Breast cancer is the most commonly diagnosed cancer, and the second leading cause of cancer-related deaths, among women world-wide. Improved understanding of breast tumourigenesis may facilitate the development of more effective therapies. Peroxisome proliferator-activated receptor (PPAR)γ is a transcription factor that regulates the genes involved in insulin sensitivity and adipogenesis. In vitro and in vivo studies also suggest that PPARγ suppresses breast tumour progression; however, the mechanisms remain to be clarified. In the current study, I investigated the mammary epithelial cell-specific role of PPARγ in 7,12-dimethylbenz[a]anthracene (DMBA)-mediated breast tumourigenesis. Mammary epithelial cell-specific PPARγ knockout (PPARγ-MG KO) mice and their congenic, wild-type controls (PPARγ-WT) were treated with either DMBA alone or in combination with a PPARγ ligand (rosiglitazone)-supplemented diet, and followed for tumour formation. DMBA-mediated mammary tumour multiplicity decreased 4.5-fold among PPARγ-WT, but only 1.2-fold in PPARγ-MG KO mice upon co-treatment with rosiglitazone. Similarly, compared to respective DMBA alone groups, mammary tumour volumes were decreased, and onset was delayed, more among DMBA + Rosiglitazone treated PPARγ-WT versus PPARγ-MG KO mice. To assess whether DMBA could alter cell growth, in vitro studies using two human breast cancer cell lines were performed. Human MCF-7 and MDA-MB-231 cells were treated with DMBA, rosiglitazone or both, and assessed for changes in proliferation, apoptosis and target gene expression. DMBA exerted minimal effects on proliferation; whereas, treatments induced apoptosis in MCF-7, and necrosis in MDA-MB-231, cells. The expression of MCF-7 PPARγ1 protein increased with all treatments, while MDA-MB-231 PPARγ2 protein and BRCA1 mRNA expression increased following rosiglitazone or co-treatment. This work advances our understanding of the mammary epithelial cell-specific role of PPARγ signaling in DMBA-mediated breast tumourigenesis, and supports a role for PPARγ activation in the suppression of breast tumour progression. These findings may assist with the development of more effective anti-breast cancer agents. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2008-09-04 11:45:16.472

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The purpose of this research was to determine whether adipocytes express ACE2 and its role in obesity-associated hypertension with diet-induced obesity. To determine if ACE2 was expressed in adipose tissue and its regulation in the setting of diet-induced obesity, we fed male mice either a low fat (LF) or high fat (HF) diet acutely (1 week) or chronically ( 4 months). We demonstrated that ACE2 was regulated specifically in adipose tissue with consumption of a HF diet. However, with chronic HF feeding adipose ACE2 was dysregulated resulting in activation of the systemic RAS and increased blood pressure. To determine the role of ACE2 in obesity-associated hypertension, we used ACE2 deficient male and female mice. Wild type and ACE2 deficient mice were chronically fed either a LF or HF diet. Metabolic parameters were measured during the entire course of the study and blood pressure was measured by telemetry at the end of the study. Results from these studies demonstrate that HF diet promotes obesity-associated hypertension in male mice which is further augmented with ACE2 deficiency. Further, ACE2 deficiency resulted in marked glucose intolerance suggesting that stimulation of ACE2 may blunt the progression of obesity-associated diabetes. In contrast to the males, females are protected against obesity-associated hypertension. However, this protection in the females is lost with ovariectomy and ACE2 deficiency. These results suggest that female sex hormones protect the females against obesity-associated hypertension by regulating ACE2. To define mechanisms for HF diet-induced regulation of ACE2 in adipose tissue we examined various fatty acids for their ability to regulate ACE2 mRNA abundance in 3T3-L1 adipocytes. We revealed that omega-3 fatty acids, known regulators of PPARγ, increased ACE2 mRNA abundance in adipocytes. Therefore, we examined in vitro and in vivo regulation of ACE2 in 3T3-L1 cells and adipose tissue by PPARγ receptor ligands (TZDs). Results demonstrate regulatory effects of PPARγ to promote ACE2 gene transcription. These effects were associated with changes in glucose tolerance. Taken together, these results demonstrate that adipose ACE2 plays a protective role against obesity-associated hypertension in male and female mice and is regulated by natural and synthetic ligands of PPARγ.

Bisphenol A (BPA) is an estrogen receptor binding chemical, widely used in the plastics industry, and as such commonly encountered from plastic containers etc. Even at very low doses, BPA is believed to induce obesity and to have various endocrine disruptive effects. The purpose of this study was to determine possible gene expression changes in gonadal and inguinal adipose tissue from rats perinatally exposed to BPA. The method used was quantitative real-time PCR, and genes found to be up-regulated were PLZF, adiponectin, RXRa and Tcf21, while down-regulated genes were PPARγ, Tmem26, EsR1, Resistin, LPL, Chemerin, Serpina6, TFAM and Ahr. This is so far largely unsupported by other studies, and more research is needed.

Indiana University-Purdue University Indianapolis (IUPUI) / It has recently been shown that the gamma subtype of the peroxisome proliferator-activated receptor (PPARγ) is a target of ultraviolet B (290-320 nm; UVB) irradiation, and that PPARγ activation is necessary for full UVB-induced cyclooxygenase-2 (COX-2) induction. However, the biological significance of PPARγ activation in cutaneous photobiology is unknown. Acute UVB irradiation results in a characteristic series of events in the epidermis which includes: an initial edema response and subsequent inflammation, COX-2 induction, apoptosis, and a delayed hyperproliferative response. Therefore, the regulatory role of PPARγ activation was examined in this acute photoresponse using a topical application of the potent, irreversible PPARγ antagonist, GW9962. GW9662 was applied to the epidermis of SKH1 hairless albino mice at increasing doses (0.01-1.0mM) prior to UVB irradiation. The photobiological responses were examined through RT-PCR, skin thickness measurements, and immunohistochemistry, at 24 and 72 hours after UVB-irradiation. At the highest dose, GW9622 significantly inhibited UVB-induced inflammation, as measured by COX-2 induction at both 24 and 72 hrs. Inflammation assessed by skin thickness measurements indicated that lower doses mildly increased inflammation at 72 hrs, but suppressed inflammation at the highest dose. In contrast, GW9662 treatment dose dependently augmented UVB-induced apoptosis at 24 hours, while affecting the delayed hyperproliferative response at 72 hours in an inverse dose-response manner. The results from this study suggest that PPARγ is a key regulator of these photobiological responses. Because these responses are well known to be involved in tumor development and progression, this study also suggests a potential role for PPARγ in UVB-induced skin cancers.

Des cellules immunitaires appartenant à l’immunité de type 3 sont localisées dans muqueuse digestive. Les micro-organismes du microbiote stimulent le système immunitaire pour le maintenir en veille face à de potentiels agents infectieux, tout en ayant une tolérance pour les micro-organismes commensaux. Des études montrent des anomalies du microbiote intestinal chez les patients arthritiques. Ceci suggère une dérégulation de l’immunité mucosale digestive dans la survenue de l’inflammation articulaire. Des cellules de l’immunité mucosale pourraient migrer vers les sites articulaires, notamment grâce à des récepteurs aux chimiokines. Le facteur RORγt, indispensable à la différenciation des cellules de l’immunité de type 3, est régulé négativement par le récepteur PPARγ. A l’aide de modèle murins, nous avons étudié l’impact de l’invalidation de PPARγ et de l’inhibition du récepteur aux chimiokines CCR3 sur les relations entre sphères digestive et articulaire. Nous avons montré que l’induction d’une colite au cours d’une arthrite au collagène modifie le microbiote intestinal, retarde l’apparition et diminue la sévérité des lésions articulaires. Nous avons démontré que les souris PPARγ-/- développent spontanément une inflammation articulaire associées à des anomalies dans la répartition des cellules immunitaires de type 3 de la muqueuse digestive. Ces souris sont dysbiotiques avec une flore enrichie notamment en entérobactéries, mais non-arthritogène. Enfin, l’inhibition de CCR3 au cours du développement d’une arthrite au collagène retarde et diminue la sévérité de la pathologie et altère la répartition des leucocytes dans les articulations et de la muqueuse digestive / Numerous type 3 immune cells (Th17 and ILC3) are physiologically located in lamina propria of the intestine. Microbial agents within the gut shape the immune system to make it efficient against threats but peaceful with commensals. Recent studies demonstrated changes in gut microbiota composition (dysbiosis) in chronic inflammatory rheumatism. These results suggest a role for mucosal immunity alteration in articular inflammation occurrence. Indeed, some type 3 immune cells once activated by microbiota, are thought to migrate to joints, involving notably chemokines receptors. Transcription factor RORγt, the master regulator of type 3 immune cells, could be negatively regulated by nuclear receptor PPARγ. Using experimental murine models, we studied the consequence of PPARγ deficiency and consequence of the chemokine receptor CCR3 inhibition on the joint-gut axis. Firstly, we demonstrated that experimental colitis induces microbiota changes, delays and reduces collagen-induced arthritis severity. Secondly, we showed that PPARγ deficient mice display spontaneous joint inflammation associated with abnormal type 3 distribution within the gut. Dysbiosis with enrichment in facultative anaerobic Enterobacteriaceae was found in these mice. Fecal microbiota transfer demonstrated this microbiota is non-arthritogenic. Finally, we demonstrated that CCR3 inhibition has profound anti-arthritic potencies associated with changes in leukocytes distribution within the joint-gut axis

Mesenchymale Stammzellen können sich durch den Einfluss verschiedener Transkriptionsfaktor zu Osteoblasten, Adipozyten, Chondrozyten oder Myoblasten differenzieren. Während sie sich unter Runx2-Einfluss entlang der osteoblastären Linie differenzieren, entwickeln sie sich bei vorliegendem PPARγ entlang des adipogenen Differenzierungswegs. Das Gleichgewicht zwischen beiden Faktoren und ihr Zusammenspiel stellen einen wichtigen Bereich in der Osteoporoseforschung dar. In dieser Dissertation wurde durch Runx2-Suppression bzw. Runx2-Überexpression die Rolle dieses Faktors in pHOB und SCP1-Zellen erfasst und die Interaktion zwischen Runx2 und PPARγ untersucht. Der Runx2-Knockdown’ erfolgte mittels RNA-Interferenz, die Runx2-Überexpression durch ein Runx2 exprimierendes Plasmid. In RT-PCRs wurden mRNA-Messungen durchgeführt. Die Proteinbestimmung erfolgte im ‚Westernblot’. Der funktionelle Einfluss der Runx2-Überexpression auf die PPARγ-Transkription wurde durch Kotransfektion des an Luziferase gekoppelten PPARg-Promotorgens erfasst. Die funktionelle Aktivität des PPARg-Proteins wurde durch die Transfektion des an Luziferase gekoppelten PPRE-Gens gemessen. Promotoraktivität und Funktionalität der Proteine wurden in Luziferase-Reportergenassays erfasst. Unter basalen Kulturbedingungen differenzierten sich pHOB osteogen. Durch zweimalige siRunx2-Transfektion gelang auf mRNA-Ebene eine suffiziente Runx2-Suppression über 29 Tage auf durchschnittlich 10,1%. Neben einer Steigerung der PPARγ-mRNA nach sieben Tagen konnte darunter auch eine Suppression der osteogenen Differenzierungsmarker OC und AP beobachtet werden. Ein ‚Rescue’ der supprimierten Runx2-Genexpression konnte durch osteogene Stimulation nicht erreicht werden. In den Runx2-/PPARγ-Interaktionsversuchen wurden SCP1-Zellen adipogen stimuliert, um die PPARγ2-mRNA und PPARγ-Promotoraktivität zu erhöhen. Darunter konnte ebenfalls eine gesteigerte Funktionalität des PPARγ-Proteins beobachtet werden. Durch Runx2-Überexpression wurde in SCP1-Zellen die PPARγ-Promotoraktivität und somit der Beginn der mRNA-Synthese gehemmt. Die PPARγ2-mRNA hingegen blieb unbeeinflusst. Die zentrale Rolle des Runx2 in der osteogenen Differenzierung scheint durch den Einfluss auf die osteogenen Marker OC und AP in pHOB bestätigt zu werden. Auch der Einfluss auf die adipogene Differenzierung erfolgt über Runx2. Im Rahmen dieser Dissertation konnte erstmalig die Hemmung des PPARγ-Promotors durch Runx2 beschrieben werden. Hierdurch werden die PPARγ-Transkription und somit voraussichtlich die Interaktion zwischen Adipogenese und Osteogenese beeinflusst.

Chaque année, la grippe provoque des centaines de milliers de décès dans le monde. Dans le cas d’infections sévères, il a été démontré que la génération excessive de molécules inflammatoires telles que les cytokines et les chimiokines, la sécrétion d’espèces réactives dérivées de l'oxygène ainsi que l’afflux massif de cellules immunitaires innées et adaptatives dans les voies respiratoires contribuent à la génération de dommages pulmonaires aigus et contribuent à l'immunopathologie reliée à l’infection. Tenant compte de ce fait, le défi actuel dans le traitement de la grippe est de contrôler la réponse inflammatoire tout en inhibant la réplication virale afin de permettre à l'organisme de se défendre contre les infections sévères à l'influenza. Des études récentes ont montré que l’activation du récepteur nucléaire PPARγ par ses ligands, tel que la 15d-PGJ[indice inférieur 2], diminuait l’inflammation pulmonaire et améliorait la survie des souris infectées avec des doses létales du virus influenza. Mis à part ses effets sur PPARγ, le ligand 15d-PGJ[indice inférieur 2] est aussi connu pour activer le facteur nucléaire antioxydant Nrf2. Il a été montré que Nrf2 réduit la réplication du virus influenza. Cependant, son mode d'action dans cette fonction nécessite une clarification. De manière intéressante, une étude a montré que Nrf2 réduit l’inflammation pulmonaire en régulant l’expression de PPARγ et ceci dans un modèle murin du syndrome de détresse respiratoire aigu. Les résultats de ces études précédentes mènent à l’hypothèse que les voies de PPARγ et Nrf2 interagissent fonctionnellement et qu'elles sont impliquées dans la réduction de l’inflammation induite lors d'infections sévères causées par l'influenza. L’objectif général de cette étude est donc de mieux comprendre les mécanismes protecteurs de PPARγ et Nrf2 dans la régulation de l’inflammation et la réplication virale suite à une infection par le virus influenza. Nos résultats ont démontré premièrement que le fait de cibler les deux voies moléculaires PPARγ et Nrf2, permet une inhibition significative de l’inflammation et de la morbidité liée à l’infection. Dans un deuxième temps, nos résultats dévoilent le mécanisme antiviral de Nrf2 et démontrent que l’activation de cette voie réduit la réplication du virus influenza d’une façon dépendante de l’expression de l’antiprotéase SLPI.

Prostate Apoptosis Response-4 (Par-4) is a conserved and ubiquitous tumor-suppressor factor which can selectively induce apoptosis in tumor cells, while leaving normal cells unaffected. While Par-4 is well established as a tumor-suppressor, there have yet been no formal investigations as to whether it has a physiologic role in normal tissues. Early observations of Par-4 knockout mouse lines yielded that the adult mice displayed significant weight gain and fat accumulation compared to their wild-type counterparts while on a conventional chow diet. Interestingly, obese mouse and human subjects were found to exhibit reduced expression of Par-4 in adipose tissue as well as lower levels of secreted Par-4 in their plasma, compared to samples collected from lean human subjects. Subsequent in vitro experiments would show that loss of Par-4 has significant impact upon adipogenesis. Mechanistically, Par-4 loss during adipogenesis in cell culture correlated inversely with expression of the adipogenic transcription factor PPARγ. Subsequent experiments would demonstrate that Par-4 transcriptionally represses PPARγ at the promoter level. Thereby, we conclude that Par-4 regulates adipogenesis and lipid accumulation through transcriptional repression of the PPARγ promoter. This research utilizes novel models and may be used as the basis for Par-4-mediated therapies for obesity and metabolic disease.

PKC βII is alternatively spliced during acute insulin stimulation in L6 skeletal muscle cells. This PKC βII isoform is critical in propagating GLUT4 translocation. PKC β protein and promoter dysfunction correlate with human insulin resistance. TZD treatment ameliorates whole-body insulin-resistance. Its primary target is adipocyte PPAR γ, which it activates upon binding. This causes both altered circulating serum FFA concentrations and adipokine secretion profile. How TZDs affect the intracellular signaling of skeletal muscle cells is unknown. RT-PCR and Western blot analysis showed that TZDs elevated PKC βII by a process that involves co-transcriptional splicing. PGC1 α overexpression most closely resembled TZD treatment by increasing PKCβII protein levels and keeping PKC βI levels relatively constant. Use of a heterologous PKCβ promoter driven PKC β minigene demonstrated that PPARγ could regulate the PKCβ promoter, but whether this is direct or indirect is unclear. SRp40 splicing factor has been shown to dock onto the PGC1 α CTD and influence splicing. SRp40, through overexpression and silencing, appears to play a part in PKC β promoter regulation. PKC β promoter regulation was also studied in 3T3-L1 cells. TZDs were experimentally shown to have no role in PKC β promoter regulation despite PPARγ activation. Chromatin immunoprecipitation assays revealed PU.1 as a putative PKC β transcription factor that can cross-talk with the spliceosome, possibly through SRp40 which was also associated with the PKC β promoter. 3T3-L1 adipocyte differentiation revealed a novel developmentally-regulated switch from PKC βI to PKCβ II, using western blot and Real-Time PCR analysis. Pharmacological inhibition of PKC β II using CGP53353 and LY379196 blocked [ 3 H]2-deoxyglucose uptake and revealed a functional role for PKC β II in adipocyte ISGT. CGP53353 specifically inhibited phosphorylation of PKC β II Serine 660 and not other critical upstream components of the insulin signaling pathway. Subcellular fractionation and PM sheet assay pointed to PKC β II-mediated regulation of GLUT4 translocation to the PM. Co-immunoprecipitation between PKC β II and GLUT4 allude to possible direct interaction. Western blot and immunofluorescence assays show PKC β II activity is linked with Akt Serine 473 phosphorylation, thus full Akt activity. Western blot and co-immunoprecipitation suggested that insulin caused active mTORC2 to directly activate PKC βII. Data support a model whereby PKCβ II is downstream of mTORC2 yet upstream of Akt, thereby regulating GLUT4 translocation.