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Activation of TLR3 in keratinocytes increases expression of genes involved in formation of the epidermis, lipid accumulation, and epidermal organelles.

Borkowski AW, Park K, Uchida Y, Gallo RL - J. Invest. Dermatol. (2013)

Bottom Line: Injury to the skin, and the subsequent release of noncoding double-stranded RNA (dsRNA) from necrotic keratinocytes, has been identified as an endogenous activator of Toll-like receptor 3 (TLR3).Additionally, treatment with dsRNA resulted in increases in sphingomyelin and morphologic changes including increased epidermal lipid staining by Oil Red O and TLR3-dependent increases in lamellar bodies and keratohyalin granules.These observations show that dsRNA can stimulate some events in keratinocytes that are important for skin barrier repair and maintenance.

View Article: PubMed Central - PubMed

Affiliation: Division of Dermatology, University of California, San Diego, San Diego, California 92093-0869, USA.

ABSTRACT
Injury to the skin, and the subsequent release of noncoding double-stranded RNA (dsRNA) from necrotic keratinocytes, has been identified as an endogenous activator of Toll-like receptor 3 (TLR3). As changes in keratinocyte growth and differentiation follow injury, we hypothesized that TLR3 might trigger some elements of the barrier repair program in keratinocytes. dsRNA was observed to induce TLR3-dependent increases in human keratinocyte mRNA abundance for ABCA12 (ATP-binding cassette, sub-family A, member 12), glucocerebrosidase, acid sphingomyelinase, and transglutaminase 1. Additionally, treatment with dsRNA resulted in increases in sphingomyelin and morphologic changes including increased epidermal lipid staining by Oil Red O and TLR3-dependent increases in lamellar bodies and keratohyalin granules. These observations show that dsRNA can stimulate some events in keratinocytes that are important for skin barrier repair and maintenance.

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TLR3 activation is required for dsRNA-induced changes in gene expression(a) TLR3 was silenced in NHEK for 48 h before treatment with 1 μg/ml Poly (I:C) for 24 h. Real-time PCR was used to quantify mRNA levels and fold change values are calculated relative and normalized to GAPDH expression. *P < 0.05. Two tailed T-test. (b) NHEK were treated with 5 nM Bafilomycin A1 for 1 h prior to treatment with 1 μg/ml Poly (I:C) for 24 h. ***P < 0.001. One-way ANOVA. (c) TLR3 was silenced in NHEK for 48 h before treatment with 1 μg/ml Poly (I:C) for 24 h. Real-time PCR was used to quantify mRNA levels and fold change values are calculated relative to and normalized to GAPDH expression. *P < 0.05. Two tailed T-test. Data are mean ±SEM, n = 3, and are representative of at least three independent experiments.
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Figure 3: TLR3 activation is required for dsRNA-induced changes in gene expression(a) TLR3 was silenced in NHEK for 48 h before treatment with 1 μg/ml Poly (I:C) for 24 h. Real-time PCR was used to quantify mRNA levels and fold change values are calculated relative and normalized to GAPDH expression. *P < 0.05. Two tailed T-test. (b) NHEK were treated with 5 nM Bafilomycin A1 for 1 h prior to treatment with 1 μg/ml Poly (I:C) for 24 h. ***P < 0.001. One-way ANOVA. (c) TLR3 was silenced in NHEK for 48 h before treatment with 1 μg/ml Poly (I:C) for 24 h. Real-time PCR was used to quantify mRNA levels and fold change values are calculated relative to and normalized to GAPDH expression. *P < 0.05. Two tailed T-test. Data are mean ±SEM, n = 3, and are representative of at least three independent experiments.

Mentions: To determine if the increases in ABCA12, GBA, SMPD1, and TGM1 mRNA after Poly (I:C) treatment were dependent on TLR3 activation, we used siRNA to knockdown TLR3 in NHEK. When TLR3 was significantly decreased in keratinocytes, Poly (I:C) failed to induce a significant increase in mRNA for the barrier repair genes ABCA12, GBA, SMPD1, TGM1 and TNF (Figure 3a). Since TLR3 signaling is dependent on proper acidification and maturation of endosomes (Matsushima et al., 2004), we used Bafilomycin A1 (BafA1), a specific inhibitor of the V-type ATPase required for acidification of endosomes and lysosomes, to inhibit TLR3 signaling. BafA1 blocked Poly (I:C)-induced increases in ABCA12, GBA, and SMPD1 mRNA as well as increases in mRNA of the inflammatory cytokines TNF and IL-6 (Figure 3b). Similar effects on gene expression were seen when TRIF, a key signaling molecule downstream of TLR3, was knocked down (Supplementary Figure S4). Unlike silencing of TLR3 or TRIF, knocking down MAVS, a key signaling molecule for RIG-I-like receptors that recognizes cytosolic dsRNA, had no significant effect on Poly (I:C)-induced expression of ABCA12, GBA, SMPD1, TGM1 (Supplemental Figure S5). Although TLR3 activation was important for Poly (I:C)-induced increases in UGCG mRNA, alterations in mRNA for several lipid synthesis genes was largely independent of TLR3 (Figure 3c).


Activation of TLR3 in keratinocytes increases expression of genes involved in formation of the epidermis, lipid accumulation, and epidermal organelles.

Borkowski AW, Park K, Uchida Y, Gallo RL - J. Invest. Dermatol. (2013)

TLR3 activation is required for dsRNA-induced changes in gene expression(a) TLR3 was silenced in NHEK for 48 h before treatment with 1 μg/ml Poly (I:C) for 24 h. Real-time PCR was used to quantify mRNA levels and fold change values are calculated relative and normalized to GAPDH expression. *P < 0.05. Two tailed T-test. (b) NHEK were treated with 5 nM Bafilomycin A1 for 1 h prior to treatment with 1 μg/ml Poly (I:C) for 24 h. ***P < 0.001. One-way ANOVA. (c) TLR3 was silenced in NHEK for 48 h before treatment with 1 μg/ml Poly (I:C) for 24 h. Real-time PCR was used to quantify mRNA levels and fold change values are calculated relative to and normalized to GAPDH expression. *P < 0.05. Two tailed T-test. Data are mean ±SEM, n = 3, and are representative of at least three independent experiments.
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Related In: Results  -  Collection

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Figure 3: TLR3 activation is required for dsRNA-induced changes in gene expression(a) TLR3 was silenced in NHEK for 48 h before treatment with 1 μg/ml Poly (I:C) for 24 h. Real-time PCR was used to quantify mRNA levels and fold change values are calculated relative and normalized to GAPDH expression. *P < 0.05. Two tailed T-test. (b) NHEK were treated with 5 nM Bafilomycin A1 for 1 h prior to treatment with 1 μg/ml Poly (I:C) for 24 h. ***P < 0.001. One-way ANOVA. (c) TLR3 was silenced in NHEK for 48 h before treatment with 1 μg/ml Poly (I:C) for 24 h. Real-time PCR was used to quantify mRNA levels and fold change values are calculated relative to and normalized to GAPDH expression. *P < 0.05. Two tailed T-test. Data are mean ±SEM, n = 3, and are representative of at least three independent experiments.
Mentions: To determine if the increases in ABCA12, GBA, SMPD1, and TGM1 mRNA after Poly (I:C) treatment were dependent on TLR3 activation, we used siRNA to knockdown TLR3 in NHEK. When TLR3 was significantly decreased in keratinocytes, Poly (I:C) failed to induce a significant increase in mRNA for the barrier repair genes ABCA12, GBA, SMPD1, TGM1 and TNF (Figure 3a). Since TLR3 signaling is dependent on proper acidification and maturation of endosomes (Matsushima et al., 2004), we used Bafilomycin A1 (BafA1), a specific inhibitor of the V-type ATPase required for acidification of endosomes and lysosomes, to inhibit TLR3 signaling. BafA1 blocked Poly (I:C)-induced increases in ABCA12, GBA, and SMPD1 mRNA as well as increases in mRNA of the inflammatory cytokines TNF and IL-6 (Figure 3b). Similar effects on gene expression were seen when TRIF, a key signaling molecule downstream of TLR3, was knocked down (Supplementary Figure S4). Unlike silencing of TLR3 or TRIF, knocking down MAVS, a key signaling molecule for RIG-I-like receptors that recognizes cytosolic dsRNA, had no significant effect on Poly (I:C)-induced expression of ABCA12, GBA, SMPD1, TGM1 (Supplemental Figure S5). Although TLR3 activation was important for Poly (I:C)-induced increases in UGCG mRNA, alterations in mRNA for several lipid synthesis genes was largely independent of TLR3 (Figure 3c).

Bottom Line: Injury to the skin, and the subsequent release of noncoding double-stranded RNA (dsRNA) from necrotic keratinocytes, has been identified as an endogenous activator of Toll-like receptor 3 (TLR3).Additionally, treatment with dsRNA resulted in increases in sphingomyelin and morphologic changes including increased epidermal lipid staining by Oil Red O and TLR3-dependent increases in lamellar bodies and keratohyalin granules.These observations show that dsRNA can stimulate some events in keratinocytes that are important for skin barrier repair and maintenance.

View Article: PubMed Central - PubMed

Affiliation: Division of Dermatology, University of California, San Diego, San Diego, California 92093-0869, USA.

ABSTRACT
Injury to the skin, and the subsequent release of noncoding double-stranded RNA (dsRNA) from necrotic keratinocytes, has been identified as an endogenous activator of Toll-like receptor 3 (TLR3). As changes in keratinocyte growth and differentiation follow injury, we hypothesized that TLR3 might trigger some elements of the barrier repair program in keratinocytes. dsRNA was observed to induce TLR3-dependent increases in human keratinocyte mRNA abundance for ABCA12 (ATP-binding cassette, sub-family A, member 12), glucocerebrosidase, acid sphingomyelinase, and transglutaminase 1. Additionally, treatment with dsRNA resulted in increases in sphingomyelin and morphologic changes including increased epidermal lipid staining by Oil Red O and TLR3-dependent increases in lamellar bodies and keratohyalin granules. These observations show that dsRNA can stimulate some events in keratinocytes that are important for skin barrier repair and maintenance.

Show MeSH
Related in: MedlinePlus