Anoctamin pharmacology.

TMEM16 proteins, also known as anoctamins, are a family of ten membrane proteins with various tissue expression and subcellular localization. TMEM16A (anoctamin 1) is a plasma membrane protein that acts as a calcium-activated chloride channel. It is expressed in many types of epithelial cells, smooth muscle cells and some neurons. In airway epithelial cells, TMEM16A expression is particularly enhanced by inflammatory stimuli that also promote goblet cell metaplasia and mucus hypersecretion. Therefore, pharmacological modulation of TMEM16A could be beneficial to improve mucociliary clearance in chronic obstructive respiratory diseases. However, the correct approach to modulate TMEM16A activity (activation or inhibition) is still debated. Pharmacological inhibitors of TMEM16A could also be useful as anti-hypertensive agents given the TMEM16A role in smooth muscle contraction. In contrast to TMEM16A, TMEM16F (anoctamin 6) behaves as a calcium-activated phospholipid scramblase, responsible for the externalization of phosphatidylserine on cell surface. Inhibitors of TMEM16F could be useful as anti-coagulants and anti-viral agents. The role of other anoctamins as therapeutic targets is still unclear since their physiological role is still to be defined.

Resveratrol alleviates acute lung injury through regulating PLSCR-3-mediated mitochondrial dysfunction and mitophagy in a cecal ligation and puncture model.

Sepsis is considered as a life-threatening organ dysfunction caused by a dysregulated response of the host to an infection. Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is a life-threatening condition, and is the type of organ injury that is most commonly induced by sepsis. Resveratrol (RSV) has been shown to exert a wide range of therapeutic effects due to its anti-inflammatory and anti-oxidant properties. The present study aimed to investigate whether RSV could mitigate sepsis-induced ALI/ARDS, and also to unravel the underlying mechanism. The model of sepsis was established by applying the cecal ligation and puncture (CLP) method, and mitochondria from the lung tissue were isolated to assess mitochondrial function, as determined from measuring mitochondrial superoxide production using MitoSOX red mitochondrial superoxide indicator and the membrane potential. It was found that RSV could exert a protective role in CLP-induced ALI/ARDS, as evidenced by moderate levels of inflammatory cell infiltration and interstitial edema, as well as decreased levels of C-reactive protein (P<0.01), interleukin (IL)-6 (P<0.01), IL-1ß (P<0.01) and tumor necrosis factor-α (P<0.01). Moreover, phospholipid scramblase 3 (PLSCR-3)-mediated mitochondrial dysfunction and mitophagy were shown to contribute towards the CLP-caused lung damage, which was reversed upon RSV administration, as demonstrated by improved mitochondrial function and markedly reduced increases in the protein levels of autophagy related (ATG)5 (P<0.01), ATG7 (P<0.05) and microtubule-associated protein 1A/1B-light chain 3 (LC3-Ⅰ/Ⅱ) (P<0.01), and a significantly increased expression of P62 (P<0.05). In addition, with regard to the CLP-induced lung injury in the mouse model, overexpression of PLSCR-3 was found to remove the beneficial effects observed upon RSV treatment. Taken together, the results of the present study have uncovered a novel molecular mechanism through which RSV may alleviate ALI/ARDS via regulating PLSCR-3-mediated mitochondrial dysfunction and mitophagy in CLP-induced mouse model.

ANO9 regulates PD-L2 expression and binding ability to PD-1 in gastric cancer.

The function of ANO9 in gastrointestinal cancer remains unclear. We investigated the biological behaviors and clinical prognostic values of ANO9 in gastric cancer (GC). Knockdown experiments were performed on human GC cell lines using ANO9 siRNA. Eighty-four primary tissue samples from patients with advanced GC were examined immunohistochemically (IHC). Knockdown of ANO9 reduced the progression of cancer cells in MKN7 and MKN74 cells. A microarray analysis revealed that ANO9 regulated PD-L2 via interferon (IFN)-related genes. We confirmed using flow cytometry that the depletion of ANO9 reduced the binding ability to PD-1 by downregulating the expression of PD-L2 in MKN7 and MKN74 cells. IHC revealed a correlation between the expression of ANO9 and PD-L2 and also that the strong expression of ANO9 was an independent poor prognostic factor in patients with advanced GC. The present results indicate that ANO9 regulates PD-L2 and binding ability to PD-1 via IFN-related genes in GC. Therefore, ANO9 has potential as a biomarker and target of immune checkpoint blockage (ICB) for GC.

Evidence that polyphenols do not inhibit the phospholipid scramblase TMEM16F.

TMEM16 Ca2+-activated phospholipid scramblases (CaPLSases) mediate rapid transmembrane phospholipid flip-flop and as such play essential roles in various physiological and pathological processes such as blood coagulation, skeletal development, viral infection, cell-cell fusion, and ataxia. Pharmacological tools specifically targeting TMEM16 CaPLSases are urgently needed to understand these novel membrane transporters and their contributions to health and disease. Tannic acid (TA) and epigallocatechin gallate (EGCG) were recently reported as promising TMEM16F CaPLSase inhibitors. However, our present study shows that TA and EGCG do not inhibit the phospholipid-scrambling or ion conduction activities of the dual-functional TMEM16F. Instead, we found that TA and EGCG mainly acted as fluorescence quenchers that rapidly suppress the fluorophores conjugated to annexin V, a phosphatidylserine-binding probe commonly used to report on TMEM16 CaPLSase activity. These data demonstrate the false positive effects of TA and EGCG on inhibiting TMEM16F phospholipid scrambling and discourage the use of these polyphenols as CaPLSase inhibitors. Appropriate controls as well as a combination of both fluorescence imaging and electrophysiological validation are necessary in future endeavors to develop TMEM16 CaPLSase inhibitors.

PITPNA-AS1 abrogates the inhibition of miR-876-5p on WNT5A to facilitate hepatocellular carcinoma progression.

LncRNA PITPNA-AS1 was a newly identified lncRNA which has never been studied in cancers. Whether PITPNA-AS1 participated in the development of hepatocellular carcinoma (HCC) is obscure. Given the coaction of lncRNAs and miRNAs to carcinogenesis, the purpose of the present research is to inquire how PITPNA-AS1 affects HCC progression. Firstly, PITPNA-AS1 was observed to be heightened in HCC tissues. Then function assays proved that overexpressing or silencing PITPNA-AS1 could manipulate the proliferation and motility of HCC cells. Besides, PITPNA-AS1 was located in the cytoplasm. Among the candidate miRNAs of PITPNA-AS1, miR-876-5p was an obvious target. Moreover, mechanism experiments validated that PITPNA-AS1 modulated WNT5A expression by targeting miR-876-5p. Rescue experiments affirmed that WNT5A silencing rescued the miR-876-5p suppression-induced cellular processes in PITPNA-AS1-silenced Hep3B cells. And in vivo experiments determined that PITPNA-AS1 regulated HCC progression in vivo via miR-876-5p/WNT5A pathway. In conclusion, this work shed lights on the modulatory mechanism of PITPNA-AS1/miR-876-5p/WNT5A axis in HCC, which might be pivotal for exploring effective diagnostic biomarkers and treatment strategies for HCC patients.

TMEM16F inhibition limits pain-associated behavior and improves motor function by promoting microglia M2 polarization in mice.

Spinal cord injury (SCI) leads to sensorimotor deficits and autonomic changes. Macrophages and microglia could be polarized into the classically activated pro-inflammatory M1 phenotype or the alternatively activated anti-inflammatory M2 phenotype. Transmembrane protein with unknown function 16F (TMEM16F) exhibits functional diversity and may contribute to microglial function. However, the effects of TMEM16F on the modulation of macrophage/microglial polarization are still not fully understood. In the study, TMEM16F up-regulation was detected after SCI in mice, and TMEM16F protein was found in macrophages/microglia in injured spinal cord sections. Depletion of TMEM16F improved motor function in male mice with SCI. M1-type macrophages/microglia accumulated in lower numbers in the injured spinal cord of TMEM16F-knockout (KO) mice. M2 polarization inhibited by SCI was improved in mice with TMEM16F deficiency. TMEM16F deletion also attenuated microglial/macrophage pro-inflammatory response. Furthermore, significant down-regulation of A disintegrin and metalloprotease 17 (ADAM17) was observed in TMEM16F-KO mice. Importantly, TMEM16F-promoted M1 polarization and -inhibited M1 polarization were largely associated with the suppression of ADAM17. Overall, our findings provided new insights into the regulatory mechanisms of macrophage/microglial polarization, thereby possibly facilitating the development of new therapeutic strategies for SCI through the regulation of TMEM16F/ADAM17 signaling.

Identification of Novel Phospholipid Transfer Protein Inhibitors by High-Throughput Screening.

Atherogenesis has been recognized as a risk factor for lethal cardiovascular diseases. Plasma low-density lipoprotein levels are correlated to the occurrence of atherosclerosis, and their control is critical for both the prevention and treatment of these diseases. Phospholipid transfer protein (PLTP) is one of the key regulators of lipoprotein metabolism; PLTP-deficient mice exhibit decreased apolipoprotein B (apoB)-containing lipoprotein secretion and atherosclerosis, indicating the validity of PLTP as a promising therapeutic target. Here, we demonstrate a high-throughput screening (HTS) method to identify a novel chemotype of PLTP inhibitors. Instead of using recombinant proteins, we used human plasma as a source of enzymes in the first screening, so as to efficiently exclude promiscuous inhibitors. The selected compounds were further confirmed to target PLTP both biochemically and biophysically and were shown to inhibit apoB secretion from hepatic cells with no apparent toxicity. We believe that our approach is suitable for filtering out nonspecific inhibitors at an earlier stage of screening campaigns and that these compounds should have potential to be developed into drugs to treat dyslipidemia.

Herpes simplex viruses activate phospholipid scramblase to redistribute phosphatidylserines and Akt to the outer leaflet of the plasma membrane and promote viral entry.

Herpes simplex virus (HSV) entry is associated with Akt translocation to the outer leaflet of the plasma membrane to promote a complex signaling cascade. We hypothesized that phospholipid scramblase-1 (PLSCR1), a calcium responsive enzyme that flips phosphatidylserines between membrane leaflets, might redistribute Akt to the outside during entry. Confocal imaging, biotinylation of membrane proteins and flow cytometric analysis demonstrated that HSV activates PLSCR1 and flips phosphatidylserines and Akt to the outside shortly following HSV-1 or HSV-2 exposure. Translocation was blocked by addition of a cell permeable calcium chelator, pharmacological scramblase antagonist, or transfection with small interfering RNA targeting PLSCR1. Co-immunoprecipitation and proximity ligation studies demonstrated that PLSCR1 associated with glycoprotein L at the outer leaflet and studies with gL deletion viruses indicate that this interaction facilitates subsequent restoration of the plasma membrane architecture. Ionomycin, a calcium ionophore, also induced PLSCR1 activation resulting in Akt externalization, suggesting a previously unrecognized biological phenomenon.

Anoctamin 6 Contributes to Cl- Secretion in Accessory Cholera Enterotoxin (Ace)-stimulated Diarrhea: AN ESSENTIAL ROLE FOR PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE (PIP2) SIGNALING IN CHOLERA.

Accessory cholera enterotoxin (Ace) of Vibrio cholerae has been shown to contribute to diarrhea. However, the signaling mechanism and specific type of Cl- channel activated by Ace are still unknown. We have shown here that the recombinant Ace protein induced ICl of apical plasma membrane, which was inhibited by classical CaCC blockers. Surprisingly, an Ace-elicited rise of current was neither affected by ANO1 (TMEM16A)-specific inhibitor T16A(inh)-AO1(TAO1) nor by the cystic fibrosis transmembrane conductance regulator (CFTR) blocker, CFTR inh-172. Ace stimulated whole-cell current in Caco-2 cells. However, the apical ICl was attenuated by knockdown of ANO6 (TMEM16F). This impaired phenotype was restored by re-expression of ANO6 in Caco-2 cells. Whole-cell patch clamp recordings of ANO currents in HEK293 cells transiently expressing mouse ANO1-mCherry or ANO6-GFP confirmed that Ace induced Cl- secretion. Application of Ace produced ANO6 but not the ANO1 currents. Ace was not able to induce a [Ca2+]i rise in Caco-2 cells, but cellular abundance of phosphatidylinositol 4,5-bisphosphate (PIP2) increased. Identification of the PIP2-binding motif at the N-terminal sequence among human and mouse ANO6 variants along with binding of PIP2 directly to ANO6 in HEK293 cells indicate likely PIP2 regulation of ANO6. The biophysical and pharmacological properties of Ace stimulated Cl- current along with intestinal fluid accumulation, and binding of PIP2 to the proximal KR motif of channel proteins, whose mutagenesis correlates with altered binding of PIP2, is comparable with ANO6 stimulation. We conclude that ANO6 is predominantly expressed in intestinal epithelia, where it contributes secretory diarrhea by Ace stimulation in a calcium-independent mechanism of RhoA-ROCK-PIP2 signaling.

Phospholipid Scramblase 1 Modulates FcR-Mediated Phagocytosis in Differentiated Macrophages.

Phospholipid Scramblase 1 (PLSCR1) was initially characterized as a type II transmembrane protein involved in bilayer movements of phospholipids across the plasma membrane leading to the cell surface exposure of phosphatidylserine, but other cellular functions have been ascribed to this protein in signaling processes and in the nucleus. In the present study, expression and functions of PLSCR1 were explored in specialized phagocytic cells of the monocyte/macrophage lineage. The expression of PLSCR1 was found to be markedly increased in monocyte-derived macrophages compared to undifferentiated primary monocytes. Surprisingly, this 3-fold increase in PLSCR1 expression correlated with an apparent modification in the membrane topology of the protein at the cell surface of differentiated macrophages. While depletion of PLSCR1 in the monocytic THP-1 cell-line with specific shRNA did not inhibit the constitutive cell surface exposure of phosphatidylserine observed in differentiated macrophages, a net increase in the FcR-mediated phagocytic activity was measured in PLSCR1-depleted THP-1 cells and in bone marrow-derived macrophages from PLSCR1 knock-out mice. Reciprocally, phagocytosis was down-regulated in cells overexpressing PLSCR1. Since endogenous PLSCR1 was recruited both in phagocytic cups and in phagosomes, our results reveal a specific role for induced PLSCR1 expression in the modulation of the phagocytic process in differentiated macrophages.

Proton myo-inositol cotransporter is a novel γ-secretase associated protein that regulates Aß production without affecting Notch cleavage.

γ-Secretase is a transmembrane protease complex that is responsible for the processing of a multitude of type 1 transmembrane proteins, including the amyloid precursor protein and Notch. γ-Secretase processing of amyloid precursor protein results in the release of the amyloid ß-peptide (Aß), which is involved in the pathogenesis in Alzheimer's disease. Processing of Notch leads to the release of its intracellular domain, which is important for cell development. γ-Secretase associated proteins (GSAPs) could be of importance for substrate selection, and we have previously shown that affinity purification of γ-secretase in combination with mass spectrometry can be used for finding such proteins. In the present study, we used this methodology to screen for novel GSAPs from human brain, and studied their effect on Aß production in a comprehensive gene knockdown approach. Silencing of probable phospholipid-transporting ATPase IIA, brain-derived neurotrophic factor/neurotrophin-3 growth factor receptor precursor and proton myo-inositol cotransporter (SLC2A13) showed a clear reduction of Aß and these proteins were selected for further studies on Aß production and Notch cleavage using small interfering RNA-mediated gene silencing, as well as an overexpression approach. Silencing of these reduced Aß secretion in a small interfering RNA dose-dependent manner. Interestingly, SLC2A13 had a lower effect on Notch processing. Furthermore, overexpression of SLC2A13 increased Aß40 generation. Finally, the interaction between γ-secretase and SLC2A13 was confirmed using immunoprecipitation and a proximity ligation assay. In summary, SLC2A13 was identified as a novel GSAP that regulates Aß production without affecting Notch cleavage. We suggest that SLC2A13 could be a target for Aß lowering therapy aimed at treating Alzheimer's disease.

Anoctamin 6 mediates effects essential for innate immunity downstream of P2X7 receptors in macrophages.

Purinergic P2X7 receptors (P2X7R) are fundamental to innate immune response. In macrophages, transient stimulation of P2X7R activates several transport mechanisms and induces the scrambling of phospholipids with subsequent membrane blebbing and apoptosis. These processes support phagocytosis and subsequent killing of phagocytosed bacteria. Here we demonstrate that the stimulation of P2X7 receptors activates anoctamin 6 (ANO6, TMEM16F), a protein that functions as Ca(2+) dependent phospholipid scramblase and Ca(2+)-activated Cl(-) channel. Inhibition or knockdown of ANO6 attenuates ATP-induced cell shrinkage, cell migration and phospholipid scrambling. In mouse macrophages, Ano6 produces large ion currents by stimulation of P2X7 receptors and contributes to ATP-induced membrane blebbing and apoptosis, which is largely reduced in macrophages from Ano6-/- mice. ANO6 supports bacterial phagocytosis and killing by mouse and human THP-1 macrophages. Our data demonstrate that anoctamin 6 is an essential component of the immune defense by macrophages.

Calcium-activated and apoptotic phospholipid scrambling induced by Ano6 can occur independently of Ano6 ion currents.

Immune cells and platelets maintain plasma membrane phospholipid asymmetry. Upon activation, this asymmetry is disrupted by phospholipid scrambling (PS), which is a major step during activation of immune cells, hemostasis and apoptosis. Anoctamin 6 (Ano6; TMEM16F) causes chloride (Cl(-)) and cation currents and is required for Ca(2+)-dependent PS. It is defective in blood cells from patients with Scott syndrome, a rare bleeding disorder. We examined if Cl(-) currents and PS are related, whether both processes are Ca(2+) dependent, and whether Ca(2+)-independent scrambling during intrinsic and extrinsic apoptosis is controlled by Ano6. Ca(2+) increase by ionomycin activated Ano6 Cl(-) currents and PS in normal lymphocytes, but not in B-lymphocytes from two different patients with Scott syndrome. Fas ligand (FasL) did not increase intracellular Ca(2+), but activated Cl(-) currents in normal but not in Scott lymphocytes. Whole-cell currents were inhibited by Cl(-) channel blockers and by siRNA knockdown of Ano6. In contrast, intrinsic mitochondrial apoptosis by ABT-737 did not induce Cl(-) currents in lymphocytes. PS was not inhibited by blockers of Ano6 or removal of Cl(-) ions. Remarkably, Ca(2+)-independent scrambling due to extrinsic (FasL) or intrinsic (ABT-737) apoptosis was unchanged in Scott cells. We conclude that: (i) Ano6 Cl(-) currents are activated by increase in cytosolic Ca(2+), or Ca(2+) independent by stimulation of Fas receptors; (ii) Ca(2+)-dependent PS induced by Ano6 does not require Cl(-) currents; (iii) Ca(2+)-independent PS does not require Ano6; (iv) Ano6 is necessary for Ca(2+)-dependent PS, but not by increasing intracellular Ca(2+).

Blockade of phospholipid scramblase 1 with its N-terminal domain antibody reduces tumorigenesis of colorectal carcinomas in vitro and in vivo.

BACKGROUND: Membrane-bound phospholipid scramblase 1 (PLSCR1) is involved in both lipid trafficking and cell signaling. Previously, we showed that PLSCR1 is overexpressed in many colorectal carcinomas (CRCs). In the present study, we investigated the tumorigenic role of PLSCR1 in CRC and suggest that it is a potential therapeutic target. METHODS: To identify PLSCR1 as a therapeutic target, we studied the tumorigenic properties of CRC cell lines treated with a monoclonal antibody (NP1) against the N-terminus of PLSCR1 in vitro and in vivo. We also investigated cell cycle status and epidermal growth factor receptor-related pathways and downstream effectors of PLSCR1 after blocking its function with NP1. RESULTS: Treating CRC cells with NP1 in vitro and in vivo decreased cell proliferation, anchorage-independent growth, migration, and invasion. Adding NP1 to the CRC cell line HT29 caused arrest at G1/S. Treating HT29 cells with NP1 significantly decreased the expression of cyclin D1 and phosphorylation levels of Src, the adaptor protein Shc, and Erks. The reduced level of cyclin D1 led to an increase in the activated form of the tumor suppressor retinoblastoma protein via dephosphorylation. These actions led to attenuation of tumorigenesis. CONCLUSIONS: Therefore, PLSCR1 may serve as a potential therapeutic target for CRC.

Ribavirin-induced externalization of phosphatidylserine in erythrocytes is predominantly caused by inhibition of aminophospholipid translocase activity.

Ribavirin in combination with interferon-α is the standard treatment for chronic hepatitis C, but often induces severe anemia forcing discontinuation of the therapy. Whereas suppression of bone marrow by interferon may impact on the production of erythrocytes, it has been suggested that accumulation of ribavirin in erythrocytes induces alterations causing an early removal of these cells by the mononuclear phagocytic system. Externalization of phosphatidylserine, which is exclusively present in the cytoplasmic leaflet of the plasma membrane, is a recognition signal for phagocytosis in particular of apoptotic cells. Here, we demonstrate that surface exposure of phosphatidylserine upon prolonged treatment of erythrocytes with ribavirin results mainly from inactivation of the aminophospholipid translocase, an ATP-dependent lipid pump, which specifically transports phosphatidylserine from the outer to the inner leaflet of the plasma membrane. Inactivation is due to severe ATP depletion, although competitive inhibition by ribavirin or its phosphorylated derivatives cannot be excluded. Phospholipid scramblase, responsible for collapse of lipid asymmetry, appears to be of minor importance as erythrocytes of patients with the Scott syndrome, lacking Ca(2+)-induced lipid scrambling, are equally sensitive to ribavirin treatment. Neither the antioxidant N-acetylcysteine nor the pan-caspase inhibitor Q-VD-OPH did affect ribavirin-induced phosphatidylserine exposure, suggesting that oxidative stress or apoptotic-related mechanisms are not involved in this process. In conclusion, we propose that spontaneous loss of lipid asymmetry, not corrected by aminophospholipid translocase activity, is the mechanism for ribavirin-induced phosphatidylserine exposure that may contribute to ribavirin-induced anemia.

Homocysteine induces phosphatidylserine exposure in cardiomyocytes through inhibition of Rho kinase and flippase activity.

AIMS: Increased levels of homocysteine (Hcy) form an independent risk factor for cardiovascular disease. In a previous study we have shown that Hcy induced phosphatidylserine (PS) exposure to the outer leaflet of the plasma membrane in cardiomyocytes, inducing a pro-inflammatory phenotype. In the present study the mechanism(s) involved in Hcy-induced PS exposure were analyzed. METHODS: H9c2 rat cardiomyoblasts were subjected to 2.5 mM D,L-Hcy and analyzed for RhoA translocation and activity, Rho Kinase (ROCK) activity and expression and flippase activity. In addition, the effect of ROCK inhibition with Y27632 on Hcy-induced PS exposure and flippase activity was analyzed. Furthermore, GTP and ATP levels were determined. RESULTS: Incubation of H9c2 cells with 2.5 mM D,L-Hcy did not inhibit RhoA translocation to the plasma membrane. Neither did it inhibit activation of RhoA, even though GTP levels were significantly decreased. Hcy did significantly inhibit ROCK activation, but not its expression, and did inhibit flippase activity, in advance of a significant decrease in ATP levels. ROCK inhibition via Y27632 did not have significant added effects on this. CONCLUSION: Hcy induced PS exposure in the outer leaflet of the plasma membrane in cardiomyocytes via inhibition of ROCK and flippase activity. As such Hcy may induce cardiomyocytes vulnerable to inflammation in vivo in hyperhomocysteinaemia patients.

Identification and characterization of dual inhibitors for phospholipid transfer protein and microsomal triglyceride transfer protein.

Phospholipid transfer protein (PLTP) plays an important role in atherogenesis and lipoprotein metabolism. PLTP exerts its functions intracellularly and extracellularly. Both PLTP and microsomal triglyceride transfer protein (MTP) have been shown to regulate the secretion of apolipoprotein B (apoB) in hepatocytes. We have previously reported the characterization of inhibitors that selectively inhibit PLTP activity and reduce apoB secretion in hepatocytes. In the present study, we identified more compounds that inhibit both PLTP and MTP activity to various extents. These dual inhibitors are structurally different from the PLTP-selective inhibitors. In human hepatoma cell lines, dual inhibitors seem to be more effective in reducing apoB secretion than selective PLTP or MTP inhibitors. Furthermore, the dual inhibitors markedly reduced triglyceride secretion from hepatocytes. In the absence of PLTP, the dual inhibitors can further reduce apoB secretion, whereas selective PLTP inhibitors had no effect. We conclude that MTP and PLTP may work coordinately in the process of hepatic apoB assembly and secretion. To avoid liver toxicity mediated by MTP inhibition, selective PLTP inhibitors should be pursued.

Pharmacologic inhibition of phospholipid transfer protein activity reduces apolipoprotein-B secretion from hepatocytes.

Phospholipid transfer protein (PLTP) plays an important role in atherogenesis, and its function goes well beyond that of transferring phospholipids between lipoprotein particles. Previous studies showed that genetic deficiency of PLTP in mice causes a substantially impaired hepatic secretion of apolipoprotein-B (apoB), the major protein of atherogenic lipoproteins. To understand whether the impaired apoB secretion is a direct result from lack of PLTP activity, in this study, we further investigated the function of PLTP in apoB secretion by using PLTP inhibitors. We identified a series of compounds containing a 3-benzazepine core structure that inhibit PLTP activity. Compound A, the most potent inhibitor, was characterized further and had little cross-reactivity with microsomal triglyceride transfer protein. Compound A reduced apoB secretion in human hepatoma cell lines and mouse primary hepatocytes. Furthermore, we confirmed that the reduction of apoB secretion mediated by compound A is PLTP-dependent, because the PLTP inhibitor had no effect on apoB secretion from PLTP-deficient hepatocytes. These studies provided evidence that PLTP activity regulates apoB secretion and pharmacologic inhibition of PLTP may be a new therapy for dyslipidemia by reducing apoB secretion.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induced mitochondrial pathway to apoptosis and caspase activation is potentiated by phospholipid scramblase-3.

Tumor Necrosis Factor (TNF)-Related Apoptosis-Inducing Ligand (TRAIL) initiate pathways of cell death in which caspase activation is mediated either directly (without mitochondrial amplification), or indirectly via the release of apoptogenic factors from mitochondria. Phospholipid scramblases (PLS) are enzymes that play a key role in cellular function by inducing bidirectional movement of membrane lipids. Changes in mitochondrial membrane lipids, cardiolipin, are critical for mediating apoptotic response in many cell-types. PLS3 is a phospholipid scramblase that is localized to mitochondria and is thought to be involved in the regulation of apoptotic signals. Here we report that exogenous-expression of PLS3 enhances apoptotic death induced by TRAIL. This is acheived by potentiating the mitochondrial arm of the death pathway. Thereby, PLS3 expression facilitates changes in mitochondrial membrane lipids that promote the release of apoptogenic factors and consequent full activation and processing of the caspase-9 and effector caspase-3. Moreover, we show that knock-down of endogenous PLS3 suppresses TRAIL-induced changes in cardiolipin. Finally, we demonstrate that TRAIL-induced activation of PKC-delta mediates regulation of the PLS3-induced changes in cardiolipin.

Wogonin induces the granulocytic differentiation of human NB4 promyelocytic leukemia cells and up-regulates phospholipid scramblase 1 gene expression.

Previous studies have firmly demonstrated that wogonin, a naturally occurring monoflavonoid extracted from the root of the Chinese herb medicine Scutellaria baicalensis, could effectively inhibit the proliferation of several cancer cell lines. However, little is known about the effect of wogonin on differentiation induction of leukemic cells. Here we investigate the potential role of wogonin in the proliferation and differentiation of NB4, a human promyelocytic leukemia cell line derived from a patient with acute promyelocytic leukemia. Our results indicated that wogonin significantly suppressed the proliferation and efficiently induced the differentiation of NB4 cells. NB4 cell growth was inhibited by 55-60% after treatment with 50 microM wogonin for a period of 5 days. The results of the nitroblue tetrazolium (NBT) reduction test (with 67.13% positive cells by 50 microM wogonin for 5 days), Giemsa staining (with 67.24% positive cells by 50 microM wogonin for 5 days), and the expression of mature-related cell-surface differentiation antigens CD11b and CD14 (with 70.94% CD11b(+) and 5.82% CD14(+) cells by 50 microM wogonin for 5 days) demonstrated an increase in the differentiation-inducing action of wogonin on the NB4 cells, which was accompanied by an increase in mRNA and protein expression of phospholipids scramblase 1 (PLSCR1). Meanwhile, the level of phosphorylated PKC delta (Ser643) was dramatically increased in wogonin treated NB4 cells. Interestingly, wogonin treatment displayed little effect on the apoptosis of NB4 cells. Taken together, the results reported here demonstrated that wogonin could promote the granulocytic differentiation of NB4 cells by up-regulating the expression of PLSCR1 gene.