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Comparative Study
. 2015 Jun;34(6):391-9.
doi: 10.1089/dna.2014.2711. Epub 2015 Mar 24.

Differential responses of normal human melanocytes to intra- and extracellular dsRNA

Suiquan Wang  1 , Dongyin Liu  1 , Rong Jin  1 , Yiping Zhu  1 , Aie Xu  1
Affiliations
Comparative Study

Differential responses of normal human melanocytes to intra- and extracellular dsRNA

Suiquan Wang et al. DNA Cell Biol. 2015 Jun.

Abstract

Viral factor has been implicated in the etiopathogenesis of vitiligo. To elucidate the effects of viral double-stranded RNA (dsRNA) on melanocytes and to explore the underlying mechanisms, primary cultured normal human melanocytes were treated with synthetic viral dsRNA analog poly(I:C). The results demonstrated that poly(I:C)-triggered apoptosis when transfected into melanocytes, while extracellular poly(I:C) did not have that effect. Intracellular poly(I:C)-induced melanocyte death was decreased by RIG-I or MDA5 siRNA, but not by TLR3 siRNA. Both intracellular and extracellular poly(I:C) induced the expression of IFNB, TNF, IL6, and IL8. However, extracellular poly(I:C) demonstrated a much weaker induction capacity of cytokine genes than intracellular poly(I:C). Further analysis revealed that phosphorylation of TBK1, IRF3, IRF7, and TAK1 was differentially induced by intra- or extracellular poly(I:C). NFκB inhibitor Bay 11-7082 decreased the induction of all the cytokines by poly(I:C), suggesting the ubiquitous role of NFκB in the process. Poly(I:C) treatment also induced the phosphorylation of p38 and JNK in melanocytes. Both JNK and p38 inhibitors showed suppression on the cytokine induction by intra- or extracellular poly(I:C). However, only the JNK inhibitor decreased the intracellular poly(I:C)-induced melanocyte death. Taken together, this study provides the possible mechanism of viral factor in the pathogenesis of vitiligo.

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Figures

<b>FIG. 1.</b>
FIG. 1.
Apoptosis induction by intracellular poly(I:C) in normal human melanocytes. Melanocytes were transfected with 1 μg/mL of poly(I:C) (TR) or treated with various concentrations of poly(I:C) (0, 1, 10, or 100 μg/mL) in the culture medium for 24 h. (A) Lactate dehydrogenase (LDH) contents in the supernatants of cell culture were measured to evaluate cell death. (B) Cells were harvested and cell lysates were subjected to SDS-PAGE and analyzed by western blotting with antibodies against cleaved Caspase-8 and Caspase-3. β-actin was probed as the loading control. (C) Melanocytes were untreated (Un), mock transfected (MT), transfected with 1 μg/mL poly(I:C) (TR), or treated with 100 μg/mL of poly(I:C) (NT) in the culture medium for 24 h. Cells were then stained with Annexin V-PI and examined by a flow cytometer. One experiment representative of three experiments. SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.
<b>FIG. 2.</b>
FIG. 2.
Roles of different receptors in the intracellular poly(I:C)-induced melanocyte death. (A) Melanocytes were transfected with 1 μg/mL of poly(I:C) (TR) or treated with various concentrations of poly(I:C) (0, 1, 10, or 100 μg/mL) in the culture medium for 24 h. Protein levels of RIG-I, MDA5, and TLR3 were examined by western blotting. β-actin was probed as loading control. (B) Melanocytes were transfected with siRNAs targeting RIG-I, MDA5, TLR3, or a nonsilencing control, respectively. After 72 h, cells were harvested and the effects of siRNA were confirmed by western blotting. (C) Melanocytes were transfected with corresponding siRNAs. After 72 h, cells were mock transfected or transfected with 1 μg/mL poly(I:C). Cell death was evaluated by LDH release 24 h later. Results are presented as mean±SD from three experiments. *p<0.05 versus control siRNA.
<b>FIG. 3.</b>
FIG. 3.
Induction of cytokine genes by intra- or extracellular poly(I:C) in normal human melanocytes. Melanocytes were transfected with 1 μg/mL of poly(I:C) (TR) or treated with various concentrations of poly(I:C) (0, 1, 10, or 100 μg/mL) in the culture medium. Total RNA was extracted at 8 h after transfection. Quantitative real-time PCR was performed to measure the mRNA levels of IFNB (A), TNF (B), IL6 (C), and IL8 (D). Results are presented as mean±SD from three experiments.
<b>FIG. 4.</b>
FIG. 4.
Phosphorylation profile of cytokine expression-related genes in normal human melanocytes in response to intra- or extracellular poly(I:C). Melanocytes were transfected with 1 μg/mL of poly(I:C) (TR) or treated with 100 μg/mL of poly(I:C) in the medium (NT) for 0.5, 1, 2, or 24 h. Cell lysates were processed for western blotting with antibodies against p-TAK1, TAK1, p-TBK1, TBK1, p-IRF3, IRF3, p-IRF7, and IRF7. β-actin was probed as loading control.
<b>FIG. 5.</b>
FIG. 5.
Involvement of NFκB activity in the induction of cytokine genes by intra- or extracellular poly(I:C). Untreated or pretreated with 10 μM BAY 11-7082 for 1 h, melanocytes were then stimulated with 1 μg/mL of intracellular poly(I:C) (TR) or 100 μg/mL of extracellular poly(I:C) (NT). RNA was extracted after 8 h and quantitative real-time PCR was performed to measure the expression of IFNB and TNF (A, C) or IL6 and IL8 (B, D). Mock, Mock transfection. Un, Untreated control. Results are presented as mean±SD from three experiments. *p<0.05 versus TR, #p<0.05 versus NT.
<b>FIG. 6.</b>
FIG. 6.
Effects of p38 MAPK and JNK activation on cell death and cytokine production in normal human melanocytes. (A) Melanocytes were treated with 1 μg/mL of intracellular poly(I:C) (TR) or 100 μg/mL of extracellular poly(I:C) (NT) for 0.5, 1, 2, or 24 h. Cell lysates were processed for western blotting with corresponding antibodies. (B) Untreated or pretreated with various MAPK inhibitors, melanocytes were mock transfected or transfected with 1 μg/mL of poly(I:C). After 24 h, LDH release was measured to evaluate cell death. (C–F) Melanocytes were pretreated with various MAPK inhibitors for 1 h and then stimulated with 1 μg/mL of intracellular poly(I:C) (TR) or 100 μg/mL of extracellular poly(I:C) (NT). RNA was extracted after 8 h and quantitative real-time PCR was performed to measure the expression of IFNB and TNF (C, E) or IL6 and IL8 (D, F). Results are presented as mean±SD from three experiments. *p<0.05 versus TR, #p<0.05 versus NT.
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