Project description:Toll like receptor 4 (TLR4), an innate immunity gene, is involved in responses to several pulmonary agonists including endotoxin (LPS; Poltorak et al.,1998), ozone (O3 ,Kleeberger et. al., 2001), Pseudomonas aeruginosa (Faure et al, 2004), and hyperoxia (Zhang et al, 2005). TLR4 appears to partially mediate the response to LPS- and O3-induced lung injury, however, TLR4 is protective for prevention of injury in Pseudomonas aeruginosa infection and against acute lung injury (hyperoxia). The mechanism behind this protection is unclear. We previously demonstrated that TLR4 was also protective against BHT-induced chronic inflammation and tumor promotion (Bauer et al, 2005). C.C3H-Tlr4Lps-d (BALBLps-d) mice, congenic for a 10 cM region of C3H/HeJ chromosome 4 that contains Tlr4 (Vogel et al, 1994), have a missence mutation that renders TLR4 dysfunctional. The Tlr4 mutation likely abrogates signaling by disrupting a direct point of contact with other signaling molecules (Akira S, Takeda K. Toll-like receptor signalling. Nat Rev Immunol 2004;4(7):499-511.). Bronchoalveolar lavage fluid (BALF) alveolar macrophages, lymphocytes, and total protein content were significantly elevated in BALBLps-d mice compared to BALB/c (BALB; Tlr4 sufficient) mice following chronic BHT (Bauer et al., 2005). BALBLps-d mice also had a significant increase in tumor multiplicity (60%) over that of BALB mice in response to an MCA/BHT tumor promotion protocol. While this was the first model to demonstrate a protective role for TLR4 in chronic lung inflammation and tumorigenesis, the downstream mechanism regulating this protective response remains unknown. Using Affymetrix microarray analysis followed by GeneSpring and Ingenuity pathway analyses, we herein identified known and novel downstream pathways and their interactions that may be involved in the protective mechanism elicited by TLR4. We therefore hypothesize that these pathways and interactions amongst the genes identified during the tumor promotion/chronic inflammation stage are in part influencing the differential strain response observed during tumorigenesis. Keywords: time course, tumor study Protocol 1 - 3 biological replicates after chronic dosing in each mouse strain Protocol 2 - multiple replicates after MCA/BHT tumor progression model used

Project description:Toll like receptor 4 (TLR4), an innate immunity gene, is involved in responses to several pulmonary agonists including endotoxin (LPS; Poltorak et al.,1998), ozone (O3 ,Kleeberger et. al., 2001), Pseudomonas aeruginosa (Faure et al, 2004), and hyperoxia (Zhang et al, 2005). TLR4 appears to partially mediate the response to LPS- and O3-induced lung injury, however, TLR4 is protective for prevention of injury in Pseudomonas aeruginosa infection and against acute lung injury (hyperoxia). The mechanism behind this protection is unclear. We previously demonstrated that TLR4 was also protective against BHT-induced chronic inflammation and tumor promotion (Bauer et al, 2005). C.C3H-Tlr4Lps-d (BALBLps-d) mice, congenic for a 10 cM region of C3H/HeJ chromosome 4 that contains Tlr4 (Vogel et al, 1994), have a missence mutation that renders TLR4 dysfunctional. The Tlr4 mutation likely abrogates signaling by disrupting a direct point of contact with other signaling molecules (Akira S, Takeda K. Toll-like receptor signalling. Nat Rev Immunol 2004;4(7):499-511.). Bronchoalveolar lavage fluid (BALF) alveolar macrophages, lymphocytes, and total protein content were significantly elevated in BALBLps-d mice compared to BALB/c (BALB; Tlr4 sufficient) mice following chronic BHT (Bauer et al., 2005). BALBLps-d mice also had a significant increase in tumor multiplicity (60%) over that of BALB mice in response to an MCA/BHT tumor promotion protocol. While this was the first model to demonstrate a protective role for TLR4 in chronic lung inflammation and tumorigenesis, the downstream mechanism regulating this protective response remains unknown. Using Affymetrix microarray analysis followed by GeneSpring and Ingenuity pathway analyses, we herein identified known and novel downstream pathways and their interactions that may be involved in the protective mechanism elicited by TLR4. We therefore hypothesize that these pathways and interactions amongst the genes identified during the tumor promotion/chronic inflammation stage are in part influencing the differential strain response observed during tumorigenesis. Keywords: time course, tumor study

Project description:Constitutive androstane receptor (CAR) agonists, such as TCPOBOP, are known to cause robust hepatocyte proliferation and hepatomegaly in mice along with induction of drug metabolism genes, without any associated liver injury. Yes-associated protein (YAP) is a key transcription regulator that tightly controls organ size including that of liver. Ours and other previous studies suggested increased nuclear localization and activation of YAP after TCPOBOP treatment in mice and potential role of YAP in CAR-driven proliferative response. Here, we investigated a direct role of YAP in CAR-driven hepatomegaly and hepatocyte proliferation using hepatocyte-specific YAP-KO mice. AAV8-TBG-CRE vector was injected to YAP-floxed mice for achieving hepatocyte-specific YAP deletion followed by TCPOBOP treatment. YAP deletion did not alter protein expression of CAR or CAR-driven induction of drug metabolism genes (including Cyp2b10, Cyp2c55 and UGT1a1). However, YAP deletion substantially reduced TCPOBOP-induced hepatocyte proliferation. TCPOBOP-driven cell cycle activation was disrupted in YAP-KO mice due to delayed (and decreased) induction of cyclin D1 and higher expression of p21, resulting in decreased phosphorylation of retinoblastoma (Rb) protein. Further, induction of other cyclins, which are sequentially involved in progression through cell cycle (including cyclin E1, A2 and B1) and important mitotic regulators (such as aurora B kinase and polo-like kinase 1) was remarkably reduced in YAP-KO mice. Microarray analysis revealed that 26% of TCPOBOP‐responsive genes mainly related to proliferation, but not to drug metabolism, were altered by YAP deletion. YAP regulated these proliferation genes via alerting expression of cMyc and FOXM1, two critical transcriptional regulators of CAR-mediated hepatocyte proliferation. Conclusion: Our study revealed an important role of YAP signaling in CAR-driven hepatocyte proliferation; however, CAR-driven induction of drug metabolism genes was independent of YAP. We used microarrays to detail the global programme of gene expression in livers of hepatocyte-specific YAP KO mice following TCPOBOP treatment

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Constitutive androstane receptor in macrophages regulates Toll-like receptor 4-mediated innate immune responses during endotoxemic liver injury

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