Eung-Gook Kim

Runx2 is a common target of transforming growth factor beta1 and bone morphogenetic protein 2, and cooperation between Runx2 and Smad5 induces osteoblast-specific gene expression in the pluripotent mesenchymal precursor cell line C2C12.

ABSTRACT When C2C12 pluripotent mesenchymal precursor cells are treated with transforming growth factor β1 (TGF-β1), terminal differentiation into myotubes is blocked. Treatment with bone morphogenetic protein 2 (BMP-2) not only blocks myogenic differentiation of C2C12 cells but also induces osteoblast differentiation. The molecular mechanisms governing the ability of TGF-β1 and BMP-2 to both induce ligand-specific responses and inhibit myogenic differentiation are not known. We identified Runx2/PEBP2αA/Cbfa1, a global regulator of osteogenesis, as a major TGF-β1-responsive element binding protein induced by TGF-β1 and BMP-2 in C2C12 cells. Consistent with the observation that Runx2 can be induced by either TGF-β1 or BMP-2, the exogenous expression of Runx2 mediated some of the effects of TGF-β1 and BMP-2 but not osteoblast-specific gene expression. Runx2 mimicked common effects of TGF-β1 and BMP-2 by inducing expression of matrix gene products (for example, collagen and fibronectin), suppressing MyoD expression, and inhibiting myotube formation of C2C12 cells. For osteoblast differentiation, an additional effector, BMP-specific Smad protein, was required. Our results indicate that Runx2 is a major target gene shared by TGF-β and BMP signaling pathways and that the coordinated action of Runx2 and BMP-activated Smads leads to the induction of osteoblast-specific gene expression in C2C12 cells.

Involvement of Tyrosine Phosphorylation and Protein Kinase C in the Activation of Phospholipase D by H2O2 in Swiss 3T3 Fibroblasts

We have investigated the mechanisms involved in H2O2-mediated phospholipase D (PLD) activation in Swiss 3T3 fibroblasts. In the presence of vanadate, H2O2 induced tyrosine phosphorylation of PLD as well as the platelet-derived growth (PDGF) factor receptor, protein kinase Cα (PKCα), and a 62-kDa protein in rat brain PLD1 (rPLD1) immune complexes. PDGF also induced tyrosine phosphorylation of PLD, but this was abolished by catalase, indicating that it was mediated by H2O2 generation. Interestingly, PLD was found to be constitutively associated with the PDGF receptor and PKCα. Stimulation by H2O2 showed a concentration- and time-dependent tyrosine phosphorylation of the proteins in rPLD1 immunoprecipitates and activation of PLD in the cells. Pretreatment of the cells with the protein-tyrosine kinase inhibitors genistein and herbimycin A resulted in a concentration-dependent inhibition of H2O2-induced tyrosine phosphorylation and PLD activation. Activation of PLD by H2O2 was also inhibited dose-dependently by the PKC inhibitors Ro 31-8220 and calphostin C. Down-regulation of PKC by prolonged treatment with 4β-phorbol 12-myristate 13-acetate also abolished H2O2-stimulated PLD activity. H2O2 or vanadate alone did not induce tyrosine phosphorylation of proteins in the rPLD1 immune complex or PLD activation. Reduction of intracellular H2O2levels by pretreatment of the cells with catalase dramatically abrogated tyrosine phosphorylation of proteins in the rPLD1 immune complex and PLD activation, suggesting the potential role of intracellular H2O2 in H2O2-mediated PLD signaling. Taken together, these results suggest that both protein-tyrosine kinase(s) and protein kinase C participate in H2O2-induced PLD activation in Swiss 3T3 cells.

Increased caveolin-1, a cause for the declined adipogenic potential of senescent human mesenchymal stem cells

Mesenchymal stem cell (MSC) has drawn much attention in the aspect of tissue renewal and wound healing because of its multipotency. We initially observed that bone marrow-derived human MSCs (hMSCs) divided poorly and took flat and enlarged morphology after expanded in culture over a certain number of cell passage, which resembled characteristic features of senescent cells, well-studied in human diploid fibroblasts (HDFs). More interestingly, adipogenic differentiation potential of hMSCs sharply declined as they approached the end of their proliferative life span. In this study, altered hMSCs were verified to be senescent by their senescence-associated beta-galactosidase (SA-beta-gal) activity and the increased expression of cell cycle regulating proteins (p16(INK4a), p21(Waf1) and p53). Similar as in HDFs, basal phosphorylation level of ERK was also significantly increased in senescent hMSCs, implying altered signal paths commonly shared by the senescent cells. Insulin, a major component of adipogenesis inducing medium, did not phosphorylate ERK 1/2 more in senescent hMSCs after its addition whereas it did in young cells. In senescent hMSCs, we also found a significant increase of caveolin-1 expression, previously reported as a cause for the attenuated response to growth factors in senescent HDFs. When we overexpressed caveolin-1 in young hMSC, not only insulin signaling but also adipogenic differentiation was significantly suppressed with down-regulated PPARgamma2. These data indicate that loss of adipogenic differentiation potential in senescent hMSC is mediated by the over-expression of caveolin-1.

p21-Activated kinase 4 promotes prostate cancer progression through CREB.

Prostate cancer is initially androgen-dependent but, over time, usually develops hormone- and chemo-resistance. The present study investigated a role for p21-activated kinase 4 (PAK4) in prostate cancer progression. PAK4 activation was markedly inhibited by H89, a specific protein kinase A (PKA) inhibitor, and PAK4 was activated by the elevation of cAMP. The catalytic subunit of PKA interacted with the regulatory domain of PAK4, and directly phosphorylated PAK4 at serine 474 (S474). Catalytically active PAK4 enhanced the transcriptional activity of CREB independent of S133 phosphorylation. Stable knockdown of PAK4 in PC-3 and DU145 prostate cancer cells inhibited tumor formation in nude mice. Decreased tumorigenicity correlated with decreased expression of CREB and its targets, including Bcl-2 and cyclin A1. Additionally, in androgen-dependent LNCap-FGC cells, PAK4 regulated cAMP-induced neuroendocrine differentiation, which is known to promote tumor progression. Finally, PAK4 enhanced survival and decreased apoptosis following chemotherapy. These results suggested that PAK4 regulates progression toward hormone- and chemo-resistance in prostate cancer, and this study identified both a novel activation mechanism and potential downstream effector pathways. Therefore, PAK4 may be a promising therapeutic target in prostate cancer.

c-Jun N-terminal kinase is involved in motility of endothelial cell

Cell motility is essential for a wide range of cellular activities including anigogenesis as well as metastasis of tumor cells. Ras has been implicated in cell migration and invasion, and functions at upstream of mitogen-activated protein kinase (MAPK) families, which include extracellular-signal regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAPK. In the present study, we examined the role of JNK in endothelial cell motility using stable transfectant (DAR-ECV) of ECV304 endothelial cells expressing previously established oncogenic H-Ras (leu 61). DAR-ECV cells showed an enhanced angiogenic potential and motility (∼2-fold) compared to ECV304 cells. Western blot analysis revealed constitutive activation of JNK in DAR-ECV cells. Pretreatment of JNK specific inhibitors, curcumin and all trans-retinoic acid, decreased the basal motility of DAR-ECV cells in a dose-dependent manner. These inhibitors also suppressed the motility stimulated by known JNK agonists such as TNFα and anisomycin. To further confirm the role of JNK, ECV304 cells expressing dominant active SEK1 (DAS-ECV) were generated. Basal non-stimulated levels of the cellular migration were greater in DAS-ECV clones than those in control ECV304 cells. These results suggest that Ras-SEK1-JNK pathway regulates motility of endothelial cells during angiogenesis.

Runx3 Inactivation Is a Crucial Early Event in the Development of Lung Adenocarcinoma

Targeted inactivation of Runx3 in mouse lung induced mucinous and nonmucinous adenomas and markedly shortened latency of adenocarcinoma formation induced by oncogenic K-Ras. RUNX3 was frequently inactivated in K-RAS mutated human lung adenocarcinomas. A functional genetic screen of a fly mutant library and molecular analysis in cultured cell lines revealed that Runx3 forms a complex with BRD2 in a K-Ras-dependent manner in the early phase of the cell cycle; this complex induces expression of p14(ARF)/p19(Arf) and p21(WAF/CIP). When K-Ras was constitutively activated, the Runx3-BRD2 complex was stably maintained and expression of both p14(ARF) and p21(WAF/CIP) was prolonged. These results provide a missing link between oncogenic K-Ras and the p14(ARF)-p53 pathway, and may explain how cells defend against oncogenic K-Ras.

Up-regulation and co-expression of fibroblast growth factor receptors in human gastric cancer

Abstract Fibroblast growth factor (FGF), a key regulatory factor of cell growth and differentiation, is involved in embryonic development, angiogenesis, and tumorigenesis. To date, four different FGF receptors (FGFRs) have been cloned and characterized. We examined the expression of four FGFRs in human gastric cancer tissues and cell lines using Northern analysis, ribonuclease protection assay, and immunohistochemistry. The mRNAs of FGFR-1 (10/14), FGFR-2 (9/14), and FGFR-4 (9/14) were up-regulated in cancer compared with normal tissues. FGFR-3 mRNAs were barely detectable in both normal and cancer tissues. These FGFR mRNAs were co-expressed in various combinations of two or three in the same tissue. Immunohistochemistry confirmed specific staining of multiple FGFRs, except FGFR-3, in the cancer specimens. To investigate the functional significance of FGFR co-expression we examined the invasive property of SNU-16 cells, which exhibited gene amplification of FGFR-2, -3, and -4 as well as over-expression of keratinocyte growth factor receptor (KGFR), a splice variant of FGFR-2, and FGFR-4 mRNA. KGF plus acidic FGF (aFGF), KGF, and aFGF treatment enhanced the invasive potential of SNU-16 cells over the control by 100%, 107%, and 47%, respectively, indicating that neither additive nor synergistic effect was induced by stimulation with aFGF plus KGF. These results suggest that co-expression of FGFRs in various combinations may cause subtle changes in the progression of gastric cancer.

Myosin II directly binds and inhibits Dbl family guanine nucleotide exchange factors: a possible link to Rho family GTPases

The activity of Rho GTPases in migrating cells is regulated by binding of myosin II to GEFs.

Age-related changes in the distribution of nitrotyrosine in the cerebral cortex and hippocampus of rats.

A wealth of indirect evidence implicates oxidative damage of cellular constituents in aging, as well as in the pathogenesis of the neurodegenerative diseases of later years. In the present study, we have determined age-related changes in the distribution of 3-nitrotyrosine (3-NT) in the cerebral cortex and hippocampus of rats. In adult rats, no 3-NT-immunoreactive cells were found in the cerebral cortex and hippocampus, whereas 3-NT immunoreactivity was significantly increased in aged rats. Some pyramidal cells of CA3 area and granule cells of the dentate gyrus highly expressed 3-NT in aged rats. Many interneurons located within stratum pyramidale and stratum oriens of CA1 were strongly immunoreactive for 3-NT. Our first demonstrations of increased 3-NT in the cerebral cortex and hippocampus during aging implicate these areas as sites for functionally significant oxidative damage. The mechanisms underlying the increased immunoreactivity for 3-NT, and the functional implications of this increase, require elucidation.

Inhibitory effect of epigallocatechin 3-O-gallate on vascular smooth muscle cell hypertrophy induced by angiotensin II

Recent evidence indicates that epigallocatechin 3-O-gallate (EGCG), the major catechin derived from green tea leaves, lowers the risk of cardiovascular diseases such as atherosclerosis and hypertension. However, a precise mechanism for this biologic function has not yet been clearly delineated. Angiotensin II (Ang II) stimulates vascular smooth muscle cell (VSMC) hypertrophy, which is a critical event in the development of atherosclerosis, hypertension, and angioplasty-induced restenosis. In the present study, we show that EGCG inhibits Ang II-stimulated VSMC hypertrophy, as determined by [3H]leucine incorporation into VSMC. Since mitogen-activated protein kinase (MAPK) families are involved in cell growth, we determined whether EGCG affects them. EGCG pretreatment did not exert any significant changes in Ang II-stimulated activation of extracellular signal-regulated kinase (ERK) and p38 MAPK. EGCG only inhibited Ang II-stimulated activation of c-Jun N-terminal kinase (JNK). Moreover, EGCG suppressed Ang II-induced c-jun mRNA expression. In contrast, EGC, a structural analogue of EGCG, did not inhibit the JNK activity or c-jun mRNA expression. In addition, a specific JNK inhibitor, SP600125, dose-dependently suppressed Ang II-stimulated VSMC hypertrophy. These results suggest that the effect of EGCG on Ang II-induced VSMC hypertrophy is due to specific inhibition of the JNK signaling pathway at both transcriptional and posttranslational levels, which may underlie its beneficial effect on the cardiovascular diseases.