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dc.contributor.supervisor Dixon, Ian (Physiology and Pathophysiology) Jassal, Davinder (Physiology and Pathophysiology) en_US
dc.contributor.author Zeglinski, Matthew
dc.date.accessioned 2016-09-09T20:33:13Z
dc.date.available 2016-09-09T20:33:13Z
dc.date.issued 2016 en_US
dc.identifier.uri http://hdl.handle.net/1993/31668
dc.description.abstract Transforming growth factor-β1 (TGFβ1) is a mediator of the fibrotic response through activation of quiescent cardiac fibroblasts to hypersynthetic myofibroblasts. Scleraxis (Scx) is a pro-fibrotic transcription factor that is induced by TGFβ1-3 and works synergistically with Smads to promote collagen expression. Ski is a negative regulator of TGFβ/Smad signaling through its interactions with Smad proteins at the promoter region of TGFβ regulated genes. To date, no studies have examined the direct DNA:protein transcriptional mechanisms that regulate Scx expression by TGFβ1-3 or Ski, nor the mechanisms that govern Ski expression by Scx. We hypothesize that Ski and Scx regulate one another, and form a negative feedback loop that represses gene expression and is a central regulator of the fibrotic response in cardiac myofibroblasts. Primary adult rat cardiac myofibroblasts were isolated via retrograde Langendorff perfusion. First passage (P1) cells were infected with adenovirus encoding HA-Ski, HA-Scx, or LacZ at the time of plating. Twenty-four hours later, cells were harvested for Western blot, quantitative real-time PCR (qPCR), and electrophoretic gel shift assays (EMSA). NIH-3T3 or Cos7 cells were transfected with equal quantities of plasmid DNA for 24 hours prior to harvesting for luciferase, qPCR, and EMSA analysis. Ski overexpression in P1 myofibroblasts resulted in a reduction in both Scx mRNA and protein levels. Overexpression of Scx had no effect on Ski expression. Luciferase reporter assays demonstrated that Scx was induced by TGFβ1 treatment in a concentration dependent manner. However, ectopic Smad2/3 expression was unable to transactivate the Scx promoter in a luciferase reporter assay. Inhibition of p44/42-MAPK signaling modestly counteracted the effect of TGFβ1 on Scx expression. Scx had no effect on Ski promoter expression, however, both tumor necrosis factor-α (TNFα) and p65 expression repressed the Ski promoter and correlated with reduced Ski mRNA levels. We conclude that Ski is a repressor of Scx and that Scx expression is partially mediated through a Smad-independent, p44/42-MAPK pathway in cardiac myofibroblasts. Furthermore, this study proposes a role for TNFα/p65 NF-κΒ signaling in the regulation of Ski gene expression in the cardiac myofibroblast. en_US
dc.subject Ski en_US
dc.subject Scleraxis en_US
dc.subject Myofibroblasts en_US
dc.subject Cardiac Fibrosis en_US
dc.subject p65 en_US
dc.subject Smad en_US
dc.subject MAPK en_US
dc.subject Erk1/2 en_US
dc.subject TGFβ1 en_US
dc.subject TNFα en_US
dc.title Transcriptional regulation of ski and scleraxis in primary cardiac myofibroblasts en_US
dc.degree.discipline Physiology and Pathophysiology en_US
dc.contributor.examiningcommittee Czubryt, Michael (Physiology and Pathophysiology) Kirshenbaum, Lorrie (Physiology and Pathophysiology) Wigle, Jeffrey (Biochemistry and Medical Genetics) Allen, Bruce (University of Montreal) en_US
dc.degree.level Doctor of Philosophy (Ph.D.) en_US
dc.description.note October 2016 en_US


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