Protective Effects of Metallothionein-III on Oxidative Stress-Induced Neuronal Cell Death
- Issued Date
- 2007
- Abstract
- Metallothionein-III protects against 6-hydroxydopamine-induced oxidative stress by increasing expression of heme oxygenase-1 in a PI3K and ERK/Nrf2 dependent manner
Parkinson's disease (PD) is a neurodegenerative movement disorder characterized by cell loss in the substantia nigra resulting in striatal dopamine depletion. Enhanced oxidative stress is implicated in the pathogenesis of PD. One mechanism by which cells defend themselves against oxidative stress is through the transcriptional upregulation of cytoprotective genes. Under oxidative stress conditions, the transcription factor NF-E2-related factor (Nrf2) binds to the antioxidant response element (ARE) to induce antioxidant and phase II detoxification enzymes, such as heme oxygenase-1 (HO-1). The catecholaminergic neurotoxin 6-hydroxydopamine (6-OHDA) induces the production of reactive oxygen species (ROS), leading to neuronal cell death. Metallothioneins (MTs) are low molecular weight, metal-binding proteins with established antioxidant capabilities. The expression and induction of MTs have been associated with protection against DNA damage, oxidative stress and apoptosis. However, the underlying mechanism for their effects is unclear. The present study demonstrated that MT-III prevents the accumulation of ROS, caspase-3 activation, and subsequent cell death in dopaminergic SH-SY5Y cells challenged with the PD-related neurotoxin 6-OHDA. The mechanism involves phosphatidylinositol 3-kinase (PI3K)/Akt and ERK1/2-dependent induction of the stress response protein HO-1. Pretreatment of SH-SY5Y cells with MT-III significantly reduced 6-OHDA-induced generation of ROS, caspase-3 activation, and subsequent cell death. MT-III up-regulates HO-1 expression and this expression confers neuroprotection against oxidative injury induced by 6-OHDA. Moreover, MT-III induces Nrf2 nuclear translocation, which is upstream of MT-III-induced HO-1 expression, and PI3K/Akt and ERK1/2 activation, a pathway that is involved in induced Nrf2 nuclear translocation, HO-1 expression and neuroprotection. Taken together, these results suggest that MT-III augments the cellular antioxidant defense capacity by inducing HO-1 via the PI3K and ERK/Nrf2 signaling pathway, thereby protecting cells from oxidative stress. Collectively, these data provide evidence that MT-III prevents the degeneration of dopaminergic neurons by 6-OHDA and may prove useful in the treatment of PD.
- Alternative Author(s)
- Hwang Yong Pil
- Affiliation
- 조선대학교 약학대학
- Department
- 일반대학원 약학과
- Awarded Date
- 2008-02
- Table Of Contents
- Contents
Contents ---------------------------------------------------------------------------------------- i
List of Figures ------------------------------------------------------------------------------- vi
List of Abbreviations ---------------------------------------------------------------------- xii
Abstract
1. Neuroprotecion by metallothionein-III involved activation of
nuclear factor-??B through phosphatidylinositol-3 kinase signaling pathway ----- 1
2. Metallothionein-III protected against 6-hydroxydopamine-induced
oxidative stress by increasing expression of heme oxygenase-1 in the
PI3K and ERK/Nrf2 dependent manner ------------------------------------------------ 3
I. Introduction -------------------------------------------------------------------------------5
1. Neuroprotecion by metallothionein-III involved activation of
nuclear factor-??B through phosphatidylinositol-3 kinase signaling pathway ----- 5
2. Metallothionein-III protected against 6-hydroxydopamine-induced
oxidative stress by increasing expression of heme oxygenase-1 in the
PI3K and ERK/Nrf2 dependent manner ----------------------------------------------- 9
II. Materials & Methods ------------------------------------------------------------------15
1. Materials ---------------------------------------------------------------------------------- 15
2. Expression and purification of human MT-III---------------------------------------- 16
3. Cell culture and cell viability assay --------------------------------------------------- 16
4. ROS production -------------------------------------------------------------------------- 17
5. Electrophoretic mobility shift assay ---------------------------------------------------18
6. Luciferase and ?]-galactosidase assays ------------------------------------------------ 19
7. Reverse transcriptase-polymerase chain reaction ------------------------------------19
8. HO-1 assay ------------------------------------------------------------------------------- 20
9. Nuclear and cytosolic lysate preparation --------------------------------------------- 20
10. Western immunoblot analysis -------------------------------------------------------- 21
11. Immunocytochemistry ----------------------------------------------------------------- 22
12. Determination of caspase-3 activity ------------------------------------------------- 23
13. TUNEL assay --------------------------------------------------------------------------- 24
14. Statistical analysis --------------------------------------------------------------------- 24
III. Results -----------------------------------------------------------------------------------25
Neuroprotecion by metallothionein-III involved activation of
nuclear factor-??B through phosphatidylinositol-3 kinase signaling pathway
1. Expression and purification of MT-III ------------------------------------------------ 25
2. MT-III enhanced neuronal survival --------------------------------------------------- 29
3. MT-III reduced the activation of caspase-3 activation ----------------------------- 34
4. Activation of NF-?eB contributed to the anti-apoptotic effect of MT-III --------- 39
5. MT-III activated NF-?eB through PI3K signaling pathway ------------------------ 47
6. Inhibition of PI3K/Akt signaling pathway reduced the anti-apoptotic
effect of MT-III --------------------------------------------------------------------------- 53
Metallothionein-III protected against 6-hydroxydopamine-induced
oxidative stress by increasing expression of heme oxygenase-1
in the PI3K and ERK/Nrf2 dependent manner
7. MT-III protected against 6-OHDA-induced cell death ----------------------------- 61
8. MT-III reduced on 6-OHDA-induced the activation of caspase-3 ---------------- 66
9. MT-III reduced 6-OHDA-induced ROS generation -------------------------------- 68
10. MT-III up-regulated HO-1 expression ---------------------------------------------- 71
11. MT-III induced Nrf2 nuclear translocation ----------------------------------------- 76
12. Involvement of PI3K and MAPK pathway in HO-1 expression by MT-III ---- 79
13. HO-1 activity inhibitor suppressed the protective effect of MT-III on
6-OHDA-induced cell death ---------------------------------------------------------- 85
14. PI3K and ERK1/2 pathway were necessary and sufficient to attenuate
6-OHDA-induced cell death ---------------------------------------------------------- 88
IV. Discussion ------------------------------------------------------------------------------ 93
1. Neuroprotecion by metallothionein-III involved activation of
nuclear factor-??B through phosphatidylinositol-3 kinase signaling pathway --- 93
2. Metallothionein-III protected against 6-hydroxydopamine-induced
oxidative stress by increasing expression of heme oxygenase-1
in the PI3K and ERK/Nrf2 dependent manner -------------------------------------- 98
V. References ----------------------------------------------------------------------------- 102
VI. Abstract in Korean ----------------------------------------------------------------- 117
List of Figures
Neuroprotecion by metallothionein-III involved activation of
nuclear factor-??B through phosphatidylinositol-3 kinase signaling pathway
Fig. 1. Stereo drawing of the metallothionein crystal structure and electrostatic potential map of the surface of metallothionein ---------------------------- 14
Fig. 2. Construct of human MT-III expression vector, construction of
pGEX4T-hMT-III --------------------------------------------------------------- 27
Fig. 3. Purification of recombinant human MT-III protein -------------------------28
Fig. 4. MT-III prevented H2O2-induced cell death ---------------------------------- 30
Fig. 5. MT-III prevented Dox-induced cell death ----------------------------------- 31
Fig. 6. Apo- and Zn-MT-III prevented Dox-induced cell death ------------------- 32
Fig. 7. MT-III inhibited H2O2-induced apoptosis ----------------------------------- 33
Fig. 8. MT-III reduced on Dox-induced the activation of caspase-3 ------------- 35
Fig. 9. MT-III reduced on Dox-induced the activation of caspase-9 ------------- 36
Fig. 10 MT-III up-regulated the expression of Bcl-2 ------------------------------- 37
Fig. 11. MT-III reduced the release of cytochrome c from
the mitochondria -------------------------------------------------------------- 38
Fig. 12. MT-III increased the transcriptional activity of NF-?eB in
Neuro2A cells in a dose dependent manner ------------------------------- 41
Fig. 13. MT-III increased the transcriptional activity of NF-?eB in
SH-SY5Y cells in a dose dependent manner ------------------------------ 42
Fig. 14. MT-III increased the DNA binding activity of NF-?eB:
Dose response ----------------------------------------------------------------- 43
Fig. 15. MT-III increased the DNA binding activity of NF-?eB:
Time course -------------------------------------------------------------------- 44
Fig. 16. MT-III increased the nuclear translocation of NF-?eB -------------------- 45
Fig. 17. MT-III induced I?eB degradation -------------------------------------------- 46
Fig. 18. MT-III-mediated activation of NF-?eB was inhibited by
PI3K inhibitors, LY294002 and Wortmannin, or Akt inhibitor -------- 49
Fig. 19. MT-III increased the phosphorylation of Akt ----------------------------- 50
Fig. 20. PI3K inhibitors inhibited MT-III-induced nuclear translocation
of NF-?eB: Western immunoblot analysis ---------------------------------- 51
Fig. 21. PI3K inhibitors inhibited MT-III-induced nuclear translocation
of NF-?eB: Immunocytochemistry ------------------------------------------ 52
Fig. 22. PI3K and NF-?eB inhibitors diminished the anti-apoptotic
effect of MT-III ------------------------------------------------------------------55
Fig. 23. Akt and p65 inhibitors diminished the anti-apoptotic
effect of MT-III: Cell viability ------------------------------------------------- 56
Fig. 24. Akt and p65 inhibitors diminished the anti-apoptotic
effect of MT-III: TUNEL assay --------------------------------------------- 57
Fig. 25. PI3K inhibitor reduced the anti-apoptotic effect of MT-III ----------------- 58
Fig. 26. MT-III inhibited H2O2-induced apoptosis in the primary neuronal cells - 59
Fig. 27. MT-III increased the nuclear translocation of NF-??B
in the primary neuronal cells --------------------------------------------------- 60
Metallothionein-III protects against 6-hydroxydopamine-induced
oxidative stress by increasing expression of heme oxygenase-1
in the PI3K and ERK/Nrf2 dependent manner
Fig. 28. MT-III prevented 6-OHDA-induced cell death in SH-SY5Y cells:
Cell viability ---------------------------------------------------------------------- 62
Fig. 29. MT-III prevented 6-OHDA-induced cell death in SH-SY5Y cells:
Morphological analysis --------------------------------------------------------- 63
Fig. 30. MT-III prevented 6-OHDA-induced cell death in SH-SY5Y cells:
TUNEL assays -------------------------------------------------------------------- 64
Fig. 31. MT-III prevented 6-OHDA-induced cell death in SH-SY5Y cells:
LDH leakage assay -------------------------------------------------------------- 65
Fig. 32. MT-III reduced on 6-OHDA-induced the activation of caspase-3 and -9 - 67
Fig. 33. MT-III reduced on 6-OHDA-induced cellular ROS production ----------- 69
Fig. 34. Prevention of 6-OHDA-induced ROS by MT-III depended
on the length of the MT-III pretreatment ------------------------------------- 70
Fig. 35. MT-III induced HO-1 expression: Dose-dependent induction of HO-1 -- 72
Fig. 36. MT-III induced HO-1 expression: Time-dependent induction of HO-1 -- 73
Fig. 37. MT-III increased HO-1 activity ------------------------------------------------ 74
Fig. 38. MT-III induced HO-1 expression ----------------------------------------------- 75
Fig. 39. MT-III increased Nrf2 translocation ------------------------------------------- 77
Fig. 40. MT-III increased the ARE-driven luciferase activities and
ARE-binding activity of Nrf2 ---------------------------------------------------- 78
Fig. 41. MT-III activated the PI3K and ERK1/2 pathways --------------------------- 80
Fig. 42. PI3K and MAPK inhibitors attenuated the MT-III-induced
HO-1 expression by MT-III ------------------------------------------------------81
Fig. 43. PI3K and MAPK inhibitors attenuated the MT-III-induced
HO-1-ARE-luciferase activity ------------------------------------------------- 82
Fig. 44. PI3K and MAPK inhibitors attenuated the MT-III-induced
nuclear translocation of Nrf2 --------------------------------------------------- 83
Fig. 45. PI3K and MAPK inhibitors attenuated the MT-III-induced
the ARE-binding activity of Nrf2 ---------------------------------------------- 84
Fig. 46. A HO-1 enzyme inhibitor ZnPP reversed the protective effect of
MT-III against 6-OHDA-induced cell death: Cell viability ---------------- 86
Fig. 47. ZnPP reversed the protective effect of MT-III against
6-OHDA-induced cell death: Morphological analysis -----------------------87
Fig. 48. PI3K and ERK1/2 inhibitors attenuated the anti-apoptotic
effect of MT-III: Cell viability --------------------------------------------------- 89
Fig. 49. PI3K and ERK1/2 inhibitors attenuated the anti-apoptotic
effect of MT-III: Caspase-3 activity -------------------------------------------- 90
Fig. 50. PI3K and ERK1/2 inhibitors attenuated the anti-apoptotic
effect of MT-III: TUNEL assay ------------------------------------------------- 91
Fig. 51. A proposed pathway for MT-III induced HO-1 expression via upregulation
of PI3K, ERK and Nrf2, which explains neuroprotection against
oxidative stress in dopaminergic SH-SY5Y cells ----------------------------- 92
List of Abbreviations
6-OHDA 6-hydroxydopamine
AD Alzheimer's disease
ARE Antioxidant response element
BSA Bovine serum albumin
DMEM Dulbecco…s modified Eagle…s Medium
DMSO Dimethylsulfoxide
Dox Doxorubicin
EMSA Electrophoretic mobility shift analysis
ERK1/2 Extracellular signal-related kinase1/2
ESI-TOF MS Electrospray Ionization Time-of-flight Mass Spectrometry
FBS Fetal bovine serum
GIF Growth inhibitory factor
GST Glutathione-S transferase
HO-1 Heme oxygenase-1
HPLC High performance liquid chromatography
ICP- AES Inductively Coupled Plasma-Atomic Emisssion Spectrometer
IKK1 I??B kinase 1
I?eB Inhibitor ?eB
JNK1/2 c-Jun N-terminal kinase1/2
MAPK Mitogen-activated protein kinase
MT-III Metallothionein-III
MTT 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide
NF-?eB Nuclear factor ?eB
Nrf2 NF-E2-related factor 2
PBS Phosphate-buffered saline
PD Parkinson's disease
PDGF Platelet-derived growth factor
PI3K Phosphatidylinositol 3-kinase
PKC Protein kinase C
PMA Phorbolmyristate acetate
ROS Reactive oxygen species
RT-PCR Reverse transcriptase polymerase chain reaction
SDS-PAGE Sodium dodecyl sulfate-polyacryamide gel electrophoresis
TFA Trifluoroacetic acid
TUNEL Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labeling
- Degree
- Doctor
- Publisher
- 조선대학교
- Citation
- 황용필. (2007). Protective Effects of Metallothionein-III on Oxidative Stress-Induced Neuronal Cell Death.
- Type
- Dissertation
- URI
- https://oak.chosun.ac.kr/handle/2020.oak/7005
http://chosun.dcollection.net/common/orgView/200000235904
- Appears in Collections:
- General Graduate School > 4. Theses(Ph.D)
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