Sperm-Guiding Unconventional Prostaglandins in C. elegans: Synthesis and Signaling

Prostaglandins comprise a family of lipid signaling molecules derived from polyunsaturated fatty acids and are involved in a wide array of biological processes, including fertilization. Prostaglandin-endoperoxide synthase (a.k.a. cyclooxygenase or Cox) initiates prostaglandin synthesis from 20-carbon polyunsaturated fatty acids, such as arachidonic acid. Oocytes of Caenorhabditis elegans (C. elegans) have been shown to secrete sperm-guidance cues prostaglandins, independent of Cox enzymes. Both prostaglandin synthesis and signal transduction in C. elegans are environmentally modulated pathways that regulate sperm guidance to the fertilization site. Environmental factors such as food triggers insulin and TGF-β secretion and their levels regulate tissue-specific prostaglandin synthesis in C. elegans. This novel PG pathway is abundant in mouse and human ovarian follicular fluid, where their functions, mechanism of synthesis and pathways remain to be established. Given the importance of prostaglandins in reproductive processes, a better understanding of how diets and other environmental factors influence their synthesis and function may lead to new strategies towards improving fertility in mammals.

Esrrγa regulates nephron development and ciliogenesis by controlling prostaglandin synthesis and cooperation with Ppargc1a

Cilia are essential for the ontogeny and function of many tissues, including the kidney. In mammals, Esrrγ has been previously established as a significant determinant of renal health, with decreased expression linked to age related dysfunction, cyst formation, and kidney disease. Here, we report that the Esrrγ vertebrate ortholog estrogen related receptor gamma a (esrrγa) is essential for proper cell fate choice within kidney functional units (nephrons) as well as ciliogenesis. Deficiency of esrrγa resulted in nephrons with alterations in proximodistal segmentation and a decreased multiciliated epithelial cell populace. Surprisingly, esrrγa deficiency disrupted renal ciliogenesis and caused a similar abrogation within the developing node and otic vesicle—all defects that occurred independently of changes in cell polarity or basal body organization. These phenotypes were consistent with interruptions in prostaglandin signaling, and we found that ciliogenesis was rescued in esrrγa deficient embryos with exogenous PGE2 or through overexpression of the cyclooxygenase enzyme Ptgs1. Through genetic interaction studies, we found that peroxisome proliferator–activated receptor gamma, coactivator 1 alpha (ppargc1a), which acts upstream of Ptgs1–mediated prostaglandin synthesis, has a synergistic relationship with esrrγa in the ciliogenic pathway. These data position Esrrγa as a novel link between ciliogenesis and nephrogenesis through regulation of prostaglandin signaling and cooperation with Ppargc1a, and highlight Esrrγa as a potential new therapeutic target for ciliopathies.

923 Hedgehog signaling drives epithelial-to-mesenchymal transition, immune evasion, and anti-PD-1 resistance through coordinated upregulation of Wnt ligands and PGE2 synthesis

BackgroundImmunotherapy resistance has been correlated with epithelial-to-mesenchymal transition (EMT),1 2 however our understanding of tumor-intrinsic mechanisms driving this immune evasive phenotype is lacking. We have previously shown that Wnt ligands are upregulated in anti-PD-1 resistant melanomas,3 and postulated that upstream transcriptional regulation of select EMT pathways may underpin these findings. The hedgehog signaling (HH) transcription factor Gli2 promotes EMT.MethodsGli2 was constitutively activated (Gli2CA ) in a BRAFV600EPTEN-/- murine cell line via an N-terminal truncating mutation and silenced using CRISPR-Cas9. Multi-parameter flow cytometry and RNAseq was utilized to evaluate the impact of Gli2 on the tumor immune microenvironment. Anti-PD-1 resistance studies were performed in Gli2CA and control tumors. Bioinformatics studies were conducted using the melanoma TCGA and Hugo et al databases.2ResultsWe found upregulation of Gli2 targets in patients with anti-PD-1-refractory metastatic melanoma as well as in an autochthonous BRAFV600EPTEN-/- melanoma model after escape from anti-PD-1. RNAseq and Western blot studies demonstrated Gli2CA to promote EMT and Wnt ligand production in addition to upregulated COX2 in BRAFV600EPTEN-/- melanoma. This finding was reversed by genetic ablation and pharmacologic inhibition of Gli2, implicating a previously undescribed role for Gli2 in modulating COX2. These data were consistent with a notable correlation between a Gli2 signature and a prostaglandin synthesis signature in human melanoma TCGA database. Flow cytometry analysis showed exclusion of cytolytic T and NK cells, a shift from cDC1s to cDC2s, and enhanced MDSC recruitment in Gli2CA tumors. Consistent with these findings, whole tumor RNAseq of Gli2CA tumors demonstrated a decrease in Cd3e, Prf1, and Xcr1 with a concomitant increase in Cxcl1, Cxcl2, Ccl2, Ptgs2, and Arg1 relative to control tumors. RNAseq of FACS-sorted DCs from Gli2CA tumors demonstrated a loss of cDC1-associated genes including Xcr1, Wdfy4, and Clec9a compared to DCs derived from control tumors. In-line with our previous results showing that Wnt5a promotes MDSC recruitment in a Yap-dependent manner,4 we found that Yap inhibition or Wnt5a deletion in the BRAFV600EPTEN-/-Gli2CA cell line diminished MDSC-recruiting chemokines. Further consistent with these findings, Gli2CA tumors resist anti-PD-1 antibody therapy.Abstract 923 Figure 1Gli2 in tumors promotes Wnt and prostaglandin signaling, generating an immunosuppressive microenvironmentConclusionsOur data demonstrates that the HH transcription factor Gli2 drives the development of a tolerogenic tumor microenvironment unfavorable to anti-PD-1 immunotherapy by coordinating the upregulation of Wnt ligand expression and prostaglandin synthesis (figure 1). We propose that HH gene signatures are worthy of further study as a guide for selecting Wnt ligand and prostaglandin inhibitors in future immunotherapy studies.AcknowledgementsThe authors would like to acknowledge the Duke Cancer Institute Flow Cytometry Core.ReferencesBagaev A, et al. Conserved pan-cancer microenvironment subtypes predict response to immunotherapy. Cancer Cell 2021.Hugo W, et al. Genomic and transcriptomic features of response to anti-PD-1 therapy in metastatic melanoma. Cell 2016;165(1):35–44.DeVito NC, et al. Pharmacological Wnt ligand inhibition overcomes key tumor-mediated resistance pathways to anti-PD-1 immunotherapy. Cell Rep 2021;35(5):109071.Theivanthiran B, et al. A tumor-intrinsic PD-L1/NLRP3 inflammasome signaling pathway drives resistance to anti-PD-1 immunotherapy. J Clin Invest 2020;130(5):2570–2586.

Change of Urinary Nitrite Excretion in Primary Enuresis after Indomethacin Treatment

Various treatment modalities have been used in primary Nocturnal Enuresis (PNE). Inhibition of prostaglandin synthesis may have value in the management of PNE. The effect of PGs on the urinary system are similar to those of Nitrous oxide (No), so there might be a link between No production and PNE. We can use nitrite as a good indicator of both PG and No because it is a stable metabolite of No. Our objective in this study was to assess urinary nitrite excretion in patients with enuresis and to evaluate the effect of indomethacin (a potent prostaglandin synthesis inhibitor) on urinary nitrite excretion. Sixty children participated in this study with age range 5-14 years and were divided into three groups: Group A comprised 20 children with PNE and were given 50 mg indomethacin suppositories each night for 1 month, group B comprised also 20 children with PNE not receiving treatment. Both groups were assessed by frequency of bed wetting episodes as well as by measuring urinary nitrites. In addition, 20 normal comparable controls were assessed as regards their urinary nitrites to show the difference in its values between enuretics and normal individuals. The results showed increase in nitric acid level in enuretic children than controls with marked decrease in its levels after receiving Indomethacin and marked improvement in the frequency of bed-wetting.

Combined COX-2/PPARγ Expression as Independent Negative Prognosticator for Vulvar Cancer Patients

Vulvar cancer incidence numbers have been rising steadily over the past decades. Especially the number of young patients with vulvar cancer increased recently. Therefore, the need to identify new prognostic factors for vulvar carcinoma is more apparent. Cyclooxygenase-2 (COX-2) has long been an object of scientific interest in the context of carcinogenesis. This enzyme is involved in prostaglandin synthesis and the latter binds to nuclear receptors like PPARγ. Therefore, the aim of this study was to investigate COX-2- and PPARγ- expression in tissues of vulvar carcinomas and to analyze their relevance as prognostic factors. The cytoplasmatic expression of COX-2 as well as PPARγ is associated with a significantly reduced survival, whereas nuclear expression of PPARγ results in a better survival. Especially the combined expression of both COX-2 and PPARγ in the cytoplasm is an independent negative prognosticator for vulvar cancer patients.

Role and Regulation of Mechanotransductive HIF-1α Stabilisation in Periodontal Ligament Fibroblasts

Orthodontic tooth movement (OTM) creates compressive and tensile strain in the periodontal ligament, causing circulation disorders. Hypoxia-inducible factor 1α (HIF-1α) has been shown to be primarily stabilised by compression, but not hypoxia in periodontal ligament fibroblasts (PDLF) during mechanical strain, which are key regulators of OTM. This study aimed to elucidate the role of heparan sulfate integrin interaction and downstream kinase phosphorylation for HIF-1α stabilisation under compressive and tensile strain and to which extent downstream synthesis of VEGF and prostaglandins is HIF-1α-dependent in a model of simulated OTM in PDLF. PDLF were subjected to compressive or tensile strain for 48 h. In various setups HIF-1α was experimentally stabilised (DMOG) or destabilised (YC-1) and mechanotransduction was inhibited by surfen and genistein. We found that HIF-1α was not stabilised by tensile, but rather by compressive strain. HIF-1α stabilisation had an inductive effect on prostaglandin and VEGF synthesis. As expected, HIF-1α destabilisation reduced VEGF expression, whereas prostaglandin synthesis was increased. Inhibition of integrin mechanotransduction via surfen or genistein prevented stabilisation of HIF-1α. A decrease in VEGF expression was observed, but not in prostaglandin synthesis. Stabilisation of HIF-1α via integrin mechanotransduction and downstream phosphorylation of kinases seems to be essential for the induction of VEGF, but not prostaglandin synthesis by PDLF during compressive (but not tensile) orthodontic strain.

COX and PTGDS Gene Expression Levels in PGD2 Synthesis Pathway are Correlated with miR-520 in Patients with Vessel Restenosis

Background: The vessel restenosis is related to the inflammatory events in subendothelial space. It is proposed that the inflammatory agents affect the prostaglandin synthesis pathway. In this study, we investigated the COX-1, COX-2, PTGDS and miRNA-520a-5p expression levels and the serum 15-Deoxy-Δ12,14-PGJ2 metabolite values in patients with the stenosed and re-stenosed vessels. Furthermore, the associations between genes and miR-520 were evaluated in the monocyte transfection studies. Methods: The subjects (n=60) were included three groups; healthy subjects (control (stenosis < 5%), stent no restenosis (SNR, restenosis < 5%) and in-stent restenosis (ISR, restenosis > 70%)). The miRNA and gene expression levels were measured by RT-qPCR technique. 15-Deoxy-Δ12,14-PGJ2 values were measured by the ELISA technique. The miR-520 was transfected into myocytes using PEI polymer. Results: The monocyte COX-1, COX-2 and PTGDS gene expression levels and the serum 15-Deoxy- Δ12,14-PGJ2 values increased significantly in the patients. Furthermore, the miR-520 correlated conversely with the COX-1, and PTGDS gene expression levels. Conclusion: The results showed that the PGD2 synthesis pathway is active in the patients and, miR- 520 may be involved in the function of this pathway.

Duktus Arteriosus pada Bayi Prematur

Abstract—Ductus arteriosus (DA) is a connecting vessel between proximal descending aorta and pulmonary artery. This important structure normally close after birth. The opened ductus causes increasing of pulmonary blood flow and decreasing of certain organ blood flow (intestine, skin, muscle, and renal) causing complications such as heart failure, metabolic acidosis, necrotizing enterocolitis, and pulmonary edema/bleeding. Prevalence of DA is 0,2/1000 live birth. In under 1500 g babies the proportion of DA is 25%. Surgery and medicine are the treatment modalities of DA closure. Modalities of medicine are indometacine, ibuprofen, and paracetamol. These three modalities work by inhibiting cyclooxygenase enzime causing blockade of prostaglandin synthesis. Drug adverse event can be minimized by carefull in making treatment choice. Keywords: ductus arteriosus, complication, treatment Abstrak—Duktus arteriosus (DA) merupakan pembuluh darah yang menghubungkan aorta desendens proksimal dan arteri pulmonalis. Struktur yang penting pada janin tersebut secara normal menutup setelah lahir. Duktus yang masih terbuka tersebut mengakibatkan peningkatan aliran darah paru dan penurunan aliran darah ke organ usus, kulit, otot, dan ginjal sehingga menyebabkan komplikasi seperti gagal jantung, asidosis metabolik, necrotizing enterocolitis (NEC), serta edema paru/perdarahan. Prevalensi DA yang masih terbuka adalah 0,2 per 1000 kelahiran hidup. Proporsi bayi yang bergejala dengan DA yang masih terbuka kurang lebih 25% bayi dengan berat badan lahir di bawah 1500g. Pilihan terapi penutupan DA adalah cara bedah dan medis. Cara medis memiliki beberapa pilihan yaitu indometasin, ibuprofen, dan parasetamol. Ketiga modalitas terapi tersebut bekerja melalui penghambatan enzim siklooksigenase sehingga sintesis prostaglandin terhambat. Beberapa hal perlu diperhatikan dalam membuat pilihan terapi sehingga komplikasi yang berhubungan dengan efek samping obat dapat dihindari.. Kata kunci: ductus arteriosus, komplikasi, terapi