Browsing by Author "Kim, Seung Joon"

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    Differential Activation and Inhibition of RhoA by Fluid Flow Induced Shear Stress in Chondrocytes
    (Wiley, 2013) Wan, Qiaoqiao; Kim, Seung Joon; Yokota, Hiroki; Na, Sungsoo; Biomedical Engineering, Purdue School of Engineering and Technology
    Physical force environment is a major factor that influences cellular homeostasis and remodelling. It is not well understood, however, as a potential role of force intensities in the induction of cellular mechanotransduction. Using a fluorescence resonance energy transfer-based approach, we asked whether activities of GTPase RhoA in chondrocytes are dependent on intensities of flow-induced shear stress. We hypothesized that RhoA activities can be either elevated or reduced by selecting different levels of shear-stress intensities. The result indicates that C28/I2 chondrocytes have increased RhoA activities in response to high shear stress (10 or 20 dyn/cm(2) ), whereas a decrease in activity was seen with an intermediate shear stress of 5 dyn/cm(2) . No changes were seen under low shear stress (2 dyn/cm(2) ). The observed two-level switch of RhoA activities is closely linked to the shear-stress-induced alterations in actin cytoskeleton and traction forces. In the presence of constitutively active RhoA (RhoA-V14), intermediate shear stress suppressed RhoA activities, while high shear stress failed to activate them. In chondrocytes, expression of various metalloproteinases is, in part, regulated by shear and normal stresses through a network of GTPases. Collectively, the data suggest that intensities of shear stress are critical in differential activation and inhibition of RhoA activities in chondrocytes.
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    Effects of Collagen Gel Stiffness on Cdc42 Activities of Endothelial Colony Forming Cells during Early Vacuole Formation
    (2013-08-14) Kim, Seung Joon; Na, Sungsoo; Xie, Dong; Li, Jiliang
    Recent preclinical reports have provided evidence that endothelial colony forming cells (ECFCs), a subset of endothelial progenitor cells, significantly improve vessel formation, largely due to their robust vasculogenic potential. While it has been known that the Rho family GTPase Cdc42 is involved in this ECFC-driven vessel formation process, the effect of extracellular matrix (ECM) stiffness on its activity during vessel formation is largely unknown. Using a fluorescence resonance energy transfer (FRET)-based Cdc42 biosensor, we examined the spatio-temporal activity of Cdc42 of ECFCs in three-dimensional (3D) collagen matrices with varying stiffness. The result revealed that ECFCs exhibited an increase in Cdc42 activity in a soft (150 Pa) matrix, while they were much less responsive in a rigid (1 kPa) matrix. In both soft and rigid matrices, Cdc42 was highly activated near vacuoles. However, its activity is higher in a soft matrix than that in a rigid matrix. The observed Cdc42 activity was closely associated with vacuole formation. Soft matrices induced higher Cdc42 activity and faster vacuole formation than rigid matrices. However, vacuole area is not dependent on the stiffness of matrices. Time courses of Cdc42 activity and vacuole formation data revealed that Cdc42 activity proceeds vacuole formation. Collectively, these results suggest that matrix stiffness is critical in regulating Cdc42 activity in ECFCs and its activation is an important step in early vacuole formation.
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    Matrix rigidity regulates spatiotemporal dynamics of Cdc42 activity and vacuole formation kinetics of endothelial colony forming cells
    (Elsevier B.V., 2014-01-24) Kim, Seung Joon; Wan, Qiaoqiao; Cho, Eunhye; Han, Bumsoo; Yoder, Mervin C.; Voytik-Harbin, Sherry L.; Na, Sungsoo; Department of Biomedical Engineering, School of Engineering and Technology
    Recent evidence has shown that endothelial colony forming cells (ECFCs) may serve as a cell therapy for improving blood vessel formation in subjects with vascular injury, largely due to their robust vasculogenic potential. The Rho family GTPase Cdc42 is known to play a primary role in this vasculogenesis process, but little is known about how extracellular matrix (ECM) rigidity affects Cdc42 activity during the process. In this study, we addressed two questions: Does matrix rigidity affect Cdc42 activity in ECFC undergoing early vacuole formation? How is the spatiotemporal activation of Cdc42 related to ECFC vacuole formation? A fluorescence resonance energy transfer (FRET)-based Cdc42 biosensor was used to examine the effects of the rigidity of three-dimensional (3D) collagen matrices on spatiotemporal activity of Cdc42 in ECFCs. Collagen matrix stiffness was modulated by varying the collagen concentration and therefore fibril density. The results showed that soft (150 Pa) matrices induced an increased level of Cdc42 activity compared to stiff (1 kPa) matrices. Time-course imaging and colocalization analysis of Cdc42 activity and vacuole formation revealed that Cdc42 activity was colocalized to the periphery of cytoplasmic vacuoles. Moreover, soft matrices generated faster and larger vacuoles than stiff matrices. The matrix-driven vacuole formation was enhanced by a constitutively active Cdc42 mutant, but significantly inhibited by a dominant-negative Cdc42 mutant. Collectively, the results suggest that matrix rigidity is a strong regulator of Cdc42 activity and vacuole formation kinetics, and that enhanced activity of Cdc42 is an important step in early vacuole formation in ECFCs.
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    Polyphenon E Effects on Gene Expression in PC-3 Prostate Cancer Cells
    (MDPI, 2022-11-18) Carastro, L. Michael; Vallebuona, Ethan J.; Cordova, Ricardo; Gannon, Ashely N.; Kim, Seung Joon; Costello, Corrine M.; Declet-Bauzo, Ricardo A.; Kumar, Nagi; Park, Jong Y.; Biochemistry and Molecular Biology, School of Medicine
    Polyphenon E (Poly E) is a standardized, caffeine-free green tea extract with defined polyphenol content. Poly E is reported to confer chemoprotective activity against prostate cancer (PCa) progression in the TRAMP model of human PCa, and has shown limited activity against human PCa in human trials. The molecular mechanisms of the observed Poly E chemopreventive activity against PCa are not fully understood. We hypothesized that Poly E treatment of PCa cells induces gene expression changes, which could underpin the molecular mechanisms of the limited Poly E chemoprevention activity against PCa. PC-3 cells were cultured in complete growth media supplemented with varied Poly E concentrations for 24 h, then RNA was isolated for comparative DNA microarray (0 vs. 200 mg/L Poly E) and subsequent TaqMan qRT-PCR analyses. Microarray data for 54,613 genes were filtered for >2-fold expression level changes, with 8319 genes increased and 6176 genes decreased. Eight genes involved in key signaling or regulatory pathways were selected for qRT-PCR. Two genes increased expression significantly, MXD1 (13.98-fold; p = 0.0003) and RGS4 (21.98-fold; p = 0.0011), by qRT-PCR. MXD1 and RGS4 significantly increased gene expression in Poly E-treated PC-3 cells, and the MXD1 gene expression increases were Poly E dose-dependent.
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    STIFFNESS OF 3D COLLAGEN MATRICES REGULATES CDC42 ACTIVITY OF ENDOTHELIAL COLONY FORMING CELLS DURING EARLY VACUOLE
    (Office of the Vice Chancellor for Research, 2012-04-13) Kim, Seung Joon; Voytik-Harbin, Sherry; Yoder, Mervin; Na, Sungsoo
    Recent preclinical reports have provided evidence that endothelial colony forming cells (ECFCs), a subset of endothelial progenitor cells, significantly improve vessel formation, largely due to their robust vasculogenic potential. While it has been known that the Rho family GTPase Cdc42 is involved in this ECFC-driven vessel formation process, the effect of extracellular matrix (ECM) stiffness on its activity during vessel formation is largely unknown. Using a fluorescence resonance energy transfer (FRET)-based Cdc42 biosen-sor, we examined the spatio-temporal activity of Cdc42 of ECFCs in three-dimensional (3D) collagen matrices with varying stiffness. The result re-vealed that ECFCs exhibited an increase in Cdc42 activity in a soft (150 Pa) matrix, while they were much less responsive in a stiff (1000 Pa) matrix. In both soft and stiff matrices, Cdc42 was highly activated near vacuoles; how-ever, its activity is higher in a soft matrix than that in a stiff matrix. The ob-served Cdc42 activity was closely associated with vacuole area. Soft matri-ces induced higher Cdc42 activity, faster vacuole formation, and larger vac-uole area than stiff matrices. Time courses of Cdc42 activity and vacuole formation data revealed that Cdc42 activity proceeds vacuole formation. Collectively, these results suggest that matrix stiffness is critical in regulat-ing Cdc42 activity in ECFCs and its activation is an important step in early vacuole formation.