(Pro)renin receptor (ATP6AP2) depletion arrests As4.1 cells in the G0/G1 phase thereby increasing formation of primary cilia.

The (pro)renin receptor [(P)RR, ATP6AP2] is a multifunctional transmembrane protein that activates local renin-angiotensin systems, but also interacts with Wnt pathways and vacuolar H+ -ATPase (V-ATPase) during organogenesis. The aim of this study was to characterize the role of ATP6AP2 in the cell cycle in more detail. ATP6AP2 down-regulation by siRNA in renal As4.1 cells resulted in a reduction in the rate of proliferation and a G0/G1 phase cell cycle arrest. We identified a number of novel target genes downstream of ATP6AP2 knock-down that were related to the primary cilium (Bbs-1, Bbs-3, Bbs-7, Rabl5, Ttc26, Mks-11, Mks-5, Mks-2, Tctn2, Nme7) and the cell cycle (Pierce1, Clock, Ppif). Accordingly, the number of cells expressing the primary cilium was markedly increased. We found no indication that these effects were dependent of V-ATPase activity, as ATP6AP2 knock-down did not affect lysosomal pH and bafilomycin A neither influenced the ciliary expression pattern nor the percentage of ciliated cells. Furthermore, ATP6AP2 appears to be essential for mitosis. ATP6AP2 translocated from the endoplasmatic reticulum to mitotic spindle poles (pro-, meta- and anaphase) and the central spindle bundle (telophase) and ATP6AP2 knock-down results in markedly deformed spindles. We conclude that ATP6AP2 is necessary for cell division, cell cycle progression and mitosis. ATP6AP2 also inhibits ciliogenesis, thus promoting proliferation and preventing differentiation.

Associations of circulating plasma microRNAs with age, body mass index and sex in a population-based study.

BACKGROUND: Non-cellular blood circulating microRNAs (plasma miRNAs) represent a promising source for the development of prognostic and diagnostic tools owing to their minimally invasive sampling, high stability, and simple quantification by standard techniques such as RT-qPCR. So far, the majority of association studies involving plasma miRNAs were disease-specific case-control analyses. In contrast, in the present study, plasma miRNAs were analysed in a sample of 372 individuals from a population-based cohort study, the Study of Health in Pomerania (SHIP). METHODS: Quantification of miRNA levels was performed by RT-qPCR using the Exiqon Serum/Plasma Focus microRNA PCR Panel V3.M covering 179 different miRNAs. Of these, 155 were included in our analyses after quality-control. Associations between plasma miRNAs and the phenotypes age, body mass index (BMI), and sex were assessed via a two-step linear regression approach per miRNA. The first step regressed out the technical parameters and the second step determined the remaining associations between the respective plasma miRNA and the phenotypes of interest. RESULTS: After regressing out technical parameters and adjusting for the respective other two phenotypes, 7, 15, and 35 plasma miRNAs were significantly (q < 0.05) associated with age, BMI, and sex, respectively. Additional adjustment for the blood cell parameters identified 12 and 19 miRNAs to be significantly associated with age and BMI, respectively. Most of the BMI-associated miRNAs likely originate from liver. Sex-associated differences in miRNA levels were largely determined by differences in blood cell parameters. Thus, only 7 as compared to originally 35 sex-associated miRNAs displayed sex-specific differences after adjustment for blood cell parameters. CONCLUSIONS: These findings emphasize that circulating miRNAs are strongly impacted by age, BMI, and sex. Hence, these parameters should be considered as covariates in association studies based on plasma miRNA levels. The established experimental and computational workflow can now be used in future screening studies to determine associations of plasma miRNAs with defined disease phenotypes.

A multi-omics approach identifies key hubs associated with cell type-specific responses of airway epithelial cells to staphylococcal alpha-toxin.

Responsiveness of cells to alpha-toxin (Hla) from Staphylococcus aureus appears to occur in a cell-type dependent manner. Here, we compare two human bronchial epithelial cell lines, i.e. Hla-susceptible 16HBE14o- and Hla-resistant S9 cells, by a quantitative multi-omics strategy for a better understanding of Hla-induced cellular programs. Phosphoproteomics revealed a substantial impact on phosphorylation-dependent signaling in both cell models and highlights alterations in signaling pathways associated with cell-cell and cell-matrix contacts as well as the actin cytoskeleton as key features of early rHla-induced effects. Along comparable changes in down-stream activity of major protein kinases significant differences between both models were found upon rHla-treatment including activation of the epidermal growth factor receptor EGFR and mitogen-activated protein kinases MAPK1/3 signaling in S9 and repression in 16HBE14o- cells. System-wide transcript and protein expression profiling indicate induction of an immediate early response in either model. In addition, EGFR and MAPK1/3-mediated changes in gene expression suggest cellular recovery and survival in S9 cells but cell death in 16HBE14o- cells. Strikingly, inhibition of the EGFR sensitized S9 cells to Hla indicating that the cellular capacity of activation of the EGFR is a major protective determinant against Hla-mediated cytotoxic effects.

Distinct transcriptional networks in quiescent myoblasts: a role for Wnt signaling in reversible vs. irreversible arrest.

Most cells in adult mammals are non-dividing: differentiated cells exit the cell cycle permanently, but stem cells exist in a state of reversible arrest called quiescence. In damaged skeletal muscle, quiescent satellite stem cells re-enter the cell cycle, proliferate and subsequently execute divergent programs to regenerate both post-mitotic myofibers and quiescent stem cells. The molecular basis for these alternative programs of arrest is poorly understood. In this study, we used an established myogenic culture model (C2C12 myoblasts) to generate cells in alternative states of arrest and investigate their global transcriptional profiles. Using cDNA microarrays, we compared G0 myoblasts with post-mitotic myotubes. Our findings define the transcriptional program of quiescent myoblasts in culture and establish that distinct gene expression profiles, especially of tumour suppressor genes and inhibitors of differentiation characterize reversible arrest, distinguishing this state from irreversibly arrested myotubes. We also reveal the existence of a tissue-specific quiescence program by comparing G0 C2C12 myoblasts to isogenic G0 fibroblasts (10T1/2). Intriguingly, in myoblasts but not fibroblasts, quiescence is associated with a signature of Wnt pathway genes. We provide evidence that different levels of signaling via the canonical Wnt pathway characterize distinct cellular states (proliferation vs. quiescence vs. differentiation). Moderate induction of Wnt signaling in quiescence is associated with critical properties such as clonogenic self-renewal. Exogenous Wnt treatment subverts the quiescence program and negatively affects clonogenicity. Finally, we identify two new quiescence-induced regulators of canonical Wnt signaling, Rgs2 and Dkk3, whose induction in G0 is required for clonogenic self-renewal. These results support the concept that active signal-mediated regulation of quiescence contributes to stem cell properties, and have implications for pathological states such as cancer and degenerative disease.

Effects of irbesartan on gene expression revealed by transcriptome analysis of left atrial tissue in a porcine model of acute rapid pacing in vivo.

BACKGROUND: Atrial fibrillation (AF) is characterized by electrical and structural remodeling of the atria with atrial fibrosis being one hallmark. Angiotensin II (AngII) is a major contributing factor and blockage of its type I receptor (AT1R) prevents remodeling to some extent. Here we explored the effects of the AT1R antagonist irbesartan on global gene expression and profibrotic signaling pathways after induction of rapid atrial pacing (RAP) in vivo in pigs. METHODS AND RESULTS: Microarray-based RNA profiling was used to screen left atrial (LA) tissue specimens for differences in atrial gene expression in a model of acute RAP. RAP caused an overall expression profile that reflected AngII-induced ROS production, tissue remodeling, and energy depletion. Of special note, the mRNA levels of EDN1, SGK1, and CTGF encoding pro-endothelin, stress- and glucocorticoid activated kinase-1, and of connective tissue growth factor were identified to be significantly increased after 7h of rapid pacing. These specific expression changes were additionally validated by RT-qPCR or immunoblot analyses in LA, RA, and partly in LV samples. All RAP-induced differential gene expression patterns were partially attenuated in the presence of irbesartan. Similar results were obtained after RAP of HL-1 cardiomyocytes in vitro. Furthermore, exogenously added endothelin-1 (ET1) induced CTGF expression concomitant to the transcriptional activation of SGK1 in HL-1 cells. CONCLUSIONS: RAP provokes substantial changes in atrial and ventricular myocardial gene expression that could be partly reversed by irbesartan. ET1 contributes to AF-dependent atrial fibrosis by synergistic activity with AngII to stimulate SGK1 expression and enhance phosphorylation of the SGK1 protein which, in turn, induces CTGF. The latter has been consistently associated with tissue fibrosis. These findings suggest ETR antagonists as being beneficial in AF treatment.

Primary cultures of glomerular parietal epithelial cells or podocytes with proven origin.

Parietal epithelial cells (PECs) are crucially involved in the pathogenesis of rapidly progressive glomerulonephritis (RPGN) as well as in focal and segmental glomerulosclerosis (FSGS). In this study, transgenic mouse lines were used to isolate pure, genetically tagged primary cultures of PECs or podocytes using FACsorting. By this approach, the morphology of primary glomerular epithelial cells in culture could be resolved: Primary podocytes formed either large cells with intracytoplasmatic extensions or smaller spindle shaped cells, depending on specific culture conditions. Primary PECs were small and exhibited a spindle-shaped or polygonal morphology. In the very early phases of primary culture, rapid changes in gene expression (e.g. of WT-1 and Pax-2) were observed. However, after prolonged culture primary PECs and podocytes still segregated clearly in a transcriptome analysis--demonstrating that the origin of primary cell cultures is important. Of the classical markers, synaptopodin and podoplanin expression were differentially regulated the most in primary PEC and podocyte cultures. However, no expression of any endogenous gene allowed to differentiate between the two cell types in culture. Finally, we show that the transcription factor WT1 is also expressed by PECs. In summary, genetic tagging of PECs and podocytes is a novel and necessary tool to derive pure primary cultures with proven origin. These cultures will be a powerful tool for the emerging field of parietal epithelial cell biology.