Borbála Kiss

PARP-1 Inhibition Increases Mitochondrial Metabolism through SIRT1 Activation

SIRT1 regulates energy homeostasis by controlling the acetylation status and activity of a number of enzymes and transcriptional regulators. The fact that NAD(+) levels control SIRT1 activity confers a hypothetical basis for the design of new strategies to activate SIRT1 by increasing NAD(+) availability. Here we show that the deletion of the poly(ADP-ribose) polymerase-1 (PARP-1) gene, encoding a major NAD(+)-consuming enzyme, increases NAD(+) content and SIRT1 activity in brown adipose tissue and muscle. PARP-1(-/-) mice phenocopied many aspects of SIRT1 activation, such as a higher mitochondrial content, increased energy expenditure, and protection against metabolic disease. Also, the pharmacologic inhibition of PARP in vitro and in vivo increased NAD(+) content and SIRT1 activity and enhanced oxidative metabolism. These data show how PARP-1 inhibition has strong metabolic implications through the modulation of SIRT1 activity, a property that could be useful in the management not only of metabolic diseases, but also of cancer.

Investigation of micronized titanium dioxide penetration in human skin xenografts and its effect on cellular functions of human skin-derived cells

Abstract:  Titanium dioxide (TiO2) nanoparticles are ubiquitously used materials in everyday life (e.g. paints, household products and plastic goods). However, despite the wide array of common applications, their pathogenetic role was also suggested under certain conditions (e.g. pulmonary neoplasias and lung fibrosis). From a dermatological point of view, it is also of great importance that TiO2 also serves as a physical photoprotective agent in sunscreens and is widely used in various cosmetic products. However, the effect of TiO2 on human cutaneous functions is still unknown. Therefore, in the current study, we investigated the in vivo penetration of TiO2 via human skin transplanted to immunodeficient mice and, furthermore, we measured the in vitro effects of nanoparticles on various functional properties of numerous epidermal and dermal cells in culture. Hereby, using various nuclear microscopy methods, we provide the first evidence that TiO2 nanoparticles in vivo do not penetrate through the intact epidermal barrier. However, we also report that TiO2, when exposed directly to cell cultures in vitro, exerts significant and cell‐type dependent effects on such cellular functions as viability, proliferation, apoptosis and differentiation. Therefore, our novel findings will hopefully inspire one to systemically explore in future, clinically oriented trials whether there is indeed a risk from micronized TiO2‐containing products on skin with an impaired stratum corneum barrier function.

Peroxisome Proliferator-activated Receptor (PPAR)-2 Controls Adipocyte Differentiation and Adipose Tissue Function through the Regulation of the Activity of the Retinoid X Receptor/PPARγ Heterodimer

The peroxisome proliferator-activated receptor-γ (PPARγ, NR1C3) in complex with the retinoid X receptor (RXR) plays a central role in white adipose tissue (WAT) differentiation and function, regulating the expression of key WAT proteins. In this report we show that poly(ADP-ribose) polymerase-2 (PARP-2), also known as an enzyme participating in the surveillance of the genome integrity, is a member of the PPARγ/RXR transcription machinery. PARP-2-/- mice accumulate less WAT, characterized by smaller adipocytes. In the WAT of PARP-2-/- mice the expression of a number of PPARγ target genes is reduced despite the fact that PPARγ1 and -γ2 are expressed at normal levels. Consistent with this, PARP-2-/- mouse embryonic fibroblasts fail to differentiate to adipocytes. In transient transfection assays, PARP-2 small interference RNA decreases basal activity and ligand-dependent activation of PPARγ, whereas PARP-2 overexpression enhances the basal activity of PPARγ, although it does not change the maximal ligand-dependent activation. In addition, we show a DNA-dependent interaction of PARP-2 and PPARγ/RXR heterodimer by chromatin immunoprecipitation. In combination, our results suggest that PARP-2 is a novel cofactor of PPARγ activity.

Is there penetration of titania nanoparticles in sunscreens through skin? A comparative electron and ion microscopy study

We report on a comparative study by Transmission Electron Microscopy (HRTEM) and Scanning Transmission Ion Microscopy (STIM) combined with Rutherford Backscattering Spectrometry (RBS) and Particle Induced X-Ray Emission (PIXE) on ultra-thin and thin cross-sections, respectively, of various skin samples (porcine skin, healthy human skin, human skin grafted on a severe combined immuno-deficient mouse model) to which we applied topically various formulations containing titanium dioxide (TiO2) nanoparticles with primary particle sizes in the range from 20–100 nm. Whereas the HRTEM and STIM/PIXE images reveal clear differences – mainly related to the different thickness of the cross-sections – they unambiguously show that penetration of TiO2 nanoparticles is restricted to the topmost 3–5 corneocyte layers of the stratum corneum (SC).

Poly(ADP-ribose) polymerase-2 depletion reduces doxorubicin-induced damage through SIRT1 induction

AIMS Doxorubicin (DOX) is widely used in cytostatic treatments, although it may cause cardiovascular dysfunction as a side effect. DOX treatment leads to enhanced free radical production that in turn causes DNA strand breakage culminating in poly(ADP-ribose) polymerase (PARP) activation and mitochondrial and cellular dysfunction. DNA nicks can activate numerous enzymes, such as PARP-2. Depletion of PARP-2 has been shown to result in a protective phenotype against free radical-mediated diseases, suggesting similar properties in the case of DOX-induced vascular damage. METHODS AND RESULTS PARP-2(+/+) and PARP-2(-/-) mice and aortic smooth muscle (MOVAS) cells were treated with DOX (25 mg/kg or 3 μM, respectively). Aortas were harvested 2-day post-treatment while MOVAS cells were treated with DOX for 7 hours. Aortas from PARP-2(-/-) mice displayed partial protection against DOX toxicity, and the protection depended on the conservation of smooth muscle but not on the conservation of endothelial function. DOX treatment evoked free radical production, DNA breakage and PARP activation. Importantly, depletion of PARP-2 did not quench any of these phenomena, suggesting an alternative mechanism. Depletion of PARP-2 prevented DOX-induced mitochondrial dysfunction through SIRT1 activation. Genetic deletion of PARP-2 resulted in the induction of the SIRT1 promoter and consequently increased SIRT1 expression both in aortas and in MOVAS cells. SIRT1 activation enhanced mitochondrial biogenesis, which provided protection against DOX-induced mitochondrial damage. CONCLUSION Our data identify PARP-2 as a mediator of DOX toxicity by regulating vascular SIRT1 activity and mitochondrial biogenesis. Moreover, to the best of our knowledge, this is the first report of SIRT1 as a protective factor in the vasculature upon oxidative stress.

Deletion of PARP-2 induces hepatic cholesterol accumulation and decrease in HDL levels

Poly(ADP-ribose) polymerase-2 (PARP-2) is acknowledged as a DNA repair enzyme. However, recent investigations have attributed unique roles to PARP-2 in metabolic regulation in the liver. We assessed changes in hepatic lipid homeostasis upon the deletion of PARP-2 and found that cholesterol levels were higher in PARP-2(-/-) mice as compared to wild-type littermates. To uncover the molecular background, we analyzed changes in steady-state mRNA levels upon the knockdown of PARP-2 in HepG2 cells and in murine liver that revealed higher expression of sterol-regulatory element binding protein (SREBP)-1 dependent genes. We demonstrated that PARP-2 is a suppressor of the SREBP1 promoter, and the suppression of the SREBP1 gene depends on the enzymatic activation of PARP-2. Consequently, the knockdown of PARP-2 enhances SREBP1 expression that in turn induces the genes driven by SREBP1 culminating in higher hepatic cholesterol content. We did not detect hypercholesterolemia, higher fecal cholesterol content or increase in serum LDL, although serum HDL levels decreased in the PARP-2(-/-) mice. In cells and mice where PARP-2 was deleted we observed decreased ABCA1 mRNA and protein expression that is probably linked to lower HDL levels. In our current study we show that PARP-2 impacts on hepatic and systemic cholesterol homeostasis. Furthermore, the depletion of PARP-2 leads to lower HDL levels which represent a risk factor to cardiovascular diseases.

Genetic Ablation of PARP-1 Protects Against Oxazolone-Induced Contact Hypersensitivity by Modulating Oxidative Stress

Contact hypersensitivity (CHS) reaction is a form of delayed-type of hypersensitivity caused by contact allergens such as oxazolone (OXA). In previous studies it has been shown that poly(ADP-ribose) polymerase (PARP) inhibition reduces the extent of inflammation in CHS. We aimed to shed light on the molecular events causing the protective effect of PARP inhibitors. PARP-1 and -2 knockout mice were sensitized by abdominal delivery of OXA, and a week later CHS was induced by applying OXA on the ears of the mice. PARP-1(-/-) mice were protected against OXA-induced CHS in contrast to PARP-2(-/-) mice. In PARP-1(-/-) mice, neutrophil infiltration was reduced in line with the suppressed expression of proinflammatory cytokines, cell adhesion factors, and matrix metalloproteinase-9, which is likely because of impaired activation of NF-κB p65 and activating transcription factor-2, the two redox-sensitive transcription factors. Moreover, reduced nitrosative and oxidative stress was observed under inflammatory conditions in the PARP-1(-/-) mice when compared with PARP-1(+/+). In conclusion, PARP-1 activation is necessary for proinflammatory gene expression through which PARP-1 enhances neutrophil infiltration and hence oxidative/nitrosative stress, forming a vicious circle, and further aggravating the inflammatory process. Our data identify PARP-1 as a possible target in CHS.

Combined Treatment of MCF-7 Cells with AICAR and Methotrexate, Arrests Cell Cycle and Reverses Warburg Metabolism through AMP-Activated Protein Kinase (AMPK) and FOXO1

Cancer cells are characterized by metabolic alterations, namely, depressed mitochondrial oxidation, enhanced glycolysis and pentose phosphate shunt flux to support rapid cell growth, which is called the Warburg effect. In our study we assessed the metabolic consequences of a joint treatment of MCF-7 breast cancer cells with AICAR, an inducer of AMP-activated kinase (AMPK) jointly with methotrexate (MTX), a folate-analog antimetabolite that blunts de novo nucleotide synthesis. MCF7 cells, a model of breast cancer cells, were resistant to the individual application of AICAR or MTX, however combined treatment of AICAR and MTX reduced cell proliferation. Prolonged joint application of AICAR and MTX induced AMPK and consequently enhanced mitochondrial oxidation and reduced the rate of glycolysis. These metabolic changes suggest an anti-Warburg rearrangement of metabolism that led to the block of the G1/S and the G2/M transition slowing down cell cycle. The slowdown of cell proliferation was abolished when mitotropic transcription factors, PGC-1α, PGC-1β or FOXO1 were silenced. In human breast cancers higher expression of AMPKα and FOXO1 extended survival. AICAR and MTX exerts similar additive antiproliferative effect on other breast cancer cell lines, such as SKBR and 4T1 cells, too. Our data not only underline the importance of Warburg metabolism in breast cancer cells but nominate the AICAR+MTX combination as a potential cytostatic regime blunting Warburg metabolism. Furthermore, we suggest the targeting of AMPK and FOXO1 to combat breast cancer.

Long-Term Followup of Dermal Substitution with Acellular Dermal Implant in Burns and Postburn Scar Corrections

Full-thickness burn and other types of deep skin loss will result in scar formation. For at least partial replacement of the lost dermal layer, there are several options to use biotechnologically derived extracellular matrix components or tissue scaffolds of cadaver skin origin. In a survey, we have collected data on 18 pts who have previously received acellular dermal implant Alloderm. The age of these patients at the injury varied between 16 months and 84 years. The average area of the implants was 185 cm2. Among those, 15 implant sites of 14 patients were assessed at an average of 50 months after surgery. The scar function was assessed by using the modified Vancouver Scar Scale. We have found that the overall scar quality and function was significantly better over the implanted areas than over the surrounding skin. Also these areas received a better score for scar height and pliability. Our findings suggest that acellular dermal implants are especially useful tools in the treatment of full-thickness burns as well as postburn scar contractures.

AMP-Activated Kinase (AMPK) Activation by AICAR in Human White Adipocytes Derived from Pericardial White Adipose Tissue Stem Cells Induces a Partial Beige-Like Phenotype.

Beige adipocytes are special cells situated in the white adipose tissue. Beige adipocytes, lacking thermogenic cues, morphologically look quite similar to regular white adipocytes, but with a markedly different response to adrenalin. White adipocytes respond to adrenergic stimuli by enhancing lipolysis, while in beige adipocytes adrenalin induces mitochondrial biogenesis too. A key step in the differentiation and function of beige adipocytes is the deacetylation of peroxisome proliferator-activated receptor (PPARγ) by SIRT1 and the consequent mitochondrial biogenesis. AMP-activated protein kinase (AMPK) is an upstream activator of SIRT1, therefore we set out to investigate the role of AMPK in beige adipocyte differentiation using human adipose-derived mesenchymal stem cells (hADMSCs) from pericardial adipose tissue. hADMSCs were differentiated to white and beige adipocytes and the differentiation medium of the white adipocytes was supplemented with 100 μM [(2R,3S,4R,5R)-5-(4-Carbamoyl-5-aminoimidazol-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl dihydrogen phosphate (AICAR), a known activator of AMPK. The activation of AMPK with AICAR led to the appearance of beige-like morphological properties in differentiated white adipocytes. Namely, smaller lipid droplets appeared in AICAR-treated white adipocytes in a similar fashion as in beige cells. Moreover, in AICAR-treated white adipocytes the mitochondrial network was more fused than in white adipocytes; a fused mitochondrial system was characteristic to beige adipocytes. Despite the morphological similarities between AICAR-treated white adipocytes and beige cells, functionally AICAR-treated white adipocytes were similar to white adipocytes. We were unable to detect increases in basal or cAMP-induced oxygen consumption rate (a marker of mitochondrial biogenesis) when comparing control and AICAR-treated white adipocytes. Similarly, markers of beige adipocytes such as TBX1, UCP1, CIDEA, PRDM16 and TMEM26 remained the same when comparing control and AICAR-treated white adipocytes. Our data point out that in human pericardial hADMSCs the role of AMPK activation in controlling beige differentiation is restricted to morphological features, but not to actual metabolic changes.