Holger Summer

Charcot-Marie-Tooth disease CMT4A: GDAP1 increases cellular glutathione and the mitochondrial membrane potential

Mutations in GDAP1 lead to recessively or dominantly inherited peripheral neuropathies (Charcot-Marie-Tooth disease, CMT), indicating that GDAP1 is essential for the viability of cells in the peripheral nervous system. GDAP1 contains domains characteristic of glutathione-S-transferases (GSTs), is located in the outer mitochondrial membrane and induces fragmentation of mitochondria. We found GDAP1 upregulated in neuronal HT22 cells selected for resistance against oxidative stress. GDAP1 over-expression protected against oxidative stress caused by depletion of the intracellular antioxidant glutathione (GHS) and against effectors of GHS depletion that affect the mitochondrial membrane integrity like truncated BH3-interacting domain death agonist and 12/15-lipoxygenase. Gdap1 knockdown, in contrast, increased the susceptibility of motor neuron-like NSC34 cells against GHS depletion. Over-expression of wild-type GDAP1, but not of GDAP1 with recessively inherited mutations that cause disease and reduce fission activity, increased the total cellular GHS content and the mitochondrial membrane potential up to a level where it apparently limits mitochondrial respiration, leading to reduced mitochondrial Ca(2+) uptake and superoxide production. Fibroblasts from autosomal-recessive CMT4A patients had reduced GDAP1 levels, reduced GHS concentration and a reduced mitochondrial membrane potential. Thus, our results suggest that the potential GST GDAP1 is implicated in the control of the cellular GHS content and mitochondrial activity, suggesting an involvement of oxidative stress in the pathogenesis of CMT4A.

TOX3 is a neuronal survival factor that induces transcription depending on the presence of CITED1 or phosphorylated CREB in the transcriptionally active complex

TOX3 is a nuclear protein containing a high mobility group (HMG)-box domain, which regulates Ca2+-dependent transcription in neurons through interaction with the cAMP-response-element-binding protein (CREB). TOX3 appears to be associated with breast cancer susceptibility and was previously shown to be expressed downstream of a cytoprotective cascade together with CITED1, a transcriptional regulator that does not bind directly to DNA. In the present study we show that TOX3 is predominantly expressed in the brain, forms homodimers and interacts with CITED1. TOX3 overexpression protects neuronal cells from cell death caused by endoplasmic reticulum stress or BAX overexpression through the induction of anti-apoptotic transcripts and repression of pro-apoptotic transcripts, which correlates with enhanced transcription involving isolated estrogen-responsive elements and estrogen-responsive promoters. However, both functions cannot be inhibited with the anti-estrogen fulvestrant and are only attenuated by mutation of estrogen-responsive elements. TOX3 also interacts with native CREB and induces the CREB-responsive BCL-2 promoter, which can be inhibited by coexpression of CITED1. Coexpression of CREB, by contrast, abolishes TOX3-mediated transcription from the estrogen-responsive complement C3 promoter. Our results suggest that TOX3 can enhance transcriptional activation from different cytoprotective promoters and that this is dependent on the predominance of either phosphorylated CREB or CITED1 within the transcriptionally active complex.

BAY 87-2243, a highly potent and selective inhibitor of hypoxia-induced gene activation has antitumor activities by inhibition of mitochondrial complex I

The activation of the transcription factor hypoxia‐inducible factor‐1 (HIF‐1) plays an essential role in tumor development, tumor progression, and resistance to chemo‐ and radiotherapy. In order to identify compounds targeting the HIF pathway, a small molecule library was screened using a luciferase‐driven HIF‐1 reporter cell line under hypoxia. The high‐throughput screening led to the identification of a class of aminoalkyl‐substituted compounds that inhibited hypoxia‐induced HIF‐1 target gene expression in human lung cancer cell lines at low nanomolar concentrations. Lead structure BAY 87‐2243 was found to inhibit HIF‐1α and HIF‐2α protein accumulation under hypoxic conditions in non‐small cell lung cancer (NSCLC) cell line H460 but had no effect on HIF‐1α protein levels induced by the hypoxia mimetics desferrioxamine or cobalt chloride. BAY 87‐2243 had no effect on HIF target gene expression levels in RCC4 cells lacking Von Hippel–Lindau (VHL) activity nor did the compound affect the activity of HIF prolyl hydroxylase‐2. Antitumor activity of BAY 87‐2243, suppression of HIF‐1α protein levels, and reduction of HIF‐1 target gene expression in vivo were demonstrated in a H460 xenograft model. BAY 87‐2243 did not inhibit cell proliferation under standard conditions. However under glucose depletion, a condition favoring mitochondrial ATP generation as energy source, BAY 87‐2243 inhibited cell proliferation in the nanomolar range. Further experiments revealed that BAY 87‐2243 inhibits mitochondrial complex I activity but has no effect on complex III activity. Interference with mitochondrial function to reduce hypoxia‐induced HIF‐1 activity in tumors might be an interesting therapeutic approach to overcome chemo‐ and radiotherapy‐resistance of hypoxic tumors.

Myocardial transcriptome analysis of human arrhythmogenic right ventricular cardiomyopathy

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited cardiomyopathy primarily of the right ventricle characterized through fibrofatty replacement of cardiomyocytes. The genetic etiology in ARVC patients is most commonly caused by dominant inheritance and high genetic heterogeneity. Though histological examinations of ARVC-affected human myocardium reveals fibrolipomatous replacement, the molecular mechanisms leading to loss of cardiomyocytes are largely unknown. We therefore analyzed the transcriptomes of six ARVC hearts and compared our findings to six nonfailing donor hearts (NF). To characterize the ARVC-specific transcriptome, we compared our findings to samples from seven patients with idiopathic dilated cardiomyopathy (DCM). The myocardial DCM and ARVC samples were prepared from hearts explanted during an orthotopic heart transplantation representing myocardium from end-stage heart failure patients (NYHA IV). From each heart, left (LV) and right ventricular (RV) myocardial samples were analyzed by Affymetrix HG-U133 Plus 2.0 arrays, adding up to six sample groups. Unsupervised cluster analyses of the groups revealed a clear separation of NF and cardiomyopathy samples. However, in contrast to the other samples, the analyses revealed no distinct expression pattern in LV and RV of myocardial ARVC samples. We further identified differentially expressed transcripts using t-tests and found transcripts separating diseased and NF ventricular myocardium. Of note, in failing myocardium only ~15-16% of the genes are commonly regulated compared with NF samples. In addition both cardiomyopathies are clearly distinct on the transcriptome level. Comparison of the expression patterns between the failing RV and LV using a paired t-test revealed a lack of major differences between LV and RV gene expression in ARVC hearts. Our study is the first analysis of specific ARVC-related RV and LV gene expression patterns in terminal failing human hearts.

Expression of pro-inflammatory genes in human endothelial cells: Comparison of rivaroxaban and dabigatran

INTRODUCTION In addition to its central role in coagulation, thrombin is involved in non-hemostatic activities such as inflammation. Direct inhibition of thrombin activity (e.g. with dabigatran) or reducing its generation by inhibition of Factor Xa (e.g. with rivaroxaban) may therefore have anti-inflammatory effects. MATERIALS AND METHODS Microarray experiments were performed to identify transcriptome-wide changes in mRNA expression levels induced by thrombin in the presence and absence of the PAR-1 antagonist vorapaxar in primary human umbilical vein endothelial cells (HUVECs). On this basis, HUVECs were incubated with recalcified plasma, with or without rivaroxaban (0.3-3000nM), dabigatran (0.3-10,000nM), or vorapaxar (0.3-10nM). Expression levels of preselected pro-inflammatory genes were quantified by real-time PCR. RESULTS Vorapaxar abolished 67 of the 69 transcripts altered by more than twofold on addition of thrombin to HUVECs. ELAM-1, VCAM-1, ICAM-1, MCP-1, IL-8, CXCL1, and CXCL2 were among the genes most strongly induced by thrombin. Inflammatory gene expression after stimulation of thrombin generation was concentration-dependently suppressed by vorapaxar, dabigatran, and rivaroxaban. However, dabigatran at low concentrations (3-300nM) increased significantly the expression levels of CXCL1, CXCL2, IL-8, ELAM-1, MCP-1, and tissue factor. CONCLUSION In HUVECs, plasma-induced transcriptional changes are mediated by thrombin-induced PAR-1 activation. Rivaroxaban downregulated the expression of pro-inflammatory markers and tissue factor to a similar extent to dabigatran.

Genomic profiling of developing cardiomyocytes from recombinant murine embryonic stem cells reveals regulation of transcription factor clusters.

Cardiomyocytes derived from pluripotent embryonic stem cells (ESC) have the advantage of providing a source for standardized cell cultures. However, little is known on the regulation of the genome during differentiation of ESC to cardiomyocytes. Here, we characterize the transcriptome of the mouse ESC line CM7/1 during differentiation into beating cardiomyocytes and compare the gene expression profiles with those from primary adult murine cardiomyocytes and left ventricular myocardium. We observe that the cardiac gene expression pattern of fully differentiated CM7/1-ESC is highly similar to adult primary cardiomyocytes and murine myocardium, respectively. This finding is underlined by demonstrating pharmacological effects of catecholamines and endothelin-1 on ESC-derived cardiomyocytes. Furthermore, we monitor the temporal changes in gene expression pattern during ESC differentiation with a special focus on transcription factors involved in cardiomyocyte differentiation. Thus, CM7/1-ESC-derived cardiomyocytes are a promising new tool for functional studies of cardiomyocytes in vitro and for the analysis of the transcription factor network regulating pluripotency and differentiation to cardiomyocytes.