TAT&RGD Peptide-Modified Naringin-Loaded Lipid Nanoparticles Promote the Osteogenic Differentiation of Human Dental Pulp Stem Cells.

Background: Naringin is a naturally occurring flavanone that promotes osteogenesis. Owing to the high lipophilicity, poor in vivo bioavailability, and extensive metabolic alteration upon administration, the clinical efficacy of naringin is understudied. Additionally, information on the molecular mechanism by which it promotes osteogenesis is limited. Methods: In this study, we prepared TAT & RGD peptide-modified naringin-loaded nanoparticles (TAT-RGD-NAR-NPs), evaluated their potency on the osteogenic differentiation of human dental pulp stem cells (hDPSCs), and studied its mechanism of action through metabolomic analysis. Results: The particle size and zeta potential of TAT-RGD-NAR-NPs were 160.70±2.05 mm and -20.77±0.47mV, respectively. The result of cell uptake assay showed that TAT-RGD-NAR-NPs could effectively enter hDPSCs. TAT-RGD-NAR-NPs had a more significant effect on cell proliferation and osteogenic differentiation promotion. Furthermore, in metabolomic analysis, naringin particles showed a strong influence on the glycerophospholipid metabolism pathway of hDPSCs. Specifically, it upregulated the expression of PLA2G3 and PLA2G1B (two isozymes of phospholipase A2, PLA2), increased the biosynthesis of lysophosphatidic acid (LPA). Conclusion: These results suggested that TAT-RGD-NPs might be used for transporting naringin to hDPSCs for modulating stem cell osteogenic differentiation. The metabolomic analysis was used for the first time to elucidate the mechanism by which naringin promotes hDPSCs osteogenesis by upregulating PLA2G3 and PLA2G1B.

Secreted PLA2-III is a possible therapeutic target to treat neuropathic pain.

Lysophosphatidic acid (LPA) plays a critical role in developing and maintaining chronic pain in various animal models. Previous studies have reported that cytosolic and calcium-independent phospholipase A2 (PLA2) is involved in the LPA receptor-mediated amplification of LPA production in the spinal dorsal horn (SDH) after nerve injury, while the involvement of secreted PLA2 (sPLA2) remains unclear. The present study revealed that only sPLA2 -III among 11 species of PLA2 showed a significant upregulation of gene expression in the SDH. Intraspinal injection of adeno-associated virus-miRNA targeting sPLA2-III prevented hyperalgesia and unique hypoalgesia in mice treated with partial sciatic nerve ligation. In addition, intrathecal treatment with antisense oligodeoxynucleotide or siRNA targeting sPLA2-III significantly reversed the established thermal hyperalgesia. In the high-throughput screening of sPLA2-III inhibitors from the chemical library, we identified two hit compounds. Through in vitro characterization of PLA2 inhibitor profiles and in vivo assessment of the anti-hyperalgesic effects of known PLA2 inhibitors as well as hit compounds, sPLA2-III was found to be a novel therapeutic target molecule for the treatment of Neuropathic pain.

Enhanced Phospholipase A2 Group 3 Expression by Oxidative Stress Decreases the Insulin-Degrading Enzyme.

Oxidative stress has a ubiquitous role in neurodegenerative diseases and oxidative damage in specific regions of the brain is associated with selective neurodegeneration. We previously reported that Alzheimer disease (AD) model mice showed decreased insulin-degrading enzyme (IDE) levels in the cerebrum and accelerated phenotypic features of AD when crossbred with alpha-tocopherol transfer protein knockout (Ttpa-/-) mice. To further investigate the role of chronic oxidative stress in AD pathophysiology, we performed DNA microarray analysis using young and aged wild-type mice and aged Ttpa-/- mice. Among the genes whose expression changed dramatically was Phospholipase A2 group 3 (Pla2g3); Pla2g3 was identified because of its expression profile of cerebral specific up-regulation by chronic oxidative stress in silico and in aged Ttpa-/- mice. Immunohistochemical studies also demonstrated that human astrocytic Pla2g3 expression was significantly increased in human AD brains compared with control brains. Moreover, transfection of HEK293 cells with human Pla2g3 decreased endogenous IDE expression in a dose-dependent manner. Our findings show a key role of Pla2g3 on the reduction of IDE, and suggest that cerebrum specific increase of Pla2g3 is involved in the initiation and/or progression of AD.

Sphingomyelin Regulates the Activity of Secretory Phospholipase A2 in the Plasma Membrane.

We examined the effect of the cellular sphingolipid level on the release of arachidonic acid (AA) and the activity of secretory phospholipase A2 (sPLA2 ) using two Chinese hamster ovary (CHO)-K1 cell mutants, LY-B and LY-A cells, deficient in sphingolipid synthesis. In LY-B cells, deficiency of sphingolipids enhanced the release of AA induced by bee venom sPLA2-III or human sPLA2-V. These alterations were reversed by replenishment of exogenous sphingomyelin (SM). In LY-A cells, deficiency of SM increased the release of AA induced by sPLA2. In CHO-K1 cells, decrease and increase of SM level in the plasma membrane by pharmacological methods increased and inhibited the release of AA, respectively. SM inhibited the activity of sPLA2 in vitro. Niemann-Pick disease type C (NPC) is a lysosomal storage disorder caused by mutation of either the NPC1 or NPC2 gene, and is characterized by accumulation of cholesterol and sphingolipids including SM in late endosomes/lysosomes. Increased levels of AA and sPLA2 activity are involved in various neurodegenerative diseases. In CHO cells lacking NPC1 (A101 cells), SM level was lower in the plasma membrane, while it was higher in late endosomes/lysosomes. The release of AA induced by sPLA2 was increased in A101 cells than that in parental cells (JP17 cells), which was attenuated by adding exogenous SM. In addition, sPLA2 -III-induced cytotoxicity in A101 cells was much higher than that in JP17 cells. These results suggest that SM in the plasma membrane plays important roles in regulating sPLA2 activity and the enzyme-induced cytotoxicity in A101 cells.

Secreted phospholipase A2 and mast cells.

Phospholipase A2s (PLA2s) are a group of enzymes that hydrolyze the sn-2 position of phospholipids to release (typically unsaturated) fatty acids and lysophospholipids, which serve as precursors for a variety of bioactive lipid mediators. Among the PLA2 superfamily, secreted PLA2 (sPLA2) enzymes comprise the largest subfamily that includes 11 isoforms with a conserved His-Asp catalytic dyad. Individual sPLA2 enzymes exhibit unique tissue and cellular localizations and specific enzymatic properties, suggesting their distinct biological roles. Recent studies using transgenic and knockout mice for individual sPLA2 isofoms have revealed their involvement in various pathophysiological events. Here, we overview the current state of knowledge about sPLA2s, specifically their roles in mast cells (MCs) in the context of allergology. In particular, we highlight group III sPLA2 (PLA2G3) as an "anaphylactic sPLA2" that promotes MC maturation and thereby anaphylaxis through a previously unrecognized lipid-orchestrated circuit.

Somatic mutations in arachidonic acid metabolism pathway genes enhance oral cancer post-treatment disease-free survival.

The arachidonic acid metabolism (AAM) pathway promotes tumour progression. Chemical inhibitors of AAM pathway prolong post-treatment survival of cancer patients. Here we test whether non-synonymous somatic mutations in genes of this pathway, acting as natural inhibitors, increase post-treatment survival. We identify loss-of-function somatic mutations in 15 (18%) of 84 treatment-naïve oral cancer patients by whole-exome sequencing, which we map to genes of AAM pathway. Patients (n = 53) who survived ≥ 12 months after surgery without recurrence have significantly (P = 0.007) higher proportion (26% versus 3%) of mutations than those who did not (n = 31). Patients with mutations have a significantly (P = 0.003) longer median disease-free survival (24 months) than those without (13 months). Compared with the presence of a mutation, absence of any mutation increases the hazard ratio for death (11.3) significantly (P = 0.018). The inferences are strengthened when we pool our data with The Cancer Genome Atlas (TCGA) data. In patients with AAM pathway mutations, some downstream pathways, such as the PI3K-Akt pathway, are downregulated.

Secreted phospholipases A2are differentially expressed and epigenetically silenced in human breast cancer cells.

Secreted phospholipases A2 (sPLA2s) have recently been associated with several cancers, but their role in breast cancer is unknown. Here we demonstrate that mRNA expression of group IIA, III and X sPLA2s differs both in vivo in tumour biopsies and in breast cancer cells in vitro. Their expression is differentially regulated by DNA methylation and histone acetylation and, significantly, all three genes are silenced in aggressive triple negative cells due to both mechanisms. The transcription start site promoter region and the upstream CpG islands, exclusive to the group X sPLA2 gene, have variable roles in the regulation of sPLA2 expression. Our results suggest that the differential expression of hGIIA, hGIII and hGX sPLA2s in breast cancer cells is a consequence of various degrees of epigenetic silencing due to DNA hypermethylation and histone deacetylation.

Mast cell maturation is driven via a group III phospholipase A2-prostaglandin D2-DP1 receptor paracrine axis.

Microenvironment-based alterations in phenotypes of mast cells influence the susceptibility to anaphylaxis, yet the mechanisms underlying proper maturation of mast cells toward an anaphylaxis-sensitive phenotype are incompletely understood. Here we report that PLA2G3, a mammalian homolog of anaphylactic bee venom phospholipase A2, regulates this process. PLA2G3 secreted from mast cells is coupled with fibroblastic lipocalin-type PGD2 synthase (L-PGDS) to provide PGD2, which facilitates mast-cell maturation via PGD2 receptor DP1. Mice lacking PLA2G3, L-PGDS or DP1, mast cell-deficient mice reconstituted with PLA2G3-null or DP1-null mast cells, or mast cells cultured with L-PGDS-ablated fibroblasts exhibited impaired maturation and anaphylaxis of mast cells. Thus, we describe a lipid-driven PLA2G3-L-PGDS-DP1 loop that drives mast cell maturation.

Human group III PLA2 as a drug target: structural analysis and inhibitor binding studies.

Group III phospholipase A(2) is a known mediator of inflammation, atherosclerosis and cancer in mammals. This enzyme, therefore, is a potential drug target. The availability of the human group III phospholipase A(2) (hIIIPLA(2)) amino acid sequence offers an opportunity to study its structural features by modeling. The monomeric hIII PLA(2) model is based on the 44% identity it has with the bee venom PLA(2), the only known representative structure of this group. The overall structure comprises of three α-helices, a ß-wing and the calcium binding loop which is present at the N-terminus of the enzyme. However, the unique structural features of hIIIPLA(2) in comparison to the other well known group I/II PLA(2)s are: (1) the replacement of the 'conserved' tyrosine residue by phenylalanine at position 87 in the active site; (2) a decrease in the volume of the substrate binding hydrophobic channel and (3) presence of a C-terminal extension which has a close proximity to the third helix. Docking studies of the enzyme with small molecules gives a detailed insight into the participating residues of the enzyme and also the possible type of interactions with the drug molecules. The ligand molecules have binding affinities predicted to range from micromolar to nanomolar range, thereby making them either potential lead molecules or potent drugs. This analysis paves the way for possible therapeutic applications in pathological states caused by this enzyme.

Hypoxic preconditioning suppresses group III secreted phospholipase A2-induced apoptosis via JAK2-STAT3 activation in cortical neurons.

Our previous studies show that group III secreted phospholipases A(2) (sPLA(2)s III) induces extensive neuronal apoptosis in brain cortical cultures. However, the molecular mechanisms underlying sPLA(2) III-induced neuronal injury/death are still unknown. Also it is not clear whether hypoxic pre-conditioning (HPC) is able to protect neurons from the sPLA(2) III insult. In this report, we demonstrate that sPLA(2) III significantly decreased production of Bcl-xl and the ratio of Bcl-xl/Bax, and increased expression of Bax, cleaved caspase 3, and cleaved alpha-Fodrin in primary neuronal culture. HPC prevented the sPLA(2) III-induced decreases in production of Bcl-xl and the ratio of Bcl-xl/Bax, and increases in expression of Bax, cleaved caspase 3, and alpha-Fodrin. However, the HPC-produced neuronal protection was eliminated or attenuated by AG490, rapamycin, and STAT3 shRNA. Our results suggest that sPLA(2) III-induced neuronal apoptosis is likely because of its alterations in expression and activity of Bcl-xl, Bax, caspase 3, and its target gene fodrin; and that HPC-produced neuroprotection against the sPLA(2) III toxicity is mediated via JAK-STAT signal pathways that regulate the expression of Bcl-xl, Bax, and cleaved caspase 3 in cultured cortical neurons.

Group III secreted phospholipase A2 regulates epididymal sperm maturation and fertility in mice.

Although lipid metabolism is thought to be important for the proper maturation and function of spermatozoa, the molecular mechanisms that underlie this dynamic process in the gonads remains incompletely understood. Here, we show that group III phospholipase A2 (sPLA2-III), a member of the secreted phospholipase A2 (sPLA2) family, is expressed in the mouse proximal epididymal epithelium and that targeted disruption of the gene encoding this protein (Pla2g3) leads to defects in sperm maturation and fertility. Although testicular spermatogenesis in Pla2g3-/- mice was grossly normal, spermatozoa isolated from the cauda epididymidis displayed hypomotility, and their ability to fertilize intact eggs was markedly impaired. Transmission EM further revealed that epididymal spermatozoa in Pla2g3-/- mice had both flagella with abnormal axonemes and aberrant acrosomal structures. During epididymal transit, phosphatidylcholine in the membrane of Pla2g3+/+ sperm underwent a dramatic shift in its acyl groups from oleic, linoleic, and arachidonic acids to docosapentaenoic and docosahexaenoic acids, whereas this membrane lipid remodeling event was compromised in sperm from Pla2g3-/- mice. Moreover, the gonads of Pla2g3-/- mice contained less 12/15-lipoxygenase metabolites than did those of Pla2g3+/+ mice. Together, our results reveal a role for the atypical sPLA2 family member sPLA2-III in epididymal lipid homeostasis and indicate that its perturbation may lead to sperm dysfunction.

Isolation and functional characterization of a new acidic PLA(2) Ba SpII RP4 of the Bothrops alternatus snake venom from Argentina.

An acidic protein with phospholipase A(2) activity was purified to homogeneity from the venom of the Northeast Argentinian viperid Bothrops alternatus by two chromatographic steps: a conventional gel filtration on Sephadex G-75 and reversed phase on C18 HPLC column. A molecular mass of 14185.48 Da was determined by mass spectrometry, displaying a homodimer conformation. The kinetic assay demonstrated a catalytically active phospholipase A(2) in correspondence with Asp49 PLA(2) group. The enzyme designated Ba SpII RP4 contains an amino acid composition of 121 residues and a calculated theoretical pI value of 4.88. Amino acid sequence alignments with other Bothrops PLA(2) revealed a high degree of homology sequence (90-56%). Ba SpII RP4 did not show myotoxic activity upon muscular fibers at doses up to 100 microg i.m. route injection or lethal response when it was i.p. injected at the hightest dose of 200 microg. This toxin generates slight biological activities like paw edema inflammation and a delay in the clotting time, although Ba SpII RP4 exhibited catalytic activity. The primary amino acid sequence, determined a quadruple-time of flight (Q-TOF) hybrid mass spectrometer Q-TOF Ultima from Micromass (Manchester, UK) equipped with a nano Zspray source operating in a positive ion mode and tandem mass spectrum, an ESI/MS mass spectrum (TOF MS mode) "de novo amino acid sequencing", also provides more database about the small group of the non-myotoxic PLA(2)s isolated up to the present.

Secretory phospholipase A2 type III enhances alpha-secretase-dependent amyloid precursor protein processing through alterations in membrane fluidity.

In the non-amyloidogenic pathway, amyloid precursor protein (APP) is cleaved by alpha-secretases to produce alpha-secretase-cleaved soluble APP (sAPP(alpha)) with neuroprotective and neurotrophic properties; therefore, enhancing the non-amyloidogenic pathway has been suggested as a potential pharmacological approach for the treatment of Alzheimer's disease. Here, we demonstrate the effects of type III secretory phospholipase A(2) (sPLA(2)-III) on sAPP(alpha) secretion. Exposing differentiated neuronal cells (SH-SY5Y cells and primary rat neurons) to sPLA(2)-III for 24 h increased sAPP(alpha) secretion and decreased levels of Abeta(1-42) in SH-SY5Y cells, and these changes were accompanied by increased membrane fluidity. We further tested whether sPLA(2)-III-enhanced sAPP(alpha) release is due in part to the production of its hydrolyzed products, including arachidonic acid (AA), palmitic acid (PA), and lysophosphatidylcholine (LPC). Addition of AA but neither PA nor LPC mimicked sPLA(2)-III-induced increases in sAPP(alpha) secretion and membrane fluidity. Treatment with sPLA(2)-III and AA increased accumulation of APP at the cell surface but did not alter total expressions of APP, alpha-secretases, and beta-site APP cleaving enzyme. Taken together, these results support the hypothesis that sPLA(2)-III enhances sAPP(alpha) secretion through its action to increase membrane fluidity and recruitment of APP at the cell surface.

Group III secreted phospholipase A2 transgenic mice spontaneously develop inflammation.

PLA2 (phospholipase A2) group III is an atypical sPLA2 (secretory PLA2) that is homologous with bee venom PLA2 rather than with other mammalian sPLA2s. In the present paper, we show that endogenous group III sPLA2 (PLA2G3) is expressed in mouse skin and that Tg (transgenic) mice overexpressing human PLA2G3 spontaneously develop skin inflammation. Pla2g3-Tg mice over 9 months of age frequently developed dermatitis with hyperkeratosis, acanthosis, parakeratosis, erosion, ulcer and sebaceous gland hyperplasia. The dermatitis was accompanied by infiltration of neutrophils and macrophages and by elevated levels of pro-inflammatory cytokines, chemokines and prostaglandin E2. In addition, Pla2g3-Tg mice had increased lymph aggregates and mucus in the airway, lymphocytic sialadenitis, hepatic extramedullary haemopoiesis, splenomegaly with increased populations of granulocytes and monocytes/macrophages, and increased serum IgG1. Collectively, these observations provide the first demonstration of spontaneous development of inflammation in mice with Tg overexpression of mammalian sPLA2.

Analyses of group III secreted phospholipase A2 transgenic mice reveal potential participation of this enzyme in plasma lipoprotein modification, macrophage foam cell formation, and atherosclerosis.

Among the many mammalian secreted phospholipase A2 (sPLA2) enzymes, PLA2G3 (group III secreted phospholipase A2) is unique in that it possesses unusual N- and C-terminal domains and in that its central sPLA2 domain is homologous to bee venom PLA2 rather than to other mammalian sPLA2s. To elucidate the in vivo actions of this atypical sPLA2, we generated transgenic (Tg) mice overexpressing human PLA2G3. Despite marked increases in PLA2 activity and mature 18-kDa PLA2G3 protein in the circulation and tissues, PLA2G3 Tg mice displayed no apparent abnormality up to 9 months of age. However, alterations in plasma lipoproteins were observed in PLA2G3 Tg mice compared with control mice. In vitro incubation of low density (LDL) and high density (HDL) lipoproteins with several sPLA2s showed that phosphatidylcholine was efficiently converted to lysophosphatidylcholine by PLA2G3 as well as by PLA2G5 and PLA2G10, to a lesser extent by PLA2G2F, and only minimally by PLA2G2A and PLA2G2E. PLA2G3-modified LDL, like PLA2G5- or PLA2G10-treated LDL, facilitated the formation of foam cells from macrophages ex vivo. Accumulation of PLA2G3 was detected in the atherosclerotic lesions of humans and apoE-deficient mice. Furthermore, following an atherogenic diet, aortic atherosclerotic lesions were more severe in PLA2G3 Tg mice than in control mice on the apoE-null background, in combination with elevated plasma lysophosphatidylcholine and thromboxane A2 levels. These results collectively suggest a potential functional link between PLA2G3 and atherosclerosis, as has recently been proposed for PLA2G5 and PLA2G10.

Distinct expression pattern of the full set of secreted phospholipases A2 in human colorectal adenocarcinomas: sPLA2-III as a biomarker candidate.

Recent studies suggest that secreted phospholipases A2 (sPLA2s) represent attractive potential tumour biomarkers and therapeutic targets for various cancers. As a first step to address this issue in human colorectal cancer, we examined the expression of the full set of sPLA2s in sporadic adenocarcinomas and normal matched mucosa from 21 patients by quantitative PCR and immunohistochemistry. In normal colon, PLA2G2A and PLA2G12A were expressed at high levels, PLA2G2D, PLA2G5, PLA2G10 and PLA2G12B at moderate levels, and PLA2G1B, PLA2G2F and PLA2G3 at low levels. In adenocarcinomas from left and right colon, the expression of PLA2G3 was increased by up to 40-fold, while that of PLA2G2D and PLA2G5 was decreased by up to 23- and 14-fold. The variations of expression for sPLA2-IID, sPLA2-III and sPLA2-V were confirmed at the protein level. The expression pattern of these sPLA2s appeared to be linked respectively to the overexpression of interleukin-8, defensin alpha6, survivin and matrilysin, and downregulation of SFRP-1 and RLPA-1, all these genes being associated to colon cancer. This original sPLA2 profile observed in adenocarcinomas highlights the potential role of certain sPLA2s in colon cancer and suggests that sPLA2-III might be a good candidate as a novel biomarker for both left and right colon cancers.

Human group III secreted phospholipase A2 promotes neuronal outgrowth and survival.

Human sPLA2-III [group III secreted PLA2 (phospholipase A2)] is an atypical sPLA2 isoenzyme that consists of a central group III sPLA2 domain flanked by unique N- and C-terminal domains. In the present study, we found that sPLA2-III is expressed in neuronal cells, such as peripheral neuronal fibres, spinal DRG (dorsal root ganglia) neurons and cerebellar Purkinje cells. Adenoviral expression of sPLA2-III in PC12 cells (pheochromocytoma cells) or DRG explants facilitated neurite outgrowth, whereas expression of a catalytically inactive sPLA2-III mutant or use of sPLA2-III-directed siRNA (small interfering RNA) reduced NGF (nerve growth factor)-induced neuritogenesis. sPLA2-III also suppressed neuronal death induced by NGF deprivation. Lipid MS revealed that sPLA2-III overexpression increased the cellular level of lysophosphatidylcholine, a PLA2 reaction product with neuritogenic and neurotropic activities, whereas siRNA knockdown reduced the level of lysophosphatidylcholine. These observations suggest the potential contribution of sPLA2-III to neuronal differentiation and its function under certain conditions.