A balancing act: RNA binding protein HuR/TTP axis in endometriosis patients

Endometriosis, a major reproductive pathology affecting 8-10% of women is characterized by chronic inflammation and immune dysfunction. Human antigen R (HuR) and Tristetraprolin (TTP) are RNA binding proteins that competitively bind to cytokines involved in inflammation including: tumor necrosis factor alpha (TNF-α), granulocyte macrophage colony stimulating factor (GM-CSF), interleukin 6 (IL-6) among others, and stabilize and destabilize them, respectively. The aim of this study was to examine RNA binding protein (RNABP) HuR/TTP axis in endometriosis patients compared to menstrual stage matched healthy fertile controls in hopes of better understanding their contribution to the pathogenesis of endometriosis. Additionally, using a targeted in vitro siRNA approach, we examined whether knock-down of TTP can play a functional role on other RNABPs that competitively bind to inflammatory targets of TTP in both endometriotic and endometrial epithelial cell lines. Our results suggest that RNABPs TTP and HuR are dysregulated in endometriotic lesions compared to matched eutopic patient samples as well endometrium from healthy controls. Silencing of TTP in endometriotic and endometrial epithelial cells revealed differential response to inflammatory cytokines and other RNABPs. Our results suggest potential involvement of HuR/TTP RNA binding protein axis in regulation of inflammation in endometriosis

Estrogen mediates inflammatory role of mast cells in endometriosis pathophysiology

Endometriosis is an estrogen dependent, chronic inflammatory disease characterized by the growth of endometrial lining outside of the uterus. Mast cells have emerged as key players in regulating not only allergic responses but also other mechanisms such as angiogenesis, fibrosis, and pain. The influence of estrogen on mast cell function has also been recognized as a potential factor driving disease pathophysiology in number of allergic and chronic inflammatory conditions. However, precise information is lacking on the cross talk between endocrine and immune factors within the endometriotic lesions and whether that contributes to the involvement of mast cells with disease pathophysiology. In this study, we observed a significant increase in mast cell numbers within endometriotic lesions compared to matched eutopic endometrium from the same patients. Compared to eutopic endometrium, endometriotic lesions had significantly higher levels of stem cell factor (SCF), a potent growth factor critical for mast cell expansion, differentiation, and survival for tissue resident mast cells. Targeted mRNA Q-PCR array revealed that the endometriotic lesions harbour microenvironment (upregulation of CPA3, VCAM1, CCL2, CMA1, CCR1, and KITLG) that is conducive to mast cells recruitment and subsequent differentiation. To examine cross-talk of mast cells within the endometriotic lesion microenvironment, endometriotic epithelial cells (12Z) and endometrial stromal cells (hESC) incubated with mast cell-conditioned media showed significantly increased production of pro-inflammatory and chemokinetic cytokines. To further understand the impact of estrogen on mast cells in endometriosis, we induced endometriosis in C57BL/6 mice. Mature mast cells were significantly higher in peritoneal fluid of estrogen-treated mice compared to untreated mice within the sham operated groups. Mouse endometriotic lesion tissue revealed several genes (qRT-PCR) relevant in mast cell biology significantly upregulated in the estrogen treated, endometriosis-induced group compared to control endometrium. The endometriotic lesions from estrogen treated mice also had significantly higher density of Alcian blue stained mast cells compared to untreated lesions or control endometrium. Collectively, these findings suggest that endometriotic lesions provide a microenvironment necessary for recruitment and differentiation of mast cells. In turn, mast cells potentially release pro-inflammatory mediators that contribute to chronic pelvic pain and endometriosis disease progression

The microRNAome of pregnancy: deciphering miRNA networks at the maternal-fetal interface.

MicroRNAs (miRNAs) post-transcriptionally regulate a vast network of genes by inhibiting mRNA translation. Aberrant miRNA expression profiles have been implicated in pathologies and physiological processes including pregnancy and angiogenesis. Using our established model of implantation failure and spontaneous fetal loss in pigs (Sus scrofa), 236 miRNAs were profiled and compared between 1) non-pregnant and pregnant endometrium, 2) maternal and fetal tissues, and 3) viable and growth-arrested conceptus attachment sites by microarray and Real-Time PCR. Many significant differences in miRNA expression were observed between each of the aforementioned comparisons, and several were validated by PCR. Results indicated which miRNAs were important during pregnancy, which were elevated on the maternal or fetal side of the maternal-fetal interface, and they implicated the maternal expression of miR-10a, 27a, 29c, 323, 331-5p, 339-3p, 374b-5p, and 935 in the spontaneous loss observed in pigs. Several putative mRNA targets of the miRNAs (elevated in endometrium associated with arresting conceptuses) were assessed by quantitative Real-Time PCR and were depressed, supporting their regulation by miRNAs. Finally, targets were clustered by function to obtain ranked lists of gene networks that indicated which pathways/physiological processes might be important in non-pregnant (extracellular matrix factors) versus pregnant endometrium (nuclear transcription factor regulation), maternal (blood vessel development) versus fetal (neuronal differentiation) tissue, and healthy (extracellular matrix factors) versus arresting (GRAM domain) conceptus attachment sites. Overall, we demonstrate the presence of miRNAs on both sides of the maternal-fetal interface, implicate them in spontaneous fetal loss, and present a unique glimpse into the vast microRNAome of pregnancy

mRNAs Assessed by Real-Time PCR.

<p>mRNAs Assessed by Real-Time PCR.</p

miRNA Expression during Porcine Pregnancy.

<p>Expression levels of miRNAs detected by microarray which were significantly different (<i>p</i><0.05) between non-pregnant endometrium (n = 4) as compared to endometrium associated with healthy conceptus attachment sites (A, n = 3), and arresting conceptus attachment sites (B, n = 3). Real-Time PCR validation was performed for several miRNAs, and significant fold changes are listed for comparison. A positive fold change indicates that the miRNA was elevated in non-pregnant endometrium. A negative fold change indicates a decrease in the miRNA in the non-pregnant endometrium. N/A: not assessed, N.S.: not significant.</p

miRNA Expression in Endometrium and Trophoblast.

<p>Expression levels of miRNAs detected by microarray which were significantly different (<i>p</i><0.05) between endometrium (n = 3) and trophoblast (n = 3) from isolated from healthy (A) and arresting conceptus attachment sites (B). Real-Time PCR validation was performed for several miRNAs, and significant fold changes are listed for comparison. A positive fold change indicates that the miRNA was elevated in endometrium. A negative fold change indicates a decrease in the miRNA in endometrium. N/A: not assessed, N.S.: not significant.</p

miRNAs Assessed by Real-Time PCR.

<p>miRNAs Assessed by Real-Time PCR.</p

Assessment of Putative miRNA Target Genes by Real-Time PCR.

<p>Eleven mRNA targets of the miRNAs that were significantly elevated in arresting as compared to healthy endometrium in the microarray experiment were selected for quantification by Real-Time PCR. Each mRNA was relatively quantified against the control gene GAPDH. As miRNAs negatively regulate their mRNA targets, a decrease in mRNA transcripts in endometrium from arresting conceptus attachment sites was expected. Endometrium from healthy attachment sites (white bars, n = 6) was compared to endometrium from arresting sites (black bars, n = 6) by t-test. Bars on the graphs represent the mean plus the SEM. Data is presented on a logarithmic scale to display all genes on one graph. No statistically significant differences were observed, however each of the 11 mRNAs quantified was decreased in arresting endometrium. Statistical analyses revealed the power of the t-tests to be low, and this may have muted differences between the tissues.</p