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Toll-like receptor 3 activation is required for normal skin barrier repair following UV damage.

Borkowski AW, Kuo IH, Bernard JJ, Yoshida T, Williams MR, Hung NJ, Yu BD, Beck LA, Gallo RL - J. Invest. Dermatol. (2014)

Bottom Line: UV damage to the skin leads to the release of noncoding RNA (ncRNA) from necrotic keratinocytes that activates Toll-like receptor 3 (TLR3).This release of ncRNA triggers inflammation in the skin following UV damage.Combined, these data further validate the conclusion that recognition of endogenous RNA by TLR3 is an important step in the program of skin barrier repair.

View Article: PubMed Central - PubMed

Affiliation: Division of Dermatology, Department of Medicine, University of California, San Diego, La Jolla, California, USA.

ABSTRACT
UV damage to the skin leads to the release of noncoding RNA (ncRNA) from necrotic keratinocytes that activates Toll-like receptor 3 (TLR3). This release of ncRNA triggers inflammation in the skin following UV damage. Recently, TLR3 activation was also shown to aid wound repair and increase the expression of genes associated with permeability barrier repair. Here, we sought to test whether skin barrier repair after UVB damage is dependent on the activation of TLR3. We observed that multiple ncRNAs induced expression of skin barrier repair genes, that the TLR3 ligand Poly (I:C) also induced expression and function of tight junctions, and that the ncRNA U1 acts in a TLR3-dependent manner to induce expression of skin barrier repair genes. These observations were shown to have functional relevance as Tlr3-/- mice displayed a delay in skin barrier repair following UVB damage. Combined, these data further validate the conclusion that recognition of endogenous RNA by TLR3 is an important step in the program of skin barrier repair.

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small nuclear RNAs stimulate skin barrier genes(a) Structures of snRNA species generated using RNAfold and VARNA applet. (b) Normal human epidermal keratinocytes were treated with 1 μg/ml in vitro transcribed snRNAs for 24 hours in the presence of a Dharmafect 1. Real-time PCR was used to quantify mRNA levels and fold change values are calculated relative to and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression and then to NHEK that have been treated with a control in vitro transcribed RNA. Data are mean +/Ȓ SEM, n = 3, and are representative of at least three independent experiments. * = P < 0.05, ** = P < 0.01, *** = P < 0.001. Two-tailed t-test.
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Figure 4: small nuclear RNAs stimulate skin barrier genes(a) Structures of snRNA species generated using RNAfold and VARNA applet. (b) Normal human epidermal keratinocytes were treated with 1 μg/ml in vitro transcribed snRNAs for 24 hours in the presence of a Dharmafect 1. Real-time PCR was used to quantify mRNA levels and fold change values are calculated relative to and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression and then to NHEK that have been treated with a control in vitro transcribed RNA. Data are mean +/Ȓ SEM, n = 3, and are representative of at least three independent experiments. * = P < 0.05, ** = P < 0.01, *** = P < 0.001. Two-tailed t-test.

Mentions: TLR3 is activated by dsRNA and it has been proposed that the double stranded stem loops of U1 RNA serve as the TLR3 ligand (Bernard et al. 2012). However, in addition to U1, numerous other noncoding snRNAs were observed to have increases in their read frequency after UVB exposure (Bernard et al. 2012). To determine whether additional ncRNAs could act in a similar manner to U1, we synthesized the spliceosomal RNAs U2, U4, U6, and minor spliceosomal RNA U12 (U2 RNA, U4 RNA, U6 RNA, and U12 RNA), as well as the small Cajol Body-specific RNAs 9 and 18 (scaRNA9 and scaRNA18). All of these snRNAs are predicted to contain double stranded regions using RNAfold software and the VARNA applet (Figure 4a) (Gruber et al. 2008; Blin et al. 2009). Treatment of NHEK for 24 hours with these synthetic snRNAs also resulted in an increase in mRNA abundance of ABCA12, GBA, SMPD1, TGM1, TNFα, and IL-6 (Figure 4b).


Toll-like receptor 3 activation is required for normal skin barrier repair following UV damage.

Borkowski AW, Kuo IH, Bernard JJ, Yoshida T, Williams MR, Hung NJ, Yu BD, Beck LA, Gallo RL - J. Invest. Dermatol. (2014)

small nuclear RNAs stimulate skin barrier genes(a) Structures of snRNA species generated using RNAfold and VARNA applet. (b) Normal human epidermal keratinocytes were treated with 1 μg/ml in vitro transcribed snRNAs for 24 hours in the presence of a Dharmafect 1. Real-time PCR was used to quantify mRNA levels and fold change values are calculated relative to and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression and then to NHEK that have been treated with a control in vitro transcribed RNA. Data are mean +/Ȓ SEM, n = 3, and are representative of at least three independent experiments. * = P < 0.05, ** = P < 0.01, *** = P < 0.001. Two-tailed t-test.
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Related In: Results  -  Collection

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Figure 4: small nuclear RNAs stimulate skin barrier genes(a) Structures of snRNA species generated using RNAfold and VARNA applet. (b) Normal human epidermal keratinocytes were treated with 1 μg/ml in vitro transcribed snRNAs for 24 hours in the presence of a Dharmafect 1. Real-time PCR was used to quantify mRNA levels and fold change values are calculated relative to and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression and then to NHEK that have been treated with a control in vitro transcribed RNA. Data are mean +/Ȓ SEM, n = 3, and are representative of at least three independent experiments. * = P < 0.05, ** = P < 0.01, *** = P < 0.001. Two-tailed t-test.
Mentions: TLR3 is activated by dsRNA and it has been proposed that the double stranded stem loops of U1 RNA serve as the TLR3 ligand (Bernard et al. 2012). However, in addition to U1, numerous other noncoding snRNAs were observed to have increases in their read frequency after UVB exposure (Bernard et al. 2012). To determine whether additional ncRNAs could act in a similar manner to U1, we synthesized the spliceosomal RNAs U2, U4, U6, and minor spliceosomal RNA U12 (U2 RNA, U4 RNA, U6 RNA, and U12 RNA), as well as the small Cajol Body-specific RNAs 9 and 18 (scaRNA9 and scaRNA18). All of these snRNAs are predicted to contain double stranded regions using RNAfold software and the VARNA applet (Figure 4a) (Gruber et al. 2008; Blin et al. 2009). Treatment of NHEK for 24 hours with these synthetic snRNAs also resulted in an increase in mRNA abundance of ABCA12, GBA, SMPD1, TGM1, TNFα, and IL-6 (Figure 4b).

Bottom Line: UV damage to the skin leads to the release of noncoding RNA (ncRNA) from necrotic keratinocytes that activates Toll-like receptor 3 (TLR3).This release of ncRNA triggers inflammation in the skin following UV damage.Combined, these data further validate the conclusion that recognition of endogenous RNA by TLR3 is an important step in the program of skin barrier repair.

View Article: PubMed Central - PubMed

Affiliation: Division of Dermatology, Department of Medicine, University of California, San Diego, La Jolla, California, USA.

ABSTRACT
UV damage to the skin leads to the release of noncoding RNA (ncRNA) from necrotic keratinocytes that activates Toll-like receptor 3 (TLR3). This release of ncRNA triggers inflammation in the skin following UV damage. Recently, TLR3 activation was also shown to aid wound repair and increase the expression of genes associated with permeability barrier repair. Here, we sought to test whether skin barrier repair after UVB damage is dependent on the activation of TLR3. We observed that multiple ncRNAs induced expression of skin barrier repair genes, that the TLR3 ligand Poly (I:C) also induced expression and function of tight junctions, and that the ncRNA U1 acts in a TLR3-dependent manner to induce expression of skin barrier repair genes. These observations were shown to have functional relevance as Tlr3-/- mice displayed a delay in skin barrier repair following UVB damage. Combined, these data further validate the conclusion that recognition of endogenous RNA by TLR3 is an important step in the program of skin barrier repair.

Show MeSH
Related in: MedlinePlus