Han Na Suh

Collagen I regulates the self-renewal of mouse embryonic stem cells through α2β1 integrin- and DDR1-dependent Bmi-1.

Adhesion of cells to extracellular matrix (ECM) influences vital aspects of anchorage‐dependent cell behavior including survival, proliferation, and differentiation. However, the role of collagen I in mouse embryonic stem cells (mESCs) is not well‐known. Therefore, in the present study we examined the effect of collagen I on mESC self‐renewal and related signal pathways. Collagen I (10 µg/ml) maintained mESCs in an undifferentiated state (Nanog, OCT4, and SSEA‐1) and did not affect differentiation (GATA4, Tbx5, Fgf5, and Cdx2) in the presence of leukemia inhibitory factor (LIF). Treatment with collagen I bound both α2β1 integrin and discoidin domain receptor 1 (DDR1), and stimulated intracellular signaling pathways. Collagen I‐bound α2β1 integrin increased integrin‐linked kinase (ILK) phosphorylation, cleaved Notch protein expression in the nuclear fraction, and Gli‐1 mRNA expression. In addition, collagen I‐bound DDR1 increased GTP‐bound Ras, phosphoinositide 3‐kinase (PI3K) p85α catalytic subunit protein expression, and Akt and ERK phosphorylation. Importantly, collagen I increased Bmi‐1 protein expression in the nucleus which was blocked by small interfering RNA (siRNA) specific for Gli‐1 and ERK, showing that parallel pathways of integrins and DDR1 merge at Bmi‐1. Furthermore, collagen I‐induced p16 decrease and p‐Rb increase were reversed by Bmi‐1‐specific siRNA. Moreover, Bmi‐1 silencing abolished the collagen I‐induced increase of proliferation indices and undifferentiation markers. These results indicate that collagen I stimulates the self‐renewal of mESCs mediated by Bmi‐1 through α2β1 integrin‐dependent ILK, Notch, Gli‐1, and DDR1‐dependent Ras, PI3K/Akt, and ERK. J. Cell. Physiol. 226: 3422–3432, 2011.

Linoleic acid stimulates gluconeogenesis via Ca2+/PLC, cPLA2, and PPAR pathways through GPR40 in primary cultured chicken hepatocytes

Fatty acids serve vital functions as sources of energy, building materials for cellular structures, and modulators of physiological responses. Therefore, this study examined the effect of linoleic acid on glucose production and its related signal pathways in primary cultured chicken hepatocytes. Linoleic acid (double-unsaturated, long chain) increased glucose production in a dose (> or =10(-4) M)- and time (> or =8 h)-dependent manner. Both oleic acid (monounsaturated, long chain) and palmitic acid (saturated, long chain) also increased glucose production, whereas caproic acid (saturated, short chain) failed to increase glucose production. Linoleic acid increased G protein-coupled receptor 40 (GPR40; also known as free fatty acid receptor-1) protein expression and glucose production that was blocked by GPR40-specific small interfering RNA. Linoleic acid increased intracellular calcium concentration, which was blocked by EGTA (extracellular calcium chelator)/BAPTA-AM (intracellular calcium chelator), U-73122 (phospholipase C inhibitor), nifedipine, or methoxyverapamil (L-type calcium channel blockers). Linoleic acid increased cytosolic phospholipase A(2) (cPLA(2)) phosphorylation and the release of [(3)H]-labeled arachidonic acid. Moreover, linoleic acid increased the level of cyclooxygenase-2 (COX-2) protein expression, which stimulated the synthesis of prostaglandin E(2) (PGE(2)). The increase in PGE(2) production subsequently stimulated peroxisome proliferator-activated receptor (PPAR) expression, and MK-886 (PPAR-alpha antagonist) and GW-9662 (PPAR-delta antagonist) inhibited glucose-6-phosphatase and phosphoenolpyruvate carboxykinase. In addition, linoleic acid-induced glucose production was blocked by inhibition of extracellular and intracellular calcium, cPLA(2), COX-2, or PPAR pathways. In conclusion, linoleic acid promoted glucose production via Ca(2+)/PLC, cPLA(2)/COX-2, and PPAR pathways through GPR40 in primary cultured chicken hepatocytes.

Effect of BSA-induced ER stress on SGLT protein expression levels and α-MG uptake in renal proximal tubule cells

Recent studies demonstrated that endoplasmic reticulum (ER) stress regulates glucose homeostasis and that ER stress preconditioning which induces an adaptive, protective unfolded protein response (UPR) offers cytoprotection against nephrotoxins. Thus the aim of the present study was to use renal proximal tubule cells (PTCs) to further elucidate the link between the BSA-induced ER stress and alpha-methyl-d-glucopyranoside (alpha-MG) uptake and to identify related signaling pathways. Among ER stress inducers such as high glucose, BSA, H2O2, or tumicamycin, BSA pretreatment ameliorated the reduction of Na(+)-glucose cotransporter (SGLT) expression and alpha-MG uptake by gentamicin or cyclosporine A. Immunofluorescence studies revealed that BSA (10 mg/ml) stimulated the expression of glucose-regulated protein 78 (GRP78), an ER stress biomarker. In addition, BSA increased levels of GRP78 protein expression and eukaryotic initiation factor 2alpha (eIF2alpha) phosphorylation in a time-dependent manner. Furthermore, transfection with a GRP78-specific small interfering RNA (siRNA) inhibited BSA-stimulated SGLT expression and alpha-MG uptake. In experiments designed to unravel the mechanisms underlying BSA-induced ER stress, BSA stimulated the production of cellular reactive oxygen species (ROS), and antioxidants such as ascorbic acid or N-acetylcysteine (NAC) blocked BSA-induced increases in GRP78 activation, eIF2alpha phosphorylation, SGLT expression, and alpha-MG uptake. Moreover, the cells upregulated peroxisome proliferator-activated receptor-gamma (PPARgamma) mRNA levels in response to BSA or troglitazone (a PPARgamma agonist), but BSA was ineffective in the presence of GW9662 (a PPARgamma antagonist). In addition, both BSA and troglitazone stimulated GRP78 and eIF2alpha activation, SGLT expression, and alpha-MG uptake, whereas GW9662 inhibited the effects of BSA. BSA also stimulated phosphorylation of JNK and NF-kappaB, and GW9662 or GRP78 siRNA attenuated this response. Moreover, SP600125 or SN50 effectively blocked SGLT expression and alpha-MG uptake in BSA- or PPARgamma agonists (troglitazone or PGJ2)-treated PTCs. We conclude that BSA induces ER stress through ROS production and PPARgamma activation, which subsequently activates JNK/NF-kappaB signaling to enhance glucose uptake in renal PTCs.

Laminin regulates mouse embryonic stem cell migration: involvement of Epac1/Rap1 and Rac1/cdc42

Laminin is the first extracellular matrix (ECM) component to be expressed in the developing mammalian embryo. However, the roles of laminin or the related signal pathways are not well known in mouse embryonic stem cells (mESCs). Presently, we examined the effect of laminin on mESC migration. Laminin (10 microg/ml) decreased cell aggregation, whereas migration was increased. Laminin bound alpha6beta1 integrin and laminin receptor 1 (LR1), decreasing their mRNA levels. Laminin increased focal adhesion kinase (FAK) and paxillin phosphorylation, cAMP intracellular concentration, and the protein levels of exchange factor directly activated by cAMP (Epac1) and Rap1. These increases were completely blocked by alpha6beta1 integrin and LR1 neutralizing antibody, indicating that laminin-bound LR1 assists laminin-induced alpha6beta1 integrin activity and initiates signal. As a downstream signal molecule, laminin activated small G protein such as Rac1/cdc42 and its effector protein p21-activated kinase (PAK). Subsequently, laminin stimulated E-cadherin complex disruption. Inhibition of each pathway such as those for alpha6beta1 integrin and LR1, FAK, Rap1, and PAK1 blocked laminin-induced migration. We conclude that laminin binds both alpha6beta1 integrin and LR1 and induces signaling FAK/paxillin and cAMP/Epac1/Rap1. These signaling merge at Rac1/cdc42 subsequently activate PAK1. Activated PAK1 enhances E-cadherin complex disruption and finally increases mESCs migration.

Midkine prevented hypoxic injury of mouse embryonic stem cells through activation of Akt and HIF-1α via low-density lipoprotein receptor-related protein-1.

Stem cell functions are dramatically altered by oxygen in tissue culture, which means the antioxidant/oxidant balance is critical for protection as well as toxicity. This study examined the effect of the heparin‐binding growth factor midkine (MK) on hypoxia‐induced apoptosis and related signal pathways in mouse embryonic stem cells (mESCs). Hypoxia (60 h) increased lactate dehydrogenase release and apoptosis, and reduced cell viability and proliferation. These effects were reversed by MK (100 ng/ml). MK also reversed hypoxia‐induced increases of intracellular reactive oxygen species, c‐Jun N‐terminal kinase (JNK), and p38 mitogen‐activated protein kinase (MAPK) phosphorylation. Blockage of JNK and p38 MAPK using small interference (si)RNAs produced a decrease in apoptosis. A loss of mitochondrial membrane potential, increases of cytochrome c release from mitochondria to cytosol, and cleaved caspase‐3 expression, as well as decreases in cIAP‐2 and Bcl‐2 were also reversed by MK. Hypoxia alone and hypoxia with MK increased low‐density lipoprotein receptor‐related protein‐1 (LRP‐1) mRNA and protein expression. Hypoxia with MK rapidly increased serine/threonine protein kinase (Akt) phosphorylation which reversed by LRP‐1 Ab (0.1 µg/ml) and prolonged heme oxygenase‐1 (HO‐1) expression. In addition, hypoxia with MK increased the expression of hypoxia‐inducible factor‐1α (HIF‐1α). Moreover, inhibition of Akt, HO‐1, and HIF‐1α signaling pathways abolished the MK‐induced blockage of apoptosis. In conclusion, MK partially prevented hypoxic injury of mESCs through activation of Akt, HO‐1, and HIF‐1α via LRP‐1. J. Cell. Physiol. 227: 1731–1739, 2012.

High glucose induced translocation of Aquaporin8 to chicken hepatocyte plasma membrane: Involvement of cAMP, PI3K/Akt, PKC, MAPKs, and microtubule

Aquaporin8 (AQP8) is a transmembrane water channel that is found mainly in hepatocytes. The direct involvement of AQP8 in high glucose condition has not been established. Therefore, this study examined the effects of high glucose on AQP8 and its related signal pathways in primary cultured chicken hepatocytes. High glucose increased the movement of AQP8 from the intracellular membrane to plasma membrane in a 30 mM glucose concentration and in a time‐ (≥10 min) dependent manner. On the other hand, 30 mM mannitol did not affect the translocation of AQP8, which suggested the absence of osmotic effect. Thirty millimolar glucose increased intracellular cyclic adenosine 3, 5‐monophosphate (cAMP) level. Moreover, high glucose level induced Akt phosphorylation, protein kinase C (PKC) activation, p44/42 mitogen‐activated protein kinases (MAPKs), p38 MAPK, and c‐jun NH2‐terminal kinase (JNK) phosphorylation. On the other hand, inhibition of each pathway by SQ 22536 (adenylate cyclase inhibitor), LY 294002 (PI3‐K phosphatidylinositol 3‐kinase inhibitor), Akt inhibitor, staurosporine (PKC inhibitor), PD 98059 (MEK inhibitor), SB 203580 (p38 MAPK inhibitor), or SP 600125 (JNK inhibitor) blocked 30 mM glucose‐induced AQP8 translocation, respectively. In addition, inhibition of microtubule movement with nocodazole blocked high glucose‐induced AQP8 translocation. High glucose level also increased the level of kinesin light chain and dynein protein expression. In conclusion, high glucose level stimulates AQP8 via cAMP, PI3‐K/Akt, PKC, and MAPKs pathways in primary cultured chicken hepatocytes. J. Cell. Biochem. 103: 1089–1100, 2008.

Role of interleukin-6 in the control of DNA synthesis of hepatocytes: involvement of PKC, p44/42 MAPKs, and PPARdelta.

Interleukin-6 (IL-6) is a pleiotropic cytokine with a pivotal role in normal hepatic growth and liver regeneration. Therefore, in the present study, we examined the effect of IL-6 on cell proliferation and the related signaling pathways in primary cultured chicken hepatocytes. IL-6 increased the level of [3H]thymidine incorporation in a time (≥ 6 hr)- and a dose (≥ 0.1 ng/ml)-dependent manner. Indeed, IL-6 increased the number of BrdU-positive cells and the total number of cells. IL-6 (10 ng/ml) increased the level of IL-6Rα and glycoprotein (gp) 130 (IL-6Rβ) protein expression, Janus Kinase (JAK) 2, signal transducer and activator of transcription (STAT) 3, PKC, p44/42 MAPKs phosphorylation, and PPARδ protein expression. Inhibition of each pathways blocked IL-6-induced [3H]thymidine incorporation increase. IL-6 increased c-fos, c-jun, and c-myc proto-oncogene mRNA levels and the percentage of cells in the S phase according to fluorescence-activated cell sorter (FACS) analysis. IL-6-induced G1/S phase progression was inhibited by AG 490 (2x10-5 M, JAK2 inhibitor), a STAT3 inhibitor peptide (10-5 M), bisindolylmaleimide I (10-6 M, PKC inhibitor), PD 98059 (10-5 M, p44/42 MAPKs blocker), or PPARδ-specific small interfering RNAs (siRNAs). In conclusion, IL-6 stimulates the proliferation of primary cultured chicken hepatocytes through PKC, p44/42 MAPKs, and PPARδ pathways.

Interleukin-6 promotes 2-deoxyglucose uptake through p44/42 MAPKs activation via Ca2+/PKC and EGF receptor in primary cultured chicken hepatocytes

Interleukin‐6 (IL‐6) is involved in a variety of biological responses, including the glucose metabolism and cell growth, which is a critical physiological function requiring multiple metabolic pathways. Therefore, in the present study, we examined the effect of IL‐6 on 2‐deoxyglucose (2‐DG) uptake and the related signaling pathways in primary cultured chicken hepatocytes. IL‐6 increased 2‐DG uptake in a time‐ (≥4 h) and a dose ‐(≥5 ng/ml) dependent manner. Indeed, IL‐6 increased GLUT‐2 mRNA and protein expression as well as 2‐DG uptake, which were blocked by actinomycin D (AD, transcription inhibitor) and cycloheximide (CHX, translation inhibitor). IL‐6 (10 ng/ml) increased the level of IL‐6Rα and glycoprotein (gp) 130 (IL‐6Rβ) protein expressions. IL‐6 increased Janus Kinase (JAK)‐2, signal transducer and activator of transcription (STAT)‐3 phosphorylation, intracellular Ca2+ concentration, and PKC phosphorylation. IL‐6‐induced increase of 2‐DG uptake and GLUT‐2 protein expression were blocked by JAK2‐specific siRNA, a STAT3 inhibitor, staurosporine, and bisindolylmaleimide I (PKC inhibitors). In addition, IL‐6 increased EGFR/src/FAK, PI3K/Akt phosphorylation and 2‐DG uptake as well as GLUT‐2 protein expression, which were blocked by AG 1478 (EGF receptor inhibitor), PP2 (src family of tyrosine kinase inhibitor), PI3K‐specific siRNA, and a Akt inhibitor. Furthermore, IL‐6 increased p44/42 MAPKs phosphorylation and p44 and p42 MAPK‐specific siRNA mixture blocked IL‐6‐induced increase of 2‐DG uptake and GLUT‐2 protein expression. In conclusion, IL‐6 stimulates the 2‐DG uptake through p44/42 MAPKs activation via Ca2+/PKC and EGF receptor in primary cultured chicken hepatocytes. J. Cell. Physiol. 218: 643–652, 2009.

Effectiveness of (99m)Tc-tetrofosmin for assessment of heart functions in micropigs.

This study examined the suitability of a nuclear imaging technique using 99mTc-tetrofosmin as an agent to assess the heart functions of healthy micropigs. The mean age of the pigs was 360 days (male), and the mean body weight was 35.3 kg ranging from 34.5-36 kg. There were no significant perfusion defects in any of the reconstructed images. Gated single-photon emission computed tomography imaging can be used to calculate the ventricular volume and ejection fraction (EF). In this case, an EF of 79% was calculated from the ventricular volume of the end-systolic image (10 ml) subtracted from that of the end-diastolic volume (49 ml). A perfusion defect (particularly the apex, lateral wall) is unlikely because of the presence of a preserved wall motion in a segment with a defect. It is concluded that quantitative cardiac scintigraphy, using 99mTc-tetrofosmin is an adequate technique for estimating the heart functions of healthy micropigs.