FBXO32, encoding a member of the SCF complex, is mutated in dilated cardiomyopathy

Ubiquitination defect in cells expressing mutant FBXO32. a Co-immunopricipitation analysis. HEK293 cells were transfected with the indicated plasmids and immunoblot analysis was performed from total cell lysates using a specific anti-ubiquitin antibody. FBXO32 expression is shown as well as GAPDH. The blot is representative of three independent experiments. b Immunoblot analysis of the ubiquitination in cardiomyocytes. Cells were transfected with the Flag-FBXO32-WT or Flag-FBXO32-Mutant and whole cell extracts were analyzed by immunoblotting using the indicated antibodies. (TIF 1928 kb

Characterization of the expression, regulation and function of the Nuclear Hormone Receptors Peroxisom Proliferator Activated Receptor delta in humane T-lymphocytes

Peroxisome proliferator-activated receptor delta (PPARδ) ist ein nukleärer Rezeptor, der der Superfamilie der Steroidhormon-Rezeptoren angehört. Seine biologischen Funktionen sind vielfältig und reichen von der β-Oxidation von Fettsäuren über die Proliferation der Keratinozyten bis hin zur Differenzierung von epithelial Zellen. Die Rolle von PPARδ innerhalb des Immunsystems wurde bislang nicht genau aufgeklärt. Eine Vielzahl von Indizien weisen auf eine zentrale Rolle von PPARδ bei Psoriasis hin. Psoriasis (Schuppenflechte) ist eine häufig auftretende chronisch verlaufende entzündliche Erkrankung der Haut mit einer Prävalenz von 2% - 4% in den westlichen Industrienationen. Da die Psoriasis durch übermäßige Keratinozytenproliferation und Differenzierung gekennzeichnet ist, beschränkte sich die Forschung bislang auf die Bedeutung von PPARδ in Keratinozyten. An der Psoriasispathogenese sind jedoch auch aktivierte T-Zellen beteiligt. Daher wurde im Rahmen dieser Arbeit die Expression und die funktionelle Bedeutung von PPARδ in humanen T-Zellen untersucht. Die Expression konnte sowohl in aktivierten humanen T-Zellen, die aus peripheralen Blut isoliert wurden, als auch in T-Zellen der suprabasalen-Schicht psoritatischer Haut gezeigt werden. Weiterhin wurde PPARδ in T-Zellen durch Typ 1 Interferon (IFN) induziert. Funktionell verstärkt PPARδ die Proliferation primärer T-Zellen und schützt diese sowohl vor Typ 1 IFN-induzierter als auch vor Serumentzug-induzierter Apoptose. Die Untersuchungen ergaben, dass diese zellulären Funktionen durch Aktivierung von ERK1/2 vermittelt werden. Die hier gezeigten Resultate weisen auf eine direkte molekulare Verbindung zwischen den Typ 1 IFN-Signalwegen und PPARδ hin und die funktionelle Rolle von PPARδ in humanen T-Zellen wird deutlich aufgezeigt. Dies deutet daraufhin, dass die Typ 1 IFN vermittelte Induktion von PPARδ möglicherweise für das Ausharren der aktivierten T-Zellen in läsionaler Haut bei Psoriasis verantwortlich ist. Für Experimente im Rahmen der Untersuchung der Funktionen von PPARδ in TZellen wurde die Expression des PPARδ-Gens mittels siRNA inhibiert. Die siRNA wurde dabei von einem Lentivektor exprimiert. Aufgrund ihrer Charakteristika stellen die Lentiviren besonders geeignete Vektoren für den Gentransfer dar. Replikationsinkompetente lentivirale Vektoren sind in der Lage ruhende und sich nicht teilende Zellen zu infizieren und ihr genetisches Material (shRNA) in das Zielzellgenom zu integrieren. Dadurch wird die siRNA in den infizierten Zellen und deren Tochterzellen stabil exprimiert. Für experimentelle Anwendungen lentiviraler Vektoren sind ein hoher Titer der Virusüberstände und effiziente Virus- Konzentrationen wichtige Faktoren. Aus diesem Grund war das Ziel zu Beginn dieser Arbeit die Produktion und Aufkonzentrierung von Lentivektoren zu optimieren. Dazu wurden mehrere bereits in der Literatur veröffentlichte Protokolle unter Verwendung verschiedener Transfervektoren, die sich in der Größe unterscheiden (7.5-13.2 kb), evaluiert. In dieser Arbeit konnte ein modifiziertes Virusproduktionsprotokoll, das anwendbare Titer (von bis zu 107 V/ml) für eine Auswahl unterschiedlicher Lentivektoren mit Inserts bis zu 7.5 kb liefert, etabliert werden. Ferner zeigten die Resultate, dass die Ausbeute an Viren nach der Ultrazentrifugation abhängig ist von der Größe des im Vektor beeinhaltenden Inserts und dass die Ausbeute stark abnimmt bei großen Inserts. Auch wurde hier ein schnelles (4h) und leicht anwendbares Zentrifugationsprotokoll präsentiert, welches eine hohe Ausbeute auch für große und fragile Vektoren ermöglicht.Peroxisome proliferator-activated receptor delta (PPARδ) is a nuclear hormone receptor regulating diverse biological processes, including β-oxidation of fatty acid, proliferation of keratinocytes and epithelial cell differentiation. The role of PPARδ in the immune system has not been thoroughly studied to date. Several lines of evidence support a role for PPARδ in psoriasis pathogenesis. Psoriasis is a chronic inflammatory skin disease with an estimated prevalence of 2% - 4% in the western civilization. Given that psoriasis is characterized by keratinocyte hyperproliferation and aberrant terminal differenctiation of keratinocytes, research has been conducted on the role of PPARδ in keratinocytes. However, a large amount of clinical and experimental evidence supports a main role for T cells in pathogenesis of psoriasis. Therefore, the present work aimed at characterizing the expression and function of PPARδ in human T cells. In the course of this work, the expression of PPARδ was shown in activated human T cells purified from peripheral blood, as well as in T cells isolated from affected psoriasis skin lesions. Furthermore, PPARδ is induced in T cells upon stimulation with type 1 interferon (IFN). Functionally, PPARδ enhances proliferation of primary T cells and blocks apoptosis induced by type 1 IFN and by serum deprivation. It was shown that these cellular functions are mediated by activation of ERK1/2 signaling. The results presented in this work establish a direct molecular link between type 1 IFN signaling and PPARδ, define a functional role for PPARδ in human T cells, and suggest that the induction of PPARδ by type 1 IFN contributes to the persistence of activated T cells in psoriasis skin lesions. To study the function of PPARδ in T cells the suppression of PPARδ gene expression was necessary. This was accomplished using siRNA expressed from a lentiviral vector. Vectors based on lentiviruses are highly efficient vehicles for gene transfer approaches and are widely used in basic research. The use of replication–deficient pseudotyped HIV-derived lentiviral vectors allows the efficient transduction of primary cells including human T cells. Generation of high titer lentiviral stocks and efficient virus concentration are central to maximize the utility of lentiviral technology. Therefore, the present work initlally established protocols for optimizing the production and concentration of lentivectors. Published protocols for lentivirus production on a range of transfer vectors differing in size (7.5–13.2 kb) were evaluated. Based on these, a modified virus production protocol was developed robustly yielding useful titers (up to 107 / ml) for a range of different transfer vectors containing packaging inserts up to 7.5 kb. The results also showed that the recovery of virus after concentration by ultracentrifugation depends on the size of the packaged inserts, heavily decreasing for large packaged inserts. To address this technical limitation, a fast (4h) centrifugation protocol at reduced speed allowing high virus recovery even for large and fragile lentivirus vectors was developed

Nonsteroidal Anti-Inflammatory Drugs Induce Apoptosis in Cutaneous T-Cell Lymphoma Cells and Enhance Their Sensitivity for TNF-Related Apoptosis-Inducing Ligand

Cutaneous T-cell lymphomas (CTCL) form a heterogeneous group of non-Hodgkin's lymphomas of the skin. In previous studies, we had characterized CTCL cells as resistant to the death ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which correlated to pronounced expression of the caspase-8/-10 inhibitor c-FLIP. For identification of proapoptotic strategies in CTCL cells and for overcoming their death ligand resistance, we investigated the effects of nonsteroidal anti-inflammatory drugs (NSAIDs) such as acetylsalicylic acid, sodium salicylate, and diclofenac (DF). These drugs strongly enhanced apoptosis, as well as decreased CTCL cell proliferation and vitality, and DF furthermore sensitized for TRAIL-induced apoptosis. Full activation of the caspase cascade (caspase-3, -8, -9) and decreased mitochondrial membrane potential were characteristic for NSAID treatment, whereas cytochrome c release was seen only for DF. Downregulation of Mcl-1 and enhanced surface expression of TRAIL were seen in response to NSAIDs. Most characteristic for apoptosis induction was the downregulation of c-FLIP. In agreement with the critical role of c-FLIP for apoptosis deficiency of CTCL cells, its overexpression decreased NSAID-mediated apoptosis and its downregulation by small hairpin RNA-enhanced apoptosis. The study provides a rationale for the use of NSAIDs as a new therapeutic option for CTCL patients. Supporting this concept, ex vivo lymphoma cells of CTCL patients also revealed significant sensitivity for NSAID treatment

Control of histone H3 phosphorylation by CaMKIIδ in response to haemodynamic cardiac stress.

Heart failure is associated with the reactivation of a fetal cardiac gene programme that has become a hallmark of cardiac hypertrophy and maladaptive ventricular remodelling, yet the mechanisms that regulate this transcriptional reprogramming are not fully understood. Using mice with genetic ablation of calcium/calmodulin-dependent protein kinase II δ (CaMKIIδ), which are resistant to pathological cardiac stress, we show that CaMKIIδ regulates the phosphorylation of histone H3 at serine-10 during pressure overload hypertrophy. H3 S10 phosphorylation is strongly increased in the adult mouse heart in the early phase of cardiac hypertrophy and remains detectable during cardiac decompensation. This response correlates with up-regulation of CaMKIIδ and increased expression of transcriptional drivers of pathological cardiac hypertrophy and of fetal cardiac genes. Similar changes are detected in patients with end-stage heart failure, where CaMKIIδ specifically interacts with phospho-H3. Robust H3 phosphorylation is detected in both adult ventricular myocytes and in non-cardiac cells in the stressed myocardium, and these signals are abolished in CaMKIIδ-deficient mice after pressure overload. Mechanistically, fetal cardiac genes are activated by increased recruitment of CaMKIIδ and enhanced H3 phosphorylation at hypertrophic promoter regions, both in mice and in human failing hearts, and this response is blunted in CaMKIIδ-deficient mice under stress. We also document that the chaperone protein 14-3-3 binds phosphorylated H3 in response to stress, allowing proper elongation of fetal cardiac genes by RNA polymerase II (RNAPII), as well as elongation of transcription factors regulating cardiac hypertrophy. These processes are impaired in CaMKIIδ-KO mice after pathological stress. The findings reveal a novel in vivo function of CaMKIIδ in regulating H3 phosphorylation and suggest a novel epigenetic mechanism by which CaMKIIδ controls cardiac hypertrophy

Advancing male age differentially alters levels and localization patterns of PLCzeta in sperm and testes from different mouse strains

Sperm-specific phospholipase C zeta (PLC) initiates intracellular calcium (Ca2+) transients which drive a series of concurrent events collectively termed oocyte activation. Numerous investigations have linked abrogation and absence/reduction of PLC with forms of male infertility in humans where oocyte activation fails. However, very few studies have examined potential relationships between PLC and advancing male age, both of which are increasingly considered to be major effectors of male fertility. Initial efforts in humans may be hindered by inherent PLC variability within the human population, alongside a lack of sufficient controllable repeats. Herein, utilizing immunoblotting, immunofluorescence, and quantitative reverse transcription PCR (qRT-PCR) we examined for the first time PLC protein levels and localization patterns in sperm, and PLC mRNA levels within testes, from mice at 8 weeks, 12 weeks, 24 weeks, and 36 weeks of age, from two separate strains of mice, C57BL/6 (B6; inbred) and CD1 (outbred). Collectively, advancing male age generally diminished levels and variability of PLC protein and mRNA in sperm and testes, respectively, when both strains were examined. Furthermore, advancing male age altered the predominant pattern of PLC localization in mouse sperm, with younger mice exhibiting predominantly post-Acrosomal, and older mice exhibiting both post-Acrosomal and acrosomal populations of PLC. However, the specific pattern of such decline in levels of protein and mRNA was strain-specific. Collectively, our results demonstrate a negative relationship between advancing male age and PLC levels and localization patterns, indicating that aging male mice from different strains may serve as useful models to investigate PLC in cases of male infertility and subfertility in humans

Additional file 1: Table S1. of FBXO32, encoding a member of the SCF complex, is mutated in dilated cardiomyopathy

Clinical characteristics of the studied family. (DOCX 82 kb

Additional file 3: Figure S2. of FBXO32, encoding a member of the SCF complex, is mutated in dilated cardiomyopathy

Autozygome analysis in the studied family. Autozygosity mapping was performed using AgileMultideogram. The result is showing the single shared ROH (runs of homozygosity) on chromosome 8 between the four affected members (dark blue). (TIF 4051 kb

Additional file 5: Figure S3. of FBXO32, encoding a member of the SCF complex, is mutated in dilated cardiomyopathy

Mutation in FBXO32 abrogates binding to SKP1 within the SCF complex. a Schematic representation of the SCF complex. The FBXO32 mutation represented as a black triangle is within the F-Box domain which normally mediates binding to SKP1. b Immunoprecipitation showing reduced interaction of mutant FBXO32 with CUL1 after co-transfection of HEK293 cells with WT-FBXO32 or mutant-FBXO32 and CUL1, ROC1, and SKP1. c Quantitative analysis of (b) from three independent experiments. (TIF 2185 kb

Additional file 4: Table S2. of FBXO32, encoding a member of the SCF complex, is mutated in dilated cardiomyopathy

Coverage and sequencing depth of the exome data. (DOCX 17 kb

Additional file 2: Figure S1. of FBXO32, encoding a member of the SCF complex, is mutated in dilated cardiomyopathy

Echocardiographic images of patients from the studied family. Echocardiography performed on two of the affected siblings index patients IV.5 (a) and IV.7 (b) prior to heart transplantation. M-mode echo and parasternal long-axis view showing dilatation of the left ventricle and of the left atria with severe hypokinesia. (TIF 5479 kb