Lipocalin Prostaglandin D Synthase Research Articles

L-PGDS deletion accelerates the development of aging-associated osteoarthritis

Purpose: Lipocalin prostaglandin D synthase (L-PGDS) is responsible for the biosynthesis of PGD2, which has been implicated in the regulation of inflammation and cartilage biology. This study aimed to evaluate the effect of L-PGDS deletion on the development of aging-associated osteoarthritis (OA) in mice. Methods: Cartilage degradation was evaluated by histology. The expression of MMP-13 and ADAMTS-5 was assessed by immunohistochemistry. Bone changes were determined by microcomputed tomography (μ-CT). Pain related behaviours were assessed using the von Frey and the open-field assays. Results: L-PGDS deletion promoted cartilage degradation during aging, which was associated with enhanced expression of extracellular matrix degrading enzymes MMP-13 and ADAMTS-5, and their breakdown products, C1,2C, VDIPEN and NITEG. Moreover, L-PGDS deletion enhanced subchondral bone changes, but had no effect on its angiogenesis. Additionally, L-PGDS deletion increased mechanical sensitivity and reduced spontaneous locomotor activity. Finally, we showed that the expression of L-PGDS was elevated in aged mice. Conclusions: Together, these findings indicate an important role for L-PGDS in aging-associated OA. They also suggest that L-PGDS may constitute a new efficient therapeutic target in OA.

L-PGDS deficiency accelerated the development of naturally occurring age-related osteoarthritis.

Osteoarthritis (OA) is the most common musculoskeletal disorder among the elderly. It is characterized by progressive cartilage degradation, synovial inflammation, subchondral bone remodeling and pain. Lipocalin prostaglandin D synthase (L-PGDS) is responsible for the biosynthesis of PGD2, which has been implicated in the regulation of inflammation and cartilage biology. This study aimed to evaluate the effect of L-PGDS deficiency on the development of naturally occurring age-related OA in mice.OA-like structural changes were assessed by histology, immunohistochemistry, and micro–computed tomography. Pain related behaviours were assessed using the von Frey and the open-field assays.L-PGDS deletion promoted cartilage degradation during aging, which was associated with enhanced expression of extracellular matrix degrading enzymes, matrix metalloprotease 13 (MMP-13) and a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS-5), and their breakdown products, C1,2C, VDIPEN and NITEG. Moreover, L-PGDS deletion enhanced subchondral bone changes, but had no effect on its angiogenesis. Additionally, L-PGDS deletion increased mechanical sensitivity and reduced spontaneous locomotor activity. Finally, we showed that the expression of L-PGDS was elevated in aged mice. Together, these findings indicate an important role for L-PGDS in naturally occurring age-related OA. They also suggest that L-PGDS may constitute a new efficient therapeutic target in OA.

Mass spectrometric quantification of prostaglandin D2 levels in choroid plexus, cerebrospinal fluid and rostral ventrolateral medulla link PGD2 signaling to the development of neurogenic hypertension (1140.10)

Roles of Prostaglandin (PG) D2 signaling in blood pressure (BP) control are obscure. PGD2 is synthesized from precursor arachidonic acid and PGH2 by the action of enzyme lipocalin‐prostaglandin D synthase (L‐PGDS). Choroid plexus lining the third ventricle (3VCP) and cerebrospinal fluid (CSF) from AngII‐salt hypertensive (AngII‐HS‐HTN) rats express high mRNA and protein levels of L‐PGDS. Blocking the L‐PGDS enzyme centrally with its antagonist AT56 attenuates AngII‐salt‐HTN development. So we hypothesized that increased PGD2 synthesis in the brain is central to AngII‐salt mediated increase in BP.Method: Two sets of experiments were performed. In one set of high salt (HS, 2%) fed rats, vehicle or AngII (150 ng/kg/min, sc) was infused. In another set of HS fed rats, L‐PGDS was first blocked chronically with different doses (nmol‐h‐1, sc) of AT56 (0.6,6,23) and then AngII was infused. From both studies, brain tissue and CSF were collected on day 4 of AngII infusion for ultra performance liquid chromatography‐tandem mass spectrometric analysis of substrate arachidonic acid (AA) and metabolites PGD2, PGJ2, delta12‐PGJ2 and PGE2.Result: HS and AngII compared to vehicle controls (p<0.05) increased the levels of substrate AA in 3VCP. HS and AngII significantly increased the metabolite PGD2 in 3VCP, CSF and rostral ventrolateral medulla (RVLM) compared to vehicle controls. This increase in PGD2 was inhibited in the RVLM of rats treated with AT56 at doses (6 and 23 nmol‐h‐1, sc) that were previously observed to attenuate AngII‐HS‐HTN. There was with no change in the levels of metabolites PGJ2, delta12‐PGJ2 and PGE2 with HS and AngII treatment.Conclusion: Increased PGD2 synthesis and signaling in the 3VCP, CSF and RVLM mediate development of AngII‐salt HTN. AA‐3VCP(ng/region ± SE) PGD2‐3VCP(ng/region ± SE) PGD2‐RVLM(ng/region ± SE) PGD2‐CSF (pg/ml ± SE) HS Vehicle 333 ± 80 0.8 ± 0.4 0.3 ± 0.1 193 ± 39 HS AngII 3057 ± 1163 10.3 ± 4 9 ± 1 418 ± 51 Grant Funding Source: RO1 HL 076312 and RCO HL 060363

Nrf2 is essential for the expression of lipocalin–prostaglandin D synthase induced by prostaglandin D2

Nrf2 is a transcription factor that protects against inflammatory diseases, but the underlying mechanism of this effect remains unclear. Here, we report that Nrf2 uses lipocalin–prostaglandin D synthase (L-PGDS) as a mechanism for suppressing inflammation. Exogenously added prostaglandin D2 (PGD2) induced L-PGDS expression in bone-marrow-derived macrophages (BMDMs), suggesting a positive feedback loop between L-PGDS expression and PGD2. Unlike lipopolysaccharide (LPS)-induced L-PGDS expression, PGD2-mediated expression was independent of MAPK, PU.1, or TLR4. Sequence analysis located a putative Nrf2 binding site in the murine L-PGDS promoter, to which Nrf2 bound when treated with PGD2. Chemical activation, or overexpression, of Nrf2 was sufficient to induce L-PGDS expression in macrophages, BMDMs, or lungs of Nrf2-knockout (KO) mice, but treatment with PGD2 failed to do so, suggesting a pivotal role for Nrf2 in the expression of L-PGDS. Consistent with this, expression of Nrf2 in the lungs of Nrf2-KO mice was sufficient to induce the expression of L-PGDS and to reduce neutrophilic lung inflammation elicited by LPS. Furthermore, expression of L-PGDS in mouse lungs decreased neutrophilic infiltration, ameliorating lung inflammation in mice. Together, our results show that Nrf2, activated by PGD2, induced L-PGDS expression, resulting in decreased inflammation. We suggest that the positive feedback induction of L-PGDS by PGD2 is part of the mechanism by which Nrf2 regulates inflammation.

Structural and dynamic insights into substrate binding and catalysis of human lipocalin prostaglandin D synthase

Lipocalin prostaglandin D synthase (L-PGDS) regulates synthesis of an important inflammatory and signaling mediator, prostaglandin D2 (PGD2). Here, we used structural, biophysical, and biochemical approaches to address the mechanistic aspects of substrate entry, catalysis, and product exit of this enzyme. Structure of human L-PGDS was solved in a complex with a substrate analog (SA) and in ligand-free form. Its catalytic Cys 65 thiol group was found in two different conformations, each making a distinct hydrogen bond network to neighboring residues. These help in elucidating the mechanism of the cysteine nucleophile activation. Electron density for ligand observed in the active site defined the substrate binding regions, but did not allow unambiguous fitting of the SA. To further understand ligand binding, we used NMR spectroscopy to map the binding sites and to show the dynamics of protein-substrate and protein-product interactions. A model for ligand binding at the catalytic site is proposed, showing a second binding site involved in ligand exit and entry. NMR chemical shift perturbations and NMR resonance line-width alterations (observed as changes of intensity in two-dimensional cross-peaks in [¹H,¹⁵N]-transfer relaxation optimization spectroscopy) for residues at the Ω loop (A-B loop), E-F loop, and G-H loop besides the catalytic sites indicate involvement of these residues in ligand entry/egress.

Prostaglandin D2 ameliorates acute lung inflammation by activating Nrf2 independent of 15-deoxy-Δ12,14-PGJ2. (P5040)

Abstract Since anti-inflammatory activity of prostaglandin D2 (PGD2) is attributed to 15-deoxy-Δ12,14-PGJ2, a byproduct of PGD2, we explored the hypothesis that PGD2, independent of 15-deoxy-Δ12,14-PGJ2, activates NF-E2-related factor 2 (Nrf2), an anti-inflammatory transcription factor, contributing to suppression of lung inflammation in acute lung injury (ALI). Intratracheal delivery of PGD2 decreased neutrophilic infiltration, MPO activity, and the production of pro-inflammatory cytokines in LPS-treated lungs. In contrast, lipocalin-prostaglandin D synthase (L-PGDS) KO mice that produce less PGD2 showed prolonged lung inflammation, highlighting the importance of L-PGDS and PGD2 in suppressing lung inflammation. Two log10 less of PGD2 than 15-deoxy-Δ12,14-PGJ2 was sufficient for activating Nrf2 in RAW 264.7. Similarly, PGD2 was more potent than 15-deoxy-Δ12,14-PGJ2 in expressing Nrf2 dependent genes in mouse lungs. While overexpression of DP1, a receptor of PGD2, enhanced PGD2-mediated Nrf2 activation, siRNA silencing DP1 expression reduced its activation, suggesting that PGD2 uses DP1 in activating Nrf2. Furthermore, PGD2 synergistically suppressed neutrophil infiltration when nrf2 was functionally complemented to the lung of the nrf2-/- mice. Our results show that PGD2 activated Nrf2 independent of 15-deoxy-Δ12,14-PGJ2 and involved DP1. We suggest that PGD2 has a potent anti-inflammatory activity, which is at least in part mediated by activating Nrf2.

A new role for lipocalin prostaglandin d synthase in the regulation of brown adipose tissue substrate utilization.

In this study, we define a new role for lipocalin prostaglandin D synthase (L-PGDS) in the control of metabolic fuel utilization by brown adipose tissue (BAT). We demonstrate that L-PGDS expression in BAT is positively correlated with BAT activity, upregulated by peroxisome proliferator–activated receptor γ coactivator 1α or 1β and repressed by receptor-interacting protein 140. Under cold-acclimated conditions, mice lacking L-PGDS had elevated reliance on carbohydrate to provide fuel for thermogenesis and had increased expression of genes regulating glycolysis and de novo lipogenesis in BAT. These transcriptional differences were associated with increased lipid content in BAT and a BAT lipid composition enriched with de novo synthesized lipids. Consistent with the concept that lack of L-PGDS increases glucose utilization, mice lacking L-PGDS had improved glucose tolerance after high-fat feeding. The improved glucose tolerance appeared to be independent of changes in insulin sensitivity, as insulin levels during the glucose tolerance test and insulin, leptin, and adiponectin levels were unchanged. Moreover, L-PGDS knockout mice exhibited increased expression of genes involved in thermogenesis and increased norepinephrine-stimulated glucose uptake to BAT, suggesting that sympathetically mediated changes in glucose uptake may have improved glucose tolerance. Taken together, these results suggest that L-PGDS plays an important role in the regulation of glucose utilization in vivo.

Lipocalin prostaglandin D synthase and PPARγ2 coordinate to regulate carbohydrate and lipid metabolism in vivo.

Mice lacking Peroxisome Proliferator-Activated Receptor γ2 (PPARγ2) have unexpectedly normal glucose tolerance and mild insulin resistance. Mice lacking PPARγ2 were found to have elevated levels of Lipocalin prostaglandin D synthase (L-PGDS) expression in BAT and subcutaneous white adipose tissue (WAT). To determine if induction of L-PGDS was compensating for a lack of PPARγ2, we crossed L-PGDS KO mice to PPARγ2 KO mice to generate Double Knock Out mice (DKO). Using DKO mice we demonstrated a requirement of L-PGDS for maintenance of subcutaneous WAT (scWAT) function. In scWAT, DKO mice had reduced expression of thermogenic genes, the de novo lipogenic program and the lipases ATGL and HSL. Despite the reduction in markers of lipolysis in scWAT, DKO mice had a normal metabolic rate and elevated serum FFA levels compared to L-PGDS KO alone. Analysis of intra-abdominal white adipose tissue (epididymal WAT) showed elevated expression of mRNA and protein markers of lipolysis in DKO mice, suggesting that DKO mice may become more reliant on intra-abdominal WAT to supply lipid for oxidation. This switch in depot utilisation from subcutaneous to epididymal white adipose tissue was associated with a worsening of whole organism metabolic function, with DKO mice being glucose intolerant, and having elevated serum triglyceride levels compared to any other genotype. Overall, L-PGDS and PPARγ2 coordinate to regulate carbohydrate and lipid metabolism.

Lipopolysaccharide-dependent interaction between PU.1 and cJun determines production of lipocalin-type prostaglandin D synthase and prostaglandin D2 in macrophages

Previously, we reported that expression of lipocalin-prostaglandin D synthase (L-PGDS) is inducible in macrophages and protects from Pseudomonas pneumonia. Here, we investigated the mechanism by which L-PGDS gene expression is induced in macrophages. A promoter analysis of the murine L-PGDS promoter located a binding site of PU.1, a transcription factor essential for macrophage development and inflammatory gene expression. A chromatin immunoprecipitation assay showed that PU.1 bound to the cognate site in the endogenous L-PGDS promoter in response to LPS. Overexpression of PU.1, but not of PU.1(S148A), a mutant inert to casein kinase II (CKII) or NF-kappaB-inducing kinase (NIK), induced L-PGDS in RAW 264.7 cells. Conversely, siRNA silencing of PU.1 expression blunted productions of L-PGDS and prostaglandin D2 (PGD(2)). LPS treatment induced formation of the complex of PU.1 and cJun on the PU.1 site, but inactivation of cJun by treatment with JNK or p38 kinase inhibitor abolished the complex, and suppressed PU.1 transcriptional activity for L-PGDS gene expression. Together, these results show that PU.1, activated by CKII or NIK, cooperates with MAPK-activated cJun to maximally induce L-PGDS expression in macrophages following LPS treatment, and suggest that PU.1 participates in innate immunity through the production of L-PGDS and PGD(2).

Estradiol suppresses rapid eye movement sleep and activation of sleep‐active neurons in the ventrolateral preoptic area

Studies from multiple species, including humans, suggest that gonadal hormones, and ovarian hormones in particular, influence the physiology of sleep, but the mechanisms by which these hormones influence sleep behaviors are unknown. Previously, we demonstrated a 50% reduction in lipocalin-prostaglandin D synthase (L-PGDS) transcript levels, following estradiol treatment, at the level of the ventrolateral preoptic area (VLPO), a putative sleep-active nucleus. Catalytic activity of L-PGDS produces prostaglandin D(2) (PGD(2)), an endogenous somnogen. Based on our previous studies, we hypothesized that estradiol is acting via PGD(2) to suppress neuronal activity in the VLPO of females. To begin to test whether this is true, we quantified the number of Fos-immunopositive cells in hormonally manipulated male and female rats. We found that in females during the light phase, estradiol suppressed Fos expression in VLPO neurons. Interestingly, protein expression of L-PGDS followed the same pattern. Surprisingly, changes in the hormonal milieu of males had no effect. Using telemetry to record electroencephalograms from gonadally intact females, we found, in the light phase of proestrus when estradiol levels are high, a marked reduction in rapid eye movement (REM) sleep compared with the other days of the estrous cycle. However, during the dark phase of proestrus when estrogen and progesterone levels are elevated, significantly less time was spent in both non-REM and REM sleep. Thus, it seems that hormones in females play a major role in the regulation of sleep and arousal via activation of neurons in key sleep and arousal centers.

Molecular characterization and developmental expression pattern of the chicken apolipoprotein D gene: Implications for the evolution of vertebrate lipocalins

The insect Lazarillo and the mammalian apolipoprotein D (ApoD) are orthologous members of the lipocalin protein family. We report the cloning and embryonic expression of chicken ApoD, the first molecularly characterized nonmammalian ApoD. We also report the ApoD expression in mouse during postnatal development and some novel aspects of the expression of the paralogous lipocalin prostaglandin D-synthase (PGDS) and discuss these results in view of the lipocalin family evolution in vertebrates. ApoD is expressed in subsets of central nervous system (CNS) neurons and glia during late chicken embryogenesis. Contrary to mouse ApoD, no expression appears in neural crest-derived cephalic mesenchyme and blood vessel pericytes. Also, ApoD is expressed in developing chicken feathers. These expressions are corroborated by quantitative reverse transcriptase-polymerase chain reaction profiles. ApoD is expressed during mouse postnatal development in a subset of CNS neurons, astrocytes and oligodendrocytes, but also in meninges and pericytes. Chicken PGDS is expressed in brain meninges and perivascular cells. Our results suggest that the amniote last common ancestor expressed ApoD and PGDS in the brain during embryogenesis. ApoD appears restricted to ectodermal derivatives, whereas PGDS is expressed by derivatives of the three germ layers.