Jung Sun Heo

Sonic hedgehog stimulates mouse embryonic stem cell proliferation by cooperation of Ca2+/protein kinase C and epidermal growth factor receptor as well as Gli1 activation.

Hedgehog signaling has an essential role in the control of stem cell growth in embryonic tissues. Therefore, this study examined the effect of sonic hedgehog (Shh) on the self‐renewal of mouse embryonic stem (ES) cells and its related mechanisms. Shh increased DNA synthesis blocked by the inhibition of the smoothened receptor. Shh required Gli1 activation to induce the increases in Notch/Hes‐1 and Wnt/β‐catenin. Shh increased the intracellular calcium concentration ([Ca2+]i) and protein kinase C (PKC) activity. We show that the Shh‐induced increase in the Gli1 mRNA level requires [Ca2+]i and PKC. Shh increased the phosphorylation of epidermal growth factor receptor (EGFR), which is blocked by the matrix metalloproteinase inhibitor. Subsequently, Shh increased the nuclear factor (NF)‐κB p65 phosphorylation, which was inhibited by blocking PKC and EGFR tyrosine kinase. Shh also increased the level of the cell cycle regulatory proteins in a dose‐dependent manner. However, Shh decreased the levels of the cyclin‐dependent kinase inhibitory proteins. The effect of Shh on these proteins was inhibited by blocking PKC, EGFR, and NF‐κB as well as transfection of Gli1 small interfering RNA (siRNA). Finally, Shh‐induced progression of the G1/S‐phase was blocked by the inhibition of PKC and EGFR tyrosine kinase. In conclusion, Shh stimulates mouse ES cell proliferation through Gli1 activation as well as Ca2+/PKC and EGFR.

High glucose increase cell cycle regulatory proteins level of mouse embryonic stem cells via PI3-K/Akt and MAPKs signal pathways.

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.

Arachidonic acid release by H2O2 mediated proliferation of mouse embryonic stem cells: involvement of Ca2+/PKC and MAPKs-induced EGFR transactivation.

Reactive oxygen species (ROS) generated by a variety of endogenous factors and roles in embryonic stem (ES) cells has yet to be identified. Thus, we examined role of arachidonic acid (AA) in H2O2‐indued proliferation of mouse ES cells and its related signaling molecules. AA release was maximally increased in response to 10−4 M H2O2 for 1 h. In addition, H2O2 increased intracellular Ca2+ concentration ([Ca2+]i) and the phosphorylation of protein kinase C (PKC), p44/42, p38 mitogen‐activated protein kinase (MAPK), and JNK/SAPK. Moreover, H2O2 induced an increase in the phosphorylation of epidermal growth factor receptor (EGFR), which was blocked by the inhibition of p44/42 or p38 MAPKs. The inhibition of each signal molecule with specific inhibitors blocked H2O2‐induced cytosolic phospholipase A2 (cPLA2) activation and AA release. H2O2 increased NF‐κB phosphorylation to induce an increase in the levels of cyclooxygenase (COX)‐2 proteins. Subsequently, H2O2 stimulated PGE2 synthesis, which was reduced by the inhibition of NF‐κB activation. Moreover, each H2O2 or PGE2 increased DNA synthesis and the number of cells. However, H2O2‐induced increase in DNA synthesis was inhibited by the suppression of cPLA2 pathway. In conclusion, H2O2 increased AA release and PGE2 production by the upregulation of cPLA2 and COX‐2 via Ca2+/PKC/MAPKs and EGFR transactivation, subsequently proliferation of mouse ES cells. J. Cell. Biochem. 106: 787–797, 2009.

Effect of hypoxia on 2-deoxyglucose uptake and cell cycle regulatory protein expression of mouse embryonic stem cells: involvement of Ca2+ /PKC, MAPKs and HIF-1alpha.

This study investigated the signal molecules linking the alteration in 2-dexoyglucose (2-DG) uptake and DNA synthesis in mouse embryonic stem (ES) cells under hypoxia. Hypoxia increased the 2-DG uptake and GLUT-1 protein expression level while the undifferentiated state of ES cells and cell viability were not affected by the hypoxia (1 - 48h). Subsequently, [3H] thymidine incorporation was significantly increased at 12 hours of hypoxic exposure. Hypoxia increased the Ca2+ uptake and PKC β I, ε, and ζ translocation from the cytosol to the membrane fraction. Moreover, hypoxia increased the level of p44/42 mitogen-activated protein kinases (MAPKs) phosphorylation and hypoxia inducible factor-1α (HIF-1α) in a time-dependent manner. On the other hand, inhibition of these pathways blocked the hypoxia-induced increase in the 2-DG uptake and GLUT-1 protein expression level. Under hypoxia, cell cycle regulatory protein expression [cyclin D1, cyclin E, cyclin-dependent kinase (CDK) 2, and CDK 4] were increased in a time-dependent manner, which were blocked by PD 98059. pRB protein was also increased in a time-dependent manner. In conclusion, under hypoxia, there might be a parallel relationship between the expression of GLUT1 and DNA synthesis, which is mediated by the Ca2+ /PKC, MAPK, and the HIF-1α signal pathways in mouse ES cells.

Effect of dihydrotestosterone on hydrogen peroxide-induced apoptosis of mouse embryonic stem cells.

Steroid hormones have been reported to activate various signal transducers that trigger a variety of cellular responses. Among these hormones, testosterone has been identified as an antioxidant that protects against cellular damage. Therefore, using mouse embryonic stem (ES) cells as a model system, this study evaluated the effects of dihydrotestosterone (DHT), a biologically active testosterone metabolite, on H2O2‐induced apoptosis. H2O2 increased the release of lactate dehydrogenase (LDH) and DNA fragmentation but reduced the cell viability in a time‐dependent manner (≥8 h). Moreover, H2O2 decreased the level of DNA synthesis and the levels of the cell cycle regulatory proteins [cyclin D1, cyclin E, cyclin‐dependent kinase (CDK) 2, and CDK 4]. These effects of H2O2 were inhibited by a pretreatment with DHT. However, a treatment with flutamide (androgen receptor inhibitor, 10−3 M) abolished the protective effects of DHT. This result was supported by the presence of the androgen receptor in mouse ES cells. The activity of the antioxidant enzyme, catalase, was increased by the DHT treatment but not by a co‐treatment with DHT and flutamide. Using CM‐H2DCFDA (DCF‐DA) for the detection of intracellular H2O2, DHT decreased the intracellular H2O2 levels but flutamide blocked this effect. H2O2 also increased the level of p38 MAPK, JNK/SAPK, and NF‐κB phosphorylation, which were inhibited by the DHT pretreatment. Catalase inhibited the effect of H2O2 on MAPKs and NF‐κB. However, the flutamide treatment abolished the inhibitory effects of DHT on the H2O2‐induced increase in the levels of p38 MAPK, JNK/SAPK, and NF‐κB phosphorylation. DHT inhibited the H2O2‐induced increase in caspase‐3 expression and decreased the level of Bcl‐2 and the cellular inhibitor of apoptosis protein (cIAP)‐2. These effects were abolished by the flutamide treatment. In conclusion, DHT prevents the H2O2‐induced apoptotic cell death of mouse ES cells through the activation of catalase and the downregulation of p38 MAPK, JNK/SAPK, and NF‐κB via the androgen receptor. J. Cell. Physiol. 216: 269–275, 2008.

A potential role of connexin 43 in epidermal growth factor‐induced proliferation of mouse embryonic stem cells: Involvement of Ca2+/PKC, p44/42 and p38 MAPKs pathways

Abstract.  Objectives: The gap junction protein, connexin (Cx), plays an important role in maintaining cellular homeostasis and cell proliferation by allowing communication between adjacent cells. Therefore, this study has examined the effect of epidermal growth factor (EGF) on Cx43 and its relationship to proliferation of mouse embryonic stem cells. Materials and methods: Expressions of Cx43, mitogen‐activated protein kinases (MAPKs) and cell cycle regulatory proteins were assessed by Western blot analysis. Cell proliferation was assayed with [3H]thymidine incorporation. Intercellular communication level was measured by a scrape loading/dye transfer method. Results: The results showed that EGF increased the level of Cx43 phosphorylation in a time‐ (≥5 min) and dose‐ (≥10 ng/mL) dependent manner. Indeed, EGF‐induced increase in phospho‐Cx43 level was significantly blocked by either AG 1478 or herbimycin A (tyrosine kinase inhibitors). EGF increased Ca2+ influx and protein kinase C (PKC) translocation from the cytosolic compartment to the membrane compartment. Moreover, pre‐treatment with BAPTA‐AM (an intracellular Ca2+ chelator), EGTA (an extracellular Ca2+ chelator), bisindolylmaleimide I or staurosporine (PKC inhibitors) inhibited the EGF‐induced phosphorylation of Cx43. EGF induced phosphorylation of p38 and p44/42 MAPKs, and this was blocked by SB 203580 (a p38 MAPK inhibitor) and PD 98059 (a p44/42 MAPK inhibitor), respectively. EGF or 18α‐glycyrrhetinic acid (GA; a gap junction inhibitor) increased expression levels of the protooncogenes (c‐fos, c‐jun and c‐myc), cell cycle regulatory proteins [cyclin D1, cyclin E, cyclin‐dependent kinase 2 (CDK2), CDK4 and p‐Rb], [3H]thymidine incorporation and cell number, but decreased expression levels of the p21WAF1/Cip1 and p27Kip1, CDK inhibitory proteins. Transfection of Cx43 siRNA also increased the level of [3H]thymidine incorporation and cell number. EGF, 18α‐GA or transfection of Cx43 siRNA increased 2‐DG uptake and GLUT‐1 protein expression. Conclusions: EGF‐induced phosphorylation of Cx43, which was mediated by the Ca2+/PKC, p44/42 and p38 MAPKs pathways, partially contributed to regulation of mouse embryonic stem cell proliferation.

ANG II-stimulated DNA synthesis is mediated by ANG II receptor-dependent Ca2+/PKC as well as EGF receptor-dependent PI3K /Akt /mTOR /p70S6K1 signal pathways in mouse embryonic stem cells

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.

Epinephrine increases DNA synthesis via ERK1/2s through cAMP, Ca2+/PKC, and PI3K/Akt signaling pathways in mouse embryonic stem cells

Epinephrine is a catecholamine that plays important roles in regulating a wide variety of physiological systems by acting through the adrenergic receptors (ARs). The cellular responses to AR stimulation are mediated through various signaling pathways. Therefore, this study examined the effects of epinephrine on DNA synthesis and related signaling molecules in mouse embryonic stem cells (ESCs). Epinephrine increased DNA synthesis in a dose‐ and time‐dependent manner, as determined by the level of [3H]‐thymidine incorporation. AR subtypes (α1A, α2A, β1, β2, and β3) were expressed in mouse ESCs and their expression levels were increased by epinephrine. In this experiment, epinephrine increased cAMP levels, intracellular Ca2+ concentration ([Ca2+]i), and translocation of protein kinase C (PKC) from the cytosol to the membrane compartment. In addition, we observed Akt phosphorylation in response to epinephrine; this was stimulated by phosphorylation of the epidermal growth factor receptor (EGFR). Epinephrine also induced phosphorylation of ERK1/2 (p44/42 MAPKs), while inhibition of PKC or Akt blocked this phosphorylation. Epinephrine increased the mRNA levels of proto‐oncogenes (c‐fos, c‐jun, c‐myc), while inhibition of ERK1/2 decreased these mRNA levels. In experiments aimed at examining the involvement of cell cycle regulatory proteins, epinephrine increased the levels of cyclin E/cyclin‐dependent kinase 2 (CDK2) and cyclin D1/cyclin‐dependent kinase 4 (CDK4). In conclusion, epinephrine stimulates DNA synthesis via ERK1/2 through cAMP, Ca2+/PKC, and PI3K/Akt signaling pathways in mouse ESCs. J. Cell. Biochem. 104: 1407–1420, 2008.

Dopamine regulates cell cycle regulatory proteins via cAMP, Ca2+/PKC, MAPKs, and NF‐κB in mouse embryonic stem cells

This study examined the effect of dopamine on DNA synthesis and its related signal cascades in mouse embryonic stem (ES) cells. Dopamine inhibited DNA synthesis in both a dose‐ and time‐dependent manner. Dopamine, SKF 38393 (D1 receptor agonist), and quinpirole (D2 receptor agonist) decreased the level of [3H]‐thymidine incorporation. The level of cyclic adenosine 3, 5‐monophosphate (cAMP) was increased by SKF 38393 but not by quinpirole. The protein kinase C (PKC) protein was translocated from the cytosolic fraction to the membrane compartment by dopamine. Dopamine also increased [Ca2+]i, which was blocked by EGTA (an extracellular Ca2+ chelator), BAPTA‐AM (an intracellular Ca2+ chelator), nifedipine (a L‐type Ca2+ channel blocker), SQ 22536 [an adenylyl cyclase (AC) inhibitor] and neomycin [a phospholipase C (PLC) inhibitor]. Dopamine, SKF 38393, and quinpirole increased the level of p44/42 mitogen‐activated protein kinases (MAPKs), p38 MAPK, and stress‐activated protein kinase/Jun‐N‐terminal kinase (SAPK/JNK) phosphorylation. Dopamine also increased level of H2O2 formation and activated the transcription factor family NF‐κB. Moreover, SKF 38393, quinpirole, and dopamine inhibited cell cycle regulatory proteins, which is consistent with the change in the level of [3H]‐thymidine incorporation observed. The dopamine‐induced decrease in cyclin E, cyclin‐dependent protein kinase‐2 (CDK‐2), and cyclin D1, CDK‐4 were blocked by pertussis toxin (G protein inhibitor), SQ 22536, neomycin, bisindolylmaleimide I (PKC inhibitor), SB 203580 (p38 MAPK inhibitor), PD 98059 (p44/42 inhibitor), and SP 600125 (SAPK/JNK inhibitor). In conclusion, dopamine inhibits DNA synthesis in mouse ES cells via the cAMP, Ca2+/PKC, MAPKs, and NF‐κB signaling pathways. J. Cell. Physiol. 208: 399–406, 2006.

Dopamine stimulates 45Ca2+ uptake through cAMP, PLC/PKC, and MAPKs in renal proximal tubule cells.

We have examined the effect of dopamine on Ca2+ uptake and its related signaling pathways in primary renal proximal tubule cells (PTCs). Dopamine increased Ca2+ uptake in a concentration (>10−10 M) and time‐ (>8 h) dependent manner. Dopamine‐induced increase in Ca2+ uptake was prevented by SCH 23390 (a DA1 antagonist) rather than spiperone (a DA2 antagonist). SKF 38393 (a DA1 agonist) increased Ca2+ uptake unlike the case with quinpirole (a DA2 agonist). Dopamine‐induced increase in Ca2+ uptake was blocked by nifedipine and methoxyverapamil (L‐type Ca2+ channel blockers). Moreover, dopamine‐induced increase in Ca2+ uptake was blocked by pertussis toxin (a Gi protein inhibitor), protein kinase A (PKA) inhibitor amide 14/22 (a PKA inhibitor), and SQ 22536 (an adenylate cyclase inhibitor). Subsequently, dopamine increased cAMP level. The PLC inhibitors (U 73122 and neomycin), the PKC inhibitors (staurosporine and bisindolylmaleimide I) suppressed the dopamine‐induced increase of Ca2+ uptake. SB 203580 (a p38 MAPK inhibitor) and PD 98059 (a MAPKK inhibitor) also inhibited the dopamine‐induced increase of Ca2+ uptake. Dopamine‐induced p38 and p42/44 MAPK phosphorylation was blocked by SQ 22536, neomycin, and staurosporine. The stimulatory effect of dopamine on Ca2+ uptake was significantly inhibited by the NF‐κB inhibitors SN50, TLCK, and Bay 11‐7082. In addition, dopamine significantly increased the level of NF‐κB p65, which was prevented by either SQ 22536, neomycin, staurosporine, PD 98059, or SB 203580. Thus, dopamine stimulates Ca2+ uptake in PTCs, initially through by Gs coupled dopamine receptors, PLC/PKC, followed by MAPK, and ultimately by NF‐κB activation. J. Cell. Physiol. 211: 486–494, 2007.