Margaret A Park's research while affiliated with University of South Florida and other places

HIF1 (Hypoxia-inducible Factor 1) is a transcription factor that plays a crucial role in the hypoxia stress response. Its primary function is to return the cell to its homeostatic state following oxygen deprivation. However, chronic hypoxia exposure can cause irreversible physiological changes that can lead to pulmonary hypertension (PH) and the need for therapeutics to ameliorate these conditions is great and unmet. Previous studies in our lab have demonstrated that CPEB2 (cytoplasmic polyadenylation element binding protein 2) is a translational repressor of one of the HIF1 subunits: HIF1α. Our lab demonstrated that the alternatively spliced CPEB2A isoform of CPEB2 is a repressor of translation, while the CPEB2B isoform is a translational activator of HIF1α during hypoxia, suggesting a major regulatory role for CPEB2 AS in the pulmonary hypoxic response. Although it is well established that during hypoxia, HIF1α levels are dramatically upregulated due to a decrease in the degradation of this factor, we propose that during chronic hypoxia, the expression of HIF1α is maintained via a translational mechanism, likely alongside a decrease in proteolytic degradation. In this study we demonstrate that depletion of the CPEB2B splice isoform has an inhibitory effect on the translation of nascent HIF1α protein during chronic hypoxia, but not the acute phase. We further demonstrate that this pathway is dependent on the initiation factor eIF3H. Finally, we show data which indicate that CPEB2A and CPEB2B bind differentially to cytoplasmic polyadenylation element consensus sequences depending on surrounding sequence context. These findings are important, since they provide evidence for potential of CPEB2 to act as a therapeutic target for treating chronic hypoxia-related pulmonary diseases.

Type 1 diabetes (T1D) is a consequence of autoimmune β cell destruction, but the role of lipids in this process is unknown. We previously reported that activation of Ca2+-independent phospholipase A2β (iPLA2β) modulates polarization of macrophages (MΦ). Hydrolysis of the sn-2 substituent of glycerophospholipids by iPLA2β can lead to the generation of oxidized lipids (eicosanoids), pro- and antiinflammatory, which can initiate and amplify immune responses triggering β cell death. As MΦ are early triggers of immune responses in islets, we examined the impact of iPLA2β-derived lipids (iDLs) in spontaneous-T1D prone nonobese diabetic mice (NOD), in the context of MΦ production and plasma abundances of eicosanoids and sphingolipids. We find that (a) MΦNOD exhibit a proinflammatory lipid landscape during the prediabetic phase; (b) early inhibition or genetic reduction of iPLA2β reduces production of select proinflammatory lipids, promotes antiinflammatory MΦ phenotype, and reduces T1D incidence; (c) such lipid changes are reflected in NOD plasma during the prediabetic phase and at T1D onset; and (d) importantly, similar lipid signatures are evidenced in plasma of human subjects at high risk for developing T1D. These findings suggest that iDLs contribute to T1D onset and identify select lipids that could be targeted for therapeutics and, in conjunction with autoantibodies, serve as early biomarkers of pre-T1D.

Type 1 diabetes (T1D) is a consequence of autoimmune-mediated destruction of pancreatic β-cells and the leading causes for this process are incompletely understood. Our previous work suggests the involvement of lipid signaling from immune cells on T1D development. Relevant lipids were shown to be generated by the Ca2+-independent phospholipase A2β (iPLA2β), which is ubiquitously expressed and hydrolyzes membrane phospholipids at the sn-2 position to release bioactive lysophospholipids and free fatty acids such as arachidonic acid. Arachidonic acid can be metabolized to generate eicosanoids, many of which are pro-inflammatory. We found that iPLA2β activation promotes pro-inflammatory M1 macrophage phenotype and that selective inhibition of iPLA2β preserves β-cell mass and reduces T1D incidence and insulitis in the NOD mice. Herein, we report that NOD macrophages generate significantly higher pro-inflammatory lipids and reduced anti-inflammatory lipids than C57 macrophages during the prediabetic phase. Such changes in the lipidome are mitigated with reduced expression of iPLA2β. Specifically, lipidomics analyses revealed that NOD.iPLA2β+/- macrophage production of multiple pro-inflammatory lipids (PGE2, leukotrienes, 12-HETE, DHETs) is decreased, and of anti-inflammatory lipids increased, relative to NOD-derived macrophages. The mitigated inflammatory lipid signature during the prediabetic phase the NOD.iPLA2β+/- contributed to a reduced T1D incidence. These findings suggest a role for macrophage iPLA2β-derived lipids (iDLs) in T1D development. In support, adoptive transfer of NOD.iPLA2β+/- macrophages reduced T1D incidence and improved glucose tolerance; and conditional knockout of iPLA2β in macrophages reduced T1D incidence in the NOD mice. We hypothesize that iDLs produced by macrophages contribute to T1D development and that these could be targeted to prevent onset/progression of T1D. Disclosure A. Almutairi: None. Y. Gai: None. X.Y. Lei: None. M.A. Park: None. C. Chalfant: None. S. Ramanadham: None. D. Stephenson: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (R01DK110292); National Institute of Allergy and Infectious Diseases (R21AI146743)

Phospholipases A2 (PLA2s) catalyze hydrolysis of the sn-2 substituent from glycerophospholipids to yield a free fatty acid (i.e., arachidonic acid), which can be metabolized to pro- or anti-inflammatory eicosanoids. Macrophages modulate inflammatory responses and are affected by Ca2+-independent PLA2beta (iPLA2β). Here, we assessed the link between iPLA2β-derived lipids (iDLs) and macrophage polarization. Macrophages from wild-type (WT) and knockout (iPLA2β-/-) mice were classically (M1 pro-inflammatory phenotype) or alternatively (M2 anti-inflammatory phenotype) activated, and eicosanoid production was determined by ultra-performance liquid chromatography electrospray ionization MS/MS. As a genotypic control, we performed similar analyses on macrophages from RIP.iPLA2β.Tg mice with selective iPLA2β overexpression in β-cells. Compared with WT, generation of select pro-inflammatory prostaglandins was lower in iPLA2β-/- knockouts, and that of a specialized pro-resolving lipid mediator (SPM), resolvin D2, was higher; both changes are consistent with the M2 phenotype. Conversely, macrophages from RIP.iiPLA2β.Tg mice exhibited an opposite landscape, one associated with the M1 phenotype: namely, increased production of proinflammatory eicosanoids (6-keto PGF1α, PGE2, LTB4) and decreased ability to generate resolvin D2. These changes were not linked with secretory PLA2 or cytosolic PLA2α or with leakage of the transgene. Thus, we report previously unidentified links between select iPLA2β-derived eicosanoids, an SPM, and macrophage polarization. Importantly, our findings reveal for the first time that β-cell iPLA2β-derived signaling can predispose macrophage responses. These findings suggest that iDLs play critical roles in macrophage polarization, and we posit that they could be targeted therapeutically to counter inflammation-based disorders.

The sphingolipid ceramide 1-phosphate (C1P) directly binds to and activates group IVA cytosolic phospholipase A 2 (cPLA 2 α) to stimulate the production of eicosanoids. Because eicosanoids are important in wound healing, we examined the repair of skin wounds in knockout (KO) mice lacking cPLA 2 α and in knock-in (KI) mice in which endogenous cPLA 2 α was replaced with a mutant form having an ablated C1P interaction site. Wound closure rate was not affected in the KO or KI mice, but wound maturation was enhanced in the KI mice compared to that in wild-type controls. Wounds in KI mice displayed increased infiltration of dermal fibroblasts into the wound environment, increased wound tensile strength, and a higher ratio of type I:type III collagen. In vitro, primary dermal fibroblasts (pDFs) from KI mice showed substantially increased collagen deposition and migration velocity compared to pDFs from wild-type and KO mice. KI mice also showed an altered eicosanoid profile of reduced proinflammatory prostaglandins (PGE 2 and TXB 2 ) and an increased abundance of certain hydroxyeicosatetraenoic acid (HETE) species. Specifically, an increase in 5-HETE enhanced dermal fibroblast migration and collagen deposition. This gain-of-function role for the mutant cPLA 2 α was also linked to the relocalization of cPLA 2 α and 5-HETE biosynthetic enzymes to the cytoplasm and cytoplasmic vesicles. These findings demonstrate the regulation of key wound-healing mechanisms in vivo by a defined protein-lipid interaction and provide insights into the roles that cPLA 2 α and eicosanoids play in orchestrating wound repair.

Triple negative breast cancer (TNBC) has an unusually low 5-year survival rate linked to higher metastatic rates. Our laboratory recently delineated a role for the alternative RNA splicing (AS) of cytoplasmic polyadenylation element binding protein 2 (CPEB2), via inclusion/exclusion of exon 4, in the metastasis of TNBC. In these studies, the mechanism governing the inclusion/exclusion of exon 4 was examined. Specifically, the RNA trans-factor, SRSF3, was found to be explicitly associated with CPEB2 exon 4. A SRSF3 consensus sequence was identified in exon 4, and mutation of this sequence abolished the association of SRSF3. The expression of SRSF3 was upregulated in TNBC cells upon the acquisition of anoikis resistance correlating with a reduction in the CPEB2A/B ratio. Importantly, downregulation of SRSF3 in these cells by siRNA induced the exclusion of exon 4 in cells increasing the ratio of CPEB2A (exon 4 excluded) to CPEB2B (exon 4 included). Downregulation of SRSF3 also reversed the CPEB2A/B ratio of a wild-type CPEB2 exon 4 minigene and endogenous CPEB2 pre-mRNA, but not a mutant CPEB2 minigene with the SRSF3 RNA -element ablated. SRSF3 downregulation ablated the anoikis resistance of TNBC cells, which was “rescued” by ectopic expression of CPEB2B. Finally, analysis of The Cancer Genome Atlas database showed a positive relationship between SRSF3 expression and lower CPEB2A/B ratios in aggressive breast cancers. Implications: These findings demonstrate that SRSF3 modulates CPEB2 AS to induce the expression of the CPEB2B isoform that drives TNBC phenotypes correlating with aggressive human breast cancer. Visual Overview: http://mcr.aacrjournals.org/content/molcanres/17/9/1920/F1.large.jpg.

Phosphatidylethanolamine (PE) and phosphatidylcholine (PC) are highly prevalent phospholipids in mammalian membranes. There are currently no methods for detection of minute levels of these phospholipids or simultaneously with products of the utilization of these phospholipid substrates by phospholipase A 2 (PLA 2 ) enzymes. To examine the substrate utilization of PE and PC by PLA 2 , we developed a method to accurately detect and measure specific forms of PE and PC as low as 50 femtomoles. Validation of this method consisted of an enzymatic assay to monitor docosahexaenoic acid and arachidonic acid release from the hydrolysis of PE and PC by group IV phospholipase A 2 (cPLA 2 α) coupled to the generation of Lyso-PE (LPE) and Lyso-PC (LPC). In addition, the PE and PC profiles of RAW 264.7 macrophages were monitored with zymosan/lipopolysaccharide-treatment. Finally, genetic validation for the specificity of the method consisted of the downregulation of two biosynthetic enzymes responsible for the production of PE and PC, choline kinase A (CHKA) and ethanolamine kinase 1 (ETNK1). This new UPLC ESI-MS/MS method provides accurate and highly sensitive detection of PE and PC species containing AA and DHA allowing for the specific examination of the substrate utilization of these phospholipids by PLA 2 in vitro and in cells.

Type 1 diabetes (T1D) is a consequence of autoimmune destruction of β-cells and we find that the Ca2+-independent phospholipase A2β (iPLA2β) is induced in β-cells and immune cells and mitigation of iPLA2β attenuates β-cell death and T1D incidence. Activation of iPLA2β leads to generation of pro-inflammatory or anti-inflammatory bioactive lipids and we previously reported that iPLA2β activation favors M1 pro-inflammatory macrophage phenotype. Here, we utilized UPLC ESI-MS/MS protocols to assess the lipid profile generated by macrophages as a means to identify select lipids that are associated with T1D development. Macrophages were treated with DMSO (vehicle), IFNγ+LPS (to induce M1 polarization), or IL-4 (to induce M2 polarization) and at 16h, the media was collected and processed for assessment of eicosanoid class of bioactive lipids. Comparison of spontaneous diabetes-resistant C57BL/6J and spontaneous diabetes-prone NOD mice revealed that nearly all pro-inflammatory eicosanoids were elevated in NOD during the prediabetic phase, relative to C57. Theabundances increased in the NOD between 4-8 and decreased by 15 weeks, correlating with onset of insulitis. These findings are consistent with a higher inflammatory status in the NOD vs. C57. To assess contribution of iPLA2β to the lipid profile, macrophage responses in NOD were compared with those in NOD.iPLA2β-/+. Such analyses revealed that production of select pro-inflammatory and anti-inflammatory lipids were decreased and increased, respectively, from macrophages and islets of NOD.iPLA2β-/+, relative to age-matched NOD-WT, and this correlated with reduced T1D incidence in the NOD.iPLA2β-/+. We further find that iPLA2β-derived lipids (iDLs) play a critical role in alternative splicing events that favor generation of pro-apoptotic variants of several factors (Bcl-x, capsase-9, and RAGE). These findings raise the possibility that iDLs are candidates for targeting to counter T1D development. Disclosure A. Nelson: None. M.A. Park: None. C. Chalfant: None. S. Ramanadham: None.

The translational regulator cytosolic polyadenylation element binding protein 2 has two isoforms, CPEB2A and CPEB2B, derived by alternative splicing of RNA into a mature form that either includes or excludes exon 4. Previously, we reported that this splicing event is highly dysregulated in aggressive forms of breast cancers, which overexpress CPEB2B. The loss of CPEB2A with a concomitant increase in CPEB2B was also required for breast cancer cells to resist cell death due to detachment (anoikis resistance) and metastasize in vivo. To examine the mechanism by which CPEB2 isoforms mediate opposing effects on cancer-related phenotypes, we used next-generation sequencing of triple-negative breast cancer cells in which the isoforms were specifically downregulated. Downregulation of the CPEB2B isoform inhibited pathways driving the epithelial-to-mesenchymal transition and hypoxic response, whereas downregulation of the CPEB2A isoform did not have this effect. Examining key nodes of these pathways showed that CPEB2B induced the expression of regulatory DNA trans- factors (e.g., HIF1α and TWIST1). Specifically, CPEB2B functioned as a translational activator of TWIST1 and HIF1α. Functional studies showed that specific downregulation of either HIF1α or TWIST1 inhibited the ability of CPEB2B to induce the acquisition of anoikis resistance and drive metastasis. Overall, this study demonstrates that CPEB2 alternative splicing is a major regulator of key cellular pathways linked to anoikis resistance and metastasis.