The Prostaglandin F Synthase Activity of the Human Aldose Reductase AKR1B1 Brings New Lenses to Look at Pathologic Conditions

Prostaglandins are important regulators of female reproductive functions to which aldose reductases exhibiting hydroxysteroid dehydrogenase activity also contribute. Our work on the regulation of reproductive function by prostaglandins (PGs), lead us to the discovery that AKR1B5 and later AKR1B1were highly efficient and physiologically relevant PGF synthases. PGE2 and PGF2α are the main prostanoids produced in the human endometrium and proper balance in their relative production is important for normal menstruation and optimal fertility. Recent evidence suggests that PGE2/EP2 and PGF2α/FP may constitute a functional dyad with physiological relevance comparable to the prostacyclin-thromboxane dyad in the vascular system. We have recently reported that AKR1B1 was expressed and modulated in association with PGF2α production in response to IL-1β in the human endometrium. In the present study, we show that the human AKR1B1 (gene ID: 231) also known as ALDR1 or ALR2 is a functional PGF2α synthase in different models of living cells and tissues. Using human endometrial cells, prostate, and vascular smooth muscle cells, cardiomyocytes and endothelial cells we demonstrate that IL-1β is able to up regulate COX-2 and AKR1B1 proteins as well as PGF2α production under normal glucose concentrations. We show that the promoter activity of AKR1B1 gene is increased by IL-1β particularly around the multiple stress response region containing two putative antioxidant response elements adjacent to TonE and AP1. We also show that AKR1B1 is able to regulate PGE2 production through PGF2α acting on its FP receptor and that aldose reductase inhibitors like alrestatin, Statil (ponalrestat), and EBPC exhibit distinct and characteristic inhibition of PGF2α production in different cell models. The PGF synthase activity of AKR1B1 represents a new and important target to regulate ischemic and inflammatory responses associated with several human pathologies

Expression of genes related to prostaglandin synthesis or signaling in human subcutaneous and omental adipose tissue: depot differences and modulation by adipogenesis

Objectives. (1) To examine depot-specific PGE2 and PGF2α release and mRNA expression of enzymes or receptors involved in PG synthesis or signaling in human adipose tissues; (2) to identify changes in expression of these transcripts through preadipocyte differentiation; and (3) to examine associations between adipose tissue mRNA expression of these transcripts and adiposity measurements. Methods. Fat samples were obtained surgically in women. PGE2 and PGF2α release by preadipocytes and adipose tissue explants was measured. Expression levels of mRNA coding for enzymes or receptors involved in PG synthesis or signaling were measured by RT-PCR. Results. Cultured preadipocytes and explants from omental fat released more PGE2 and PGF2α than those from the subcutaneous depot and the corresponding transcripts showed consistent depot differences. Following preadipocyte differentiation, expression of PLA2G16 and PTGER3 mRNA was significantly increased whereas COX-1, COX-2, PTGIS, and PTGES mRNA abundance were decreased in both compartments ( for all). Transcripts that were stimulated during adipogenesis were those that correlated best with adiposity measurements. Conclusion. Cells from the omental fat compartment release more PGE2 and PGF2α than those from the subcutaneous depot. Obesity modulates expression of PG-synthesizing enzymes and PG receptors which likely occurs through adipogenesis-induced changes in expression of these transcripts. 1. Introductio

Production sélective et directionnelle des prostaglandines dans l'endomètre

Les prostaglandines (PG) sont impliquées dans plusieurs processus et un bon équilibre dans leur production relative est important pour la cyclicité et la fertilité à la fois chez le bovin et l’humain. Les principales PG produites dans l'endomètre sont la PGE2 et la PGF2α. Leur action est régulée au niveau de la biosynthèse, du catabolisme et de la transduction de leur signal. Le but du présent projet était d’améliorer notre compréhension des mécanismes de production du PGF2α, étant moins connus que ceux du PGE2. Tout d'abord, chez le bovin, nous avons établi la voie impliquée dans l'induction du PGF2α par l'ocytocine (OT) et constaté qu’elle nécessite la transactivation d’EGFR. Deuxièmement, nous avons démontré que la protéine 4 associée à la résistance multiple aux médicaments (MRP4) est un transporteur fonctionnel sous le contrôle de l’OT et conduit à la libération polarisée des prostaglandines. Ensuite, comme plus d’une prostaglandine F2α synthase sont dentifiées dans l’endomètre humain, nous avons vérifié quelles enzymes encore non identifiées pourraient avoir le même rôle chez le bovin. Nous avons identifié et confirmé que les protéines recombinantes d’AKR1A1 et d’AKR1B1 sont les synthases les plus puissantes dans les deux espèces. Troisièmement, nous avons décrit l’association entre AKR1B1 et l’augmentation de la production PGF2α par les cellules endométriales humaines après stimulation par l'interleukine-1β (IL-1β). Comme plusieurs synthases sont exprimées simultanément dans l'endomètre, une preuve définitive du rôle d’AKR1B1 exigeait le KO du gène. Nous avons utilisé une approche utilisant l'ARN-guidée DNase Cas9 et les courtes répétitions palindromiques interespacées par grappes régulières (CRISPR) pour abolir l'expression du gène dans les cellules stromales. Le clone généré a produit une perte d'expression d’AKR1B1 et de production du PGF2α mais maintenu sa capacité à augmenter la production de PGE2 en réponse à l'IL-1β. En testant cela, nous avons aussi constaté que la PGF2α est capable de réguler la production de PGE2 en agissant sur le récepteur FP. Nos résultats confirment qu’AKR1B1 est fortement impliqué dans la synthèse de PGF2α et que cette activité représente une nouvelle et importante cible pour réguler les réponses inflammatoires et ischémiques associées à plusieurs pathologies humaines.Prostaglandins (PG) are key regulators of reproductive processes and a good balance in the relative production of different members is important for cyclicity and fertility in females. The main PG produced in the endometrium are PGE2 and PGF2α. Their action is controlled at the level of biosynthesis, catabolism and signal transduction. The aim of the present project was to improve our understanding of the production of PGF2α as it is underdocumented. In cattle, we established that induction of PGF2α by oxytocin (OT) required the transactivation of EGFR. We also showed that multidrug resistance protein 4 (MRP4) is a functional PG carrier under the control of OT contributing to the polarized release of prostaglandins in the bovine endometrium. Since two different enzymes exhibit PGFS activity in the human endometrium, we investigagted wheter other proteins could play the same role in the bovine endometrium. We have identified and confirmed that AKR1A1 and AKR1B1 recombinant proteins were the most powerful synthases in both species. Finally, AKR1B1 is a very effective PGF2α synthase in the bovine and human endometrium. We have proposed that AKR1B1 play a leading role in PGF2α production stimulation following interleukin-1β (IL- 1β) stimulation in human endometrial cells. However, because multiple synthases are expressed simultaneously in the endometrium, a definitive proof of the role of AKR1B1 would require knockout of its gene. Accordingly, we used an editing genome approach using RNA-guided cas9 DNase and clustered regularly interspaced short palindromic repeats (CRISPR) to abolish the expression of AKR1B1 gene in stromal cells. The resulting clone exhibited a complete loss of AKR1B1 expression and PGF2α production, but maintained the ability to increase PGE2 production in response to IL-1β. While testing this, we also found that PGF2α was able to regulate the production of PGE2 through a feedback loop involving the FP receptor. Our results confirm that AKR1B1 is the leading enzyme responsible for the synthesis of PGF2α thus representing a new and important target for the treatment of inflammatory and ischemic responses associated with human diseases

Expression of Genes Related to Prostaglandin Synthesis or Signaling in Human Subcutaneous and Omental Adipose Tissue: Depot Differences and Modulation by Adipogenesis

Objectives. (1) To examine depot-specific PGE 2 and PGF 2 release and mRNA expression of enzymes or receptors involved in PG synthesis or signaling in human adipose tissues; (2) to identify changes in expression of these transcripts through preadipocyte differentiation; and (3) to examine associations between adipose tissue mRNA expression of these transcripts and adiposity measurements. Methods. Fat samples were obtained surgically in women. PGE 2 and PGF 2 release by preadipocytes and adipose tissue explants was measured. Expression levels of mRNA coding for enzymes or receptors involved in PG synthesis or signaling were measured by RT-PCR. Results. Cultured preadipocytes and explants from omental fat released more PGE 2 and PGF 2 than those from the subcutaneous depot and the corresponding transcripts showed consistent depot differences. Following preadipocyte differentiation, expression of PLA2G16 and PTGER3 mRNA was significantly increased whereas COX-1, COX-2, PTGIS, and PTGES mRNA abundance were decreased in both compartments ( ≤ 0.01 for all). Transcripts that were stimulated during adipogenesis were those that correlated best with adiposity measurements. Conclusion. Cells from the omental fat compartment release more PGE 2 and PGF 2 than those from the subcutaneous depot. Obesity modulates expression of PG-synthesizing enzymes and PG receptors which likely occurs through adipogenesis-induced changes in expression of these transcripts

Prostaglandin (PG) F2 alpha synthesis in human subcutaneous and omental adipose tissue: modulation by inflammatory cytokines and role of the human aldose reductase AKR1B1.

PGF2α may be involved in the regulation of adipose tissue function.1) To examine PGF2α release by primary preadipocytes, mature adipocytes and whole tissue explants from the subcutaneous and omental fat compartments; 2) To assess which PGF synthase is the most relevant in human adipose tissue.Fat samples were obtained by surgery in women. PGF2α release by preadipocytes, adipocytes and explants under stimulation by TNF-α, IL-1β or both was measured. Messenger RNA expression levels of AKR1B1 and AKR1C3 were measured by RT-PCR in whole adipose tissue and cytokine-treated preadipocytes. The effect of AKR1B1 inhibitor ponalrestat on PGF2α synthesis was investigated.PGF2α release was significantly induced in response to cytokines compared to control in omental (p = 0.01) and to a lesser extent in subcutaneous preadipocytes (p = 0.02). Messenger RNA of COX-2 was significantly higher in omental compared to subcutaneous preadipocytes in response to combined TNF-α and IL-1β (p = 0.01). Inflammatory cytokines increased AKR1B1 mRNA expression and protein levels (p≤0.05), but failed to increase expression levels of AKR1C3 in cultured preadipocytes. Accordingly, ponalrestat blunted PGF2α synthesis by preadipocytes in basal and stimulated conditions (p≤0.05). Women with the highest PGF2α release by omental adipocytes had a higher BMI (p = 0.05), waist circumference (p≤0.05) and HOMAir index (p≤0.005) as well as higher mRNA expression of AKR1B1 in omental (p<0.10) and subcutaneous (p≤0.05) adipose tissue compared to women with low omental adipocytes PGF2α release. Positive correlations were observed between mRNA expression of AKR1B1 in both compartments and BMI, waist circumference as well as HOMAir index (p≤0.05 for all).PGF2α release by omental mature adipocytes is increased in abdominally obese women. Moreover, COX-2 expression and PGF2α release is particularly responsive to inflammatory stimulation in omental preadipocytes. Yet, blockade of PGF synthase AKR1B1 inhibits most of the PGF2α release

Role of Dicer1-Dependent Factors in the Paracrine Regulation of Epididymal Gene Expression

<div><p>Dicer1 is an endoribonuclease involved in the biogenesis of functional molecules such as microRNAs (miRNAs) and endogenous small interfering RNAs (endo-siRNAs). These small non-coding RNAs are important regulators of post-transcriptional gene expression and participate in the control of male fertility. With the knowledge that 1) Dicer1-dependent factors are required for proper sperm maturation in the epididymis, and that 2) miRNAs are potent mediators of intercellular communication in most biological systems, we investigated the role of Dicer1-dependent factors produced by the proximal epididymis (initial segment/caput)- including miRNAs- on the regulation of epididymal gene expression in the distal epididymis regions (<i>i</i>.<i>e</i>. corpus and cauda). To this end, we performed comparative microarray and ANOVA analyses on control <i>vs</i>. <i>Defb41</i>^{<i>iCre/wt</i>}<i>;Dicer1</i>^<i>fl/fl</i> mice in which functional Dicer1 is absent from the principal cells of the proximal epididymis. We identified 35 and 33 transcripts that displayed significant expression level changes in the corpus and cauda regions (Fold change > 2 or < −2; p < 0.002), respectively. Among these transcripts, Zn-alpha 2-glycoprotein (<i>Azgp1</i>) encodes for a sperm equatorial protein whose expression in the epididymis of Dicer1 cKO mice is significantly increased compared to controls. In addition, 154 miRNAs, including <i>miR-210</i>, <i>miR-672</i>, <i>miR-191</i> and <i>miR-204</i>, showed significantly impaired biogenesis in the absence of Dicer1 from the principal cells of the proximal epididymis (Fold change > 2 or < −2; p < 0.01). These miRNAs are secreted via extracellular vesicles (EVs) derived from the DC2 epididymal principal cell line, and their expression correlates with target transcripts involved in distinct biological pathways, as evidenced by <i>in silico</i> analysis. Albeit correlative and based on <i>in silico</i> approach, our study proposes that Dicer1-dependent factors trigger- directly or not—significant genes expression changes in distinct regions of this organ. The paracrine control of functions important to post-testicular sperm maturation by Dicer1-dependent factors may open new avenues for the identification of molecular targets important to male fertility control.</p></div

PGF_2α release by omental mature adipocytes.

<p>Comparison of (<b>A</b>) omental adipocyte PGF_2α release; (<b>B</b>) subcutaneous adipocyte PGF_2α release; (<b>C</b>) omental <i>AKR1B1</i> mRNA expression; and (<b>D</b>) subcutaneous <i>AKR1B1</i> mRNA expression in women with low or high omental adipocyte PGF_2α release. Data are presented as mean ± SEM. ^† p < 0.10, *p ≤ 0.05, **p ≤ 0.005, *** p ≤ 0.0001. Expression levels relative to <i>ATP5O</i> mRNA expression. OM: omental; SC: Subcutaneous.</p

Effect of aldose reductase inhibitor on PGF_2α release by human primary preadipocytes.

<p>PGF_2α release by subcutaneous and omental preadipocytes treated for 24 h with 1 ng/ml IL-1β in the presence or absence of increasing concentrations (0-20 μM) of ponalrestat. Data are presented as mean ± SEM. Results are expressed as pg/ml*μg protein*24 h (* p≤0.05, n = 7 for all conditions).</p

<b>Characteristics of the sample (n = 46).</b>

a<p>n = 45.</p