Expression of cytosolic NADP +-dependent isocitrate dehydrogenase in melanocytes and its role as an antioxidant

Ji Young Kim, Jae Yong Shin, Miri Kim, Seung Kyung Hann, Sang Ho Oh

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

Background: Cytosolic NADP +-dependent ICDH (IDPc) has an antioxidant effect as a supplier of NADPH to the cytosol, which is needed for the production of glutathione. Objective: To evaluate the expression of IDPc in melanocytes and to elucidate its role as an antioxidant. Methods: The knock-down of IDPc expression in immortalized mouse melanocyte cell lines (melan-a) was performed using the short interfering RNA (siRNA)-targeted gene silencing method. After confirming the silencing of IDPc expression with mRNA and protein levels, viability, apoptosis and necrosis, as well as ROS production in IDPc-silenced melanocytes were monitored under conditions of oxidative stress and non-stress. Also, the ratio of oxidized glutathione to total glutathione was examined, and whether the addition of glutathione recovered cell viability, decreased by oxidant stress, was checked. Results: The expression of IDPc in both primary human melanocytes and melan-a cells was confirmed by Western blot and RT-PCR. The silencing of IDPc expression by transfecting IDPc siRNA in melan-a cells was observed by Western blotting and real-time RT-PCR. IDPc knock-down cells showed significantly decreased cell viability and an increased number of cells under apoptosis and necrosis. IDPc siRNA-treated melanocytes demonstrated a higher intensity of DCFDA after the addition of H 2O 2 compared with scrambled siRNA-treated melanocytes, and a lower ratio of reduced glutathione to oxidized glutathione were observed in IDPc siRNA transfected melanocytes. In addition, the addition of glutathione recovered cell viability, which was previously decreased after incubation with H 2O 2. Conclusions: This study suggests that decreased IDPc expression renders melanocytes more vulnerable to oxidative stress, and IDPc plays an important antioxidant function in melanocytes.

Original languageEnglish
Pages (from-to)118-125
Number of pages8
JournalJournal of Dermatological Science
Volume65
Issue number2
DOIs
Publication statusPublished - 2012 Feb 1

Fingerprint

Isocitrate Dehydrogenase
Melanocytes
NADP
Small Interfering RNA
Glutathione
Antioxidants
Cells
Oxidative stress
Glutathione Disulfide
Cell Survival
Apoptosis
Oxidants
Oxidative Stress
Necrosis
Western Blotting
Genes
isocitrate dehydrogenase (NADP+)
Messenger RNA
Gene Silencing
Cytosol

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Dermatology

Cite this

Kim, Ji Young ; Shin, Jae Yong ; Kim, Miri ; Hann, Seung Kyung ; Oh, Sang Ho. / Expression of cytosolic NADP +-dependent isocitrate dehydrogenase in melanocytes and its role as an antioxidant. In: Journal of Dermatological Science. 2012 ; Vol. 65, No. 2. pp. 118-125.
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abstract = "Background: Cytosolic NADP +-dependent ICDH (IDPc) has an antioxidant effect as a supplier of NADPH to the cytosol, which is needed for the production of glutathione. Objective: To evaluate the expression of IDPc in melanocytes and to elucidate its role as an antioxidant. Methods: The knock-down of IDPc expression in immortalized mouse melanocyte cell lines (melan-a) was performed using the short interfering RNA (siRNA)-targeted gene silencing method. After confirming the silencing of IDPc expression with mRNA and protein levels, viability, apoptosis and necrosis, as well as ROS production in IDPc-silenced melanocytes were monitored under conditions of oxidative stress and non-stress. Also, the ratio of oxidized glutathione to total glutathione was examined, and whether the addition of glutathione recovered cell viability, decreased by oxidant stress, was checked. Results: The expression of IDPc in both primary human melanocytes and melan-a cells was confirmed by Western blot and RT-PCR. The silencing of IDPc expression by transfecting IDPc siRNA in melan-a cells was observed by Western blotting and real-time RT-PCR. IDPc knock-down cells showed significantly decreased cell viability and an increased number of cells under apoptosis and necrosis. IDPc siRNA-treated melanocytes demonstrated a higher intensity of DCFDA after the addition of H 2O 2 compared with scrambled siRNA-treated melanocytes, and a lower ratio of reduced glutathione to oxidized glutathione were observed in IDPc siRNA transfected melanocytes. In addition, the addition of glutathione recovered cell viability, which was previously decreased after incubation with H 2O 2. Conclusions: This study suggests that decreased IDPc expression renders melanocytes more vulnerable to oxidative stress, and IDPc plays an important antioxidant function in melanocytes.",
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Expression of cytosolic NADP +-dependent isocitrate dehydrogenase in melanocytes and its role as an antioxidant. / Kim, Ji Young; Shin, Jae Yong; Kim, Miri; Hann, Seung Kyung; Oh, Sang Ho.

In: Journal of Dermatological Science, Vol. 65, No. 2, 01.02.2012, p. 118-125.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Expression of cytosolic NADP +-dependent isocitrate dehydrogenase in melanocytes and its role as an antioxidant

AU - Kim, Ji Young

AU - Shin, Jae Yong

AU - Kim, Miri

AU - Hann, Seung Kyung

AU - Oh, Sang Ho

PY - 2012/2/1

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N2 - Background: Cytosolic NADP +-dependent ICDH (IDPc) has an antioxidant effect as a supplier of NADPH to the cytosol, which is needed for the production of glutathione. Objective: To evaluate the expression of IDPc in melanocytes and to elucidate its role as an antioxidant. Methods: The knock-down of IDPc expression in immortalized mouse melanocyte cell lines (melan-a) was performed using the short interfering RNA (siRNA)-targeted gene silencing method. After confirming the silencing of IDPc expression with mRNA and protein levels, viability, apoptosis and necrosis, as well as ROS production in IDPc-silenced melanocytes were monitored under conditions of oxidative stress and non-stress. Also, the ratio of oxidized glutathione to total glutathione was examined, and whether the addition of glutathione recovered cell viability, decreased by oxidant stress, was checked. Results: The expression of IDPc in both primary human melanocytes and melan-a cells was confirmed by Western blot and RT-PCR. The silencing of IDPc expression by transfecting IDPc siRNA in melan-a cells was observed by Western blotting and real-time RT-PCR. IDPc knock-down cells showed significantly decreased cell viability and an increased number of cells under apoptosis and necrosis. IDPc siRNA-treated melanocytes demonstrated a higher intensity of DCFDA after the addition of H 2O 2 compared with scrambled siRNA-treated melanocytes, and a lower ratio of reduced glutathione to oxidized glutathione were observed in IDPc siRNA transfected melanocytes. In addition, the addition of glutathione recovered cell viability, which was previously decreased after incubation with H 2O 2. Conclusions: This study suggests that decreased IDPc expression renders melanocytes more vulnerable to oxidative stress, and IDPc plays an important antioxidant function in melanocytes.

AB - Background: Cytosolic NADP +-dependent ICDH (IDPc) has an antioxidant effect as a supplier of NADPH to the cytosol, which is needed for the production of glutathione. Objective: To evaluate the expression of IDPc in melanocytes and to elucidate its role as an antioxidant. Methods: The knock-down of IDPc expression in immortalized mouse melanocyte cell lines (melan-a) was performed using the short interfering RNA (siRNA)-targeted gene silencing method. After confirming the silencing of IDPc expression with mRNA and protein levels, viability, apoptosis and necrosis, as well as ROS production in IDPc-silenced melanocytes were monitored under conditions of oxidative stress and non-stress. Also, the ratio of oxidized glutathione to total glutathione was examined, and whether the addition of glutathione recovered cell viability, decreased by oxidant stress, was checked. Results: The expression of IDPc in both primary human melanocytes and melan-a cells was confirmed by Western blot and RT-PCR. The silencing of IDPc expression by transfecting IDPc siRNA in melan-a cells was observed by Western blotting and real-time RT-PCR. IDPc knock-down cells showed significantly decreased cell viability and an increased number of cells under apoptosis and necrosis. IDPc siRNA-treated melanocytes demonstrated a higher intensity of DCFDA after the addition of H 2O 2 compared with scrambled siRNA-treated melanocytes, and a lower ratio of reduced glutathione to oxidized glutathione were observed in IDPc siRNA transfected melanocytes. In addition, the addition of glutathione recovered cell viability, which was previously decreased after incubation with H 2O 2. Conclusions: This study suggests that decreased IDPc expression renders melanocytes more vulnerable to oxidative stress, and IDPc plays an important antioxidant function in melanocytes.

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