Interferon-γ induces senescence in normal human melanocytes

Abstract

Background: Interferon-γ (IFN-γ) plays an important role in the proceedings of vitiligo through recruiting lymphocytes to the lesional skin. However, the potential effects of IFN-γ on skin melanocytes and the subsequent contribution to the vitiligo pathogenesis are still unclear.

Objective: To investigate the effects of IFN-γ on viability and cellular functions of melanocytes.

Methods: Primary human melanocytes were treated with IFN-γ. Cell viability, apoptosis, cell cycle melanin content and intracellular reactive oxygen species (ROS) level were measured. mRNA expression was examined by real-time PCR. The release of interleukin 6 (IL-6) and heat shock protein 70 (HSP-70) was monitored by ELISA. β-galactosidase staining was utilized to evaluate melanocyte senescence.

Results: Persistent IFN-γ treatment induced viability loss, apoptosis, cell cycle arrest and senescence in melanocytes. Melanocyte senescence was characterized as the changes in pigmentation and morphology, as well as the increase of β-galactosidase activity. Increase of p21Cip1/Waf1 protein was evident in melanocytes after IFN-γ treatment. IFN-γ induction of senescence was attenuated by siRNAs against p21, Janus kinase 2 (JAK2) or signal transducer and activator of transcription 1 (STAT1), but not by JAK1 siRNA nor by p53 inhibitor pifithrin-α. IFN-γ treatment increased the accumulation of intracellular ROS in melanocytes, while ROS scavenger N-acetyl cysteine (NAC) effectively inhibited IFN-γ induced p21 expression and melanocyte senescence. IL-6 and HSP-70 release was significantly induced by IFN-γ treatment, which was largely inhibited by NAC. The increase of IL-6 and HSP-70 release could also be observed in senescent melanocytes.

Conclusion: IFN-γ can induce senescence in melanocytes and consequently enhance their immuno-competency, leading to a vitiligo-prone milieu.

Figure 1. IFN-γ decreased viability of melanocytes, caused apoptosis and cell cycle arrest.

Primary normal human Melanocytes were treated with various concentrations of IFN-γ (0, 100 or 1000 U/ml) for 72 h. Cell viability was then examined by MTS assay (A). Apoptosis was analyzed by flow cytometry after cells were stained with PI and Annexin V-FITC (B). (C) Cell cycle distribution of melanocytes was measured 24 h post IFN-γ treatment. Results are presented as mean ± SD from at least three independent melanocyte cultures. *P<0.05, **P<0.01, Student's t-test compared with controls.

Figure 2. Effects of IFN-γ on melanogenesis in normal melanocytes.

(A) Melanocytes were treated with various concentrations of IFN-γ (0, 100 or 1000 U/ml) for 3 or 7days before melanin content was measured. The melanin content was normalized on the basis of protein concentration. (b–g) Total RNA was extracted from melanocytes treated with or without IFN-γ for 24 hours. Real-time PCR was then performed to evaluate the relative mRNA levels of (B) tyrosinase (TYR), (C) tyrosinase-related protein 1 (TYRP1), (D) Melan-A, (E) melanocyte protein 17 (PMEL17), (F) microphthalmia-associated transcription factor (MITF), and (G) dopachrome tautomerase (DCT). The values shown represent the mean ± SD of three independent melanocyte cultures. *P<0.05 and **P<0.01.

Figure 3. IFN-γ caused senescence in melanocytes through p21 pathway.

Melanocytes were treated with or without 100/ml IFN-γ for 7 days. Senescence was evaluated based on SA-β-gal staining, and cell morphology. (A) Representative pictures of SA-β-gal-stained cells observed under bright-field microscope. Flattened and enlarged cells with blue/green stain were regarded as senescent cells (B) Quantification of SA-β-gal-positive cells based on microscopic analysis. CON represents the control cells. **P<0.01. (C) After 7 days of treatment, melanocytes were cultured in fresh medium without IFN-γ for 3 days and cell viability was examined by MTS assay. (D) Melanocytes were cultured in the presence or absence of IFN-γ for up to 7 days and cells were harvest on day 1, 3 and 7. Cell lysates were subjected to SDS-PAGE and analyzed by western blot with indicated antibodies. β-actin was probed as the loading control. (E,) Bar graphs of SA-β-gal staining results. (E) Melanocytes were transfected with scrambled control or p21 siRNAs for 48 h before IFN-γ treatment. (F) Melanocytes were treated with or without 100 U/ml IFN-γ for 7 days in the presence of DMSO or 20 µM pifithrin-α (PFT-α).

Figure 4. JAK2 and STAT1 activities are necessary for IFN-γ caused melanocyte senescence.

Melanocytes were transfected with JAK1, JAK2, STAT1 siRNAs or scrambled control siRNA (Ctrl). After 48 h, cells were treated with or without 100 U/ml IFN-γ for additional 7 days. (A) Cell viability of melancytes was measured by MTS assay. (B) Protein level of p21 was evaluated by Western blot. β-actin was probed as the loading control. (C) Percentages of SA-β-gal-positive cells were determined based on microscopic analysis.

Figure 5. Involvement of Reactive Oxygen Species (ROS) in the IFN-γ induced senescence.

(A) Melancytes were stimulated with indicated concentration of IFN-γ for 24 h. Generated ROS was detected with flow cytometer after labelled with the ROS sensor DCFH-DA. (B,C) Melanocytes were treated with or without 100 U/ml IFN-γ for 7 days in the presence of vehicle or 1 mM NAC. (B) Protein level of p21 was evaluated by Western blot. β-actin was probed as the loading control. (C) Percentages of SA-β-gal-positive cells were determined based on microscopic analysis. CON represents the cell culture without IFN-γ.

Figure 6. Release of IL-6 and hsp70 from melanocytes was enhanced after persistent IFN-γ treatment or senescence induction.

(A) Melanocytes were treated with or without 100 U/ml IFN-γ for continuous 7 days. RNA was extracted from melanocytes at day 1 and day 7. Real time PCR was performed to evaluate the transcription of IL-6 in these cells. CON represents the control cells. (B–D) Melanocytes were treated with or without 100 U/ml IFN-γ for 7 days in the presence of vehicle or 1 mM NAC. Supernatants of cell culture were collected at the indicated time points. Medium was changed 48 h before the supernatant collection. Release of IL-6 (B) or HSP-70 (C) in response to IFN-γ treatment was monitored by ELISA analysis. (D) The effect of NAC on the release of IL-6 and HSP-70 after IFN-γ treatment was evaluated. (E) Melanocytes were treated with or without 100 U/ml IFN-γ for 7 days to induce senescence. Senescent melanocytes were then cultured in normal medium for 4 days before the supernatant was collected. The medium was changed 48 h before the supernatant collection. Protein levels of released IL-6 and HSP-70 from senescent cells were compared with those from normal cells.