Tuberous sclerosis complex (TSC) is an autosomal dominant genetic syndrome characterized by the growth of benign tumors in multiple organ systems including brain, lung, kidney, skin, and heart. Kidney angiomyolipoma (AML) are benign, slow growing renal tumors that are seen in about 80% of TSC patients, but also occur sporadically. Although heterogeneous in nature, AMLs have a relatively low somatic mutation rate compared to most other cancers, with biallelic loss of either TSC1 or TSC2 gene considered as the primary and sufficient driver for tumor development. We hypothesized that epigenetic alterations of the AML chromatin landscape change the transcriptional dynamics of the underlying genetic system that supports and gives rise to the tumor-cell phenotype. Our data have identified microphthalmia-associated transcription factor (MITF) to be an orchestrating gene in AML development, as 6 out of the top 10 differentially expressed genes in AML are putative MITF-target genes. Integrative analysis of RNA Seq (n=28), H3K27ac ChIP Seq (n=25) and MITF ChIP Seq data (n=3), obtained from fresh-frozen kidney AML specimens, has enabled us to characterize components of a tumor-specific regulatory network under the transcriptional control of MITF. This novel approach has the potential to identify a variety of therapeutic targets, as well as provide unprecedented insight into the mechanisms behind angiomyolipoma development. / 2021-06-07T00:00:00Z

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Osteoactivin (OA) is a type I transmembrane glycoprotein initially identified in bone in 2002. The protein is synthesized, processed and heavily glycosylated by osteoblasts. Its expression is associated with increased osteoblast differentiation and matrix mineralization. To determine the role of OA in skeletal homeostasis in vivo. we utilized a mouse model with a natural mutation in the osteoactivin gene. This mutation is due to a premature stop codon, which results in the generation of a truncated 150 amino acid OA protein. This animal, which we will refer to as OA mutant, was shown by ìCT and histomorphometric analysis to have increased bone volume, trabecular thickness, and trabecular number compared to wild-type (WT) mice at 4 weeks of age, which is a time at which bone formation is most active. Histological analysis of long bones stained with TRAP (tartrate resistant acid phosphatase) and colorimetric analysis of serum TRAP 5b levels indicated that the numbers of osteoclasts are significantly increased in OA mutant samples. Interestingly, although the numbers of osteoclasts as compared to WT were higher in OA mutant mice, serum levels of C-telopeptide of type I collagen (CTX) and osteocalcin, biomarkers for bone resorption and bone formation respectively, were significantly decreased. These data suggested that in mice the presence of truncated OA protein results in increased osteoclast number, but that they are inefficient in resorbing bone and may in part contribute to the increase in bone volume in OA mutant mice in vivo. To further investigate the role of OA in osteoclast differentiation, osteoclasts were differentiated from hematopoietic stem cell progenitors ex vivo. HSCs were cultured in the presence of 50 ng/ml of M-CSF for two days and then with M-CSF and 100 ng/ml of RANKL in the presence or absence of 50 ng/ml recombinant OA. We observed a dramatic increase in multinucleated TRAP-positive osteoclasts and the number of nuclei per osteoclast in OA-treated cultures compared to control. Additionally, analysis of HSCs showed increased cell proliferation in response to exogenous OA treatment. When osteoclasts were differentiated in ex vivo cultures derived from OA mutant and WT mice, we observed decreased osteoclast number, size, and function in OA mutant compared to WT cultures. This decrease was abrogated when cultures were treated exogenously with recombinant OA. Quantitative PCR analysis of RNA isolated during osteoclast differentiation from WT and OA mutant mice reveal decreased gene expression of critical osteoclast differentiation and functional markers, which explains the osteoclast defect observed ex vivo. To investigate the role of OA in osteoblast differentiation, primary osteoblasts were derived from mesenchymal progenitors isolated from calvariae of WT and OA mutant neonatal pups. OA mutant osteoblasts were found to have decreased alkaline phosphatase (ALP) staining and activity at day 14 in culture. Furthermore when cultures were differentiated to 21 days to simulate matrix mineralization in vitro, OA mutant osteoblasts exhibited decreased Alizarin Red and Von Kossa staining. Quantitative measurement of calcium also showed decreased mineral deposition in OA mutant mice compared to WT. Electron microscopic and protein studies were able to eliminate the notion of ER stress or cell toxicity as a result of ER stress playing a role in the delayed osteoblast differentiation observed in OA mutant osteoblasts. Furthermore, OA mutant osteoblasts exhibited decreased proliferation and survival ex vivo. These data reveal an effect of osteoactivin in osteoblasts ex vivo. This study provided an in vivo tool to study the role of osteoactivin in bone cells and the regulation of bone formation and bone resorption by this molecule. Taken together, these findings suggest that the presence of truncated OA leads to increased bone volume due to defective interplay between bone-resorbing osteoclasts and bone-forming osteoblasts. Data presented here support the notion of osteoactivin as a novel molecule in modulating skeletal homeostasis in vivo. / Cell Biology

O câncer de mama é importante problema de saúde pública. O microambiente onde as células cancerígenas se encontram possui moléculas como a laminina e seus peptídeos bioativos, que influenciam a biologia tumoral. Estudo anterior realizado no laboratório demonstrou que o peptídeo C16, derivado da laminina, aumenta a expressão gênica de GPNMB e SPOCK1. Nesse trabalho, demonstramos que o peptídeo C16 aumentou níveis moleculares de GPNMB e SPOCK em células malignas MDA-MB-231e MCF-7, comparado com células normais MCF-10A. C16 estimulou significantemente a invasão de células MDA-MB-231. Silenciamento de GPNMB diminuiu a invasão celular desencadeada por C16. Contextualizando in vivo nossos resultados in vitro, imunohistoquímica em tissue microarrays mostrou que a presença de GPNMB e SPOCK é significantemente maior em câncer de mama. Assim, C16 regula os níveis de GPNMB e SPOCK em células mamárias malignas. C16 e GPNMB cooperam regulando a invasão de células MDA-MB-231. GPNMB e SPOCK foram mais detectados em câncer de mama comparado com mama normal. / Breast cancer is an important public health. The microenvironment in which cancer cells are found contains molecules such as laminin and its bioactive peptides that influence tumor biology. Previous study conducted in the laboratory showed that the C16 peptide derived from laminin, increases the gene expression of GPNMB and SPOCK1. In this work, we demonstrate that the C16 peptide increased molecular levels of GPNMB and SPOCK in malignant cells MDA-MB-231e MCF-7 cells compared with normal cells MCF-10A. C16 significantly stimulated invasion of MDA-MB-231 cells. GPNMB silencing decreased cell invasion triggered by C16. Contextualizing in vivo our in vitro results, tissue microarrays immunohistochemistry showed that the presence of GPNMB and SPOCK are significantly higher in breast cancer. Thus, C16 regulates the levels of GPNMB and SPOCK in malignant breast cells. C16 and cooperate GPNMB regulating the invasion of MDA-MB-231 cells. SPOCK and GPNMB were more detected in breast cancer compared to normal breast.

O câncer de mama é importante problema de saúde pública. O microambiente onde as células cancerígenas se encontram possui moléculas como a laminina e seus peptídeos bioativos, que influenciam a biologia tumoral. Estudo anterior realizado no laboratório demonstrou que o peptídeo C16, derivado da laminina, aumenta a expressão gênica de GPNMB e SPOCK1. Nesse trabalho, demonstramos que o peptídeo C16 aumentou níveis moleculares de GPNMB e SPOCK em células malignas MDA-MB-231e MCF-7, comparado com células normais MCF-10A. C16 estimulou significantemente a invasão de células MDA-MB-231. Silenciamento de GPNMB diminuiu a invasão celular desencadeada por C16. Contextualizando in vivo nossos resultados in vitro, imunohistoquímica em tissue microarrays mostrou que a presença de GPNMB e SPOCK é significantemente maior em câncer de mama. Assim, C16 regula os níveis de GPNMB e SPOCK em células mamárias malignas. C16 e GPNMB cooperam regulando a invasão de células MDA-MB-231. GPNMB e SPOCK foram mais detectados em câncer de mama comparado com mama normal. / Breast cancer is an important public health. The microenvironment in which cancer cells are found contains molecules such as laminin and its bioactive peptides that influence tumor biology. Previous study conducted in the laboratory showed that the C16 peptide derived from laminin, increases the gene expression of GPNMB and SPOCK1. In this work, we demonstrate that the C16 peptide increased molecular levels of GPNMB and SPOCK in malignant cells MDA-MB-231e MCF-7 cells compared with normal cells MCF-10A. C16 significantly stimulated invasion of MDA-MB-231 cells. GPNMB silencing decreased cell invasion triggered by C16. Contextualizing in vivo our in vitro results, tissue microarrays immunohistochemistry showed that the presence of GPNMB and SPOCK are significantly higher in breast cancer. Thus, C16 regulates the levels of GPNMB and SPOCK in malignant breast cells. C16 and cooperate GPNMB regulating the invasion of MDA-MB-231 cells. SPOCK and GPNMB were more detected in breast cancer compared to normal breast.

Das globale Phänomen Übergewicht erhöht das Risiko für die Entwicklung von Diabetes, Atherosklerose und Herz-Kreislauf-Erkrankungen. Diese sind assoziiert mit der Expression des Transmembranproteins Glycoprotein nonmetastatic melanoma protein b (Gpnmb), das von Makrophagen und dendritischen Zellen exprimiert wird. Wir haben die Rolle von Gpnmb in genetisch- und diät-induzierter Atherosklerose sowie in diät-induzierter Adipositas in Gpnmb-Knockout- und Wildtyp-Mäusen untersucht. Körpergewicht und Blutfette wurden in beiden Erkrankungen nicht von Gpnmb beeinflusst. Gpnmb wurde in Makrophagen von atherosklerotischen Läsionen stark exprimiert, jedoch hatte das Fehlen von Gpnmb keinen Einfluss auf die Größe der Aortenläsion. In Übergewicht konnten wir dagegen einen größeren Effekt von Gpnmb detektieren. Gpnmb hatte einen positiven Einfluss auf den Insulin- und Glukoseplasmaspiegel sowie auf die Leberfibrose bei adipösen Mäusen. Gpnmb-Knockout-Tiere besaßen mehr Makrophagen im epididymalen Fettgewebe. Da Gpnmb in den entsprechenden Makrophagen von Wildtyp-Mäusen stark exprimiert wurde, könnte Gpnmb eine abschwächende Rolle auf Entzündungen des Fettgewebes haben. Dies wird durch in-vitro-Daten bestätigt, wo Gpnmb hauptsächlich in reparativen, TGFβ-stimulierten Makrophagen exprimiert wurde. Diese Expression führte jedoch nur zu einer leichten entzündungshemmenden Wirkung. Eine weitere Aufgabe von Makrophagen, die Autophagie, wurde durch Gpnmb nicht beeinflusst. Dies ist überraschend, da die Expression, die Freisetzung und der Abbau von Gpnmb durch Bafilomycin, einem Inhibitor des letzten Schritts der Autophagie, auf einzigartige Weise erhöht wurde. Zusammenfassend ist die Gpnmb-Expression in voll ausgereiften Makrophagen stark induziert und kann durch lysosomale Hemmung weiter gesteigert werden. Bei Adipositas verhindert Gpnmb die Entwicklung einer Insulinresistenz, möglicherweise durch Dämpfung der Entzündung des Fettgewebes. / Obesity, an emerging global phenomenon, increases the risk for the development of diabetes, atherosclerosis and cardiovascular diseases. Those metabolic diseases have been associated with the expression of glycoprotein nonmetastatic melanoma protein b (Gpnmb), a transmembrane protein that is expressed by macrophages and dendritic cells. We studied the role of Gpnmb in genetically- and diet-induced atherosclerosis as well as diet-induced obesity in Gpnmb-knockout and respective wildtype control mice. The absence of Gpnmb did not affect body weight and blood lipid parameters in both diseases. Whereas Gpnmb was strongly expressed in atherosclerotic lesion-associated macrophages, the absence of Gpnmb did not influence the development of aortic lesion size. On the other hand, the absence of Gpnmb elicited stronger effects in obesity. We observed a positive influence of Gpnmb on insulin and glucose plasma levels as well as liver fibrosis in obese mice. Moreover, Gpnmb-knockout animals contained more macrophages in epididymal adipose tissue. Gpnmb was strongly expressed in adipose tissue macrophages in wildtype mice, suggesting an alleviating role of Gpnmb on adipose tissue inflammation. This was corroborated by in vitro data where Gpnmb was mostly expressed in reparative macrophages stimulated with transforming growth factor β (TGFβ). However, this expression resulted only in a mild anti-inflammatory effect. Another important macrophage feature, autophagy, was not influenced by Gpnmb. This is surprising as Gpnmb expression, shedding and degradation was uniquely increased by bafilomycin, an inhibitor of the last step of autophagy. Taken together, Gpnmb expression is strongly induced in fully mature macrophages and can be further increased due to lysosomal inhibition. In obesity, Gpnmb prevents the development of insulin resistance possibly by dampening adipose tissue inflammation.

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No description available.

Ischämische Herzerkrankungen stellen die weltweit häufigste Todesursache dar. Das Chemokin SDF-1 zählt zu den vielversprechendsten neuen Therapietargets. Allerdings werden die dem SDF-1 Effekt zugrunde liegenden Mechanismen kontrovers diskutiert. Um den Einfluss von SDF-1 auf die Herzregeneration aufzuklären, wurden transgene Ratten generiert, welche SDF-1 in Kardiomyozyten überexprimieren. Die basale Herzfunktion war in diesen Ratten nicht verändert, jedoch zeigte sich nach Herzinfarkt eine Verschlechterung der kardialen Funktion. Des Weiteren ließen sich eine verstärkte Fibrosebildung, ein Anstieg neutrophiler Granulozyten im Blut sowie eine erhöhte Einwanderung von Makrophagen in die Herzen transgener Ratten feststellen. Dagegen waren die Anlockung von Stammzellen und die Blutgefäßneubildung nicht verändert. Diese Daten bestätigen, dass kardiales SDF-1 eine nachteilige Wirkung ausüben kann, indem es entzündliche Prozesse im geschädigten Gewebe beeinflusst. Ferner wurde ein Microarray-basiertes Screening in kardialem Gewebe nach Herzinfarkt durchgeführt. Ziel der Studie war die Identifizierung neuer Moleküle, deren Rolle bei Herzerkrankungen bislang unbekannt ist. Das Screening brachte das Glykoprotein GPNMB hervor, welches an fibrotischen Prozessen nach Gewebeschädigung beteiligt ist. Wir untersuchten das Protein mit Hilfe des DBA/2J Mausstammes, in dem kein funktionelles GPNMB vorhanden ist. Die Untersuchung dieser Mäuse ergab keine Veränderungen der basalen Herzfunktion, nach Herzinfarkt zeigte sich jedoch eine verbesserte kardiale Funktion sowie erhöhte Hämoglobinkonzentrationen im Blut. Außerdem war die Funktion von Makrophagen verändert. Darüber hinaus fanden wir erhöhte Konzentrationen von GPNMB in Plasmaproben von Patienten nach akutem Herzinfarkt. Zusammenfassend weisen die Ergebnisse darauf hin, dass GPNMB nicht nur ein vielversprechendes Therapietarget, sondern auch einen potenziellen Biomarker für ischämische Herzerkrankungen darstellt. / Ischemic heart diseases are a major cause of death worldwide due to the postmitotic state of the heart. The chemokine SDF-1 is one of the most promising novel therapeutic targets due to its ability to attract leukocytes and stem cells. However, the role of different cardiac cell types in this process remains elusive and underlying mechanisms have been controversially discussed. To clarify the role of SDF-1 and its receptor CXCR4 in myocardial regeneration, we generated transgenic rats overexpressing SDF-1 in cardiomyocytes. The function of the heart at baseline was not altered in these rats, whereas the induction of myocardial infarction resulted in impaired cardiac function and remodeling. This finding was accompanied by increased fibrosis, neutrophil blood counts and macrophage infiltration into the heart. On the other hand, stem cell recruitment and neovascularization were not altered in SDF-1 transgenic rats. In conclusion, our findings confirm that the SDF-1/CXCR4 axis can have adverse effects by affecting the inflammatory state of the healing heart. In addition, a microarray-based screening was conducted in rat hearts after myocardial infarction with the aim to discover yet unknown molecules involved in cardiac repair. This screening yielded GPNMB, a glycoprotein known to be involved in inflammatory and fibrotic processes after tissue injury. We studied the protein further using DBA/2J mice that lack functional levels of GPNMB. While the cardiac function was normal in these mice at baseline, induction of myocardial infarction revealed a preservation of cardiac function, less dilatation as well as higher red blood cell and hemoglobin levels. Moreover, the absence of GPNMB resulted in an altered activity and distribution of macrophages. We also found increased levels of GPNMB in plasma of patients after myocardial infarction. In conclusion, our findings suggest that GPNMB might constitute a novel therapeutic target and biomarker of acute ischemic heart diseases.