Browsing by Subject "intracellular signaling pathways"
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Item Characterization of the Role of PKN in TGF-Beta 1-Mediated Cell Cycle Regulation of Vascular Smooth Muscle Cells(2005-12-01) Su, Chang; Neeraj Agarwal; Glenn Dillon; Robert MalletChang 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.Item Molecular Regulation of Cardiac Stem Cell Growth and Differentiation by Extrinsic Factors and Novel Intracellular Signaling Pathways(2008-05-01) Bartosh, T.J.; Rouel S. Roque; Harold Sheedlo; Robert WordingerT. J. Bartosh, Molecular regulation of cardiac stem cell growth and differentiation by extrinsic factors and novel intracellular signaling pathways. Doctors of Philosophy (Biomedical Sciences), May 2008, 293 pp., 4 tables, 59 illustrations, bibliography, 353 titles. Insufficient myocardial regeneration following ischemic injury provokes cardiac dysfunction, adverse tissue remodeling, and ultimately heart failure. Stem cell replacement therapy appears to be a promising strategy for improving cardiac function, however, challenges involving inadequate stem cell differentiation, engraftment, and survival following transplantation currently impede the efficacy of regeneration protocols. These limitations emphasize the significance of identifying extrinsic factors and corresponding molecular mechanisms regulating cardiac cell differentiation. Recent reports recognizing a stem cell component in the adult heart (i.e. cardiac stem cells, CSC) have provided additional targets and/or tools for myocardial repair. This investigation verified the presence of CSC in the adult dog heart, described methods to generate three dimensional (3D) cardiac microtissues (‘cardiospheres’) from CSC, and revealed features of cardiospheres potentially useful for identifying extrinsic procardiogenic factors, evaluating myocardial response to stress, and delivering functional CSC into the damaged heart. Specifically, cardiosphere formation was facilitated by culturing CSC in growth medium on a poly-L-ornithine substratum. Cardiospheres are comprised of interior cells that exhibited characteristics of CSC; differentiating cardiomyocytes at the periphery with organized contractile machinery; and/or vascular cells capable of forming vessel-like networks. Upon co-culture with neonatal cardiomycocytes, spheres developed foci of contracting regions. Furthermore, cardiospheres exhibited increased resistance to oxidative stress and survived subcutaneous injections without undergoing neoplastic transformation further supporting their ability to effectively promote myocardial regeneration. Retionic acid (RA), the active form of vitamin A, augmented expression of myocyte-specific proteins in cardiospheres, thus, RA may improve the success of cardiac regenerative therapies and provide an appropriate stimulus to model underlying mechanisms fundamental for CSC differentiation. The tendency for cultured stem cells to undergo various levels of multi-lineage commitment is, however, cumbersome for deciphering precise cues that direct cell fate decisions. In this study, the molecular pathways important for RA-induced cardiac differentiation were examined using h9c2 cardioblasts as a stable cell model. In h9c2 cells, RA treatment promoted transcriptional enhancement of the muscle-enriched gene regulatory protein MEF2C, morphological alterations indicative of differentiation, and a robust increase in expression of myocyte differentiation genes including cardiac myosin heavy chain (cMHC) and ventricular myosin light chain-2 (vMLC2). These changes were preceded by rapid events involving elevation of intracellular Ca^2+ and phosphorylation of p38 MAPK. The effects of RA were attenuated using CA^2+ buffering agents or chemical inhibitors of L-type Ca^2+ channels (LTCC) and the phosphatase calcinuerin, but not by RA receptor antagonists. Furthermore, overexpression of dominant negative (dn) MEF2C, dnp38 MAPK, or CAIN, a physiological calcineruin inhibitor, abrogated MEF2 activity and RA-induced differentiation. These results imply that RA promotes cardiomyocyte differentiation, independent of RA response element activation, via induction of CA^2+ -regulated signaling pathways by activating the LTCC-calcineurin/38MAPK-MEF2 axis. Taken together, these findings enhance our understanding of extrinsic factors and molecular mechanisms indispensable for myocyte differentiation and subsequently provide novel therapeutic targets and cellular tools for regeneration and repair of damaged myocardium.