Retinoic acid increases hypoxia-inducible factor-1α through intracrine prostaglandin E(2) signaling in human renal proximal tubular cells HK-2

We have previously shown in HK-2 cells that ATRA (all-trans-retinoic acid) up-regulates HIF-1α (hypoxia-inducible factor-1α) in normoxia, which results in increased production of renal protector VEGF-A (vascular endothelial growth factor-A). Here we investigated the role of COXs (cyclooxygenases) in these effects and we found that, i) ATRA increased the expression of COX-1 and COX-2 mRNA and protein and the intracellular levels (but not the extracellular ones) of PGE(2). Furthermore, inhibitors of COX isoenzymes blocked ATRA-induced increase in intracellular PGE(2), HIF-1α up-regulation and increased VEGF-A production. Immunofluorescence analysis found intracellular staining for EP1-4 receptors (PGE(2) receptors). These results indicated that COX activity is critical for ATRA-induced HIF-1α up-regulation and suggested that intracellular PGE(2) could mediate the effects of ATRA; ii) Treatment with PGE(2) analog 16,16-dimethyl-PGE(2) resulted in up-regulation of HIF-1α and antagonists of EP1-4 receptors inhibited 16,16-dimethyl-PGE(2)- and ATRA-induced HIF-1α up-regulation. These results confirmed that PGE(2) mediates the effects of ATRA on HIF-1α expression; iii) Prostaglandin uptake transporter inhibitor bromocresol green blocked the increase in HIF-1α expression induced by PGE(2) or by PGE(2)-increasing cytokine interleukin-1β, but not by ATRA. Therefore only intracellular PGE(2) is able to increase HIF-1α expression. In conclusion, intracellular PGE(2) increases HIF-1α expression and mediates ATRA-induced HIF-1α up-regulation.     

Protein S-glutathionylation induced by hypoxia increases hypoxia-inducible factor-1α in human colon cancer cells

Hypoxia is a common characteristic of many types of solid tumors. Intratumoral hypoxia selects for tumor cells that survive in a low oxygen environment, undergo epithelial–mesenchymal transition, are more motile and invasive, and show gene expression changes driven by hypoxia-inducible factor-1α (HIF-1α) activation. Therefore, targeting HIF-1α is an attractive strategy for disrupting multiple pathways crucial for tumor growth. In the present study, we demonstrated that hypoxia increases the S-glutathionylation of HIF-1α and its protein levels in colon cancer cells. This effect is significantly prevented by decreasing oxidized glutathione as well as glutathione depletion, indicating that S-glutathionylation and the formation of protein-glutathione mixed disulfides is related to HIF-1α protein levels. Moreover, colon cancer cells expressing glutaredoxin 1 are resistant to inducing HIF-1α and expressing hypoxia-responsive genes under hypoxic conditions. Therefore, S-glutathionylation of HIF-1α induced by tumor hypoxia may be a novel therapeutic target for the development of new drugs.     

Vasohibin induces prolyl hydroxylase-mediated degradation of hypoxia-inducible factor-1α in human umbilical vein endothelial cells

Vasohibin is thought to be an important negative feedback regulator of angiogenesis that is selectively induced in endothelial cells by VEGF. Here, we assessed the role of vasohibin on HIF-1α expression under oxidative stress induced by hydrogen peroxide (H₂O₂) in HUVEC. VEGF induced significant cell growth that was associated with an increase in vasohibin expression. Following H₂O₂-pretreatment, VEGF further increased cell growth but this was contrastingly associated with a decrease in vasohibin expression when compared with VEGF alone. Interestingly, vasohibin inhibited cell proliferation through degradation of HIF-1α expression during H₂O₂-pretreatment. Furthermore, vasohibin elevated the expression of prolyl hydroxylase (PHD). These results suggest that vasohibin plays crucial roles as a negative feedback regulator of angiogenesis through HIF-1α degradation via PHD.     

Mitochondrial reactive oxygen species mediates nicotine-induced hypoxia-inducible factor-1α expression in human non-small cell lung cancer cells

Cigarette smoking is not only a documented risk for lung carcinogenesis but also promotes lung cancer development. Nicotine, a major component of cigarette smoke but not a carcinogen by itself, has been found to induce proliferation, invasion and metastasis of non-small cell lung cancer (NSCLC). Here we reported that proinvasive effect of nicotine is analogous to that of hypoxia and involves stabilization and activation of hypoxia-inducible factor (HIF)-1α, a key factor in determining the presence of HIF-1 and expression of its downstream metastasis-associated genes. Furthermore, nicotine-induced upregulation of HIF-1α was dependent on mitochondria-derived reactive oxygen species (ROS). Ecotopic expression of mitochondrial targeted catalase effectively prevented nicotine-induced accumulation of HIF-1α protein. In addition, we demonstrated that the effect of nicotine in upregulation of HIF-1α was mediated by Dihydro-β-erythroidine (DhβE)-sensitive nicotine acetylcholine receptors (nAChRs) and required synergistic cooperation of Akt and mitogen-activated protein kinase (MAPK) pathways. These results suggest that exposure to nicotine could mimic effects of hypoxia to stimulate HIF-1α accumulation and activity that might underlie the high metastatic potential of lung cancer.     

Retinoic acid enhances the initiation of DNA synthesis stimulated by prostaglandin F2α in Swiss 3T3 cells

Retinoic acid at concentrations of 10⁻⁸–10⁻⁶M increases the initiation of DNA synthesis stimulated by prostaglandin F_2α (PGF_2α), alone or with insulin, in confluent resting Swiss mouse 3T3 cells. Analogues of retinoic acid had a similar effect. Adding retinoic acid, alone or with insulin, did not stimulate DNA synthesis. The synergistic effect of PGE₁ or PGE₂ with PGF_2α and insulin was not further enhanced by retinoic acid. Neither the synergistic effect of retinoic acid nor that of PGE₁ or PGE₂ with PGF_2α was affected by indomethacin, an inhibitor of prostaglandin synthesis. The results suggest that the synergy of retinoic acid with PGF_2α is not mediated through an increase of prostaglandin synthesis and that retinoic acid stimulates event(s) in common with those of PGE₁ and PGE₂ leading to an increase in the initiation of DNA synthesis.     

HSP47 regulates ECM accumulation in renal proximal tubular cells induced by TGF-β1 through ERK1/2 and JNK MAPK pathways

Heat shock protein (HSP)47 is a collagen-specific molecular chaperone that is essential for the biosynthesis of collagen molecules. It is likely that increased levels of HSP47 contribute to the assembly of procollagen and thereby cause an excessive accumulation of collagens in disease processes associated with fibrosis. Although HSP47 promotes renal fibrosis, the underlying mechanism and associated signaling events have not been clearly delineated. We examined the role of HSP47 in renal fibrosis using a rat unilateral ureteral obstruction model and transforming growth factor (TGF)-β(1)-treated human proximal tubular epithelial (HK-2) cells. An upregulation of HSP47 in both in vivo and in vitro models was observed, which correlated with the increased synthesis of extracellular matrix (ECM) proteins and expression of tissue-type plasminogen activator inhibitor (PAI)-1. Blockade of HSP47 by short interfering RNA suppressed the expression of ECM proteins and PAI-1. In addition, TGF-β(1)-induced HSP47 expression in HK-2 cells was attenuated by ERK1/2 and JNK MAPK inhibitors. These data suggest that ERK1/2 and JNK signaling events are involved in modulating the expression of HSP47, the chaperoning effect of which on TGF-β(1) would ultimately contribute to renal fibrosis by enhancing the synthesis and deposition of ECM proteins.     

Uric acid induced epithelial−mesenchymal transition of renal tubular cells through PI3K/p-Akt signaling pathway

The phenotypic changes of tubular epithelial cell are hallmark features of renal diseases caused by abnormal uric acid levels. We hereby intend to investigate whether PI3K/p-Akt signaling plays a role in uric-acid induced epithelial−mesenchymal transition process. The normal rat kidney cell line (NRK-52E) was used as a proximal tubular cell model in this study. NRK-52E cells were exposed to different concentrations of uric acid, or PI3K inhibitor LY294002, or both, respectively. The effects of uric acid on cell morphology were examined by phase contrast microscopy, while molecular alternations were assessed by western blot analysis and immunofluorescence staining. We found that uric acid induced visible morphological alterations in NRK-52E cells accompanied by increased expression of α-smooth muscle actin and reduced expression of E-cadherin. Moreover, phosphorylation of Akt protein was obviously increased, whereas Akt level remained stable. Furthermore, the above effects were abolished when PI3K/p-Akt pathway was blocked by the PI3K inhibitor. These findings demonstrated that high uric acid could induce phenotypic transition of cultured renal tubular cells, which was probably via activating PI3K/p-Akt signaling pathway.     

Fibroblast growth factor increases TNFα-mediated prostaglandin E2 production and TNFα receptor expression in human fibroblasts

To examine the possible role of basic fibroblast growth factor (FGF) in regulating the effects of TNF, we tested the effect of FGF on TNF-mediated PGE₂ production and TNF receptor expression in human fibroblasts. We found that, while FGF alone had no effect on PGE₂ production, it enhanced the amount of PGE₂ produced in response to TNF between 3 and 11-fold. FGF stimulated TNF-induced PGE₂ production independent of potential TNF-mediated IL-1 production, as neither anti-IL-1 mAbs nor IL-1 receptor antagonist protein (IRAP) inhibited TNF induced-PGE₂ production or the stimulatory effect of FGF. A one minute exposure of cells to FGF prior to removal was sufficient to significantly enhance TNF-induced PGE₂ production; the maximal FGF effect was reached after a 6 h preincubation. We also found that FGF significantly enhanced TNF receptor expression. Untreated fibroblasts expressed 3,900 receptors/cell, while cells treated with FGF for 6h expressed 9,500 receptors/cell, a 2.4-fold increase in receptor number; there was no apparent change in affinity for TNF (K_d 3.8×10^–11 M). The FGF-mediated increase in TNF receptor expression and TNF-mediated PGE₂ production could be abolished by FGF mAbs, indicating a specific FGF effect. These results show that FGF increases TNF receptor expression and suggest that this may account, at least in part, for the ability of FGF to enhance TNF-mediated PGE₂ production in human fibroblasts.     

Interleukin-1β, cyclooxygenase-2, and hypoxia-inducible factor-1α in asthenozoospermia

Impaired male fertility may have a variety of causes, among which asthenozoospermia. In its etiology, several bioactive substances, such as cytokines may be involved. In this context, our aim was to evaluate the expression of interleukin-1β, cyclooxygenase-2, and hypoxia-inducible factor-1α, in spermatozoa isolated from normospermic fertile donors and asthenozoospermic infertile patients. We evaluated twenty-eight infertile patients affected by idiopathic asthenozoospermia and twenty-three normospermic fertile donors, age-matched. Sperm parameters were evaluated; immunohistochemical analysis and enzyme-linked immunosorbent assay were then performed in isolated spermatozoa. Spermatozoa from the asthenozoospermic group presented an increased expression of IL-1β, COX-2, and HIF-1α compared with the normospermic fertile subjects. Our results can lead us to speculate that the increased expression of these substances may influence sperm motility. Nevertheless, further studies are needed in order to assess whether these bioactive mediators have a potential relevance as targets in future therapeutic strategies for the treatment of male infertility.     

Characterisation of the prostaglandin E2-ethanolamide suppression of tumour necrosis factor-α production in human monocytic cells

Background and purpose

Prostaglandin ethanolamides or prostamides are naturally occurring neutral lipid derivatives of prostaglandins that have been shown to be synthesised in vivo following COX-facilitated oxygenation of arachidonoyl ethanolamine (anandamide). Although the actions of prostaglandins have been extensively studied, little is known about the physiological or pathophysiological effects of prostamides. Since prostaglandin E₂ has potent immunosuppressive/immunomodulating actions, the aim of the present study was to determine whether the derivative, prostaglandin E₂ ethanolamide (PGE₂-EA), could modulate the production of the pro-inflammatory cytokine tumour necrosis factor-α in human blood and human monocytic cells and indicate whether this action involved the same receptor systems/signals as PGE₂.

Experimental approach

Whole human blood, monocytes isolated from the blood or the human monocytic cell line THP-1 was incubated with LPS and the level of TNF-α produced was measured by ELISA assay. The actions of PGE₂-EA were assessed on the LPS-induced TNF-α release. In addition, in order to ascertain the receptors involved, the levels of cyclic AMP in cells were measured in monocytes and THP-1 cells in response to PGE₂-EA and directly compared to those of PGE₂. The effect of PGE₂-EA on the binding of radiolabelled PGE₂ to cells was also measured. Cells were incubated with radiolabelled arachidonic acid and ethanolamine to estimate the production of PGE₂-EA.

Key results

PGE₂-EA potently suppressed TNF-α production in blood, monocytes and the cell line THP-1 in a concentration-dependent manner. This occurred via cyclic AMP pathways as indicated by agents which interfere with these pathways and also direct ligand binding experiments. It was also shown that the cells were able to endogenously produce PGE₂-EA.

Conclusions and implications

This study reports that PGE₂-EA can downregulate the production of TNF-α by human mononuclear cells in response to an immune stimulus, i.e. LPS-activated TLR4, and that this appears to occur via a cAMP-dependent mechanism that most likely involves binding to the EP₂ receptor.     

Lefty A attenuates the TGF-β1-induced epithelial to mesenchymal transition of human renal proximal epithelial tubular cells

The epithelial to mesenchymal transition (EMT) is a crucial event for renal fibrosis that can be elicited by TGF-β1/Smads signaling and its downstream mediator connective tissue growth factor (CTGF). As a distinct member of the TGF-β superfamily, Lefty A has been shown to be significantly downregulated in the kidneys of patients with severe ureteral obstruction, suggesting its role in renal fibrosis induced by obstructive nephropathy. In order to determine whether Lefty A prevents TGF-β1-induced EMT, human proximal tubule epithelial cells (HK-2) were stably transfected with Lefty A or control vectors and stimulated with 10 ng/ml TGF-β1 for 48 h. The results show that stimulation with TGF-β1 led to EMT including cell morphology changes, Smad2/3 signaling pathway activation, increased α-SMA, collagen type I, and CTGF expression, and decreased E-cadherin expression in mock-transfected HK-2 cells. Overexpression of Lefty A efficiently blocked p-Smad2/3 activation and attenuated all these EMT changes induced by TGF-β1. This finding suggests that Lefty A may serve as a potential new therapeutic target to inhibit or even reverse EMT during the process of renal fibrosis.     

Betulinic acid inhibits the expression of hypoxia-inducible factor 1α and vascular endothelial growth factor in human endometrial adenocarcinoma cells

Although betulinic acid (BA) is known to induce apoptosis and antiangiogenic response in tumor cells, the underlying mechanism of its action is unknown. Deregulation of tissue collagen metabolism is one of the consequences of neoplastic transformation. The final step of collagen degradation is mediated by prolidase [E.C.3.4.13.9] which may play a role in angiogenesis. The formation of new blood vessels is regulated by the hypoxia-inducible factor 1 (HIF-1). The expression of HIF-1 correlates with hypoxia-induced angiogenesis as a result of the induction of vascular endothelial cell growth factor (VEGF). Since BA evokes anticancer activity, its effect on collagen biosynthesis, HIF-1α and VEGF expressions, as well as prolidase activity and expression was studied in cultured endometrial adenocarcinoma (EA) cells. It was found that BA inhibits collagen biosynthesis in EA cells (5[³H] proline incorporation assay). It was accompanied by a parallel decrease in prolidase activity and expression and decrease in expressions of α₁ and α₂ integrins, HIF-1α, and VEGF (western immunoblot analysis) in cultured human EA cells. The data suggest that BA may have anti-angiogenic potential by inhibition of prolidase, HIF-1α and VEGF expressions, and inhibition of collagen biosynthesis.