Role of differentially expressed microRNA-139-5p in the regulation of phenotypic internal anal sphincter smooth muscle tone.

The present study focused on the role of microRNA-139-5p (miRNA-139-5p) in the regulation of basal tone in internal anal sphincter (IAS). Applying genome-wide miRNA microarrays on the phenotypically distinct smooth muscle cells (SMCs) within the rat anorectrum, we identified miRNA-139-5p as differentially expressed RNA repressor with highest expression in the purely phasic smooth muscle of anococcygeus (ASM) vs. the truly tonic smooth muscle of IAS. This pattern of miRNA-139-5p expression, previously shown to target ROCK2, was validated by target prediction using ingenuity pathway (IPA) and by qPCR analyses. Immunoblotting, immunocytochemistry (ICC), and functional assays using IAS tissues and cells subjected to overexpression/knockdown of miRNA-139-5p confirmed the inverse relationship between miRNA-139-5p and ROCK2 expressions/IAS tone. Overexpression of miRNA-139-5p caused a decrease, while knockdown by anti-miRNA-139-5p caused an increase in the IAS tone; these tissue contractile responses were confirmed by single-cell contraction using magnetic twisting cytometry (MTC). These findings suggest miRNA-139-5p is capable of significantly influencing the phenotypic tonicity in smooth muscle via ROCK2: a lack of tone in ASM may be associated with the suppression of ROCK2 by high expression of miRNA-139-5p, whereas basal IAS tone may be associated with the persistence of ROCK2 due to low expression of miRNA-139-5p

Kinase-independent role of cyclin D1 in chromosomal instability and mammary tumorigenesis

Cyclin D1 is an important molecular driver of human breast cancer but better understanding of its oncogenic mechanisms is needed, especially to enhance efforts in targeted therapeutics. Currently, pharmaceutical initiatives to inhibit cyclin D1 are focused on the catalytic component since the transforming capacity is thought to reside in the cyclin D1/CDK activity. We initiated the following study to directly test the oncogenic potential of catalytically inactive cyclin D1 in an in vivo mouse model that is relevant to breast cancer. Herein, transduction of cyclin D1(-/-) mouse embryonic fibroblasts (MEFs) with the kinase dead KE mutant of cyclin D1 led to aneuploidy, abnormalities in mitotic spindle formation, autosome amplification, and chromosomal instability (CIN) by gene expression profiling. Acute transgenic expression of either cyclin D1(WT) or cyclin D1(KE) in the mammary gland was sufficient to induce a high CIN score within 7 days. Sustained expression of cyclin D1(KE) induced mammary adenocarcinoma with similar kinetics to that of the wild-type cyclin D1. ChIP-Seq studies demonstrated recruitment of cyclin D1(WT) and cyclin D1(KE) to the genes governing CIN. We conclude that the CDK-activating function of cyclin D1 is not necessary to induce either chromosomal instability or mammary tumorigenesis

Heterozygous disruption of Flk-1 receptor leads to myocardial ischaemia reperfusion injury in mice: application of affymetrix gene chip analysis

This study addresses an important clinical issue by identifying potential candidates of vascular endothelial growth factor (VEGF) signalling through the Flk-1 receptor that trigger cardioprotective signals under ischaemic stress. Isolated working mouse hearts of both wild-type (WT) and Flk-1+/− were subjected to global ischaemia (I) for 30 min. followed by 2 hrs of reperfusion (R). Flk-1+/− myocardium displayed almost 50% reduction in Flk-1 mRNA as examined by quantitative real-time RT-PCR at the baseline level. Flk-1+/− mouse hearts displayed reduction in left ventricular functional recovery throughout reperfusion (dp/dt 605 versus 884), after 2 hrs (P < 0.05). Coronary (1.9 versus 2.4 ml) and aortic flow (AF) (0.16 versus 1.2 ml) were reduced in Flk-1+/− after 2 hrs of reperfusion. In addition, increased infarct size (38.4%versus 28.41%, P < 0.05) and apoptotic cardiomyocytes (495 versus 213) were observed in Flk-1+/− knockout (KO) mice. We also examined whether ischaemic preconditioning (PC), a novel method to induce cardioprotection against ischaemia reperfusion injury, through stimulating the VEGF signalling pathway might function in Flk-1+/− mice. We found that knocking down Flk-1 resulted in significant reduction in the cardioprotective effect by PC compared to WT. Affymetrix gene chip analysis demonstrated down-regulation of important genes after IR and preconditioning followed by ischaemia reperfusion in Flk-1+/− mice compared to WT. To get insight into the underlying molecular pathways involved in ischaemic PC, we determined the distinct and overlapping biological processes using Ingenuity pathway analysis tool. Independent evidence at the mRNA level supporting the Affymetrix results were validated using real-time RT-PCR for selected down-regulated genes, which are thought to play important roles in cardioprotection after ischaemic insult. In summary, our data indicated for the first time that ischaemic PC modifies genomic responses in heterozygous VEGFR-2/Flk-1 KO mice and abolishes its cardioprotective effect on ischaemic myocardium

Efficient mitochondrial targeting relies on co-operation of multiple protein signals in plants

To date, the most prevalent model for transport of pre-proteins to plant mitochondria is based on the activity of an N-terminal extension serving as a targeting peptide. Whether the efficient delivery of proteins to mitochondria is based exclusively on the action of the N-terminal extension or also on that of other protein determinants has yet to be defined. A novel mechanism is reported here for the targeting of a plant protein, named MITS1, to mitochondria. It was found that MITS1 contains an N-terminal extension that is responsible for mitochondrial targeting. Functional dissection of this extension shows the existence of a cryptic signal for protein targeting to the secretory pathway. The first 11 amino acids of the N-terminal extension are necessary to overcome the activity of this signal sequence and target the protein to the mitochondria. These data suggest that co-operation of multiple determinants within the N-terminal extension of mitochondrial proteins may be necessary for efficient mitochondrial targeting. It was also established that the presence of a tryptophan residue toward the C-terminus of the protein is crucial for mitochondrial targeting, as mutation of this residue results in a redistribution of MITS1 to the endoplasmic reticulum and Golgi apparatus. These data suggest a novel targeting model whereby protein traffic to plant mitochondria is influenced by domains in the full-length protein as well as the N-terminal extension

Compartment-Restricted Biotinylation Reveals Novel Features of Prion Protein Metabolism in Vivo

A selective tagging method for detecting minor alternatively-localized populations of a protein is used to study a disease-associated transmembrane form of prion protein. The analysis reveals key features of transmembrane prion protein metabolism and one way this is altered by human disease-causing mutants

The endogenous cell-fate factor dachshund restrains prostate epithelial cell migration via repression of cytokine secretion via a CXCL signaling module

© 2015 American Association for Cancer Research. Prostate cancer is the second leading form of cancer-related death in men. In a subset of prostate cancer patients, increased chemokine signaling IL8 and IL6 correlates with castrate-resistant prostate cancer (CRPC). IL8 and IL6 are produced by prostate epithelial cells and promote prostate cancer cell invasion; however, the mechanisms restraining prostate epithelial cell cytokine secretion are poorly understood. Herein, the cell-fate determinant factor DACH1 inhibited CRPC tumor growth in mice. Using Dach1fl/fl/Probasin-Cre bitransgenic mice, we show IL8 and IL6 secretion was altered by approximately 1,000-fold by endogenous Dach1. Endogenous Dach1 is shown to serve as a key endogenous restraint to prostate epithelial cell growth and restrains migration via CXCL signaling. DACH1 inhibited expression, transcription, and secretion of the CXCL genes (IL8 and IL6) by binding to their promoter regulatory regions in chromatin. DACH1 is thus a newly defined determinant of benign and malignant prostate epithelium cellular growth, migration, and cytokine abundance in vivo

Erythroid-Specific Expression of β-globin from Sleeping Beauty-Transduced Human Hematopoietic Progenitor Cells

Gene therapy for sickle cell disease will require efficient delivery of a tightly regulated and stably expressed gene product to provide an effective therapy. In this study we utilized the non-viral Sleeping Beauty (SB) transposon system using the SB100X hyperactive transposase to transduce human cord blood CD34+ cells with DsRed and a hybrid IHK–β-globin transgene. IHK transduced cells were successfully differentiated into multiple lineages which all showed transgene integration. The mature erythroid cells had an increased β-globin to γ-globin ratio from 0.66±0.08 to 1.05±0.12 (p = 0.05), indicating expression of β-globin from the integrated SB transgene. IHK–β-globin mRNA was found in non-erythroid cell types, similar to native β-globin mRNA that was also expressed at low levels. Additional studies in the hematopoietic K562 cell line confirmed the ability of cHS4 insulator elements to protect DsRed and IHK–β-globin transgenes from silencing in long-term culture studies. Insulated transgenes had statistically significant improvement in the maintenance of long term expression, while preserving transgene regulation. These results support the use of Sleeping Beauty vectors in carrying an insulated IHK–β-globin transgene for gene therapy of sickle cell disease

Kinase-independent role of cyclin D1 in chromosomal instability and mammary tumorigenesis

Cyclin D1 is an important molecular driver of human breast cancer but better understanding of its oncogenic mechanisms is needed, especially to enhance efforts in targeted therapeutics. Currently, pharmaceutical initiatives to inhibit cyclin D1 are focused on the catalytic component since the transforming capacity is thought to reside in the cyclin D1/CDK activity. We initiated the following study to directly test the oncogenic potential of catalytically inactive cyclin D1 in an in vivo mouse model that is relevant to breast cancer. Herein, transduction of cyclin D1-/- mouse embryonic fibroblasts (MEFs) with the kinase dead KE mutant of cyclin D1 led to aneuploidy, abnormalities in mitotic spindle formation, autosome amplification, and chromosomal instability (CIN) by gene expression profiling. Acute transgenic expression of either cyclin D1WT or cyclin D1KE in the mammary gland was sufficient to induce a high CIN score within 7 days. Sustained expression of cyclin D1KE induced mammary adenocarcinoma with similar kinetics to that of the wild-type cyclin D1. ChIP-Seq studies demonstrated recruitment of cyclin D1WT and cyclin D1KE to the genes governing CIN. We conclude that the CDK-activating function of cyclin D1 is not necessary to induce either chromosomal instability or mammary tumorigenesis

Cell Cycle- and Cancer-Associated Gene Networks Activated by Dsg2: Evidence of Cystatin A Deregulation and a Potential Role in Cell-Cell Adhesion

This work was supported by grants from the National Institutes of Health (Mahoney, R01AR056067; Riobo, RO1 GM088256). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript