Characterization of the Role of PKN in TGF-Beta 1-Mediated Cell Cycle Regulation of Vascular Smooth Muscle Cells

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dc.contributor.advisorNeeraj Agarwal
dc.contributor.committeeMemberGlenn Dillon
dc.contributor.committeeMemberRobert Mallet
dc.creatorSu, Chang
dc.date.accessioned2019-08-22T19:32:14Z
dc.date.available2019-08-22T19:32:14Z
dc.date.issued2005-12-01
dc.date.submitted2013-11-05T06:54:04-08:00
dc.description.abstractChang Su, Characterization of the role of PKN in TGF-beta-1 induced cell cycle inhibition in vascular smooth muscle cells. Doctor of Philosophy (Biomedical Sciences), November 2005, 173 pp, 2 tables, 34 illustrations, 225 references. Mature vascular smooth muscle cells (VSMCs) are unique in that they can switch between proliferative and differentiated phenotypes. Aberrant proliferation of VSMC is regarded as a central feature in vascular diseases such as atherosclerosis and restenosis following balloon angioplasty. Transforming growth factor-β1 (TGF-β1) is known to inhibit smooth muscle cell progression; however, the signaling pathway(s) through which this is accomplished is poorly understood. Entry into mitosis in dividing VSMCs is triggered by Cdc2/cyclin B1 complex, which is tightly controlled by phosphatase Cdc25C that dephosphorylates tyrosine-15 and threonine-14 on Cdc2 at onset of mitosis. A serine/threonine protein kinase, PKN, was recently reported to inhibit Cdc25C activity. PKN has been identified as a downstream target for TGF-β1 signaling in VSMCs. Therefore we hypothesize that PKN mediates TGF-β1-delayed cell cycle progression by inhibiting Cdc25C. In this study, TGF-β1 is shown to delay G2/M phase progression timing in PAC-1 VSMCs. This effect is blocked by pretreatment of cells with either HA1077 of Y-27632, two pharmacological inhibitors of PKN, as well as by reduced expression of PKN by RNA interference (RNAi). Oscillation of PKN activity temporally correlates with G2/M phase progression. Co-immunoprecipitation suggests that Cdc25C and PKN physically associate with each other. Immunocytochemistry demonstrate that PKN and Cdc25C co-localize in the nuclei and peri-nuclear region of only dividing (M phase) cells but not in the interphase cells. Additionally, PKN phosphorylates Cdc25C in PAC-1 cell cultures. Finally, TGF-β1-induced delay of Cdc2 activation is abolished by pretreating the cells with Y-27632. These data suggest that PKN inhibits G2/M progression by directly binding to Cdc25C and inhibiting its activity by phosphorylation. In addition to the PKN-Cdc25C signaling pathway, TGF-β1 strongly induces the transcriptional activity of the Smad-dependent enhancer in PAC-1 cells. This effect is attenuated by blocking PKN function by either chemical inhibitors or RNAi. Active forms of MKK3/6 alone are sufficient to increase the Smad enhancer activity, and co-expression of dominant negative MKK3/6 decreases TGF-β1-induced activation of the Smad enhancer. Lastly, the Smad reporter activity induced by TGF-β1 is also significantly attenuated by SB203580, a highly specific pharmacological inhibitor for p38 MAPK. These data demonstrate a novel mechanism of PKN-MKK3/6-p38 MAPK cascade to cross talk with the Smad pathway in PAC-1 VSMCs. Taken together, findings presented in this dissertation identify components of important intracellular signaling pathways through which TGF-β1 activates PKN to inhibit proliferation and promote differentiation of SMCs. Augmenting PKN-Cdc25C-Cdc2 signaling may provide a potential therapeutic approach to counter abnormal VSMC proliferation, prevent the clinical consequences of atherosclerosis and improve outcomes after angioplasty.
dc.format.mimetypeapplication/pdf
dc.identifier.urihttps://hdl.handle.net/20.500.12503/26114
dc.language.isoen
dc.provenance.legacyDownloads2
dc.subjectCardiology
dc.subjectCell Anatomy
dc.subjectCell and Developmental Biology
dc.subjectCell Biology
dc.subjectCells
dc.subjectLife Sciences
dc.subjectMedical Cell Biology
dc.subjectMedical Microbiology
dc.subjectMedicine and Health Sciences
dc.subjectOther Cell and Developmental Biology
dc.subjectPKN
dc.subjectTGF-beta-1
dc.subjectcell cycle inhibition
dc.subjectmature vascular smooth muscle cells
dc.subjectTGF-β1
dc.subjectimmunocytochemistry
dc.subjectCdc25C
dc.subjectRNAi
dc.subjectcell
dc.subjectintracellular signaling pathways
dc.subjectVSMC proliferation
dc.subjectatherosclerosis
dc.subjectangioplasty
dc.titleCharacterization of the Role of PKN in TGF-Beta 1-Mediated Cell Cycle Regulation of Vascular Smooth Muscle Cells
dc.typeDissertation
dc.type.materialtext
thesis.degree.departmentGraduate School of Biomedical Sciences
thesis.degree.disciplineBiomedical Sciences
thesis.degree.grantorUniversity of North Texas Health Science Center at Fort Worth
thesis.degree.nameDoctor of Philosophy

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