Actin-Mediated Gene Expression Depends on RhoA and Rac1 Signaling in Proximal Tubular Epithelial Cells

Morphological alterations of cells can lead to modulation of gene expression. An essential link is the MKL1-dependent activation of serum response factor ( SRF), which translates changes in the ratio of G-and F-actin into mRNA transcription. SRF activation is only partially characterized in non-transformed epithelial cells. Therefore, the impact of GTPases of the Rho family and changes in F-actin structures were analyzed in renal proximal tubular epithelial cells. Activation of SRF signaling was compared to the regulation of a known MKL1/ SRF target gene, connective tissue growth factor ( CTGF). In the human proximal tubular cell line HKC-8 overexpression of two actin mutants either favoring or preventing the formation of F-actin fibers regulated SRF-mediated transcription as well as CTGF expression. Only overexpression of constitutively active RhoA activated SRF-dependent gene expression whereas no effect was detected upon overexpression of Rac1 mutants. To elucidate the functional role of Rho kinases as downstream mediators of RhoA, pharmacological inhibition and genetic inhibition by transient siRNA knock down were compared. Upon stimulation with lysophosphatidic acid ( LPA) Rho kinase inhibitors partially suppressed SRF-mediated transcription, whereas interference with Rho kinase expression by siRNA reduced activation of SRF, but barely affected CTGF expression. Together with the partial inhibition of CTGF expression by the pharmacological inhibitors Y27432 and H1154, Rho kinases seem to be less important in mediating RhoA signaling related to CTGF expression in HKC8 epithelial cells. Short term pharmacological inhibition of Rac1 activity by EHT1864 reduced SRF-dependent CTGF expression in HKC-8 cells, but was overcome by a stimulatory effect after prolonged incubation after 4-6 h. Similarly, human primary cells of proximal but not of distal tubular origin showed inhibitory as well as stimulatory effects of Rac1 inhibition. Thus, RhoA signaling activates MKL1-SRF-mediated CTGF expression in proximal tubular cells, whereas Rac1 signaling is more complex with adaptive cellular responses

A novel inhibitory mechanism of MRTF-A/B on the ICAM-1 gene expression in vascular endothelial cells

The roles of myocardin-related transcription factor A (MRTF-A) and MRTF-B in vascular endothelial cells are not completely understood. Here, we found a novel regulatory mechanism for MRTF-A/B function. MRTF-A/B tend to accumulate in the nucleus in arterial endothelial cells in vivo and human aortic endothelial cells (HAoECs) in vitro. In HAoECs, nuclear localization of MRTF-A/B was not significantly affected by Y27632 or latrunculin B, primarily due to the reduced binding of MRTF-A/B to G-actin and in part, to the low level of MRTF-A phosphorylation by ERK. MRTF-A/B downregulation by serum depletion or transfection of siRNA against MRTF-A and/or MRTF-B induced ICAM-1 expression in HAoECs. It is known that nuclear import of nuclear factor-kappa B (NF-kappa B) plays a key role in ICAM-1 gene transcription. However, nuclear accumulation of NF-kappa B p65 was not observed in MRTF-A/B-depleted HAoECs. Our present findings suggest that MRTF-A/B inhibit ICAM-1 mRNA expression by forming a complex with NF-kappa B p65 in the nucleus. Conversely, downregulation of MRTF-A/B alleviates this negative regulation without further translocation of NF-kappa B p65 into the nucleus. These results reveal the novel roles of MRTF-A/B in the homeostasis of vascular endothelium

Accuracy and Completeness of Drug Information in Wikipedia: A Comparison with Standard Textbooks of Pharmacology

The online resource Wikipedia is increasingly used by students for knowledge acquisition and learning. However, the lack of a formal editorial review and the heterogeneous expertise of contributors often results in skepticism by educators whether Wikipedia should be recommended to students as an information source. In this study we systematically analyzed the accuracy and completeness of drug information in the German and English language versions of Wikipedia in comparison to standard textbooks of pharmacology. In addition, references, revision history and readability were evaluated. Analysis of readability was performed using the Amstad readability index and the Erste Wiener Sachtextformel. The data on indication, mechanism of action, pharmacokinetics, adverse effects and contraindications for 100 curricular drugs were retrieved from standard German textbooks of general pharmacology and compared with the corresponding articles in the German language version of Wikipedia. Quantitative analysis revealed that accuracy of drug information in Wikipedia was 99.7%+/- 0.2% when compared to the textbook data. The overall completeness of drug information in Wikipedia was 83.8 +/- 1.5% (p<0.001). Completeness varied in-between categories, and was lowest in the category "pharmacokinetics'' (68.0% +/- 4.2%;p<0.001) and highest in the category "indication'' (91.3%+/- 2.0%) when compared to the textbook data overlap. Similar results were obtained for the English language version of Wikipedia. Of the drug information missing in Wikipedia, 62.5% was rated as didactically non-relevant in a qualitative re-evaluation study. Drug articles in Wikipedia had an average of 14.6 +/- 1.6 references and 262.8 +/- 37.4 edits performed by 142.7 +/- 17.6 editors. Both Wikipedia and textbooks samples had comparable, low readability. Our study suggests that Wikipedia is an accurate and comprehensive source of drug-related information for undergraduate medical education

TRPM7 kinase activity is essential for T cell colonization and alloreactivity in the gut

The melastatin-like transient-receptor-potential-7 protein (TRPM7), harbouring a cation channel and a serine/threonine kinase, has been implicated in thymopoiesis and cytokine expression. Here we show, by analysing TRPM7 kinase-dead mutant (Trpm7 R/R) mice, that the enzymatic activity of the receptor is not essential for thymopoiesis, but is required for CD103 transcription and gut-homing of intra-epithelial lymphocytes. Defective T cell gut colonization reduces MHCII expression in intestinal epithelial cells. Mechanistically, TRPM7 kinase activity controls TGF-β-induced CD103 expression and pro-inflammatory T helper 17, but not regulatory T, cell differentiation by modulating SMAD2. Notably, we find that the TRPM7 kinase activity promotes gut colonization by alloreactive T cells in acute graft-versus-host disease. Thus, our results unravel a function of TRPM7 kinase in T cell activity and suggest a therapeutic potential of kinase inhibitors in averting acute graft-versus-host disease

Molecular Mechanisms to Target Cellular Senescence in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) has emerged as a major cause of cancer-related death and is the most common type of liver cancer. Due to the current paucity of drugs for HCC therapy there is a pressing need to develop new therapeutic concepts. In recent years, the role of Serum Response Factor (SRF) and its coactivators, Myocardin-Related Transcription Factors A and B (MRTF-A and -B), in HCC formation and progression has received considerable attention. Targeting MRTFs results in HCC growth arrest provoked by oncogene-induced senescence. The induction of senescence acts as a tumor-suppressive mechanism and therefore gains consideration for pharmacological interventions in cancer therapy. In this article, we describe the key features and the functional role of senescence in light of the development of novel drug targets for HCC therapy with a focus on MRTFs

Detection of Cellular Senescence in Human Primary Melanocytes and Malignant Melanoma Cells In Vitro

Detection and quantification of senescent cells remain difficult due to variable phenotypes and the absence of highly specific and reliable biomarkers. It is therefore widely accepted to use a combination of multiple markers and cellular characteristics to define senescent cells in vitro. The exact choice of these markers is a subject of ongoing discussion and usually depends on objective reasons such as cell type and treatment conditions, as well as subjective considerations including feasibility and personal experience. This study aims to provide a comprehensive comparison of biomarkers and cellular characteristics used to detect senescence in melanocytic systems. Each marker was assessed in primary human melanocytes that overexpress mutant BRAFV600E, as it is commonly found in melanocytic nevi, and melanoma cells after treatment with the chemotherapeutic agent etoposide. The combined use of these two experimental settings is thought to allow profound conclusions on the choice of senescence biomarkers when working with melanocytic systems. Further, this study supports the development of standardized senescence detection and quantification by providing a comparative analysis that might also be helpful for other cell types and experimental conditions

Time-dependent effects of Rac1 inhibition by EHT1864 on CTGF regulation.

<p>(A) HKC-8 cells were transfected with SRE promoter constructs. After pre-incubation with EHT1864 (10 μM) for 30 min, the cells were stimulated with LPA (10 μM) for the times indicated. Data are means ± SD from one experiment with triplicate transfections. (B) HKC-8 cells were transfected with SRE or 4.5 kb CTGF promoter constructs. Relative luciferase activity was determined in control cells and cells pre-treated with EHT1864 (10 μM) for 30 min and then stimulated with LPA (10 μM) for 3–4 h. Data are means ± SD of 7 (SRE) and 3 (CTGF promoter) experiments. Activity in LPA-stimulated cells was set to 1 in each experiment. *** p<0.001, compared to cells stimulated with LPA, ANOVA with Dunnett’s multiple comparison test. (C) HKC-8 cells were pre-incubated with EHT1864 (10 μM) for 30 min and then stimulated with LPA (10 μM) for the times indicated. Cellular CTGF was determined in preparations of cellular homogenates by Western blotting. Tubulin was used to control for equal blotting and detection. The graph summarizes data of n = 3 experiments, stimulated with LPA for 1 h (CTGF/tubulin, means ± SD). Secreted CTGF was determined in precipitates of cell culture supernatants by Western blotting. The graph summarizes data of n = 4 experiments, stimulated with LPA for 2 h. Data were normalized to LPA-stimulated CTGF synthesis. *** p<0.001, ** p<0.01, ANOVA with Tukey’s multiple comparison test. (D) HKC-8 cells were pre-incubated with EHT1864 (10 μM) for 30 min and then stimulated with LPA (10 μM) for 4 h. Secreted CTGF (sCTGF) was determined in precipitates of cell culture supernatants and cellular CTGF (cCTGF) in cellular homogenates by Western blotting. Vinculin (vinc) served as control. Samples were detected on one blot which had to be rearranged. Data of 4–5 experiments (cellular CTGF, 4 h) and 5–7 experiments (secreted CTGF, 4–6 h) are summarized in the graphs. CTGF expression in LPA-stimulated cells was set to 1 in each experiment. * p<0.05, ***p<0.001 compared to LPA-stimulated cells, ANOVA with Tukey’s multiple comparison test. # p<0.05, paired 2-sided t-test, EHT1864-treated cells compared to control cells.</p

Cellular effects of Rac1 inhibition by EHT1864.

<p>(A) HKC-8 cells were incubated with EHT1864 (10 μM) for the times indicated. Rac1 activity was determined by pull down experiments. The blot is representative of 3 experiments with comparable results. Samples were run on one blot which had to be rearranged. pERK1/2, ERK1/2 and vinculin were detected by immunoblot procedure. The blot shows duplicate biological samples. The graph summarizes data of n = 3–6 experiments (pERK1/2 / ERK1/2 or pERK1/2 / vinculin), means ± SD, *** p<0.001, compared to control cells, ANOVA with Dunnett’s multiple comparison test. pCofilin and tubulin were detected on one blot which had to be rearranged. The graph summarizes data of n = 4 experiments (pCofilin/Tubulin), means ± SD, *** p<0.001, compared to control cells, ANOVA with Dunnett’s multiple comparison test. (B) HKC-8 cells were treated with EHT1864 (10 μM) for 30 min. F-actin was visualized with PromoFluor phalloidin. Scale bar: 20 μm. (C) HKC-8 cells were seeded around barriers. After removal of the barriers (t = 0 h) the cells were treated with EHT1864 (10 μM) for 24 h. Scale bar: 200 μm. The graph summarizes the relative migration velocity of cells treated with 5 or 10 μM EHT1864 or 10 μM Y27632. Data are means ± SEM of 3 experiments with 4 determinations each. ** p< 0.01, * p<0.05 compared to control cells.</p

Actin mutants alter CTGF expression.

<p>(A) HKC-8 cells were transfected with the flag-tagged polymerization-defective actin mutant R62D. 24 h after transfection, cells were stimulated with LPA (10 μM) for 2 h. Mutated actin (anti-flag, red) and CTGF (green) were visualized by indirect immunofluorescence; nuclei were stained with Hoechst (blue). Open arrow indicates CTGF expression in a LPA-stimulated cell; closed arrows indicate low CTGF expression in actin R62D transfected cells. Scale bar: 20 μm. (B) HKC-8 cells were transfected with the flag-tagged polymerization favoring actin mutant S14C. Cells were fixed after transfection without further stimulation. Mutated actin (anti-flag, red) and CTGF (green) were visualized by indirect immunofluorescence; nuclei were stained with Hoechst (blue). Open arrows indicate CTGF expression in transfected cells. Scale bar: 20 μm. (C): HKC-8 cells were transfected with actin expression plasmids (actin R62D and S14C) or eGFP as control and with a luciferase-coupled promoter construct comprising three SRE elements. Expression of cotransfected beta galactosidase was used as reference. Cells were stimulated with LPA (L, 10 μM) for 4 h and compared to control cells (C). Data are means ± SD of 2–4 experiments with biological duplicates. SRE activity of LPA-stimulated actin R62D-transfected cells was set to 1. # p < 0.01 compared to the respective R62D actin- or eGFP-transfected cells; * p < 0.001 analyzed separately compared to eGFP-treated cells. ANOVA with Tukey’s multiple comparison test; ++ p<0.001, + p<0.05, analysis of eGFP and R62D treated cells, LPA-stimulated cells compared to control cells. (D) Cells were treated as in (C), but co-transfected with a 4.5 kb CTGF promoter construct. # p < 0.01 compared to the respective R62D actin- or eGFP-transfected cells; * p < 0.01 analyzed separately compared to eGFP-treated cells. ANOVA with Tukey’s multiple comparison test; ++ p<0.001, analysis of eGFP and R62D treated cells, LPA-stimulated cells compared to control cells.</p

Overexpression of activated or dominant negative Rac1 alters cell morphology but not CTGF expression.

<p>(A) HKC-8 cells were transfected with dominant negative Rac1 (T17N) or constitutively active Rac1 (G12V) coupled to eGFP. 24 h after transfection, cells were stimulated with LPA (10 μM) for 1 h. F-actin fibers were visualized with PromoFluor phalloidin. Arrows indicate transfected cells. Scale bar: 20 μm. (B) HKC-8 cells were transfected with eGFP-coupled dominant negative (dn) or constitutively active (ca) Rac1, or eGFP (GFP) as control together with luciferase-coupled SRE constructs. Expression of cotransfected beta galactosidase was used as reference. Cells were stimulated with LPA (L, 10 μM) for 4 h. Data shown are means ± SD of 3–5 independent experiments performed with duplicate transfections. In each experiment the mean of the unstimulated control values was set to 1. (C) Cells were treated as in (B) but transfected with a 4.5 kb CTGF promoter construct. Data are means of 3 independent experiments with duplicate transfections. In each experiment the mean of the unstimulated control values was set to 1.</p