Interactions among peroxisome proliferator activated receptor-gamma, insulin signaling pathways, and steroidogenic acute regulatory protein in human ovarian cells

CONTEXT AND OBJECTIVE: Peroxisome proliferator activated receptor-gamma (PPAR-gamma) agonists thiazolidinediones (TZDs) are thought to ameliorate hyperandrogenism in polycystic ovary syndrome by reducing hyperinsulinemia. However, TZDs also exhibit direct effects in the human ovary. We examined interactions among PPAR-gamma, insulin signaling pathways, and steroidogenic acute regulatory (StAR) protein in human ovarian cells. MATERIALS AND METHODS: Mixed human ovarian tissue culture that contained granulosa, theca, and stromal cells, and a culture of purified granulosa cells obtained during in vitro fertilization, were established as previously described. Cells were cultured in the presence or absence of insulin, with or without 25 or 50 microm rosiglitazone or pioglitazone. Expression of PPAR-gamma, insulin receptor, or insulin receptor substrate (IRS)-1 in both cell systems and of the StAR protein in granulosa cells was measured using immunoprecipitation and immunoblotting. RESULTS: Rosiglitazone stimulated expression of PPAR-gamma, insulin receptor alpha- and beta-subunits, and IRS-1 up to 168% (P < 0.05), 679% (P < 0.006), 290% (P < 0.037), and 323% (P < 0.01) of baseline, respectively. Pioglitazone stimulated expression of PPAR-gamma, insulin receptor alpha- and beta-subunits, and IRS-1 up to 222% (P < 0.01), 362% (P < 0.001), 402% (P < 0.029), and 492% (P < 0.03), respectively. Insulin alone stimulated expression of PPAR-gamma, alpha-subunit and beta-subunit of insulin receptor, and IRS-1 up to 174% (P < 0.001), 692% (P < 0.014), 275% (P < 0.024), and 431% (P < 0.01), respectively. In purified granulosa cell culture, rosiglitazone stimulated expression of StAR protein up to 540% (P < 0.007), and pioglitazone stimulated expression of StAR protein up to 670% (P < 0.007). Insulin alone stimulated expression of StAR protein up to 600% (P < 0.012). CONCLUSIONS: Insulin and TZDs independently stimulate expression of PPAR-gamma, insulin receptor, IRS-1, and StAR protein in human ovarian cells. Thus, PPAR-gamma, insulin receptor with its signaling pathways, and StAR protein constitute a novel human ovarian regulatory system with complex interactions among its components.     

Peroxisome proliferation-activated receptor-gamma ligands ameliorate experimental autoimmune myocarditis

BACKGROUND: Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) ligands have been shown to ameliorate a variety of inflammatory conditions. The present study tested the hypothesis that PPAR-gamma ligands reduce experimental autoimmune myocarditis (EAM) associated with inhibition of the expansion and activation of T cells, as well as suppression of the expression of proinflammatory cytokines. METHODS AND RESULTS: EAM was induced in Lewis rats by immunization with porcine cardiac myosin. PPAR-gamma ligands, 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) 200 microg/kg/day i.p. and pioglitazone (PIO) 10 mg/kg/day orally, were administered for 3 weeks to rats with EAM. The results showed that enhanced PPAR-gamma expression was prominently stained in the nuclear and perinuclear regions of infiltrating inflammatory cells. Administration of PPAR-gamma ligands markedly reduced the severity of myocarditis, as shown by comparing the heart weight/body weight ratio, pericardial effusion scores, macroscopic scores and microscopic scores. PPAR-gamma ligands suppressed myocardial mRNA expression of inflammatory cytokines and the expression of interleukin (IL)-1beta protein in rats with EAM. In addition, 15d-PGJ(2) and PIO treatment suppressed the proliferative response and interferon-gamma production of T cell-enriched splenocytes from rats with EAM. Furthermore, the cytotoxic activity and myocardiogenic potential of these T cells were inhibited by 15d-PGJ(2) treatment. CONCLUSIONS: PPAR-gamma may play a role in the pathophysiology of EAM. PPAR-gamma ligands ameliorate the EAM associated with suppression of the expansion and activation of myocardiogenic T cells, as well as inhibition of the expression of proinflammatory cytokines. These results suggest that PPAR-gamma ligands such as 15d-PGJ(2) and PIO may have the potential to modulate human inflammatory heart diseases such as myocarditis.     

Regulation of aromatase P450 expression in endometriotic and endometrial stromal cells by CCAAT/enhancer binding proteins (C/EBPs): decreased C/EBPbeta in endometriosis is associated with overexpression of aromatase

In human endometriotic stromal cells, markedly high levels of aromatase P450 (P450arom) mRNA and promoter II activity are present and can be vigorously stimulated by PGE(2) via a cAMP-dependent pathway to give rise to physiologically significant estrogen biosynthesis. Stromal cells of eutopic endometrium, on the other hand, do not express sufficient levels of P450arom for detectable enzyme activity. Because P450arom is up-regulated in the ovaries of CCAAT/enhancer binding protein (C/EBP) beta knockout mice and activation of the ovarian-type P450arom promoter (II) is responsible for aberrant P450arom expression in endometriosis, we sought here to evaluate the possible roles of C/EBP isoforms in the regulation of P450arom expression in endometriotic vs. eutopic endometrial stromal cells. We previously found that the -517-bp flanking region of promoter II contained the critical cis-acting elements for baseline and cAMP (analog)-induced activity. In this study, we disrupted several potential sequences and found that mutations of a -211/-197-bp cAMP-response element (CRE) and a -317/-304-bp C/EBP binding site abolished both baseline and cAMP-induced promoter II activity. Ectopic expression of C/EBPalpha increased both baseline and cAMP-dependent promoter II activity significantly in endometriotic cells, whereas ectopic expression of C/EBPbeta or C/EBPdelta abolished promoter II activity in both untreated and cAMP-treated endometriotic stromal cells. Comparable changes in promoter II activity were observed using endometrial stromal cells, which showed, however, seemingly diminished levels of baseline and cAMP-induced promoter II activity in comparison with endometriotic cells. EMSA using a probe containing the critical -317/-304-bp C/EBP site upstream of promoter II demonstrated a distinct DNA-protein complex in endometriotic, but not in endometrial stromal cells. This specific complex, however, could not be altered using antibodies against C/EBPalpha, -beta, or -delta. Because CRE is another potential DNA motif that can bind C/EBP isoforms, we next used EMSA using a probe containing the -211/-197-bp CRE and demonstrated that specific DNA-protein complexes contained C/EBPalpha but not C/EBPbeta or C/EBPdelta in endometriotic stromal cells. In contrast, C/EBPbeta and C/EBPdelta but not C/EBPalpha were detected in DNA-protein complexes using nuclear extracts from endometrial stromal cells. Western blotting and immunohistochemistry demonstrated expression of C/EBPalpha, -beta, and -delta in human endometriotic and endometrial stroma and epithelium. Intriguingly, C/EBPbeta was expressed at increased levels in stromal cells of human eutopic endometrium compared with simultaneously biopsied endometriotic tissues. We conclude that both -317/-304 and -211/-197-bp elements in promoter II are critical for the robust cAMP-dependent induction in endometriosis. C/EBPalpha up-regulates, whereas C/EBPbeta and C/EBPdelta inhibit P450arom promoter activity via binding primarily to the -211/-197-bp CRE under in vitro conditions. In vivo down-regulation of C/EBPbeta in endometriotic stromal cells and its up-regulation in endometrial stromal cells may in part account for the induction of P450arom expression in endometriosis and its inhibition in endometrium.     

Peroxisome proliferator-activated receptor-gamma and its ligands attenuate biologic functions of human natural killer cells

Interferon-gamma (IFN-gamma) production and cytolytic activity are 2 major biologic functions of natural killer (NK) cells that are important for innate immunity. We demonstrate here that these functions are compromised in human NK cells treated with peroxisome proliferator-activated-gamma (PPAR-gamma) ligands via both PPAR-gamma-dependent and -independent pathways due to variation in PPAR-gamma expression. In PPAR-gamma-null NK cells, 15-deoxy-Delta(12,14) prostaglandin J(2) (15d-PGJ(2)), a natural PPAR-gamma ligand, reduces IFN-gamma production that can be reversed by MG132 and/or chloroquine, and it inhibits cytolytic activity of NK cells through reduction of both conjugate formation and CD69 expression. In PPARgamma-positive NK cells, PPAR-gamma activation by 15d-PGJ(2) and ciglitazone (a synthetic ligand) leads to reduction in both mRNA and protein levels of IFN-gamma. Overexpression of PPAR-gamma in PPAR-gamma-null NK cells reduces IFN-gamma gene expression. However, PPAR-gamma expression and activation has no effect on NK cell cytolytic activity. In addition, 15d-PGJ(2) but not ciglitazone reduces expression of CD69 in human NK cells, whereas CD44 expression is not affected. These results reveal novel pathways regulating NK cell biologic functions and provide a basis for the design of therapeutic agents that can regulate the function of NK cells within the innate immune response.     

Hypoxia and cAMP stimulate vascular endothelial growth factor (VEGF) in human endometrial stromal cells: potential relevance to menstruation and endometrial regeneration.

The human female reproductive tract shows unique cycle-specific changes in vascularization. Vascular endothelial growth factor (VEGF) is a specific vascular endothelial mitogen which is produced by human endometrium and is known to be regulated by steroid hormones. Vasoconstriction during menstruation leads to endometrial hypoxia, a possible stimulus for angiogenesis. In the current study we tested the hypothesis that hypoxia and cAMP, a known stimulus for endometrial decidualization, can induce VEGF in human endometrial stromal cells. Decidualized as well as non decidualized stromal cells from 6 patients were exposed to normoxia (20% oxygen) and hypoxia (2% oxygen) for up to 72h. VEGF levels were assessed by Northern analysis using a 605 bp BamHI fragment of the human VEGF cDNA, and hybridization signals were normalized to levels of 18S RNA. VEGF protein was determined by ELISA. Hypoxia stimulated VEGF mRNA in decidualized stromal cells by 10.2 fold at 48h compared to normoxic controls. VEGF protein increased 10 fold by 48h and increased further to 13 fold at 72h. In the presence of 2% oxygen VEGF mRNA in nondecidualized endometrial stromal cells was increased 1.2-8 fold between 2 and 72h of treatment. VEGF protein also increased 1.2-9 fold in this time period. cAMP regulated both VEGF mRNA and protein in non decidualized stromal cells. VEGF mRNA increased 2-4 fold in 2-72h and protein production showed a 2-6 fold increase. VEGF was seen to be regulated by both cAMP and hypoxia in an additive manner. These results demonstrate that both non-decidualized and decidualized endometrial stromal cells respond to hypoxia with increasing levels of VEGF. 8Br-cAMP, which is shown to increase VEGF levels in endometrial cells per se, has an additive effect on VEGF production under hypoxic conditions. This effect may have physiologic and pathophysiologic relevance during the process of menstruation and in post menstrual endometrial repair and angiogenesis.     

Improvement of interleukin 2 production,clonogenic capability and restoration of stromal cell function in human immunodeficiency virus-type-1 patients after highly active antiretroviral therapy

Haematological abnormalities frequently occur in patients infected by human immunodeficiency virus-type 1 (HIV-1). Increasing evidence indicates that bone marrow suppression (BM) results from viral infection of accessory cells, with impaired stromal function and alteration of haematopoietic growth factor network. We have investigated the effects of antiretroviral therapy on cytokine and chemokine production by BM cells and stromal cells in a group of HIV-1-infected subjects before and during treatment. Compared with uninfected controls, an altered cytokine and chemokine production by BM cells was observed before treatment, characterized by decreased interleukin 2 (IL-2) and elevated tumour necrosis factor-alpha, macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and RANTES (regulated on activation, normal T cell-expressed and secreted) levels, along with a defective BM clonogenic activity. Antiretroviral therapy showed increased BM clonogenic capability, associated with normalization of IL-2 production and chemokine receptors expression on CD34+ cells. Pre-therapy, BM accessory cells were represented by macrophage-like cells, in some cases positive for HIV-1 DNA, suggesting that these cells are the main target of HIV-1 infection. During therapy, the stromal cells became predominantly fibroblastoid-like, as observed in normal controls, and were negative for HIV-1 DNA. Controlling HIV-1 replication may produce amelioration of stem cell activity, and restoration of stromal cell pattern and functions, with increased IL-2 production at BM level.     

Dickkopf-1, an inhibitor of Wnt signaling, is regulated by progesterone in human endometrial stromal cells

CONTEXT: Some members of the Wnt family, including ligands, receptors, inhibitors, and signaling components, are expressed in human endometrium. Dickkopf-1 (Dkk-1), a potent inhibitor of the Wnt signaling pathway, was recently found to be up-regulated in decidualizing endometrial stromal cells during the secretory phase of the menstrual cycle, suggesting regulation by progesterone. OBJECTIVES: To test the hypothesis that progesterone regulates Dkk-1 expression in human endometrial stromal cells, we investigated the following effects on stromal cell expression of Dkk-1 mRNA and protein: decidualizing stimuli (progesterone or cAMP), RU486 (an inhibitor of progesterone action), and withdrawal of progesterone. RESULTS: Short-term treatment (up to 72 h, which corresponds to the full decidualized phenotype in response to cAMP and an early response to progesterone) did not reveal regulation of Dkk-1 mRNA or protein by cAMP but did show induction of Dkk-1 expression when the cells were treated with progesterone, an effect that was blocked by RU486. In long-term cultures (from 14 to 23 d, which corresponds to the full decidualized phenotype in response to progesterone), a significant increase in Dkk-1 mRNA and protein production was observed. Addition of RU486 or withdrawal of progesterone after long-term decidualization resulted in a decrease of Dkk-1 mRNA and protein to control levels. Estradiol alone had no effect on stromal Dkk-1 expression. CONCLUSIONS: These data strongly support regulation by progesterone of Dkk-1 mRNA synthesis and protein expression in human endometrial stromal cells and that the response is specific for progesterone and independent of cAMP and estradiol.