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SIRT1 confers protection against UVB- and H2O2-induced cell death via modulation of p53 and JNK in cultured skin keratinocytes.

Cao C, Lu S, Kivlin R, Wallin B, Card E, Bagdasarian A, Tamakloe T, Wang WJ, Song X, Chu WM, Kouttab N, Xu A, Wan Y - J. Cell. Mol. Med. (2008)

Bottom Line: Using cell culture and Western blot analysis in this study we found that SIRT1 is expressed in cultured human skin keratinocytes.We also found that SIRT1 is involved in UV-induced AMP-activated protein kinase (AMPK) and downstream acetyl-CoA carboxylase (ACC), phosphofructose kinase-2 (PFK-2) phosphorylation.Collectively, our data provide new insights into understanding of the molecular mechanisms of UV-induced skin aging, suggesting that SIRT1 activators such as resveratrol could serve as new anti-skin aging agents.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Providence College, Providence, RI 02918-0001, USA.

ABSTRACT
SIRT1 is a member of a highly conserved gene family (sirtuins) encoding nicotinamide adenine dinucleotide (NAD)(+)-dependent deacetylases, originally found to deacetylate histones leading to increased DNA stability and prolonged survival in yeast and higher organisms, including mammals. SIRT1 has been found to function as a deacetylase for numerous protein targets involved in various cellular pathways, including stress responses, apoptosis and axonal degeneration. However, the role of SIRT1 in ultraviolet (UV) signalling pathways remains unknown. Using cell culture and Western blot analysis in this study we found that SIRT1 is expressed in cultured human skin keratinocytes. Both UV radiation and H(2)O(2), two major inducers of skin cell damage, down-regulate SIRT1 in a time- and dose-dependent manner. We observed that reactive oxygen species-mediated JNK activation is involved in this SIRT1 down-regulation. SIRT1 activator, resveratrol, which has been considered as an important antioxidant, protects against UV- and H(2)O(2)-induced cell death, whereas SIRT inhibitors such as sirtinol and nicotinamide enhance cell death. Activation of SIRT1 negatively regulates UV- and H(2)O(2)-induced p53 acetylation, because nicotinamide and sirtinol as well as SIRT1 siRNA enhance UV- and H(2)O(2)-induced p53 acetylation, whereas SIRT1 activator resveratrol inhibits it. We also found that SIRT1 is involved in UV-induced AMP-activated protein kinase (AMPK) and downstream acetyl-CoA carboxylase (ACC), phosphofructose kinase-2 (PFK-2) phosphorylation. Collectively, our data provide new insights into understanding of the molecular mechanisms of UV-induced skin aging, suggesting that SIRT1 activators such as resveratrol could serve as new anti-skin aging agents.

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SIRT1 positively regulates AMPK activation in cultured skin keratinocytes. HaCaT cells were pre-treated with sirtinol (2 mM) or nicotinamide (Nico, 10 mM) for 1 hr, followed by 20 mJ/cm2 of UV for indicated time, p-AMPK (Thr 172) and total-AMPK activation were detected by Western blot (A and B). HaCaT cells were pre-treated with sirtinol (2 mM) or nicotinamide (Nico, 10 mM) for 1 hr, followed by 20 mJ/cm2 of UV and incubated for 0.5 and 2.0 hrs, p-ACC (Ser 79), p-PFK-2 (Ser 466) and β-actin were detected by Western blot (C), p-ACC was quantified in (D). HaCaT cells were treated with nicotinamide (Nico, 10 mM) or AMPK inhibitor Compound C (AMPKi, 10 μM) for 0.5, 2.0 hrs, followed by resveratrol (Rev, 10 μM) treatment for 0.5, 1.0 and 2.0 hrs, p-AMPK (Thr 172), p-ACC (Ser 79) and T-AMPK were detected by Western blot (E and F). The data in figures represent mean ± S.E. of three independent experiments. The symbol ‘#’ means P < 0.05 with UV- or H2O2-treated group.
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fig05: SIRT1 positively regulates AMPK activation in cultured skin keratinocytes. HaCaT cells were pre-treated with sirtinol (2 mM) or nicotinamide (Nico, 10 mM) for 1 hr, followed by 20 mJ/cm2 of UV for indicated time, p-AMPK (Thr 172) and total-AMPK activation were detected by Western blot (A and B). HaCaT cells were pre-treated with sirtinol (2 mM) or nicotinamide (Nico, 10 mM) for 1 hr, followed by 20 mJ/cm2 of UV and incubated for 0.5 and 2.0 hrs, p-ACC (Ser 79), p-PFK-2 (Ser 466) and β-actin were detected by Western blot (C), p-ACC was quantified in (D). HaCaT cells were treated with nicotinamide (Nico, 10 mM) or AMPK inhibitor Compound C (AMPKi, 10 μM) for 0.5, 2.0 hrs, followed by resveratrol (Rev, 10 μM) treatment for 0.5, 1.0 and 2.0 hrs, p-AMPK (Thr 172), p-ACC (Ser 79) and T-AMPK were detected by Western blot (E and F). The data in figures represent mean ± S.E. of three independent experiments. The symbol ‘#’ means P < 0.05 with UV- or H2O2-treated group.

Mentions: Some of the metabolic changes caused by resveratrol, a SIRT1 activator, mimic those observed in response to AMPK activation, and our data in this study demonstrated for the first time that UV radiation induces AMPK activation in cultured skin keratinocytes (Fig. 5A and B). We next tested the possible role of SIRT1 in AMPK activation. Our results showed that SIRT1 inhibitors sirtinol and nicotinamide inhibit UV-induced AMPK (Fig. 5A and B) and downstream PFK-2 and ACC phosphorylation (Fig. 5C and D). Furthermore, SIRT1 activator resveratrol alone also induces AMPK activation, and this induction is largely impaired by SIRT1 inhibitor (nicotinamide, or Nico) or AMPK inhibitor (compound C, or AMPKi) (Fig. 5E and F). Collectively, our data suggest that SIRT1 positively regulates AMPK activation in response to UV and resveratrol. At least some of the actions of resveratrol, such as fatty acid oxidation, are mediated by AMPK activation.


SIRT1 confers protection against UVB- and H2O2-induced cell death via modulation of p53 and JNK in cultured skin keratinocytes.

Cao C, Lu S, Kivlin R, Wallin B, Card E, Bagdasarian A, Tamakloe T, Wang WJ, Song X, Chu WM, Kouttab N, Xu A, Wan Y - J. Cell. Mol. Med. (2008)

SIRT1 positively regulates AMPK activation in cultured skin keratinocytes. HaCaT cells were pre-treated with sirtinol (2 mM) or nicotinamide (Nico, 10 mM) for 1 hr, followed by 20 mJ/cm2 of UV for indicated time, p-AMPK (Thr 172) and total-AMPK activation were detected by Western blot (A and B). HaCaT cells were pre-treated with sirtinol (2 mM) or nicotinamide (Nico, 10 mM) for 1 hr, followed by 20 mJ/cm2 of UV and incubated for 0.5 and 2.0 hrs, p-ACC (Ser 79), p-PFK-2 (Ser 466) and β-actin were detected by Western blot (C), p-ACC was quantified in (D). HaCaT cells were treated with nicotinamide (Nico, 10 mM) or AMPK inhibitor Compound C (AMPKi, 10 μM) for 0.5, 2.0 hrs, followed by resveratrol (Rev, 10 μM) treatment for 0.5, 1.0 and 2.0 hrs, p-AMPK (Thr 172), p-ACC (Ser 79) and T-AMPK were detected by Western blot (E and F). The data in figures represent mean ± S.E. of three independent experiments. The symbol ‘#’ means P < 0.05 with UV- or H2O2-treated group.
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Related In: Results  -  Collection

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fig05: SIRT1 positively regulates AMPK activation in cultured skin keratinocytes. HaCaT cells were pre-treated with sirtinol (2 mM) or nicotinamide (Nico, 10 mM) for 1 hr, followed by 20 mJ/cm2 of UV for indicated time, p-AMPK (Thr 172) and total-AMPK activation were detected by Western blot (A and B). HaCaT cells were pre-treated with sirtinol (2 mM) or nicotinamide (Nico, 10 mM) for 1 hr, followed by 20 mJ/cm2 of UV and incubated for 0.5 and 2.0 hrs, p-ACC (Ser 79), p-PFK-2 (Ser 466) and β-actin were detected by Western blot (C), p-ACC was quantified in (D). HaCaT cells were treated with nicotinamide (Nico, 10 mM) or AMPK inhibitor Compound C (AMPKi, 10 μM) for 0.5, 2.0 hrs, followed by resveratrol (Rev, 10 μM) treatment for 0.5, 1.0 and 2.0 hrs, p-AMPK (Thr 172), p-ACC (Ser 79) and T-AMPK were detected by Western blot (E and F). The data in figures represent mean ± S.E. of three independent experiments. The symbol ‘#’ means P < 0.05 with UV- or H2O2-treated group.
Mentions: Some of the metabolic changes caused by resveratrol, a SIRT1 activator, mimic those observed in response to AMPK activation, and our data in this study demonstrated for the first time that UV radiation induces AMPK activation in cultured skin keratinocytes (Fig. 5A and B). We next tested the possible role of SIRT1 in AMPK activation. Our results showed that SIRT1 inhibitors sirtinol and nicotinamide inhibit UV-induced AMPK (Fig. 5A and B) and downstream PFK-2 and ACC phosphorylation (Fig. 5C and D). Furthermore, SIRT1 activator resveratrol alone also induces AMPK activation, and this induction is largely impaired by SIRT1 inhibitor (nicotinamide, or Nico) or AMPK inhibitor (compound C, or AMPKi) (Fig. 5E and F). Collectively, our data suggest that SIRT1 positively regulates AMPK activation in response to UV and resveratrol. At least some of the actions of resveratrol, such as fatty acid oxidation, are mediated by AMPK activation.

Bottom Line: Using cell culture and Western blot analysis in this study we found that SIRT1 is expressed in cultured human skin keratinocytes.We also found that SIRT1 is involved in UV-induced AMP-activated protein kinase (AMPK) and downstream acetyl-CoA carboxylase (ACC), phosphofructose kinase-2 (PFK-2) phosphorylation.Collectively, our data provide new insights into understanding of the molecular mechanisms of UV-induced skin aging, suggesting that SIRT1 activators such as resveratrol could serve as new anti-skin aging agents.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Providence College, Providence, RI 02918-0001, USA.

ABSTRACT
SIRT1 is a member of a highly conserved gene family (sirtuins) encoding nicotinamide adenine dinucleotide (NAD)(+)-dependent deacetylases, originally found to deacetylate histones leading to increased DNA stability and prolonged survival in yeast and higher organisms, including mammals. SIRT1 has been found to function as a deacetylase for numerous protein targets involved in various cellular pathways, including stress responses, apoptosis and axonal degeneration. However, the role of SIRT1 in ultraviolet (UV) signalling pathways remains unknown. Using cell culture and Western blot analysis in this study we found that SIRT1 is expressed in cultured human skin keratinocytes. Both UV radiation and H(2)O(2), two major inducers of skin cell damage, down-regulate SIRT1 in a time- and dose-dependent manner. We observed that reactive oxygen species-mediated JNK activation is involved in this SIRT1 down-regulation. SIRT1 activator, resveratrol, which has been considered as an important antioxidant, protects against UV- and H(2)O(2)-induced cell death, whereas SIRT inhibitors such as sirtinol and nicotinamide enhance cell death. Activation of SIRT1 negatively regulates UV- and H(2)O(2)-induced p53 acetylation, because nicotinamide and sirtinol as well as SIRT1 siRNA enhance UV- and H(2)O(2)-induced p53 acetylation, whereas SIRT1 activator resveratrol inhibits it. We also found that SIRT1 is involved in UV-induced AMP-activated protein kinase (AMPK) and downstream acetyl-CoA carboxylase (ACC), phosphofructose kinase-2 (PFK-2) phosphorylation. Collectively, our data provide new insights into understanding of the molecular mechanisms of UV-induced skin aging, suggesting that SIRT1 activators such as resveratrol could serve as new anti-skin aging agents.

Show MeSH
Related in: MedlinePlus