MicroRNA-mediated Krüppel-like factor 4 upregulation induces alternatively activated macrophage-associated marker and chemokine transcription in 4,4'-methylene diphenyl diisocyanate exposed macrophages.

1. Occupational exposure to 4,4'-methylene diphenyl diisocyanate (MDI) is associated with occupational asthma (OA) development. Alveolar macrophage-induced recruitment of immune cells to the lung microenvironment plays an important role during asthma pathogenesis. Previous studies identified that MDI/MDI-glutathione (GSH)-exposure downregulates endogenous hsa-miR-206-3p/hsa-miR-381-3p. Our prior report shows that alternatively activated (M2) macrophage-associated markers/chemokines are induced by MDI/MDI-GSH-mediated Krüppel-Like Factor 4 (KLF4) upregulation in macrophages and stimulates immune cell chemotaxis. However, the underlying molecular mechanism(s) by which MDI/MDI-GSH upregulates KLF4 remain unclear.2. Following MDI-GSH exposure, microRNA(miR)-inhibitors/mimics or plasmid transfection, endogenous hsa-miR-206-3p/hsa-miR-381-3p, KLF4, or M2 macrophage-associated markers (CD206, TGM2), and chemokines (CCL17, CCL22, CCL24) were measured by either RT-qPCR, western blot, or luciferase assay.3. MDI-GSH exposure downregulates hsa-miR-206-3p/hsa-miR-381-3p by 1.46- to 9.75-fold whereas upregulates KLF4 by 1.68- to 1.99-fold, respectively. In silico analysis predicts binding between hsa-miR-206-3p/hsa-miR-381-3p and KLF4. Gain- and loss-of-function, luciferase reporter assays and RNA-induced silencing complex-immunoprecipitation (RISC-IP) studies confirm the posttranscriptional regulatory roles of hsa-miR-206-3p/hsa-miR-381-3p and KLF4 in macrophages. Furthermore, hsa-miR-206-3p/hsa-miR-381-3p regulate the expression of M2 macrophage-associated markers and chemokines via KLF4.4. In conclusion, hsa-miR-206-3p/hsa-miR-381-3p play a major role in regulation of MDI/MDI-GSH-induced M2 macrophage-associated markers and chemokines by targeting the KLF4 transcript, and KLF4-mediated regulation in macrophages.

Human Keratinocyte Response to 4,4'-Methylene Diphenyl Diisocyanate-Glutathione Conjugate Exposure.

Workplace exposure to diisocyanates like 4,4'-methylene diphenyl diisocyanate can cause occupational asthma (MDI-OA), and the underlying biological pathways are still being researched.Although uncertainty remains, evidence supports the hypothesis that dermal exposure to MDI plays an important role in the development of MDI-OA.Gene expression, proteomics, and informatics tools were utilized to characterize changes in expression of RNA and protein in cultured human HEKa keratinocyte cells following exposure to conjugates of MDI with glutathione (MDI-GSH).RT-qPCR analysis using a panel of 39 candidate primers demonstrated 9 candidate genes upregulated and 30 unchanged.HPLC-MS/MS analysis of HEKa cell lysate identified 18,540 proteins across all samples Sixty proteins demonstrate statistically significant differential expression in exposed cells, some of which suggest activation of immune and inflammatory pathways.The results support the hypothesis that dermal exposures have the potential to play an important role in the development of MDI-OA. Furthermore, proteomic and gene expression data suggest multiple immune (adaptive and innate) and inflammatory pathways may be involved in the development of MDI-OA.

4,4'-Methylene diphenyl diisocyanate exposure induces expression of alternatively activated macrophage-associated markers and chemokines partially through Krüppel-like factor 4 mediated signaling in macrophages.

Occupational exposure to the most widely used monomeric diisocyanate (dNCO), 4,4'-methylene diphenyl diisocyanate (MDI), may lead to the development of occupational asthma (OA). Alveolar macrophages with alternatively activated (M2) phenotype have been implicated in allergic airway responses and the pathogenesis of asthma. Recent in vivo studies demonstrate that M2 macrophage-associated markers and chemokines are induced by MDI-exposure, however, the underlying molecular mechanism(s) by which this proceeds is unclear.Following MDI exposure (in vivo and in vitro) M2 macrophage-associated transcription factors (TFs), markers, and chemokines were determined by RT-qPCR, western blots, and ELISA.Expression of M2 macrophage-associated TFs and markers including Klf4/KLF4, Cd206/CD206, Tgm2/TGM2, Ccl17/CCL17, Ccl22/CCL22, and CCL24 were induced by MDI/MDI-GSH exposure in bronchoalveolar lavage cells (BALCs)/THP-1 macrophages. The expression of CD206, TGM2, CCL17, CCL22, and CCL24 are upregulated by 3.83-, 7.69-, 6.22-, 6.08-, and 1.90-fold in KLF4-overexpressed macrophages, respectively. Endogenous CD206 and TGM2 were downregulated by 1.65-5.17-fold, and 1.15-1.78-fold, whereas CCL17, CCL22, and CCL24 remain unchanged in KLF4-knockdown macrophages. Finally, MDI-glutathione (GSH) conjugate-treated macrophages show increased chemotactic ability to T-cells and eosinophils, which may be attenuated by KLF4 knockdown.Our data suggest that MDI exposure may induce M2 macrophage-associated markers partially through induction of KLF4.

MicroRNA-mediated calcineurin signaling activation induces CCL2, CCL3, CCL5, IL8, and chemotactic activities in 4,4'-methylene diphenyl diisocyanate exposed macrophages.

Occupational exposure to 4,4'-methylene diphenyl diisocyanate (MDI), the most widely used monomeric diisocyanate, is one of the leading causes of occupational asthma (OA). Previously, we identified microRNA (miR)-206-3p/miR-381-3p-mediated PPP3CA/calcineurin signalling regulated iNOS transcription in macrophages and bronchoalveolar lavage cells (BALCs) after acute MDI exposure; however, whether PPP3CA/calcineurin signalling participates in regulation of other asthma-associated mediators secreted by macrophages/BALCs after MDI exposure is unknown.Several asthma-associated, macrophage-secreted mediator mRNAs from MDI exposed murine BALCs and MDI-glutathione (GSH) conjugate treated differentiated THP-1 macrophages were analysed using RT-qPCR.Endogenous IL1B, TNF, CCL2, CCL3, CCL5, and TGFB1 were upregulated in MDI or MDI-GSH conjugate exposed BALCs and macrophages, respectively. Calcineurin inhibitor tacrolimus (FK506) attenuated the MDI-GSH conjugate-mediated induction of CCL2, CCL3, CCL5, and CXCL8/IL8 but not others. Transfection of either miR-inhibitor-206-3p or miR-inhibitor-381-3p in macrophages induced chemokine CCL2, CCL3, CCL5, and CXCL8 transcription, whereas FK506 attenuated the miR-inhibitor-206-3p or miR-inhibitor-381-3p-mediated effects. Finally, MDI-GSH conjugate treated macrophages showed increased chemotactic ability to various immune cells, which may be attenuated by FK506.In conclusion, these results indicate that MDI exposure to macrophages/BALCs may recruit immune cells into the airway via induction of chemokines by miR-206-3p and miR-381-3p-mediated calcineurin signalling activation.

Circulating miRs-183-5p, -206-3p and -381-3p may serve as novel biomarkers for 4,4'-methylene diphenyl diisocyanate exposure.

BACKGROUND: Occupational exposure to the most widely used diisocyanate, 4,4'-methylene diphenyl diisocyanate (MDI), is a cause of occupational asthma (OA). Early recognition of MDI exposure and sensitization is essential for the prevention of MDI-OA. OBJECTIVE: Identify circulating microRNAs (miRs) as novel biomarkers for early detection of MDI exposure and prevention of MDI-OA. MATERIALS AND METHODS: Female BALB/c mice were exposed to one of three exposure regimens: dermal exposure to 1% MDI in acetone; nose-only exposure to 4580 ± 1497 µg/m3 MDI-aerosol for 60 minutes; or MDI dermal exposure/sensitization followed by MDI-aerosol inhalation challenge. Blood was collected and miRCURY™ miRs qPCR Profiling Service was used to profile circulate miRs from dermally exposed mice. Candidate miRs were identified and verified from mice exposed to three MDI-exposure regimens by TaqMan® miR assays. RESULTS: Up/down-regulation patterns of circulating mmu-miRs-183-5p, -206-3p and -381-3p were identified and verified. Circulating mmu-miR-183-5p was upregulated whereas mmu-miRs-206-3p and -381-3p were downregulated in mice exposed via all three MDI exposure regimens. DISCUSSION AND CONCLUSION: Upregulation of circulating miR-183-5p along with downregulation of circulating miRs-206-3p and -381-3p may serve as putative biomarkers of MDI exposure and may be considered as potential candidates for validation in exposed human worker populations.

Mass spectrometry-based analysis of murine bronchoalveolar lavage fluid following respiratory exposure to 4,4'-methylene diphenyl diisocyanate aerosol.

1. Diisocyanates are highly reactive electrophiles utilized in the manufacture of a wide range of polyurethane products and have been identified as causative agents of occupational allergic respiratory disease. However, in spite of the significant occupational health burden associated with diisocyanate-induced asthma, the mechanism of disease pathogenesis remains largely unknown. 2. To better understand the fate of inhaled diisocyanates, a nose-only aerosol exposure system was constructed and utilized to expose a BALB/c mouse model to an aerosol generated from 4,4'-methylene diphenyl diisocyanate (MDI). Tissue and bronchoalveolar lavage samples were evaluated 4 and 24 h post-exposure for evidence of diisocyanate-protein haptenation, and a label-free quantitative proteomics strategy was employed to evaluate relative changes to the protein content of the cellular fraction of the lavage fluid. 3. Following MDI aerosol exposure, expression of the number of proteins with immunological or xenobiotic metabolism relevance is increased, including endoplasmin, cytochrome P450 and argininosuccinate synthase. Western blot analysis indicated MDI-conjugated protein in the lavage fluid, which was identified as serum albumin. 4. Tandem mass spectrometry analysis of MDI-albumin revealed MDI conjugation occurs at a dilysine motif at Lys525, as well as at a glutamine-lysine motif at Lys414, in good agreement with previously published in vitro data on diisocyanate-conjugated serum albumin.

SOX9 inhibits ß-TrCP-mediated protein degradation to promote nuclear GLI1 expression and cancer stem cell properties.

The high mobility group box protein SOX9 and the GLI1 transcription factor play protumorigenic roles in pancreatic ductal adenocarcinoma (PDA). In Kras transgenic mice, each of these factors are crucial for the development of PDA precursor lesions. SOX9 transcription is directly regulated by GLI1, but how SOX9 functions downstream of GLI1 is unclear. We observed positive feedback, such that SOX9-deficient PDA cells have severely repressed levels of endogenous GLI1, attributed to loss of GLI1 protein stability. SOX9 associated with the F-box domain of the SKP1/CUL1/F-box (SCF) E3 ubiquitin ligase component, ß-TrCP (also known as F-box/WD repeat-containing protein 1A), and suppressed its association with SKP1 and GLI1, a substrate of SCF-ß-TrCP. SOX9 also tethered ß-TrCP within the nucleus and promoted its degradation. SOX9 bound to ß-TrCP through the SOX9 C-terminal PQA/S domain that mediates transcriptional activation. Suppression of ß-TrCP in SOX9-deficient PDA cells restored GLI1 levels and promoted SOX9-dependent cancer stem cell properties. These studies identify SOX9-GLI1 positive feedback as a major determinant of GLI1 protein stability and implicate ß-TrCP as a latent SOX9-bound tumor suppressor with the potential to degrade oncogenic proteins in tumor cells.

Functional Hierarchy and Cooperation of EMT Master Transcription Factors in Breast Cancer Metastasis.

Several master transcription factors (TF) can activate the epithelial-to-mesenchymal transition (EMT). However, their individual and combinatorial contributions to EMT in breast cancer are not defined. We show that overexpression of EMT-TFs individually in epithelial cells upregulated endogenous SNAI2, ZEB1/2, TCF4, and TWIST1/2 as a result of positive feedback mediated in part by suppression of their negative regulator miRNAs miR200s/203/205. We identified TCF4 as a potential new target of miR200s. Expression of ZEB1/2 strongly correlated with the mesenchymal phenotype in breast cancer cells, with the CD24-/CD44+ stemness profile, and with lower expression of core epithelial genes in human breast tumors. Knockdown of EMT-TFs identified the key role of ZEB1 and its functional cooperation with other EMT-TFs in the maintenance of the mesenchymal state. Inducible ZEB1+2 knockdown in xenograft models inhibited pulmonary metastasis, emphasizing their critical role in dissemination from primary site and in extravasation. However, ZEB1+2 depletion one-week after intravenous injection did not inhibit lung colonization, suggesting that ZEB1/2 and EMT are not essential for macrometastatic outgrowth. These results provide strong evidence that EMT is orchestrated by coordinated expression of several EMT-TFs and establish ZEB1 as a key master regulator of EMT and metastasis in breast cancer. IMPLICATIONS: The EMT program is orchestrated by coordinated expression of multiple EMT transcription factors, whereas ZEB1 integrates the EMT master regulatory network and plays the major role in promoting EMT and metastasis.

Acute 4,4'-Methylene Diphenyl Diisocyanate Exposure-Mediated Downregulation of miR-206-3p and miR-381-3p Activates Inducible Nitric Oxide Synthase Transcription by Targeting Calcineurin/NFAT Signaling in Macrophages.

Exposure to 4,4'-methylene diphenyl diisocyanate (MDI) in the occupational setting may lead to development of occupational asthma (OA), and the underlying molecular mechanisms of MDI-induced disease pathogenesis remain an active area of research. Using a nose-only mouse inhalation model, we find that circulating microRNA (miR)-206-3p and miR-381-3p are downregulated after MDI exposure; however, cellular miR-206-3p and miR-381-3p responses after MDI aerosol exposure and their pathophysiological roles in MDI-OA are unknown. We hypothesize that miR-206-3p and miR-381-3p-regulated mechanisms cause increased expression of the inducible nitric oxide synthase (iNOS) after MDI aerosol exposure. We examined cellular miR-206-3p and miR-381-3p, calcineurins, nuclear factors of activated T cells (NFATs), and iNOS levels from both nose-only exposed murine bronchoalveolar lavage cells (BALCs) and differentiated THP-1 macrophages treated with MDI-glutathione (GSH) conjugates. Both in vivo murine MDI aerosol exposure and in vitro MDI-GSH exposures in THP-1 macrophages result in downregulation of endogenous miR-206-3p and miR-381-3p and upregulation of PPP3CA and iNOS expression. Transfection of THP-1 macrophages with miR-inhibitor-206-3p and miR-inhibitor-381-3p resulted in the upregulation of PPP3CA and iNOS. Using RNA-induced silencing complex immunoprecipitation and translational reporter assays, we verified that PPP3CA, but not iNOS, is directly targeted by both miR-206-3p and miR-381-3p. Downregulation of miR-206-3p and miR-381-3p following by MDI exposure induces calcineurin/NFAT signaling-mediated iNOS transcription in macrophages and BALCs.

Distinct regulation of cyclooxygenase-2 by interleukin-1beta in normal and endometriotic stromal cells.

Aberrant production of cyclooxygenase-2 (COX-2) plays pivotal roles in many pathological processes including tumorigenesis and endometriosis, although the underlying mechanism remains obscure. Herein we report evidence to demonstrate that COX-2 is distinctly regulated by IL-1beta in normal and endometriotic stroma. Ectopic endometriotic stromal cell is at least 100 times more sensitive to IL-1beta treatment, compared with its eutopic counterpart. Induction of COX-2 expression in normal endometrial stroma by IL-1beta is primary due to enhancement of COX-2 mRNA stability. In contrast, IL-1beta not only increases COX-2 mRNA stability but also up-regulates COX-2 promoter activity in ectopic endometriotic stroma. Induction of COX-2 promoter activity by IL-1beta is mediated via MAPK-dependent phosphorylation of cAMP-responding element binding protein. Promoter activity and EMSAs demonstrate that a cAMP response element site located at -571/-564 of COX-2 promoter is critical for IL-1beta-induced COX-2 gene expression. Our results indicate that elevation of COX-2 expression in endometriotic tissues may result from increased sensitivity to proinflammatory cytokines such as IL-1beta, which is consistently present in the peritoneal fluid of endometriosis patients. Distinct regulation of COX-2 gene by IL-1beta may play a critical role in pathophysiological processes such as cancer formation and endometriosis.

KAP1 promotes proliferation and metastatic progression of breast cancer cells.

KAP1 (TRIM28) is a transcriptional regulator in embryonic development that controls stem cell self-renewal, chromatin organization, and the DNA damage response, acting as an essential corepressor for KRAB family zinc finger proteins (KRAB-ZNF). To gain insight into the function of this large gene family, we developed an antibody that recognizes the conserved zinc fingers linker region (ZnFL) in multiple KRAB-ZNF. Here, we report that the expression of many KRAB-ZNF along with active SUMOlyated KAP1 is elevated widely in human breast cancers. KAP1 silencing in breast cancer cells reduced proliferation and inhibited the growth and metastasis of tumor xenografts. Conversely, KAP1 overexpression stimulated cell proliferation and tumor growth. In cells where KAP1 was silenced, we identified multiple downregulated genes linked to tumor progression and metastasis, including EREG/epiregulin, PTGS2/COX2, MMP1, MMP2, and CD44, along with downregulation of multiple KRAB-ZNF proteins. KAP1-dependent stabilization of KRAB-ZNF required direct interactions with KAP1. Together, our results show that KAP1-mediated stimulation of multiple KRAB-ZNF contributes to the growth and metastasis of breast cancer.

MicroRNAs 206 and 21 cooperate to promote RAS-extracellular signal-regulated kinase signaling by suppressing the translation of RASA1 and SPRED1.

Despite the low prevalence of activating point mutation of RAS or RAF genes, the RAS-extracellular signal-regulated kinase (ERK) pathway is implicated in breast cancer pathogenesis. Indeed, in triple-negative breast cancer (TNBC), there is recurrent genetic alteration of pathway components. Using short hairpin RNA (shRNA) methods, we observed that the zinc finger transcription factor Krüppel-like factor 4 (KLF4) can promote RAS-ERK signaling in TNBC cells. Endogenous KLF4 bound to the promoter regions and promoted the expression of two microRNAs (miRs), miR-206 and miR-21 (i.e., miR-206/21). Antisense-mediated knockdown (anti-miR) revealed that miR-206/21 coordinately promote RAS-ERK signaling and the corresponding cell phenotypes by inhibiting translation of the pathway suppressors RASA1 and SPRED1. In TNBC cells, including cells with mutation of RAS, the suppression of either RASA1 or SPRED1 increased the levels of GTP-bound, wild-type RAS and activated ERK 1/2. Unlike the control cells, treatment of RASA1- or SPRED1-suppressed cells with anti-miR-206/21 had little or no impact on the level of activated ERK 1/2 or on cell proliferation and failed to suppress tumor initiation. These results identify RASA1 and SPRED1 mRNAs as latent RAS-ERK pathway suppressors that can be upregulated in tumor cells by anti-miR treatment. Consequently, KLF4-regulated miRs are important for the maintenance of RAS-ERK pathway activity in TNBC cells.

DEAD box protein DDX1 regulates cytoplasmic localization of KSRP.

mRNA decay mediated by the AU-rich elements (AREs) is one of the most studied post-transcriptional mechanisms and is modulated by ARE-binding proteins (ARE-BPs). To understand the regulation of K homology splicing regulatory protein (KSRP), a decay-promoting ARE-BP, we purified KSRP protein complexes and identified an RNA helicase, DDX1. We showed that down-regulation of DDX1 expression elevated cytoplasmic levels of KSRP and facilitated ARE-mediated mRNA decay. Association of KSRP with 14-3-3 proteins, that are predominately located in the cytoplasm, increased upon reduction of DDX1. We also demonstrated that KSRP associated with DDX1 or 14-3-3, but not both. These observations indicate that subcellular localization of KSRP is regulated by competing interactions with DDX1 or 14-3-3.

Applying data mining techniques to explore factors contributing to occupational injuries in Taiwan's construction industry.

Construction accident research involves the systematic sorting, classification, and encoding of comprehensive databases of injuries and fatalities. The present study explores the causes and distribution of occupational accidents in the Taiwan construction industry by analyzing such a database using the data mining method known as classification and regression tree (CART). Utilizing a database of 1542 accident cases during the period 2000-2009, the study seeks to establish potential cause-and-effect relationships regarding serious occupational accidents in the industry. The results of this study show that the occurrence rules for falls and collapses in both public and private project construction industries serve as key factors to predict the occurrence of occupational injuries. The results of the study provide a framework for improving the safety practices and training programs that are essential to protecting construction workers from occasional or unexpected accidents.

A KLF4-miRNA-206 autoregulatory feedback loop can promote or inhibit protein translation depending upon cell context.

Krüppel-like factor 4 (KLF4), a transcription factor that regulates cell fate in a context-dependent fashion, is normally induced upon growth arrest or differentiation. In many cancer cells there is dysregulation, with increased expression in proliferating cells. To identify sequence elements that mediate KLF4 suppression in normal epithelial cells, we utilized a luciferase reporter and RK3E cells, which undergo a proliferation-differentiation switch to form an epithelial sheet. A translational control element (TCE) within the KLF4 3'-untranslated region interacted with microRNAs (miRs) 206 and 344-1 to promote or inhibit KLF4 expression, respectively, in proliferating epithelial cells. Overall, the TCE suppressed expression in proliferating primary human mammary epithelial cells, but this suppressive effect was attenuated in immortalized mammary epithelial MCF10A cells, in which Dicer1 and miR-206 promoted KLF4 expression and TCE reporter activity. In contrast to MCF10A cells, in breast cancer cells the activity of miR-206 was switched, and it repressed KLF4 expression and TCE reporter activity. As miR-206 levels were KLF4 dependent, the results identify a KLF4-miR-206 feedback pathway that oppositely affects protein translation in normal cells and cancer cells. In addition, the results indicate that two distinct miRs can have opposite and competing effects on translation in proliferating cells.

Posttranscriptional control of type I interferon genes by KSRP in the innate immune response against viral infection.

Inherently unstable mRNAs contain AU-rich elements (AREs) in the 3' untranslated regions. Expression of ARE-containing type I interferon transcripts is robustly induced upon viral infection and rapidly shut off thereafter. Their transient accumulation is partly mediated through posttranscriptional regulation. Here we show that mouse embryonic fibroblasts derived from knockout mice deficient in KH-type splicing regulatory protein (KSRP), an RNA-binding protein required for ARE-mediated mRNA decay, produce higher levels of Ifna and Ifnb mRNAs in response to viral infection as a result of decreased mRNA decay. Functional analysis showed that KSRP is required for the decay of Ifna4 and Ifnb mRNAs by interaction with AREs. The increased IFN expression renders Ksrp(-)(/)(-) cells refractory to herpes simplex virus type 1 and vesicular stomatitis virus infection. These findings support a role of a posttranscriptional mechanism in the control of type I IFN expression and highlight the function of KSRP in innate immunity by negatively regulating IFN production.

Suppression of matrix metalloproteinase-9 by prostaglandin E(2) in peritoneal macrophage is associated with severity of endometriosis.

Decreased phagocytotic ability of macrophages has been reported to be associated with the severity of endometriosis, although the underlying mechanism remains uncharacterized. Expression and secretion of matrix metalloproteinase (MMP)-9 by macrophages is a means to degrade the extracellular matrix of cells that are designated for phagocytosis. Here, we describe the regulation of MMP-9 expression and activity in peritoneal macrophages of women with endometriosis. Results demonstrated that peritoneal macrophages isolated from women with endometriosis have decreased levels of protein and enzyme activity of MMP-9. Treatment of macrophages with peritoneal fluid obtained from patients with severe endometriosis inhibited MMP-9 expression and gelatinase activity. Further investigation identified prostaglandin (PG) E(2) as the major factor in the peritoneal fluid that inhibited MMP-9 activity. The inhibitory effect of PGE(2) was mediated via the EP2/EP4-dependent PKA pathway. Furthermore, expression of tissue inhibitor of metalloproteinase-1, tissue inhibitor of metalloproteinase-2, and RECK in macrophages was not affected by treatment with PGE(2), indicating the effect of PGE(2) on suppressing MMP-9 activity was not mediated by up-regulation of its inhibitor. Our results suggest that decreased phagocytotic capability of peritoneal macrophage in patients with endometriosis may be caused by PGE(2)-mediated decreases in MMP-9 expression.

Increased leptin expression in endometriosis cells is associated with endometrial stromal cell proliferation and leptin gene up-regulation.

Endometriosis is a polygenic disease with complex, multifactorial aetiologies affecting approximately 10% of women of reproductive age. Leptin is the product of the ob gene, which is related to reproductive function and immunological alteration. The angiogenic and mitogenic action of leptin may influence the formation of endometriosis. This study was aimed at determining whether leptin and leptin receptor expression differs in eutopic and ectopic endometria collected from laparoscopy and at investigating the pathophysiological role of leptin in the development of endometriosis. Leptin mRNA was undetectable in seven out of 14 eutopic endometria and only a minute amount was detected in the remaining samples. In contrast, there was a marked increase in leptin mRNA and protein expression in ectopic endometriotic lesions of patients with endometriosis (P < 0.05). Receptors for leptin were immunologically stained in eutopic endometrium as well as in ectopic endometriotic implants. However, the levels of mRNA for the long and total forms of leptin receptors were suppressed in association with the severity of endometriosis (P < 0.05). Administration of leptin stimulated its own mRNA expression in ectopic endometriotic stromal cells but decreased steady-state concentrations of mRNA encoding for leptin receptor (n = 6). In addition, leptin significantly enhanced both eutopic and ectopic endometrial stromal cell proliferation (P < 0.05). In conclusion, the differential distribution of mRNA for leptin and its receptor suggests an important autocrine and paracrine role for leptin in human endometriosis. The mitogenic and auto-augmentation effects of leptin may further contribute to the pathogenesis of endometriosis.

Distinct mechanisms regulate cyclooxygenase-1 and -2 in peritoneal macrophages of women with and without endometriosis.

Prostaglandin (PG) E2 has been shown to stimulate steroidogenesis in ectopic endometriotic stromal cells and may be involved in the development of endometriosis since this disorder is highly estrogen dependent. The biosynthesis of PGE2 is controlled by the rate-limiting enzyme termed cyclooxygenase (COX). The objective of the current study was to investigate the expression of COX in peritoneal macrophages isolated from women with and without endometriosis, and to explore the effects of pro-inflammatory agents on COX expression in peritoneal macrophages. Using quantitative RT-PCR and Western blot analyses, we found that expression of COX-2 was markedly increased (P < 0.05) in peritoneal macrophages isolated from women with early or severe endometriosis, whereas expression of COX-1 was elevated only in the severe stage (P < 0.05). On the contrary, monocytes/macrophages purified from peripheral blood of patients with endometriosis had minimal or undetectable levels of COX-2, and this was not different from disease-free women. Treatment with interleukin-1beta, tumour necrosis factor-alpha or PGE2 caused a significant increase in COX-2 (P < 0.05) but not COX-1 expression in peritoneal macrophages isolated from disease-free women. In contrast, these agents had no substantial effect on COX-1 and COX-2 expression in peritoneal macrophages from women with endometriosis. In summary, expression of COX in peritoneal macrophages was associated with the severity of endometriosis. Elevated expression of both COX-1 and COX-2 in peritoneal macrophages may contribute to the increased peritoneal fluid PGE2 concentrations and may thus play an important role in the development of endometriosis.