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Type II collagen is a major protein that maintains biological and mechanical characteristics in articular cartilage. Focal adhesion kinase (FAK) is known to play a central role in integrin signaling of cell–extracellular matrix (ECM) interactions, and chondrocyte–type II collagen interactions are very important for cartilage homeostasis. In this study, we focused on phosphorylation of FAK and MAP kinase in chondrocyte–type II collagen interaction and dedifferentiation, and the effects of FAK knockdown on chondrocyte-specific gene expression and cell proliferation were determined. The addition of exogenous type II collagen to chondrocytes increased levels of tyrosine phosphorylation, p-FAKY397, and p-ERK1/2. In contrast, expression levels of p-FAKY397 and p-ERK1/2, but not p-Smad2/3, were decreased in dedifferentiated chondrocytes with loss of type II collagen expression. Type II collagen expression was significantly increased when dedifferentiated chondrocytes were transferred to alginate beads with TGF-β1 or type II collagen, but transfected cells with small interfering RNA for FAK (FAK-siRNA) inhibited mRNA expression of type II collagen and SOX-6 compared to the control. These FAK-siRNA-transfected cells could not recover type II collagen even in the presence of TGF-β1 or type II collagen in alginate beads culture. We also found that FAK-siRNA-transfected cells decreased cell proliferation rate, but there was no effect on glycosaminoglycans (GAGs) secretion. We suggest that FAK is essentially required in chondrocyte communication with type II collagen by regulating type II collagen expression and cell proliferation. J. Cell. Physiol. 218: 623–630, 2009. © 2008 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

This study examined the synergistic effect of high glucose levels and ANG II on proliferation and its related signal pathways using mouse embryonic stem (ES) cells. The combined use of a high glucose concentration (25 mM) and ANG II increased the level of [3H]thymidine/BrdU incorporation, and the number of cells compared with either treatment alone. Each treatment with high glucose or ANG II increased the cell population in the S phase compared with control, and the combined treatment of a high glucose concentration and ANG II significantly increased the number of cells in the S phase according to FACS analysis. Moreover, the high glucose-induced increase in [3H]thymidine incorporation was blocked by inhibiting the ANG II type 1 (AT1) receptor. The combined high glucose and ANG II significantly increased the STAT3 phosphorylation compared with high glucose or ANG II alone. ANG II stimulated the influx of Ca2+ in 25 mM glucose compared with 5 mM glucose. High glucose levels increase the level of PKC α, ℇ, and ζ translocation from the cytosol to the membrane fraction. In an examination of other signal pathways, the combined treatment significantly increased the level of p44/42, p38 MAPKs phosphorylation compared with either treatment alone. Indeed, the combined treatment increased the mRNA expression level of the protooncogenes and cell cycle regulatory proteins. In conclusion, the combined treatment of a high glucose concentration and ANG II had a synergistic effect in stimulating mouse ES cell proliferation through the Ca2+/PKC, MAPKs, and the AT1 receptor. J. Cell. Physiol. 215: 374–382, 2008. © 2007 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

This study examined the effects of high glucose on cell proliferation and its related signal pathways using mouse embryonic stem (ES) cells. Here, we showed that high glucose level significantly increased [3H]thymidine incorporation, BrdU incorporation, the number of cells, [3H]leucine, and [3H]proline incorporation in a time-(>3 hr) and dose-(>25 mM) dependent manner. Moreover, high glucose level increased the cellular reactive oxygen species (ROS), Akt, and mitogen-activated protein kinases (MAPKs) phosphorylation. Subsequently, these signaling molecules involved in high glucose-induced increase of [3H]thymidine incorporation. High glucose level also increased cyclin D1, cyclin E, cyclin-dependent kinase (CDK) 2, and CDK 4 protein levels, which is cell cycle regulatory proteins acting in G1–S phase of cell cycle. Inhibition of phosphatidylinositol 3-kinase (PI3-K) (LY 294002: PI3-kinase inhibitor, 10-6 M), Akt (Akt inhibitor, 10-5 M), and p44/42 MAPKs (PD 98059: MEK inhibitor, 10-5 M) decreased these proteins. High glucose level phosphorylated the RB protein, which was decreased by inhibition of PI3-K and Akt. In conclusion, high glucose level stimulates mouse ES cell proliferation via the PI3-K/Akt and MAPKs pathways. J. Cell. Physiol. 209: 94–102, 2006. © 2006 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Here we report inhibition of phospholipase C-β1 (PLC-β1)-mediated signaling by post-translational glycosylation with β-N-acetylglucosamine (O-GlcNAc modification). In C2C12 myoblasts, isoform-specific knock-down experiments using siRNA showed that activation of bradykinin (BK) receptor led to stimulation of PLC-β1 and subsequent intracellular Ca2+ mobilization. In C2C12 myotubes, O-GlcNAc modification of PLC-β1 was markedly enhanced in response to treatment with glucosamine (GlcNH2), an inhibitor of O-GlcNAase (PUGNAc) and hyperglycemia. This was associated with more than 50% inhibition of intracellular production of IP3 and Ca2+ mobilization in response to BK. Since the abundance of PLC-β1 remained unchanged, these data suggest that O-GlcNAc modification of PLC-β1 led to inhibition of its activity. Moreover, glucose uptake stimulated by BK was significantly blunted by treatment with PUGNAc. These data support the notion that O-GlcNAc modification negatively modulates the activity of PLC-β1. J. Cell. Physiol. © 2006 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Effect of angiotensin II (ANG II) on mouse embryonic stem (ES) cell proliferation was examined. ANG II increased [3H] thymidine incorporation in a time- (>4 h) and dose- (>10-9 M) dependent manner. The ANG II-induced increase in [3H] thymidine incorporation was blocked by inhibition of ANG II type 1 (AT1) receptor but not by ANG II type 2 (AT2) receptor, and AT1 receptor was expressed. ANG II increased inositol phosphates formation and [Ca2+]i, and translocated PKC α, δ, and ζ to the membrane fraction. Consequently, the inhibition of PLC/PKC suppressed ANG II-induced increase in [3H] thymidine incorporation. The inhibition of EGF receptor kinase or tyrosine kinase prevented ANG II-induced increase in [3H] thymidine incorporation. ANG II phosphorylated EGF receptor and increased Akt, mTOR, and p70S6K1 phosphorylation blocked by AG 1478 (EGF receptor kinase blocker). ANG II-induced increase in [3H] thymidine incorporation was blocked by the inhibition of p44/42 MAPKs but not by p38 MAPK inhibition. Indeed, ANG II phosphorylated p44/42 MAPKs, which was prevented by the inhibition of the PKC and AT1 receptor. ANG II increased c-fos, c-jun, and c-myc levels. ANG II also increased the protein levels of cyclin D1, cyclin E, cyclin-dependent kinase (CDK) 2, and CDK4 but decreased the p21cip1/waf1 and p27kip1, CDK inhibitory proteins. These proteins were blocked by the inhibition of AT1 receptor, PLC/PKC, p44/42 MAPKs, EGF receptor, or tyrosine kinase. In conclusion, ANG II-stimulated DNA synthesis is mediated by ANG II receptor-dependent Ca2+/PKC and EGF receptor-dependent PI3K/Akt/mTOR/p70S6K1 signal pathways in mouse ES cells. J. Cell. Physiol. 211: 618–629, 2007. © 2007 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]