is extensively explored in cancer and other inflammatory diseases. In this study, we have investigated the inhibitory potential and binding affinity of SphK1 with cholic acid (CA), syringic acid (SA), and mangiferin (MF) using a combination of docking and molecular dynamics (MD) simulation studies followed by experimental measurements of binding affinity and enzyme inhibition assays. We observed these compounds bind to SphK1 with a significantly high affinity and eventually inhibit its kinase activity with IC50 values of 28.23 µM, 33.35 µM, and 57.2 µM for CA, SA, and MF, respectively. Further, the docking and 100 ns MD simulation studies showed that CA, SA, and MF bind with the active site residues of SphK1 with favorable energy and strong non-covalent interactions that might be accountable for inhibiting its kinase activity. Our finding indicates that CA, SA, and MF may be implicated in designing novel anti-cancer therapeutics with an improved affinity and lesser side effects by targeting SphK1.

Inositol polyphosphate multikinase modulates free fatty acids-induced insulin resistance in primary mouse hepatocytes.

Insulin resistance is a critical mediator of the development of nonalcoholic fatty liver disease (NAFLD). An excess influx of fatty acids to the liver is thought to be a pathogenic cause of insulin resistance and the development of NAFLD. Although elevated levels of free fatty acids (FFA) in plasma contribute to inducing insulin resistance and NAFLD, the molecular mechanism is not completely understood. This study aimed to determine whether inositol polyphosphate multikinase (IPMK), a regulator of insulin signaling, plays any role in FFA-induced insulin resistance in primary hepatocytes. Here, we show that excess FFA decreased IPMK expression, and blockade of IPMK decrease attenuated the FFA-induced suppression of protein kinase B (Akt) phosphorylation in primary mouse hepatocytes (PMH). Moreover, overexpression of IPMK prevented the FFA-induced suppression of Akt phosphorylation by insulin, while knockout of IPMK exacerbated insulin resistance in PMH. In addition, treatment with MG132, a proteasomal inhibitor, inhibits FFA-induced decrease in IPMK expression and Akt phosphorylation in PMH. Furthermore, treatment with the antioxidant N-acetyl cysteine (NAC) significantly attenuated the FFA-induced reduction of IPMK and restored FFA-induced insulin resistance in PMH. In conclusion, our findings suggest that excess FFA reduces IPMK expression and contributes to the FFA-induced decrease in Akt phosphorylation in PMH, leading to insulin resistance. Our study highlights IPMK as a potential therapeutic target for preventing insulin resistance and NAFLD.

Targeting sphingosine kinase 1 in p53KO thymic lymphoma.

Sphingosine kinase 1 (SK1) is a key sphingolipid enzyme that is upregulated in several types of cancer, including lymphoma which is a heterogenous group of malignancies. Treatment for lymphoma has improved significantly by the introduction of new therapies; however, subtypes with tumor protein P53 (p53) mutations or deletion have poor prognosis, making it critical to explore new therapeutic strategies in this context. SK1 has been proposed as a therapeutic target in different types of cancer; however, the effect of targeting SK1 in cancers with p53 deletion has not been evaluated yet. Previous work from our group suggests that loss of SK1 is a key event in mediating the tumor suppressive effect of p53. Employing both genetic and pharmacological approaches to inhibit SK1 function in Trp53KO mice, we show that targeting SK1 decreases tumor growth of established p53KO thymic lymphoma. Inducible deletion of Sphk1 or its pharmacological inhibition drive increased cell death in tumors which is accompanied by selective accumulation of sphingosine levels. These results demonstrate the relevance of SK1 in the growth and maintenance of lymphoma in the absence of p53 function, positioning this enzyme as a potential therapeutic target for the treatment of tumors that lack functional p53.

α-Synuclein-dependent increases in PIP5K1γ drive inositol signaling to promote neurotoxicity.

Anomalous aggregation of α-synuclein (α-Syn) is a pathological hallmark of many degenerative synucleinopathies including Lewy body dementia (LBD) and Parkinson's disease (PD). Despite its strong link to disease, the precise molecular mechanisms that link α-Syn aggregation to neurodegeneration have yet to be elucidated. Here, we find that elevated α-Syn leads to an increase in the plasma membrane (PM) phosphoinositide PI(4,5)P2, which precipitates α-Syn aggregation and drives toxic increases in mitochondrial Ca2+ and reactive oxygen species leading to neuronal death. Upstream of this toxic signaling pathway is PIP5K1γ, whose abundance and localization is enhanced at the PM by α-Syn-dependent increases in ARF6. Selective inhibition of PIP5K1γ or knockout of ARF6 in neurons rescues α-Syn aggregation and cellular phenotypes of toxicity. Collectively, our data suggest that modulation of phosphoinositide metabolism may be a therapeutic target to slow neurodegeneration for PD and other related neurodegenerative disorders.

Lipid kinase PIP5K1A regulates let-7 microRNA biogenesis through interacting with nuclear export protein XPO5.

MicroRNAs (miRNAs) are small non-coding RNAs first discovered in Caenorhabditis elegans. The let-7 miRNA is highly conserved in sequence, biogenesis and function from C. elegans to humans. During miRNA biogenesis, XPO5-mediated nuclear export of pre-miRNAs is a rate-limiting step and, therefore, might be critical for the quantitative control of miRNA levels, yet little is known about how this is regulated. Here we show a novel role for lipid kinase PPK-1/PIP5K1A (phosphatidylinositol-4-phosphate 5-kinase) in regulating miRNA levels. We found that C. elegans PPK-1 functions in the lin-28/let-7 heterochronic pathway, which regulates the strict developmental timing of seam cells. In C. elegans and human cells, PPK-1/PIP5K1A regulates let-7 miRNA levels. We investigated the mechanism further in human cells and show that PIP5K1A interacts with nuclear export protein XPO5 in the nucleus to regulate mature miRNA levels by blocking the binding of XPO5 to pre-let-7 miRNA. Furthermore, we demonstrate that this role for PIP5K1A is kinase-independent. Our study uncovers the novel finding of a direct connection between PIP5K1A and miRNA biogenesis. Given that miRNAs are implicated in multiple diseases, including cancer, this new finding might lead to a novel therapeutic opportunity.

ATF3 regulates SPHK1 in cardiomyocyte injury via endoplasmic reticulum stress.

AIM: Endoplasmic reticulum (ER) stress is common in different human pathologies, including cardiac diseases. Sphingosine kinase-1 (SPHK1) represents an important player in cardiac growth and function. Nevertheless, its function in cardiomyocyte ER stress remains vague. This study sought to evaluate the mechanism through which SPHK1 might influence ER stress during myocardial infarction (MI). METHODS: MI-related GEO data sets were queried to screen differentially expressed genes. Murine HL-1 cells exposed to oxygen-glucose deprivation (OGD) and mice with MI were induced, followed by gene expression manipulation using short hairpin RNAs and overexpression vectors. The activating transcription factor 3 (ATF3) and SPHK1 expression was examined in cells and tissues. Cell counting kit-8, TUNEL, DHE, HE, and Masson's staining were conducted in vitro and in vivo. The inflammatory factor concentrations in mouse serum were measured using ELISA. Finally, the transcriptional regulation of SPHK1 by ATF3 was validated. RESULTS: ATF3 and SPHK1 were upregulated in vivo and in vitro. ATF3 downregulation reduced the SPHK1 transcription. ATF3 and SPHK1 downregulation increased the viability of OGD-treated HL-1 cells and decreased apoptosis, oxidative stress, and ER stress. ATF3 and SPHK1 downregulation narrowed the infarction area and attenuated myocardial fibrosis in mice, along with reduced inflammation in the serum and ER stress in the myocardium. In contrast, SPHK1 reduced the protective effect of ATF3 downregulation in vitro and in vivo. CONCLUSIONS: ATF3 downregulation reduced SPHK1 expression to attenuate cardiomyocyte injury in MI.

Sphingosine kinase 1 is involved in triglyceride breakdown by maintaining lysosomal integrity in brown adipocytes.

Sphingosine 1-phosphate (S1P) has been implicated in brown adipose tissue (BAT) formation and energy consumption; however, the mechanistic role of sphingolipids, including S1P, in BAT remains unclear. Here, we showed that, in mice, BAT activation by cold exposure upregulated mRNA and protein expression of the S1P-synthesizing enzyme sphingosine kinase 1 (SphK1) and S1P production in BAT. Treatment of wild-type brown adipocytes with exogenous S1P or S1P receptor subtype-selective agonists stimulated triglyceride (TG) breakdown only marginally, compared with noradrenaline. However, genetic deletion of Sphk1 resulted in hypothermia and diminished body weight loss upon cold exposure, suggesting that SphK1 is involved in thermogenesis through mechanisms different from receptor-mediated, extracellular action of S1P. In BAT of wild-type mice, SphK1 was localized largely in the lysosomes of brown adipocytes. In the brown adipocytes of Sphk1-/- mice, the number of lysosomes was reduced and lysosomal function, including proteolytic activity, acid esterase activity, and motility, was impaired. Concordantly, nuclear translocation of transcription factor EB, a master transcriptional regulator of lysosome biogenesis, was reduced, leading to decreased mRNA expression of the lysosome-related genes in Sphk1-/- BAT. Moreover, BAT of Sphk1-/- mice showed greater TG accumulation with dominant larger lipid droplets in brown adipocytes. Inhibition of lysosomes with chloroquine resulted in a less extent of triglyceride accumulation in Sphk1-/- brown adipocytes compared with wild-type brown adipocytes, suggesting a reduced lysosome-mediated TG breakdown in Sphk1-/- mice. Our results indicate a novel role of SphK1 in lysosomal integrity, which is required for TG breakdown and thermogenesis in BAT.

Design, synthesis, antitumor activity, and molecular dynamics simulations of novel sphingosine kinase 2 inhibitors.

Targeting sphingosine kinase 2 (SphK2) has become a novel strategy for the treatment of cancer. However, potent and selective SphK2 inhibitors are rare. In our work, a series of novel SphK2 inhibitors were innovatively designed, synthesized and screened. Compound 12e showed the best inhibitory activity. Molecular dynamics simulations were carried out to analyze the detailed interactions between the SphK2 and its inhibitors. Moreover, 12e exhibited anti-proliferative activity in various cancer cells, and inhibited the migration of human breast cancer cells MCF-7.