PAR-4/LKB1 prevents intestinal hyperplasia by restricting endoderm specification in Caenorhabditis elegans embryos.

The kinase PAR-4/LKB1 is a major regulator of intestinal homeostasis, which prevents polyposis in humans. Moreover, its ectopic activation is sufficient to induce polarization and formation of microvilli-like structures in intestinal cell lines. Here, we use Caenorhabditis elegans to examine the role of PAR-4 during intestinal development in vivo. We show that it is not required to establish enterocyte polarity and plays only a minor role in brush border formation. By contrast, par-4 mutants display severe deformations of the intestinal lumen as well as supernumerary intestinal cells, thereby revealing a previously unappreciated function of PAR-4 in preventing intestinal hyperplasia. The presence of supernumerary enterocytes in par-4 mutants is not due to excessive cell proliferation, but rather to the abnormal expression of the intestinal cell fate factors end-1 and elt-2 outside the E lineage. Notably, par-4 mutants also display reduced expression of end-1 and elt-2 inside the E lineage. Our work thereby unveils an essential and dual role of PAR-4, which both restricts intestinal specification to the E lineage and ensures its robust differentiation.

A transgenic DND1GFP fusion allele reports in vivo expression and RNA-binding targets in undifferentiated mouse germ cells†.

In vertebrates, the RNA-binding protein (RBP) dead end 1 (DND1) is essential for primordial germ cell (PGC) survival and maintenance of cell identity. In multiple species, Dnd1 loss or mutation leads to severe PGC loss soon after specification or, in some species, germ cell transformation to somatic lineages. Our investigations into the role of DND1 in PGC specification and differentiation have been limited by the absence of an available antibody. To address this problem, we used CRISPR/Cas9 gene editing to establish a transgenic mouse line carrying a DND1GFP fusion allele. We present imaging analysis of DND1GFP expression showing that DND1GFP expression is heterogeneous among male germ cells (MGCs) and female germ cells (FGCs). DND1GFP was detected in MGCs throughout fetal life but lost from FGCs at meiotic entry. In postnatal and adult testes, DND1GFP expression correlated with classic markers for the premeiotic spermatogonial population. Utilizing the GFP tag for RNA immunoprecipitation (RIP) analysis in MGCs validated this transgenic as a tool for identifying in vivo transcript targets of DND1. The DND1GFP mouse line is a novel tool for isolation and analysis of embryonic and fetal germ cells, and the spermatogonial population of the postnatal and adult testis.

Delphinidin induces antiproliferation and apoptosis of endometrial cells by regulating cytosolic calcium levels and mitochondrial membrane potential depolarization.

Endometriosis is a benign gynecological disease of women of reproductive ages, wherein endometrial cells grow ectopically, decreasing their quality of life due to chronic pelvic pain and severe dysmenorrhea. Although surgery and hormone therapies are gold standards for treating endometriosis, side effects are common and the recurrence rate is nearly 50%. Recent studies are exploring phytochemicals as pharmacological adjuvants for treating endometriosis. Delphinidin is an anthocyanin with anti-inflammatory, antioxidative, and anticancerous properties. In this study, delphinidin showed antiproliferative and apoptotic effects on human endometrial cells. Additionally, treatment with delphinidin decreased the mitochondrial membrane potential and increased cytosolic calcium levels in VK2/E6E7 and End1/E6E7 cells. Delphinidin decreased the phosphorylation of proliferative signaling molecules, including ERK1/2, AKT, P70S6K, and S6, while increasing the phosphorylation of P38 MAPK and P90RSK. These results imply that delphinidin is a novel therapeutic agent for treating and managing endometriosis, and has fewer side effects.

MAPK and NF-κB signalling pathways regulate the expression of miRNA, let-7f in human endocervical epithelial cells.

MicroRNAs (miRNAs) mediate post-transcriptional gene suppression and are a critical component of the complex regulatory networks in epithelial immune responses. Transcription of miRNA genes in epithelial cells can be elaborately controlled through Toll-like receptors (TLRs), and associated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) pathways, leading to nuclear transcription factor associated-transactivation and transrepression of miRNAs. MiRNA, let-7f is involved in the regulation of innate immune responses post TLR3 stimulation in human endocervical cells (End1/E6E7) and decreased let-7f is associated with poor immune activation. Thus, expression of let-7f is under strict control. However, the mechanism by which let-7f is regulated in these cells is not known. Therefore, in the present study, we have investigated the role of MAPK and NF-κB in the transcription of let-7f. We report that signalling of TLR3, results in activation of multiple signalling pathways including MAPK/ERK, JNK, p38, and NF-κB. Of these MAPK/ p38 and JNK directly influence the expression of let-7f in End1/E6E7 cells. Inhibition of ERK and NF-κB up regulates the expression of let-7f and its transcription factor, C/EBPß. In conclusion, we have identified a system through which TLR3 mediated immune response is regulated by C/EBPß and let-7f through the temporal activation of MAPK and NF-κB in human endocervical cells.

Combined functions of two RRMs in Dead-end1 mimic helicase activity to promote nanos1 translation in the germline.

Dead-end1 (Dnd1) expression is restricted to the vertebrate germline where it is believed to activate translation of messenger RNAs (mRNAs) required to protect and promote that unique lineage. Nanos1 is one such germline mRNA whose translation is blocked by a secondary mRNA structure within the open reading frame (ORF). Dnd1 contains a canonical RNA recognition motif (RRM1) in its N-terminus but also contains a less conserved RRM2. Here we provide a mechanistic picture of the nanos1 mRNA-Dnd1 interaction in the Xenopus germline. We show that RRM1, but not RRM2, is required for binding nanos1. Similar to the zebrafish homolog, Xenopus Dnd1 possesses ATPase activity. Surprisingly, this activity appears to be within the RRM2, different from the C-terminal region where it is found in zebrafish. More importantly, we show that RRM2 is required for nanos1 translation and germline survival. Further, Dnd1 functions as a homodimer and binds nanos1 mRNA just downstream of the secondary structure required for nanos1 repression. We propose a model in which the RRM1 is required to bind nanos1 mRNA while the RRM2 is required to promote translation through the action of ATPase. Dnd1 appears to use RRMs to mimic the function of helicases.

Associations between endothelin polymorphisms and aneurysmal subarachnoid hemorrhage, clinical vasospasm, delayed cerebral ischemia, and functional outcome.

OBJECTIVE Endothelin-1, a potent vasoconstrictor, and its receptors may be involved in the pathogenesis of aneurysmal subarachnoid hemorrhage (aSAH), clinical vasospasm, delayed cerebral ischemia (DCI), and functional outcome following aSAH. In the present study, common endothelin single nucleotide polymorphisms (SNPs) and their relation to aSAH were evaluated. METHODS Blood samples from all patients enrolled in the Cerebral Aneurysm Renin Angiotensin System (CARAS) study were used for genetic evaluation. The CARAS study prospectively enrolled patients with aSAH at 2 academic institutions in the US from 2012 to 2015. Common endothelin SNPs were detected using 5' exonnuclease (TaqMan) genotyping assays. Analysis of associations between endothelin SNPs and aSAH and its clinical sequelae was performed. RESULTS Samples from 149 patients with aSAH and 50 controls were available for analysis. In multivariate logistic regression analysis, the TG (odds ratio [OR] 2.102, 95% confidence interval [CI] 1.048-4.218, p = 0.036) and TT genotypes (OR 7.884, 95% CI 1.003-61.995, p = 0.05) of the endothelin-1 T/G SNP (rs1800541) were significantly associated with aSAH. There was a dominant effect of the G allele (CG/GG genotypes; OR 4.617, 95% CI 1.311-16.262, p = 0.017) of the endothelin receptor A G/C SNP (rs5335) on clinical vasospasm. Endothelin SNPs were not associated with DCI or functional outcome. CONCLUSIONS Common endothelin SNPs were found to be associated with presentation with aSAH and clinical vasospasm. Further studies are required to elucidate the relevant pathophysiology and its potential implications in the treatment of patients with aSAH.

Transcription factor CCAAT/enhancer-binding protein-ß upregulates microRNA, let-7f-1 in human endocervical cells.

PROBLEM: In endocervical epithelial cells (End1/E6E7), miRNA let-7f plays an important role in the control of innate immunity. The underlying molecular mechanism for let-7f regulation in these cells remains largely unclear. METHODS OF STUDY: let-7f was knocked down in End1/E6E7 cells using siRNA, and differential gene expression was analyzed by microarray. Differentially expressed genes were validated by qPCR and Western blot. Expression of let-7f was studied by qPCR after inhibition of C/EBPß with betulinic acid (BA) and pCMVß plasmid and after overexpression of C/EBPß with pCMVß+ plasmid. ChIP assay was performed to confirm binding of C/EBPß to let-7f promoter. Levels of Lin28A/B were checked by qPCR after similar treatment. RESULTS: let-7f knockdown (KD) affects the expression of many transcription factors (eg, C/EBPß) which are important regulators of immune responses. We observed let-7f-1 promoter to contain 6 C/EBPß binding sites. KD of C/EBPß led to decreased let-7f expression while overexpression of C/EBPß increased its expression. Treatment of End1/E6E7 cells with TLR-3 ligand, poly(I:C) increased binding of C/EBPß at binding sites 3, 5, and 6. Expression of Lin28A/B also changed upon inhibition and overexpression of C/EBPß. Its expression is opposite to that of let-7f in End1/E6E7 cells. CONCLUSION: let-7f-1 is a direct target of transcription factor, C/EBPß in End1/E6E7 cells.

Probing and rearranging the transcription factor network controlling the C. elegans endoderm.

The ELT-2 GATA factor is the predominant transcription factor regulating gene expression in the C. elegans intestine, following endoderm specification. We comment on our previous study (Wiesenfahrt et al., 2016) that investigated how the elt-2 gene is controlled by END-1, END-3 and ELT-7, the 3 endoderm specific GATA factors that lie upstream in the regulatory hierarchy. We also discuss the unexpected result that ELT-2, if expressed sufficiently early and at sufficiently high levels, can specify the C. elegans endoderm, replacing the normal functions of END-1 and END-3.