Macrophage migration inhibitory factor is involved in a positive feedback loop increasing aromatase expression in endometriosis.

Immune-endocrine interplay may play a major role in the pathogenesis of endometriosis. In the present study, we have investigated the interaction between macrophage migration inhibitory factor (MIF), a major pro-inflammatory and growth-promoting factor markedly expressed in active endometriotic lesions, and estradiol (E(2)) in ectopic endometrial cells. Our data showed a significant increase of MIF protein secretion and mRNA expression in endometriotic cells in response to E(2). MIF production was blocked by Fulvestrant, an estrogen receptor (ER) antagonist, and induced by ERα and ERß selective agonists propyl-pyrazole-triol (PPT) and diarylpropionrile (DPN), respectively, thus demonstrating a specific receptor-mediated effect. Cell transfection with MIF promoter construct showed that E(2) significantly stimulates MIF promoter activity. Interestingly, our data further revealed that MIF reciprocally stimulates aromatase protein and mRNA expression via a posttranscriptional mRNA stabilization mechanism, that E(2) itself can upregulate aromatase expression, and that inhibition of endogenous MIF, using MIF specific siRNA, significantly inhibits E(2)-induced aromatase. Thus, the present study revealed the existence of a local positive feedback loop by which estrogen acts directly on ectopic endometrial cells to upregulate the expression of MIF, which, in turn, displays the capability of inducing the expression of aromatase, the key and rate-limiting enzyme for estrogen synthesis. Such interplay may have a considerable impact on the development of endometriosis.

Analysis of post-transcriptional regulations by a functional, integrated, and quantitative method.

In the past 10 years, transcriptome and proteome analyses have provided valuable data on global gene expression and cell functional networks. However, when integrated,these analyses revealed partial correlations between mRNA expression levels and protein abundance thus suggesting that post-transcriptional regulations may be in part responsible for this discrepancy. In the present work, we report the development of a functional, integrated, and quantitative method to measure post-transcriptional regulations that we named FunREG. This method enables (i) quantitative measure of post-transcriptional regulations mediated by selected 3-untranslated regions and exogenous small interfering-RNA or micro-RNAs and (ii) comparison of these regulatory processes in physiologically relevant systems (e.g. cancer versus primary untransformed cells). We applied FunREG to the study of liver cancer, and we demonstrate for the first time the differential regulatory mechanisms controlling gene expression at a post-transcriptional level in normal and tumoral hepatic cells. As an example, translation efficiency mediated by heparin-binding epidermal growth factor 3-untranslated region was increased 3-fold in liver cancer cells compared with normal hepatocytes, whereas stability of an mRNA containing a portion of Cyclin D1 3-untranslated region was increased more than 2-fold in HepG2 cells compared with normal hepatocytes. Consequently we believe that the method presented herein may become an important tool in fundamental and medical research. This approach is convenient and easy to perform, accessible to any investigator, and should be adaptable to a large number of cell type, functional and chemical screens, as well as genome scale analyses. Finally FunREG may represent a helpful tool to reconcile transcriptome and proteome data.

A Methodology for Concomitant Isolation of Intimal and Adventitial Endothelial Cells from the Human Thoracic Aorta.

BACKGROUND: Aortic diseases are diverse and involve a multiplicity of biological systems in the vascular wall. Aortic dissection, which is usually preceded by aortic aneurysm, is a leading cause of morbidity and mortality in modern societies. Although the endothelium is now known to play an important role in vascular diseases, its contribution to aneurysmal aortic lesions remains largely unknown. The aim of this study was to define a reliable methodology for the isolation of aortic intimal and adventitial endothelial cells in order to throw light on issues relevant to endothelial cell biology in aneurysmal diseases. METHODOLOGY/PRINCIPAL FINDINGS: We set up protocols to isolate endothelial cells from both the intima and the adventitia of human aneurysmal aortic vessel segments. Throughout the procedure, analysis of cell morphology and endothelial markers allowed us to select an endothelial fraction which after two rounds of expansion yielded a population of >90% pure endothelial cells. These cells have the features and functionalities of freshly isolated cells and can be used for biochemical studies. The technique was successfully used for aortic vessel segments of 20 patients and 3 healthy donors. CONCLUSIONS/SIGNIFICANCE: This simple and highly reproducible method allows the simultaneous preparation of reasonably pure primary cultures of intimal and adventitial human endothelial cells, thus providing a reliable source for investigating their biology and involvement in both thoracic aneurysms and other aortic diseases.

Podosomes: Multipurpose organelles?

Thirty years of research have accumulated ample evidence that podosome clusters qualify as genuine cellular organelles that are being found in more and more cell types. A podosome is a dynamic actin-based and membrane-bound microdomain and the organelle consists in an interconnected network of such basic units, forming a cytoskeletal superstructure linked to the plasma membrane. At this strategic location, podosomes are privileged sites of interactions with the pericellular environment that regulates their formation, density, lifetime, distribution, architecture and functioning. Actin polymerization is the driving force behind most podosome characteristics. In contrast to classical organelles, podosomes are not vital at the cell level but rather serve diverse and often intricate functions of which adhesion, matrix degradation and substrate sensing are the most established. These capabilities involve specific molecules, depend on podosome organization and may vary according to the cell type in which they form. Podosome-associated diseases manifest by loss or gain of podosome functions and include genetic diseases affecting podosome components and various cancers where tumor cells ectopically express podosome equivalents (invadopodia).

Importance of RhoGTPases in formation, characteristics, and functions of invadosomes.

Podosomes and invadopodia (collectively known as invadosomes) are specialized plasma-membrane actin-based microdomains that combine adhesive properties with matrix degrading and/or mechanosensor activities. These organelles have been extensively studied in vitro and current concerted efforts aim at establishing their physiological relevance and subsequent association with human diseases. Proper functioning of the bone, immune, and vascular systems is likely to depend on these structures while their occurrence in cancer cells appears to be linked to tumor metastasis. The elucidation of the mechanisms driving invadosome assembly is a prerequisite to understanding their role in vivo and ultimately to controlling their functions. Adhesive and soluble ligands act via transmembrane receptors that propagate signals to the cytoskeleton via small G proteins of the Rho family, assisted by tyrosine kinases and scaffold proteins to induce invadosome formation and rearrangements. Oncogene expression and cell-cell interactions may also trigger their assembly. Manipulation of the signals that regulate invadosome formation and dynamics could therefore be a strategy to interfere with their functions in a multitude of pathological settings, such as excessive bone breakdown, infections, vascular remodeling, transendothelial diapedesis, and metastasis.

ALK5 and ALK1 play antagonistic roles in transforming growth factor ß-induced podosome formation in aortic endothelial cells.

Transforming growth factor ß (TGF-ß) and related cytokines play a central role in the vascular system. In vitro, TGF-ß induces aortic endothelial cells to assemble subcellular actin-rich structures specialized for matrix degradation called podosomes. To explore further this TGF-ß-specific response and determine in which context podosomes form, ALK5 and ALK1 TGF-ß receptor signaling pathways were investigated in bovine aortic endothelial cells. We report that TGF-ß drives podosome formation through ALK5 and the downstream effectors Smad2 and Smad3. Concurrent TGF-ß-induced ALK1 signaling mitigates ALK5 responses through Smad1. ALK1 signaling induced by BMP9 also antagonizes TGF-ß-induced podosome formation, but this occurs through both Smad1 and Smad5. Whereas ALK1 neutralization brings ALK5 signals to full potency for TGF-ß-induced podosome formation, ALK1 depletion leads to cell disturbances not compatible with podosome assembly. Thus, ALK1 possesses passive and active modalities. Altogether, our results reveal specific features of ALK1 and ALK5 signaling with potential clinical implications.

Invadosomes: intriguing structures with promise.

Podosomes and invadopodia are highly dynamic, actin-rich adhesion structures and represent the two founding members of the invadosome family. Podosomes form spontaneously in cells of the myelomonocytic lineage but a plethora of other cells are endowed with this capacity, under appropriate stimulation, such as a soluble factor, matrix receptor, or cell stress. Related structures called invadopodia are detected in some cancer cells or appear on cells upon oncogenic transformation. In contrast to other cell adhesion devices, invadosomes harbour metalloproteases which degrade components of the extracellular matrix. Because of this distinctive feature, invadosomes have been systematically linked with invasion processes. However, it now appears that these intriguing structures are endowed with other functions and are therefore expected to contribute to a wider range of biological processes. The invadosome field has been progressing for thirty years, expanding exponentially during the last decade, where tremendous advances have been made regarding the molecular mechanism underlying their formation, dynamics and function. Invadosomes are involved in human diseases but the causative link remains to be established. To this end, 3D analysis of invadosomes is now being actively developed in ex vivo and in vivo models to demonstrate their occurrence and establish their role in pathological and physiological processes.

Macrophage migration inhibitory factor elicits an angiogenic phenotype in human ectopic endometrial cells and triggers the production of major angiogenic factors via CD44, CD74, and MAPK signaling pathways.

CONTEXT: An active angiogenesis is required for ectopic endometrial tissue growth. Our previous studies led to the identification of macrophage migration inhibitory factor (MIF), which is markedly elevated in active, vascularized, and early-stage endometriotic lesions, as a potent mitogenic factor for endothelial cells. OBJECTIVE: Our objective was to study the mechanisms by which MIF may stimulate angiogenesis in ectopic endometrial implantation sites. DESIGN: Primary cultures of ectopic endometrial cells were exposed to MIF, and the release of major angiogenic factors with targeted disruption of MIF signaling pathways was assessed. PATIENTS: Patients were women found to have endometriosis during laparoscopy. SETTING: The study was conducted at a hospital and reproduction research laboratory. INTERVENTIONS: Biopsies were removed from endometriotic lesions. MAIN OUTCOME MEASURES: Vascular endothelial cell growth factor (VEGF), IL-8, and monocyte chemotactic protein-1 (MCP-1) mRNA and protein levels and expression and small interfering RNA silencing of MIF CD74/CD44 receptor complex and phosphorylation of ERK and p38 MAPKs were evaluated. RESULTS: MIF markedly up-regulated VEGF, IL-8, and MCP-1 expression in endometriotic cells. Such an effect was abolished by (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1), a specific inhibitor of MIF, and significantly down-regulated after specific small interfering RNA silencing of CD44 or CD74. MIF treatment strongly activated ERK and p38 MAPKs, and specific inhibitors of both pathways completely blocked basal and MIF-induced VEGF, IL-8, and MCP-1 synthesis. CONCLUSIONS: These results show for the first time that MIF exerts a potent indirect angiogenic effect by interacting with ectopic endometrial cells and inducing the secretion of major angiogenic factors via CD44, CD74, and MAPK signaling pathways and provide evidence for a possible new mechanism underlying endometriosis development and pathophysiology.

Involvement of nuclear factor-kappaB in macrophage migration inhibitory factor gene transcription up-regulation induced by interleukin- 1 beta in ectopic endometrial cells.

OBJECTIVE: To investigate the involvement of the nuclear factor (NF)-kappaB in the interleukin (IL)-1 beta-mediated macrophage migration inhibitory factor (MIF) gene activation. DESIGN: Prospective study. SETTING: Human reproduction research laboratory. PATIENT(S): Nine women with endometriotic lesions. INTERVENTION(S): Endometriotic lesions were obtained during laparoscopic surgery. MAIN OUTCOME MEASURE(S): The MIF protein secretion was analyzed by ELISA, MIF mRNA expression by quantitative real-time polymerase chain reaction (PCR), NF-kappaB translocation into the nucleus by electrophoresis mobility shift assay, I kappaB phosphorylation and degradation by Western blot, and human MIF promoter activity by transient cell transfection. RESULT(S): This study showed a significant dose-dependent increase of MIF protein secretion and mRNA expression, the NF-kappaB translocation into the nucleus, I kappaB phosphorylation, I kappaB degradation, and human MIF promoter activity in endometriotic stromal cells in response to IL-1 beta. Curcumin (NF-kappaB inhibitor) significantly inhibited all these IL-1 beta-mediated effects. Analysis of the activity of deletion constructs of the human MIF promoter and a computer search localized two putative regulatory elements corresponding to NF-kappaB binding sites at positions -2538/-2528 bp and -1389/-1380 bp. CONCLUSION(S): This study suggests the involvement of the nuclear transcription factor NF-kappaB in MIF gene activation in ectopic endometrial cells in response to IL-1 beta and identifies a possible pathway of endometriosis-associated inflammation and ectopic cell growth.