상피성장인자 [epidermal growth factor (EGF)]와 transforming 성장인자[transforming growth factor α (TGF α)]는 EGF family로서 다양한 세포에서 성장 촉진 인자로 알려져 있다. 또한, 배아 (embryo) 발달 시에 EGF와 EGF 수용체 (...
상피성장인자 [epidermal growth factor (EGF)]와 transforming 성장인자[transforming growth factor α (TGF α)]는 EGF family로서 다양한 세포에서 성장 촉진 인자로 알려져 있다. 또한, 배아 (embryo) 발달 시에 EGF와 EGF 수용체 (receptor)가 높게 발현됨으로써 초기배아발달에 중요한 역할을 하게 된다. ATP는 신경전달물질로서 생물학적 작용을 할 뿐만 아니라 세포 증식과 분화에 중요한 역할을 하는 것으로 알려져 있다. 배아발달에 있어서 중요한 역할을 하는 것으로 알려진 hedgehog는 최근 배아와 성체줄기세포의 자가 증식 (self-renewal)과 분화에 관여를 하는 것으로 알려져 있다. 하지만, 배아줄기세포 [embryonic stem (ES) cells]의 기능조절에 있어서 EGF family, ATP, 그리고 hedgehog의 영향에 대해서는 아직까지 명확하게 규명되지 않았기에, 본 연구에서는 이들 성장인자와 신경전달물질이 마우스 ES 세포의 포도당 운반체 활성과 세포 증식에 미치는 영향 및 이와 관련된 신호전달기전을 알아보고자 하였다. 1. 마우스 ES 세포에서 EGF는 시간과 농도에 의존적으로 (〉12 hr, 〉10 ng/ml) [³H]-2-deoxyglucose (2-DG) uptake를 증가시켰으며, 포도당 운반체 [glucose transporter 1 (GLUT1)]의 mRNA 와 단백질의 발현을 증가시켰다. Actinomycin D와 cycloheximide는 EGF의 2-DG uptake에 대한 영향을 억제하였다. 또한, EGF에 의한 2-DG uptake 증가는 AG 1478 (EGF receptor tyrosine kinase 억제제)과 genistein, herbimycin A (tyrosine kinase 억제제)의 전처리에 의해 차단되었으며, neomycin, U 73122 [phospholipase C (PLC) 억제제]와 staurosporine, bisindolylmaleimide I [protein kinase C (PKC) 억제제]에 의해서도 EGF의 효과는 억제되었다. EGF는 inositol phosphates (IPs)의 세포 내 형성을 촉진하였으며, 세포질 (cytosol)에서 세포막 (membrane fraction)으로 PKC의 이동을 유도하였다. EGF의 2-DG uptake에 대한 효과는 SB 203580 [p38 mitogen activated protein kinase (MAPK) 억제제]와 PD 98059 (p44/42 MAPKs 억제제)에 의해 억제되었으며, EGF는 또한 p38 MAPK와 p44/42 MAPKs의 인산화 (phosphorylation)를 증가시켰으며, 이는 genistein과 bisindolylmaleimide I의 전처리에 의해 각각 억제되었다. 2. EGF는 시간 및 농도 (〉12 hr, 〉50 ng/ml) 의존적으로 DNA 합성의 지표인 [³H] thymidine과 BrdU incorporation을 증가시켰다. EGF는 또한 EGF receptor의 인산화를 촉진하였으며, AG 1478과 herbimycin A에 의한 EGF receptor의 억제, neomycin과 U 73122에 의한 PLC의 억제 및 bisindolylmaleimide I과 staurosporine에 의한 PKC의 억제, 그리고 nefedifine과 methoxyverapamil에 의한 L-type Ca²^(+) channel의 억제는 EGF에 의한 [³H] thymidine incorporation의 증가를 감소시켰다. 이들 결과는 EGF에 의한 PKC α, β_(I), γ, δ, ζ의 translocation에 의해 뒷받침 되었다. 또한, EGF는 세포내 Ca²^(+)의 유입을 증가시켰으며, 이는 EGTA (extracellular Ca²^(+) chelator), BAPTA-AM (intracellular Ca²^(+) chelator), L-type Ca²^(+) channel 억제제, tyrosine kinase 억제제, PLC 및 PKC 억제제에 의해 차단되었다. EGF에 의해 증가된 세포내 inositol phosphates의 형성이 EGF receptor tyrosine kinase 억제제에 의해 감소되었다. EGF는 또한 세포내 H₂O₂의 생성을 증가시켰으며, 이는 EGF에 의한 세포내 Ca²^(+)의 유입에 영향을 미치는 것으로 보였다. p44/42 MAPKs의 차단은 [³H] thymidine incorporation의 증가를 억제하였으나, p38 MAPK의 차단은 영향을 미치지 않았다. 또한, EGF receptor tyrosine kinase, PKC, Ca²^(+) channel, p44/42 MAPKs의 차단은 EGF에 의한 cyclin D1, cyclin E, cyclin-dependent kinase 2 (CDK 2), 그리고 CDK 4의 발현을 억제하였다. 3. TGF α는 시간과 (0 - 72 hr) 농도 (0 - 10 ng/ml) 의존적으로 [³H] thymidine과 BrdU incorporation을 증가시켰다. TGF α는 Akt, mTOR (Mammalian Target Of Rapamycin), p70S6K1 및 p44/42 MAPKs의 인산화를 촉진시켰으며, Notch와 Hes-1 그리고 Wnt1의 단백질 발현을 증가시켰다. TGF α에 의한 [³H] thymidine incorporation의 증가는 Akt 억제제 (10^(-5) M), rapamycin (mTOR 억제제, 5 × 10^(-9) M), PD 98059 (10^(-5) M), L-685,458 (Notch 억제제, 10^(-5) M)에 의해 차단되었다. TGF α는 또한 protooncogenes인 c-jun, c-myc, c-fos의 gene 발현을 증가시켰으며, 이는 LY 294002 [phosphatidylinositol 3-kinase (PI3-K) 억제제, 10^(-6) M]과 PD 98059의 전처리에 의해 발현이 억제되었다. 유세포분석기 [Fluorescence activated cell sorter (FACS)]를 통해 TGF α가 S phase에서의 세포내 군집을 증가시켰고, 이는 LY 294002와 PD 98059에 의해 억제됨을 확인하였다. TGF α는 cyclin D1/CDK 4, cyclin E/CDK 2의 단백질 수준을 증가시켰으나, 반면에 p21^(cip1/waf1)과 p27^(kip1)의 발현은 감소시켰다. 이들은 각각 Akt, mTOR, Notch의 차단에 의해 억제되었다. 4. ATP는 시간 및 농도에 의존적으로 [³H] thymidine과 BrdU incorporation을 증가시켰고, AMP-CPP (P2X1 and P2X3 효능제), ATP-γS (P2Y 효능제) 및 2-MesATP (P2X and P2Y 효능제) 또한 [³H] thymidine incorporation을 증가시켰다. 반면에, P2 purinoceptor 길항제인 suramin과 reactive blue 2 (RB2)는 ATP에 의한 [³H] thymidine incorporation 증가를 억제하였다. RT-PCR를 통해 마우스 ES 세포에서 P2X3, P2X4, P2Y1 및 P2Y2의 gene 발현을 관찰하였다. SQ 22536 (adenylate cyclase 억제제), neomycin과 U 73122, bisindolylmaleimide I과 staurosporine의 전처리 시 ATP에 의한 세포성장 효과를 억제하였다. ATP는 세포내 cAMP와 inositol phosphates를 증가시켰고, PKC α, δ, ζ의 활성을 유도하였다. 또한, ATP와 ATP 효능제는 세포내 Ca²^(+)의 유입을 증가시켰으며, EGTA와 BAPTA-AM의 병합처리는 ATP에 의한 [³H] thymidine incorporation의 증가를 억제시켰다. ATP는 Akt와 p44/42 MAPKs를 시간 의존적으로 인산화 시켰으며, suramin, RB2, wortmannin (PI3-K 억제제) 및 Akt 억제제는 p44/42 MAPKs의 인산화를 억제시켰다. 또한, ATP에 의한 [³H] thymidine incorporation의 증가는 wortmannin, Akt 억제제, PD 98059에 의해 억제되었고, ATP에 의해 증가된 cyclin D1/CDK 4, cyclin E/CDK 2의 단백질 수준은 suramin, RB2, PD 98059 및 wortmannin에 의해 감소되었다. 5. Sonic hedgehog (Shh)은 농도 의존적으로 [³H] thymidine incorporation을 증가시켰고, 이는 Smoothened 수용체 억제제인 cyclopamine에 의해 억제되었다. Shh은 세포 내 칼슘 농도를 증가시켰고, PKC α, δ, ζ의 활성을 유도하였다. 또한 이들의 활성은 Gli1의 유전자 발현을 증가시켰다. Shh은 EGF 수용체의 활성을 증가시켰으며, 이는 matrix metalloproteinase (MMP) 억제제에 의해 차단되었다. NF-κB의 활성이 유도되었으며, 이는 PKC와 EGF receptor tyrosine kinase 억제제에 의해 감소되었다. Shh은 또한 Notch/Hes-1, Wnt1/β-catenin 의 단백질 수준을 증가시켰고, siRNA에 의한 Gli1 유전자의 억제는 Shh의 효과를 차단하였다. Shh은 cyclin D1/CDK 4, cyclin E/CDK 2의 단백질 수준을 증가시켰으나, p21^(cip1/waf1)과 p27^(kip1)의 발현은 감소시켰다. 이들 단백질에 대한 Shh의 효과는 각각 PKC, EGF receptor tyrosine kinase, NF-κB을 억제하였을 때, 그리고 Gli1 유전자의 억제에 의해 차단되었다. 즉, 이상의 결과를 요약하면, 마우스 배아줄기세포에서 1) EGF는 PKC, p38 MAPK 및 p44/42 MAPKs를 통해 2-DG uptake를 증가시켰으며, 2) EGF receptor tyrosine kinase를 통한 PLC/PKC 활성, 세포내 Ca²^(+)의 유입 그리고 p44/42 MAPKs의 활성에 의해 세포의 증식을 증가시켰다. 3) TGF α는 PI3-K/Akt, p44/42 MAPKs 및 Notch를 통해 세포의 DNA 합성을 증가시켰으며, 4) ATP는 P2 수용체를 통한 PKC, PI3-K/Akt 및 MAPKs의 활성에 의해 세포 증식을 촉진하였다. 또한, 5) Shh은 PKC와 EGF 수용체를 통한 NF-κB의 활성과 Gli의 증가를 통해 세포 증식을 촉진하였다.
상피성장인자 [epidermal growth factor (EGF)]와 transforming 성장인자[transforming growth factor α (TGF α)]는 EGF family로서 다양한 세포에서 성장 촉진 인자로 알려져 있다. 또한, 배아 (embryo) 발달 시에 EGF와 EGF 수용체 (receptor)가 높게 발현됨으로써 초기배아발달에 중요한 역할을 하게 된다. ATP는 신경전달물질로서 생물학적 작용을 할 뿐만 아니라 세포 증식과 분화에 중요한 역할을 하는 것으로 알려져 있다. 배아발달에 있어서 중요한 역할을 하는 것으로 알려진 hedgehog는 최근 배아와 성체줄기세포의 자가 증식 (self-renewal)과 분화에 관여를 하는 것으로 알려져 있다. 하지만, 배아줄기세포 [embryonic stem (ES) cells]의 기능조절에 있어서 EGF family, ATP, 그리고 hedgehog의 영향에 대해서는 아직까지 명확하게 규명되지 않았기에, 본 연구에서는 이들 성장인자와 신경전달물질이 마우스 ES 세포의 포도당 운반체 활성과 세포 증식에 미치는 영향 및 이와 관련된 신호전달기전을 알아보고자 하였다. 1. 마우스 ES 세포에서 EGF는 시간과 농도에 의존적으로 (〉12 hr, 〉10 ng/ml) [³H]-2-deoxyglucose (2-DG) uptake를 증가시켰으며, 포도당 운반체 [glucose transporter 1 (GLUT1)]의 mRNA 와 단백질의 발현을 증가시켰다. Actinomycin D와 cycloheximide는 EGF의 2-DG uptake에 대한 영향을 억제하였다. 또한, EGF에 의한 2-DG uptake 증가는 AG 1478 (EGF receptor tyrosine kinase 억제제)과 genistein, herbimycin A (tyrosine kinase 억제제)의 전처리에 의해 차단되었으며, neomycin, U 73122 [phospholipase C (PLC) 억제제]와 staurosporine, bisindolylmaleimide I [protein kinase C (PKC) 억제제]에 의해서도 EGF의 효과는 억제되었다. EGF는 inositol phosphates (IPs)의 세포 내 형성을 촉진하였으며, 세포질 (cytosol)에서 세포막 (membrane fraction)으로 PKC의 이동을 유도하였다. EGF의 2-DG uptake에 대한 효과는 SB 203580 [p38 mitogen activated protein kinase (MAPK) 억제제]와 PD 98059 (p44/42 MAPKs 억제제)에 의해 억제되었으며, EGF는 또한 p38 MAPK와 p44/42 MAPKs의 인산화 (phosphorylation)를 증가시켰으며, 이는 genistein과 bisindolylmaleimide I의 전처리에 의해 각각 억제되었다. 2. EGF는 시간 및 농도 (〉12 hr, 〉50 ng/ml) 의존적으로 DNA 합성의 지표인 [³H] thymidine과 BrdU incorporation을 증가시켰다. EGF는 또한 EGF receptor의 인산화를 촉진하였으며, AG 1478과 herbimycin A에 의한 EGF receptor의 억제, neomycin과 U 73122에 의한 PLC의 억제 및 bisindolylmaleimide I과 staurosporine에 의한 PKC의 억제, 그리고 nefedifine과 methoxyverapamil에 의한 L-type Ca²^(+) channel의 억제는 EGF에 의한 [³H] thymidine incorporation의 증가를 감소시켰다. 이들 결과는 EGF에 의한 PKC α, β_(I), γ, δ, ζ의 translocation에 의해 뒷받침 되었다. 또한, EGF는 세포내 Ca²^(+)의 유입을 증가시켰으며, 이는 EGTA (extracellular Ca²^(+) chelator), BAPTA-AM (intracellular Ca²^(+) chelator), L-type Ca²^(+) channel 억제제, tyrosine kinase 억제제, PLC 및 PKC 억제제에 의해 차단되었다. EGF에 의해 증가된 세포내 inositol phosphates의 형성이 EGF receptor tyrosine kinase 억제제에 의해 감소되었다. EGF는 또한 세포내 H₂O₂의 생성을 증가시켰으며, 이는 EGF에 의한 세포내 Ca²^(+)의 유입에 영향을 미치는 것으로 보였다. p44/42 MAPKs의 차단은 [³H] thymidine incorporation의 증가를 억제하였으나, p38 MAPK의 차단은 영향을 미치지 않았다. 또한, EGF receptor tyrosine kinase, PKC, Ca²^(+) channel, p44/42 MAPKs의 차단은 EGF에 의한 cyclin D1, cyclin E, cyclin-dependent kinase 2 (CDK 2), 그리고 CDK 4의 발현을 억제하였다. 3. TGF α는 시간과 (0 - 72 hr) 농도 (0 - 10 ng/ml) 의존적으로 [³H] thymidine과 BrdU incorporation을 증가시켰다. TGF α는 Akt, mTOR (Mammalian Target Of Rapamycin), p70S6K1 및 p44/42 MAPKs의 인산화를 촉진시켰으며, Notch와 Hes-1 그리고 Wnt1의 단백질 발현을 증가시켰다. TGF α에 의한 [³H] thymidine incorporation의 증가는 Akt 억제제 (10^(-5) M), rapamycin (mTOR 억제제, 5 × 10^(-9) M), PD 98059 (10^(-5) M), L-685,458 (Notch 억제제, 10^(-5) M)에 의해 차단되었다. TGF α는 또한 protooncogenes인 c-jun, c-myc, c-fos의 gene 발현을 증가시켰으며, 이는 LY 294002 [phosphatidylinositol 3-kinase (PI3-K) 억제제, 10^(-6) M]과 PD 98059의 전처리에 의해 발현이 억제되었다. 유세포분석기 [Fluorescence activated cell sorter (FACS)]를 통해 TGF α가 S phase에서의 세포내 군집을 증가시켰고, 이는 LY 294002와 PD 98059에 의해 억제됨을 확인하였다. TGF α는 cyclin D1/CDK 4, cyclin E/CDK 2의 단백질 수준을 증가시켰으나, 반면에 p21^(cip1/waf1)과 p27^(kip1)의 발현은 감소시켰다. 이들은 각각 Akt, mTOR, Notch의 차단에 의해 억제되었다. 4. ATP는 시간 및 농도에 의존적으로 [³H] thymidine과 BrdU incorporation을 증가시켰고, AMP-CPP (P2X1 and P2X3 효능제), ATP-γS (P2Y 효능제) 및 2-MesATP (P2X and P2Y 효능제) 또한 [³H] thymidine incorporation을 증가시켰다. 반면에, P2 purinoceptor 길항제인 suramin과 reactive blue 2 (RB2)는 ATP에 의한 [³H] thymidine incorporation 증가를 억제하였다. RT-PCR를 통해 마우스 ES 세포에서 P2X3, P2X4, P2Y1 및 P2Y2의 gene 발현을 관찰하였다. SQ 22536 (adenylate cyclase 억제제), neomycin과 U 73122, bisindolylmaleimide I과 staurosporine의 전처리 시 ATP에 의한 세포성장 효과를 억제하였다. ATP는 세포내 cAMP와 inositol phosphates를 증가시켰고, PKC α, δ, ζ의 활성을 유도하였다. 또한, ATP와 ATP 효능제는 세포내 Ca²^(+)의 유입을 증가시켰으며, EGTA와 BAPTA-AM의 병합처리는 ATP에 의한 [³H] thymidine incorporation의 증가를 억제시켰다. ATP는 Akt와 p44/42 MAPKs를 시간 의존적으로 인산화 시켰으며, suramin, RB2, wortmannin (PI3-K 억제제) 및 Akt 억제제는 p44/42 MAPKs의 인산화를 억제시켰다. 또한, ATP에 의한 [³H] thymidine incorporation의 증가는 wortmannin, Akt 억제제, PD 98059에 의해 억제되었고, ATP에 의해 증가된 cyclin D1/CDK 4, cyclin E/CDK 2의 단백질 수준은 suramin, RB2, PD 98059 및 wortmannin에 의해 감소되었다. 5. Sonic hedgehog (Shh)은 농도 의존적으로 [³H] thymidine incorporation을 증가시켰고, 이는 Smoothened 수용체 억제제인 cyclopamine에 의해 억제되었다. Shh은 세포 내 칼슘 농도를 증가시켰고, PKC α, δ, ζ의 활성을 유도하였다. 또한 이들의 활성은 Gli1의 유전자 발현을 증가시켰다. Shh은 EGF 수용체의 활성을 증가시켰으며, 이는 matrix metalloproteinase (MMP) 억제제에 의해 차단되었다. NF-κB의 활성이 유도되었으며, 이는 PKC와 EGF receptor tyrosine kinase 억제제에 의해 감소되었다. Shh은 또한 Notch/Hes-1, Wnt1/β-catenin 의 단백질 수준을 증가시켰고, siRNA에 의한 Gli1 유전자의 억제는 Shh의 효과를 차단하였다. Shh은 cyclin D1/CDK 4, cyclin E/CDK 2의 단백질 수준을 증가시켰으나, p21^(cip1/waf1)과 p27^(kip1)의 발현은 감소시켰다. 이들 단백질에 대한 Shh의 효과는 각각 PKC, EGF receptor tyrosine kinase, NF-κB을 억제하였을 때, 그리고 Gli1 유전자의 억제에 의해 차단되었다. 즉, 이상의 결과를 요약하면, 마우스 배아줄기세포에서 1) EGF는 PKC, p38 MAPK 및 p44/42 MAPKs를 통해 2-DG uptake를 증가시켰으며, 2) EGF receptor tyrosine kinase를 통한 PLC/PKC 활성, 세포내 Ca²^(+)의 유입 그리고 p44/42 MAPKs의 활성에 의해 세포의 증식을 증가시켰다. 3) TGF α는 PI3-K/Akt, p44/42 MAPKs 및 Notch를 통해 세포의 DNA 합성을 증가시켰으며, 4) ATP는 P2 수용체를 통한 PKC, PI3-K/Akt 및 MAPKs의 활성에 의해 세포 증식을 촉진하였다. 또한, 5) Shh은 PKC와 EGF 수용체를 통한 NF-κB의 활성과 Gli의 증가를 통해 세포 증식을 촉진하였다.
Epidermal growth factor (EGF) and transforming growth factor α (TGF α), one of EGF family are growth-promoting factors in various cell types. It has been known that EGF and EGF receptor are highly expressed in embryo, suggesting that the EGF system is related to early embryo development in an autocr...
Epidermal growth factor (EGF) and transforming growth factor α (TGF α), one of EGF family are growth-promoting factors in various cell types. It has been known that EGF and EGF receptor are highly expressed in embryo, suggesting that the EGF system is related to early embryo development in an autocrine and/or paracrine manner. Embryonic stem (ES) cells are powerful tools for developmental biology and have been shown to respond to EGF. Adenosine 5▽ triphosphate (ATP) is potent signaling molecule that plays important biological roles in short-term signaling events during neurotransmission and long-term signaling participating in regulation of proliferation, survival, differentiation, as well as cellular death in various cell types. Sonic Hedgehog (Shh) signaling also plays a crucial role in cell survival, proliferation, differentiation, and patterning in embryonic development. However, the mechanism of EGF family, ATP, and Hedgehog (Hh) signaling in maintaining stemness and proliferation of embryonic stem cells is poorly understood. Thus, this study investigated the effect of these growth factors on self renewal and proliferation of mouse embryonic stem (ES) cells and their related signal pathways. 1. EGF significantly increased [³H]-2-deoxyglucose (2-DG) uptake in a time- and dose-dependent manner (>12 hr, >10 ng/ml) and increased mRNA and protein level of glucose transporter 1 (GLUT1) compared to control, respectively. Actinomycin D (a transcription inhibitor) and cycloheximide (a translation inhibitor) completely blocked the effect of EGF on 2-DG uptake. EGF-induced increase of 2-DG uptake was blocked by AG 1478 (an EGF receptor tyrosine kinase blocker), genistein or herbimycin (tyrosine kinase inhibitors). In addition, EGF effect was blocked by neomycin and U 73122 [phospholipase C (PLC) inhibitors] as well as staurosporine and bisindolylmaleimide I [protein kinase C (PKC) inhibitors]. EGF was also observed to increase inositol phosphates (IPs) formation and activate a PKC translocation from the cytosolic to membrane fraction, suggesting a role of PLC and PKC. SB 203580 [a p38 mitogen activated protein kinase (MAPK) inhibitor] or PD 98059 (a MEK inhibitor) blocked EGF-induced increase of 2-DG uptake. EGF also increased phosphorylation of p38 MAPK and p44/42 MAPKs, which was blocked by genistein or bisindolylmaleimide I, respectively. 2. EGF increased [³H] thymidine and BrdU incorporation in a time- and dose-dependent manner. EGF stimulated the phosphorylation of EGF receptor (EGFR). In this study, inhibition of EGFR tyrosine kinase using AG 1478 or herbimycin A, inhibition of phospholipase C using neomycin or U 73122, inhibition of protein kinase C using bisindolylmaleimide I or staurosporine, and inhibition of L-type Ca²^(+) channel using nifedifine or methyoxyverapamil prevented EGF-induced [³H] thymidine incorporation. PKC α, β_(I), γ, δ, and ξ were translocated to the membrane in response to EGF. EGF also increased [Ca²^(+)]_(i), which was blocked by EGTA (an extracellular Ca²^(+) chelating agent) and BAPTA-AM (an intracellular Ca²^(+) mobilization blocker). EGF-induced increase of [Ca²^(+)]_(i) was attenuated by inhibition of the L-type Ca²^(+) channel. Moreover, inhibition of EGFR tyrosine kinase, PLC, and PKC completely prevented EGF-induced increases in [Ca²^(+)]_(i). Inositol phosphates levels were also increased by EGF and these increases were blocked by EGFR tyrosine kinase inhibitors. Furthermore, EGF rapidly increased formation of H₂O₂, and the pretreatment of antioxidant (NAC) inhibited EGF-induced increase of [Ca²^(+)]_(i). In addition, EGF increased p44/42 MAPKs phosphorylation, but not p38 MAPK phosphorylation. Inhibition of EGFR tyrosine kinase, PLC, PKC, or Ca²^(+) channel blocked EGF-induced phosphorylation of p44/42 MAPKs. Inhibition of p44/42 MAPKs using PD 98059 (a MEK inhibitor) attenuated EGF-induced increase of [³H] thymidine incorporation, but not SB 203580 (a p38 MAPK inhibitor). Finally, inhibition of EGFR tyrosine kinase, PKC, L-type Ca²^(+) channel, or p44/42 MAPKs attenuated EGF-stimulated cyclin D1, cyclin E, cyclin-dependent kinase 2 (CDK 2), and CDK 4, respectively. 3. TGF α stimulated [³H] thymidine and BrdU incorporation, which showed the increase of DNA synthesis in a time (≥ 6 hr) and dose (≥ 0.5 ng/ml)-dependent manner. Western blot analysis demonstrated that TGF α stimulated the phosphorylation of Akt, mTOR (a Mammalian Target Of Rapamycin), p70S6K1, and p44/42 MAPKs. TGF α also increased the protein levels of Notch, Hes-1, and Wnt1. On the other hand, TGF α-induced DNA synthesis was blocked by pretreatment of Akt inhibitor (10^(-5) M), rapamycin (a mTOR inhibitor, 5 × 10^(-9) M), PD 98059 (a MEK inhibitor, 10^(-5) M), and L-685,458 (a Notch inhibitor, 10^(-5) M). TGF α also increased gene expressions of protooncogenes (c-jun, c-myc, and c-fos), which were blocked by LY 294002 [phosphatidylinositol 3-kinase (PI3-K) inhibitor, 10^(-6) M] and PD 98059. Fluorescence activated cell sorter (FACS) analysis demonstrated TGF α promoted G1/S phase progression, which was blocked by LY 294002 and PD 98059. TGF α also increased cyclin D/CDK 4 and cyclin E/CDK 2 levels while decreased p21^(cip1/waf1) and p27^(kip1), which were blocked by inhibition of Akt, mTOR, and Notch signals. 4. ATP increased the level of [³H] thymidine/BrdU incorporation and the number of cells in both a time (≥ 4 hr) and dose (≥ 10^(-6) M)-dependent manner. AMP-CPP (a P2X1 and P2X3 agonist), ATP-γS (a P2Y agonist), and 2-MesATP (a P2X and P2Y agonist) stimulated [³H] thymidine incorporation. Suramin and reactive blue 2 (RB 2) (P2 purinoceptor antagonists) inhibited the ATP-induced increase in [³H] thymidine incorporation. RT-PCR analysis revealed P2X3, P2X4, P2Y1, and P2Y2 expression in mouse ES cells. Adenylate cyclase inhibitor (SQ 22536), PLC inhibitors (neomycin or U 73122), and PKC inhibitors (bisindolylmaleimide I or staurosporine) inhibited the ATP-induced increase in [³H] thymidine incorporation. ATP increased the level of intracellular cAMP and inositol phosphates. ATP translocated PKC α, δ, and ξ from the cytosol to the membrane compartment. ATP and its agonists increased [Ca²^(+)]_(i). In addition, the ATP-induced increase in [³H] thymidine incorporation was completely inhibited by a combination of EGTA (an extracellular Ca²^(+) chelating agent) and BAPTA-AM (an intracellular Ca²^(+) mobilization blocker). ATP phosphorylated Akt and p44/42 MAPKs in a time-dependent manner, and either suramin or RB 2 blocked the ATP-induced phosphorylation of Akt. Suramin, RB 2, wortmannin (a PI3K inhibitor), or the Akt inhibitor inhibited the phosphorylation of p44/42 MAPKs. The ATP-induced increase in [³H] thymidine incorporation was inhibited by wortmannin, the Akt inhibitor, and PD 98059 (a MEK inhibitor). Suramin, RB 2, PD 98059, and wortmannin blocked the ATP-induced increase in the cyclin D1, cyclin E, CDK2, and CDK4 levels. 5. Sonic Hedgehog (Shh) signaling plays an important role in cell survival, proliferation, differentiation, and patterning in embryonic development. However, the mechanism of Hedgehog (Hh) signaling in maintaining the stemness and proliferation of embryonic stem cells is not completely understood. Therefore, this study examined the effect of sonic hedgehog (Shh) on the self-renewal of mouse embryonic stem (ES) cells. Shh increased DNA synthesis blocked by the inhibition of the smoothened (Smo) 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. This shows the Shh-induced increase in the Gli1 mRNA level requires [Ca²^(+)]_(i) and PKC. Shh increased the phosphorylation of EGF receptor (EGFR), which is blocked by the matrix metalloproteinase (MMP) inhibitor. Subsequently, Shh increased the 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 CDK (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 siRNA, Finally, Shh-induced the progression of the G1/S phase was blocked by the inhibition of PKC and EGFR tyrosine kinase. In conclusion, 1) EGF partially increased 2-DG uptake via PKC and p38 MAPK and p44/42 MAPKs, 2) EGF stimulates proliferation via PLC/PKC, Ca²^(+) influx, and p44/42 MAPKs signal pathways through EGF receptor tyrosine kinase phosphorylation, 3) TGF α regulates DNA synthesis via PI3-K/Akt, p44/42 MAPKs, and Notch pathways, 4) ATP stimulates proliferation through PKC, PI3K/Akt, and MAPKs via the P2 purinoceptors, and 5) Shh signaling regulates proliferation, which is mediated by Ca²^(+)/PKC as well as EGF receptor and Gli activation in mouse ES cells.
Epidermal growth factor (EGF) and transforming growth factor α (TGF α), one of EGF family are growth-promoting factors in various cell types. It has been known that EGF and EGF receptor are highly expressed in embryo, suggesting that the EGF system is related to early embryo development in an autocrine and/or paracrine manner. Embryonic stem (ES) cells are powerful tools for developmental biology and have been shown to respond to EGF. Adenosine 5▽ triphosphate (ATP) is potent signaling molecule that plays important biological roles in short-term signaling events during neurotransmission and long-term signaling participating in regulation of proliferation, survival, differentiation, as well as cellular death in various cell types. Sonic Hedgehog (Shh) signaling also plays a crucial role in cell survival, proliferation, differentiation, and patterning in embryonic development. However, the mechanism of EGF family, ATP, and Hedgehog (Hh) signaling in maintaining stemness and proliferation of embryonic stem cells is poorly understood. Thus, this study investigated the effect of these growth factors on self renewal and proliferation of mouse embryonic stem (ES) cells and their related signal pathways. 1. EGF significantly increased [³H]-2-deoxyglucose (2-DG) uptake in a time- and dose-dependent manner (>12 hr, >10 ng/ml) and increased mRNA and protein level of glucose transporter 1 (GLUT1) compared to control, respectively. Actinomycin D (a transcription inhibitor) and cycloheximide (a translation inhibitor) completely blocked the effect of EGF on 2-DG uptake. EGF-induced increase of 2-DG uptake was blocked by AG 1478 (an EGF receptor tyrosine kinase blocker), genistein or herbimycin (tyrosine kinase inhibitors). In addition, EGF effect was blocked by neomycin and U 73122 [phospholipase C (PLC) inhibitors] as well as staurosporine and bisindolylmaleimide I [protein kinase C (PKC) inhibitors]. EGF was also observed to increase inositol phosphates (IPs) formation and activate a PKC translocation from the cytosolic to membrane fraction, suggesting a role of PLC and PKC. SB 203580 [a p38 mitogen activated protein kinase (MAPK) inhibitor] or PD 98059 (a MEK inhibitor) blocked EGF-induced increase of 2-DG uptake. EGF also increased phosphorylation of p38 MAPK and p44/42 MAPKs, which was blocked by genistein or bisindolylmaleimide I, respectively. 2. EGF increased [³H] thymidine and BrdU incorporation in a time- and dose-dependent manner. EGF stimulated the phosphorylation of EGF receptor (EGFR). In this study, inhibition of EGFR tyrosine kinase using AG 1478 or herbimycin A, inhibition of phospholipase C using neomycin or U 73122, inhibition of protein kinase C using bisindolylmaleimide I or staurosporine, and inhibition of L-type Ca²^(+) channel using nifedifine or methyoxyverapamil prevented EGF-induced [³H] thymidine incorporation. PKC α, β_(I), γ, δ, and ξ were translocated to the membrane in response to EGF. EGF also increased [Ca²^(+)]_(i), which was blocked by EGTA (an extracellular Ca²^(+) chelating agent) and BAPTA-AM (an intracellular Ca²^(+) mobilization blocker). EGF-induced increase of [Ca²^(+)]_(i) was attenuated by inhibition of the L-type Ca²^(+) channel. Moreover, inhibition of EGFR tyrosine kinase, PLC, and PKC completely prevented EGF-induced increases in [Ca²^(+)]_(i). Inositol phosphates levels were also increased by EGF and these increases were blocked by EGFR tyrosine kinase inhibitors. Furthermore, EGF rapidly increased formation of H₂O₂, and the pretreatment of antioxidant (NAC) inhibited EGF-induced increase of [Ca²^(+)]_(i). In addition, EGF increased p44/42 MAPKs phosphorylation, but not p38 MAPK phosphorylation. Inhibition of EGFR tyrosine kinase, PLC, PKC, or Ca²^(+) channel blocked EGF-induced phosphorylation of p44/42 MAPKs. Inhibition of p44/42 MAPKs using PD 98059 (a MEK inhibitor) attenuated EGF-induced increase of [³H] thymidine incorporation, but not SB 203580 (a p38 MAPK inhibitor). Finally, inhibition of EGFR tyrosine kinase, PKC, L-type Ca²^(+) channel, or p44/42 MAPKs attenuated EGF-stimulated cyclin D1, cyclin E, cyclin-dependent kinase 2 (CDK 2), and CDK 4, respectively. 3. TGF α stimulated [³H] thymidine and BrdU incorporation, which showed the increase of DNA synthesis in a time (≥ 6 hr) and dose (≥ 0.5 ng/ml)-dependent manner. Western blot analysis demonstrated that TGF α stimulated the phosphorylation of Akt, mTOR (a Mammalian Target Of Rapamycin), p70S6K1, and p44/42 MAPKs. TGF α also increased the protein levels of Notch, Hes-1, and Wnt1. On the other hand, TGF α-induced DNA synthesis was blocked by pretreatment of Akt inhibitor (10^(-5) M), rapamycin (a mTOR inhibitor, 5 × 10^(-9) M), PD 98059 (a MEK inhibitor, 10^(-5) M), and L-685,458 (a Notch inhibitor, 10^(-5) M). TGF α also increased gene expressions of protooncogenes (c-jun, c-myc, and c-fos), which were blocked by LY 294002 [phosphatidylinositol 3-kinase (PI3-K) inhibitor, 10^(-6) M] and PD 98059. Fluorescence activated cell sorter (FACS) analysis demonstrated TGF α promoted G1/S phase progression, which was blocked by LY 294002 and PD 98059. TGF α also increased cyclin D/CDK 4 and cyclin E/CDK 2 levels while decreased p21^(cip1/waf1) and p27^(kip1), which were blocked by inhibition of Akt, mTOR, and Notch signals. 4. ATP increased the level of [³H] thymidine/BrdU incorporation and the number of cells in both a time (≥ 4 hr) and dose (≥ 10^(-6) M)-dependent manner. AMP-CPP (a P2X1 and P2X3 agonist), ATP-γS (a P2Y agonist), and 2-MesATP (a P2X and P2Y agonist) stimulated [³H] thymidine incorporation. Suramin and reactive blue 2 (RB 2) (P2 purinoceptor antagonists) inhibited the ATP-induced increase in [³H] thymidine incorporation. RT-PCR analysis revealed P2X3, P2X4, P2Y1, and P2Y2 expression in mouse ES cells. Adenylate cyclase inhibitor (SQ 22536), PLC inhibitors (neomycin or U 73122), and PKC inhibitors (bisindolylmaleimide I or staurosporine) inhibited the ATP-induced increase in [³H] thymidine incorporation. ATP increased the level of intracellular cAMP and inositol phosphates. ATP translocated PKC α, δ, and ξ from the cytosol to the membrane compartment. ATP and its agonists increased [Ca²^(+)]_(i). In addition, the ATP-induced increase in [³H] thymidine incorporation was completely inhibited by a combination of EGTA (an extracellular Ca²^(+) chelating agent) and BAPTA-AM (an intracellular Ca²^(+) mobilization blocker). ATP phosphorylated Akt and p44/42 MAPKs in a time-dependent manner, and either suramin or RB 2 blocked the ATP-induced phosphorylation of Akt. Suramin, RB 2, wortmannin (a PI3K inhibitor), or the Akt inhibitor inhibited the phosphorylation of p44/42 MAPKs. The ATP-induced increase in [³H] thymidine incorporation was inhibited by wortmannin, the Akt inhibitor, and PD 98059 (a MEK inhibitor). Suramin, RB 2, PD 98059, and wortmannin blocked the ATP-induced increase in the cyclin D1, cyclin E, CDK2, and CDK4 levels. 5. Sonic Hedgehog (Shh) signaling plays an important role in cell survival, proliferation, differentiation, and patterning in embryonic development. However, the mechanism of Hedgehog (Hh) signaling in maintaining the stemness and proliferation of embryonic stem cells is not completely understood. Therefore, this study examined the effect of sonic hedgehog (Shh) on the self-renewal of mouse embryonic stem (ES) cells. Shh increased DNA synthesis blocked by the inhibition of the smoothened (Smo) 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. This shows the Shh-induced increase in the Gli1 mRNA level requires [Ca²^(+)]_(i) and PKC. Shh increased the phosphorylation of EGF receptor (EGFR), which is blocked by the matrix metalloproteinase (MMP) inhibitor. Subsequently, Shh increased the 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 CDK (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 siRNA, Finally, Shh-induced the progression of the G1/S phase was blocked by the inhibition of PKC and EGFR tyrosine kinase. In conclusion, 1) EGF partially increased 2-DG uptake via PKC and p38 MAPK and p44/42 MAPKs, 2) EGF stimulates proliferation via PLC/PKC, Ca²^(+) influx, and p44/42 MAPKs signal pathways through EGF receptor tyrosine kinase phosphorylation, 3) TGF α regulates DNA synthesis via PI3-K/Akt, p44/42 MAPKs, and Notch pathways, 4) ATP stimulates proliferation through PKC, PI3K/Akt, and MAPKs via the P2 purinoceptors, and 5) Shh signaling regulates proliferation, which is mediated by Ca²^(+)/PKC as well as EGF receptor and Gli activation in mouse ES cells.
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