STAT5 drives abnormal proliferation in autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) leads to renal failure. The hallmark of ADPKD is increased epithelial proliferation, which has been proposed to be due to atypical signaling including abnormal JAK-STAT activity. However, the relative contribution of JAK-STAT family members in promoting proliferation in ADPKD is unknown. Here, we present siRNA JAK-STAT-focused screens discovering a previously unknown proliferative role for multiple JAK-STAT components (including STAT1, STAT2, STAT4, STAT5a, and STAT5b). Amongst these, we selected to study the growth hormone/growth hormone receptor/STAT5-axis because of its known role as a regulator of growth in nonrenal tissues. Loss of STAT5 function, facilitated by pharmacological inhibition or siRNAs, significantly reduced proliferation with an associated reduction in cyst growth in vitro. To study whether STAT5 is abnormally activated in vivo, we analyzed its expression using two independent mouse models of ADPKD. STAT5 was nuclear, thus activated, in renal epithelial cyst lining cells in both models. To test whether forced activation of STAT5 can modulate proliferation of renal cells in vivo, irrespective of the Pkd1 status, we overexpressed growth hormone. These mice showed increased STAT5 activity in renal epithelial cells, which correlated with de novo expression of cyclin D1, a STAT5 target gene. Chromatin immunoprecipitation experiments revealed that STAT5 transcriptionally activated cyclin D1 in a growth hormone-dependent fashion, thus providing a mechanism into how STAT5 enhances proliferation. Finally, we provide evidence of elevated serum growth hormone in Pkd1 mutant mice. Thus, the growth hormone/STAT5 signaling axis is a novel therapeutic target in ADPKD

Hepatic growth hormone-activated JAK2 and STAT5 signaling in liver metabolism and hepatocellular carcinoma development

Das metabolische Syndrom ist zu einer weitverbreiteten Krankheit in der westlichen Welt geworden, die mit der zunehmenden weltweiten Prävalenz von Adipositas und Diabetes zusammenhängt. Die nicht-alkoholische Fettlebererkrankung (NAFLD) gilt als hepatische Manifestation des metabolischen Syndroms und ist mit den primären Stadien der Leberkrebsentstehung assoziiert. Beim Menschen und in Mausmodellen konnte gezeigt werden, dass die Beeinträchtigung des Signalweges des hepatischen Wachstumshormons (GH)-JAK2-STAT5 mit der NAFLD-Entstehung in Verbindung steht. Allerdings bleibt die Funktion des GH-JAK2-STAT5-Signalweges in der weiteren Entwicklung der Lebererkrankung weitgehend unklar. In dieser Arbeit sollte die Funktion der hepatischen Proteine, STAT5 und JAK2, im Lipidstoffwechsel und der Leberkrebsentstehung unter Verwendung von zwei Studien untersucht werden: 1) In der ersten Studie wurde ein GH-transgenes (GHtg) Mausmodel herangezogen, welches durch erhöhte GH-Zirkulation entzündungsassoziierte hepatozelluläre Karzinome entwickelt. In diesen Tieren wurde zusätzlich STAT5 leberspezifisch deletiert (GHtgSTAT5hep). 2) In der zweiten Studie wurde JAK2 leberspezifisch in den GH-transgenen Tieren deletiert (GHtgJAK2hep) und mit GHtgSTAT5hep-Tieren verglichen. Unsere Untersuchungen haben gezeigt, dass die leberspezifische Deletion von STAT5 oder JAK2 die vorzeitige Mortalität und die hepatische/systemische Entzündung, die in dem GH- transgenen Mausmodell auftritt, verhindern konnte. Unabhängig von der erhöhten GH-Zirkulation führte die leberspezifische Deletion von STAT5 oder JAK2 zu einer bezeichnenden und frühzeitigen Leber-Steatose, die aufgrund einer ektopischen Lipidumverteilung und erhöhter de novo Lipogenese in der Leber zu Stande kam. Erstaunlicherweise hatte die STAT5-Deletion eine verfrühte Leberkrebsentstehung im GH-transgenen Modell zur Folge, während die JAK2-Deletion merklich die Entstehung und Entwicklung von Leberkrebs verlangsamen konnte. Das frühe und fortgeschrittene Entstehen von Lebertumoren in den GHtgSTAT5hep-Tieren stand im direkten Zusammenhang mit der Reduzierung von hepatoprotektiven Faktoren, der Aktivierung von onkogenem c-Jun N-terminal kinase 1 (JNK1), c-JUN und STAT3, und der Akkumulation von DNS-Schäden, herbeigeführt durch die verminderte Aktivität von p53. Auffallend war, dass die Deletion von JAK2 die onkogene Aktivierung von STAT3 verhinderte und zu reduzierten oxidativen Leberschäden führte, obwohl die Generierung von reaktiven Sauerstoffspezien (ROS) in beiden Genotypen, GHtgSTAT5hep und GHtgJAK2hep, stark erhöht war. Die reduzierten oxidativen Zellschäden in JAK2-delitierten Lebern korrelierten mit einer starken Expression und Aktivität von Glutathion-S-Transferasen (GSTs), welche auf einen erhöhten Entgiftungsmechanismus hindeuteten, der jedoch in STAT5-delitieren Lebern nicht vorhanden war. In vitro- und in vivo-Studien bestätigten weiter, dass die Hemmung von JAK2 zu einer signifikanten Hochregulation von GSTs führte und auf diese Weise vor ROS-induzierten oxidativen Zellschäden wie DNS-Schädigung, Lipidperoxidation und Proteinoxidation schützt. Zusammenfassend lassen unsere Daten vermuten, dass der GH-JAK2-STAT5-Signalweg eine hepatische Steatose verhindert. Jedoch verstärkt die Deletion von STAT5, durch den Verlust seiner hepatoprotektiven Funktion, die Entstehung von Leberkrebs, während die JAK2- Deletion vor ROS-induzierten oxidativen Schäden durch die erhöhte GST-Aktivität schützt, wodurch die Entwicklung von Leberkrebs verzögert wird.The metabolic syndrome has become a common disorder in the Western world, which is related to the increasing worldwide prevalence of obesity and diabetes mellitus. Non-alcoholic fatty liver disease (NAFLD) is considered to be a hepatic manifestation of the metabolic syndrome and is linked to the primary stages of liver cancer development. Disruption of hepatic growth hormone (GH)- janus kinase (JAK) 2- signal transducer and activator of transcription (STAT) 5 signaling is associated with NAFLD development in humans and mouse models. Yet, the role of hepatic GH-JAK2-STAT5 signal transduction in liver disease progression remains enigmatic. Here, we aimed to address the function of hepatic STAT5 and JAK2 in liver lipid metabolism and liver tumorigenesis by performing two different studies. 1) We crossed mice with hepatocyte-specific deletion of STAT5 (STAT5hep) to a mouse model of inflammation-related liver cancer caused by hyperactivated GH signaling (GHtg). 2) GHtg were crossed to mice harboring a hepatocyte-specific deletion of JAK2 (JAK2hep) and compared to STAT5 deficient mice (GHtgSTAT5hep). Our studies demonstrated that hepatic deficiency of STAT5 or JAK2 prevented premature mortality and hepatic/systemic inflammation in a GHtg background. Yet, irrespective of the hyperactivated GH signaling, STAT5 and JAK2 deficiency resulted in marked and early liver steatosis due to ectopic lipid redistribution and increased hepatic de novo lipogenesis. Remarkably, while STAT5 deficiency strongly accelerated liver tumorigenesis in the GH transgenic background, loss of JAK2 significantly decelerated liver cancer development and progression. Early and advanced liver tumorigenesis in GHtgSTAT5hep mice was linked to the abolishment of hepatoprotective factors, abnormal activation of oncogenic c-Jun N-terminal kinase 1 (JNK1), c-JUN and STAT3, and accumulation of DNA damage secondary to decreased p53 activity. Strikingly, JAK2 deficiency prevented oncogenic STAT3 signaling and was associated with diminished oxidative damage in the liver even though reactive oxygen species (ROS) generation was highly increased in both GHtgSTAT5hep and GHtgJAK2hep mice. Intriguingly, lowered oxidative damage in JAK2 deficient livers correlated with strong expression and activity of glutathione S-transferases (GSTs) suggesting an increased detoxification mechanism, which was not present in STAT5 deficient livers. In vitro and in vivo studies further confirmed that JAK2 inhibition resulted in considerable upregulation of GSTs further validating that JAK2 deficiency antagonizes ROS-induced oxidative damage, i.e. DNA damage, lipid peroxidation and protein oxidation. In conclusion, our data suggest that GH-JAK2-STAT5 signaling prevents hepatic steatosis. Yet, while STAT5 deficiency augments liver tumorigenesis caused by the loss of its hepatoprotective function, JAK2 deficiency antagonizes ROS-induced oxidative damage through GST activity, thereby delaying the development of liver cancer.submitted by Madaleine Themanns, BSc MScZusammenfassung in deutscher SpracheAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersMedizinische Universität, Dissertation, 2017OeB

Hepatic growth hormone and glucocorticoid receptor signaling in body growth, steatosis and metabolic liver cancer development

Growth hormone (GH) and glucocorticoids (GCs) are involved in the control of processes that are essential for the maintenance of vital body functions including energy supply and growth control. GH and GCs have been well characterized to regulate systemic energy homeostasis, particular during certain conditions of physical stress. However, dysfunctional signaling in both pathways is linked to various metabolic disorders associated with aberrant carbohydrate and lipid metabolism. In liver, GH-dependent activation of the transcription factor signal transducer and activator of transcription (STAT) 5 controls a variety of physiologic functions within hepatocytes. Similarly, GCs, through activation of the glucocorticoid receptor (GR), influence many important liver functions such as gluconeogenesis. Studies in hepatic Stat5 or GR knockout mice have revealed that they similarly control liver function on their target gene level and indeed, the GR functions often as a cofactor of STAT5 for CH-induced genes. Gene sets, which require physical STAT5-GR interaction, include those controlling body growth and maturation. More recently, it has become evident that impairment of GH-STAT5 signaling in different experimental models correlates with metabolic liver disease, ranging from hepatic steatosis to hepatocellular carcinoma (HCC). While GH-activated STAT5 has a protective role in chronic liver disease, experimental disruption of GC-GR signaling rather seems to ameliorate metabolic disorders under metabolic challenge. In this review, we focus on the current knowledge about hepatic CH-STAT5 and GC-GR signaling in body growth, metabolism, and protection from fatty liver disease and HCC development. (c) 2012 Elsevier Ireland Ltd. All rights reserved

Adipocyte STAT5 deficiency promotes adiposity and impairs lipid mobilisation in mice

Aims/hypothesis Dysfunction of lipid metabolism in white adipose tissue can substantially interfere with health and quality of life, for example in obesity and associated metabolic diseases. Therefore, it is important to characterise pathways that regulate lipid handling in adipocytes and determine how they affect metabolic homeostasis. Components of the Janus kinase (JAK)signal transducer and activator of transcription (STAT) pathway are involved in adipocyte physiology and pathophysiology. However, the exact physiological importance of the STAT family member STAT5 in white adipose tissue is yet to be determined. Here, we aimed to delineate adipocyte STAT5 functions in the context of lipid metabolism in white adipose tissue. Methods We generated an adipocyte specific knockout of Stat5 in mice using the Adipoq-Cre recombinase transgene followed by in vivo and in vitro biochemical and molecular studies. Results Adipocyte-specific deletion of Stat5 resulted in increased adiposity, while insulin resistance and gluconeogenic capacity was decreased, indicating that glucose metabolism can be improved by interfering with adipose STAT5 function. Basal lipolysis and fasting-induced lipid mobilisation were diminished upon STAT5 deficiency, which coincided with reduced levels of the rate-limiting lipase of triacylglycerol hydrolysis, adipose triglyceride lipase (ATGL, encoded by Pnpla2) and its coactivator comparative gene identification 58 (CGI-58). In a mechanistic analysis, we identified a functional STAT5 response element within the Pnpla2 promoter, indicating that Pnpla2 is transcriptionally regulated by STAT5. Conclusions/interpretation Our findings reveal an essential role for STAT5 in maintaining lipid homeostasis in white adipose tissue and provide a rationale for future studies into the potential of STAT5 manipulation to improve outcomes in metabolic diseases.(VLID)483223

Monogenic deficiency in murine intestinal Cdc42 leads to mucosal inflammation that induces crypt dysplasia

CDC42 controls intestinal epithelial (IEC) stem cell (IESC) division. How aberrant CDC42 initiates intestinal inflammation or neoplasia is unclear. We utilized models of inflammatory bowel diseases (IBD), colorectal cancer, aging, and IESC injury to determine the loss of intestinal Cdc42 upon inflammation and neoplasia. Intestinal specimens were collected to determine the levels of CDC42 in IBD or colorectal cancer. Cdc42 floxed mice were crossed with Villin-Cre, Villin-CreERT2 and/or Lgr5-eGFP-IRES-CreERT2, or Bmi1-CreERT2 mice to generate Cdc42 deficient mice. Irradiation, colitis, aging, and intestinal organoid were used to evaluate CDC42 upon mucosal inflammation, IESC/progenitor regenerative capacity, and IEC repair. Our studies revealed that increased CDC42 in colorectal cancer correlated with lower survival; in contrast, lower levels of CDC42 were found in the inflamed IBD colon. Colonic Cdc42 depletion significantly reduced Lgr5+ IESCs, increased progenitors' hyperplasia, and induced mucosal inflammation, which led to crypt dysplasia. Colonic Cdc42 depletion markedly enhanced irradiation- or chemical-induced colitis. Depletion or inhibition of Cdc42 reduced colonic Lgr5+ IESC regeneration. In conclusion, depletion of Cdc42 reduces the IESC regeneration and IEC repair, leading to prolonged mucosal inflammation. Constitutive monogenic loss of Cdc42 induces mucosal inflammation, which could result in intestinal neoplasia in the context of aging

Oncotarget / The ratio of STAT1 to STAT3 expression is a determinant of colorectal cancer growth

The role of STAT1 and STAT3 for colorectal carcinoma (CRC) development and progression is controversial. We evaluated 414 CRC patient samples on tissue microarrays for differential expression of STAT1 and STAT3 protein levels and correlated ratios with clinical parameters. Concomitant absence of nuclear STAT1 and STAT3 expression was associated with significantly reduced median survival by 33 months (p=0.003). To gain insight into underlying mechanisms, we generated four CRC cell lines with STAT3 knockdown. The cell lines harbor different known mutational drivers and were xenografted into SCID mice to analyze the influence of STAT3 on their tumor growth behavior. Experimental downregulation of STAT3 expression had differential, cell-line specific effects on STAT1 expression levels. STAT1 consistently showed nuclear localization irrespective of its tyrosine phosphorylation status. Two characteristic STAT1/3 expression patterns with opposite growth behavior could be distinguished: cell lines with a low STAT1/high STAT3 ratio showed faster tumor growth in xenografts. In contrast, xenografts of cell lines showing high STAT1 and low STAT3 levels grew slower. Importantly, these ratios reflected clinical outcome in CRC patients as well. We conclude that the ratio of STAT1 to STAT3 expression is a key determinant of CRC progression and that STAT1 counteracts pro-tumorigenic STAT3 signaling. Thus, we suggest that the STAT3/STAT1 ratios are better clinical predictors in CRC as compared to STAT3 or STAT1 levels alone.(VLID)240832

Oncotarget / The ratio of STAT1 to STAT3 expression is a determinant of colorectal cancer growth

The role of STAT1 and STAT3 for colorectal carcinoma (CRC) development and progression is controversial. We evaluated 414 CRC patient samples on tissue microarrays for differential expression of STAT1 and STAT3 protein levels and correlated ratios with clinical parameters. Concomitant absence of nuclear STAT1 and STAT3 expression was associated with significantly reduced median survival by 33 months (p=0.003). To gain insight into underlying mechanisms, we generated four CRC cell lines with STAT3 knockdown. The cell lines harbor different known mutational drivers and were xenografted into SCID mice to analyze the influence of STAT3 on their tumor growth behavior. Experimental downregulation of STAT3 expression had differential, cell-line specific effects on STAT1 expression levels. STAT1 consistently showed nuclear localization irrespective of its tyrosine phosphorylation status. Two characteristic STAT1/3 expression patterns with opposite growth behavior could be distinguished: cell lines with a low STAT1/high STAT3 ratio showed faster tumor growth in xenografts. In contrast, xenografts of cell lines showing high STAT1 and low STAT3 levels grew slower. Importantly, these ratios reflected clinical outcome in CRC patients as well. We conclude that the ratio of STAT1 to STAT3 expression is a key determinant of CRC progression and that STAT1 counteracts pro-tumorigenic STAT3 signaling. Thus, we suggest that the STAT3/STAT1 ratios are better clinical predictors in CRC as compared to STAT3 or STAT1 levels alone.(VLID)240832

Hepatic Deletion of Janus Kinase 2 Counteracts Oxidative Stress in Mice

Genetic deletion of the tyrosine kinase JAK2 or the downstream transcription factor STAT5 in liver impairs growth hormone (GH) signalling and thereby promotes fatty liver disease. Hepatic STAT5 deficiency accelerates liver tumourigenesis in presence of high GH levels. To determine whether the upstream kinase JAK2 exerts similar functions, we crossed mice harbouring a hepatocyte-specific deletion of JAK2 (JAK2Δhep) to GH transgenic mice (GHtg) and compared them to GHtgSTAT5Δhep mice. Similar to GHtgSTAT5Δhep mice, JAK2 deficiency resulted in severe steatosis in the GHtg background. However, in contrast to STAT5 deficiency, loss of JAK2 significantly delayed liver tumourigenesis. This was attributed to: (i) activation of STAT3 in STAT5-deficient mice, which was prevented by JAK2 deficiency and (ii) increased detoxification capacity of JAK2-deficient livers, which diminished oxidative damage as compared to GHtgSTAT5Δhep mice, despite equally severe steatosis and reactive oxygen species (ROS) production. The reduced oxidative damage in JAK2-deficient livers was linked to increased expression and activity of glutathione S-transferases (GSTs). Consistent with genetic deletion of Jak2, pharmacological inhibition and siRNA-mediated knockdown of Jak2 led to significant upregulation of Gst isoforms and to reduced hepatic oxidative DNA damage. Therefore, blocking JAK2 function increases detoxifying GSTs in hepatocytes and protects against oxidative liver damage