Iron sensitizes keratinocytes and fibroblasts to UVA-mediated matrix metalloproteinase-1 through TNF-α and ERK activation

Abstract

Oestrogen deficiency is regarded as the main causative factor in postmenopausal skin ageing and photoageing. While women after menopause experience low levels of oestrogen because of cease of ovarian function, they are also exposed to high levels of iron as a result of cessation of menstruation. In this study, we investigated whether this increase in iron presents a risk to the postmenopausal skin. Because of the lack of appropriate animal models to closely mimic the low oestrogen and high iron conditions, we tested the hypothesis in a high iron and low oestrogen culture model. Here, we showed that primary human dermal fibroblasts exposed to iron did not affect the baseline levels of matrix metalloproteinase-1 (MMP-1) activity. However, the iron-exposed fibroblasts were sensitized to UVA exposure, which resulted in a synergistic increase in MMP-1. UVA activated the three members of MAPK family: ERKs, p38, and JNKs. Additional activation of ERKs by iron contributed to the synergistic increases. Primary normal human epidermal keratinocytes (NHEK) did not respond to iron or UVA exposure as measured by MMP-1, but produced tumor necrosis factor-alpha (TNF-α) in the media, which then stimulated MMP-1 in fibroblasts. Our results indicate that iron and UVA increase MMP-1 activity in dermal fibroblasts not only directly through ERK activation but also by an indirect paracrine loop through TNF-α released by NHEK. We conclude that in addition to oestrogen deficiency, increased iron as a result of menopause could be a novel risk factor by sensitizing postmenopausal skin to solar irradiation.

Figure 1

Effects of UVA and iron on metalloproteinase (MMP)-1, 2, 9 activities in primary normal human epidermal keratinocytes (NHEK) cells and human dermal fibroblasts. (a) Primary human NHEK cells and dermal fibroblasts were grown in Ctrl (base media), Pre- (base media plus 500 ng/ml E2 and 5 μg/ml Apo-Tf) or Post-(base media plus 20 ng/ml ferritin and 5 μg/ml holo-Tf) conditions for overnight and then exposed to UVA at 50 kJ/m². After 24 h cultures, the media were collected for MMP-1 activities. (b) Fibroblasts grown under Post-condition were pretreated with deferoxamine at 50 μg/ml for 1 h before UVA exposure. (c) Twenty microlitres of cell culture media from NHEK cells and fibroblasts was loaded into a 10% non-reducing SDS–PAGE containing 2 mg/ml gelatin. After electrophoresis, the gels were washed to remove SDS and incubated overnight in an incubation buffer. Clear bands show digestion of gelatin by MMP-2 and MMP-9. * Significantly different from their respective controls (P < 0.05).

Figure 2

Participation of ERK pathway in iron- and UVA-mediated metalloproteinase-1 activities. (a) Fibroblasts grown under Post-condition were pretreated with different kinase inhibitors for 1 h before UVA exposure. DMSO was used as control, PD98059 (final concentration 10 μM) as ERK inhibitor, U0126 (20 μM) as MEK1 inhibitor, SP600126 (10 μM) as JNK inhibitor, SB202190 (10 μM) as P38 inhibitor, Wortmannin (0.5 μM) as PI-3K inhibitor. (b) Phosphorylations of ERK, P38 and JNK were measured in fibroblasts grown under Pre- and Post-conditions and post UVA exposure. *Significantly different from their respective controls (P < 0.05).

Figure 3

Increases of metalloproteinase (MMP)-1 activities in fibroblasts by media transferred from UVA-exposed normal human epidermal keratinocytes (NHEK) cells. (a) Background levels of MMP-1 were low in NHEK cells and were slightly decreased after UVA exposure. (b) Levels of MMP-1 in fibroblasts were significantly induced by media transferred from UVA-exposed NHEK cells grown under Post-condition. One millilitre of media from NHEK cells (top) were added into 2 ml of fibroblasts and 24 h later the media were collected for MMP-1 measurements. *Significantly different from the controls without UVA exposure (P < 0.05).

Figure 4

Levels of mRNA expressions of potential metalloproteinase (MMP)-1 mediators in normal human epidermal keratinocytes (NHEK) cells. NHEK cells grown under Ctrl, Pre- and Post-conditions were exposed to UVA at 50 kJ/m². After RNA extraction and reverse-transcription, the mRNA expression levels of different MMP-1 mediators were measured by real-time-PCR. *Significantly different from the controls without UVA exposure (P < 0.05).

Figure 5

Neutralizing effects of anti-tumor necrosis factor (TNF)-α antibody on normal human epidermal keratinocytes (NHEK) media-induced metalloproteinase (MMP)-1 activities in fibroblasts. Media from NHEK cells grown under Ctrl, Pre- and Post-conditions ± UVA exposure were pretreated with control IgG or TNF-α neutralizing antibody at 2 μg/ml for 1 h. After transferring the media to fibroblasts for an additional 24 h, MMP-1 activities were measured. *Significantly different from the mIg controls (P < 0.05).

Figure 6

Proposed mechanisms of iron sensitization in UVA-mediated metalloproteinase (MMP)-1 activities. Left: Iron sensitizes normal human epidermal keratinocytes cells to release tumor necrosis factor-α (TNF-α) after UVA exposure. The secreted TNF-α subsequently stimulates MMP-1 in fibroblasts, indirect effects of iron and UVA on MMP-1 of fibroblasts. Right: Iron in the epidermis sensitizes fibroblasts to UVA-mediated ERK activation and MMP-1 induction. Because iron is in place and UVA penetrates the epidermis, this pathway is considered direct effects of iron and UVA on MMP-1 of fibroblasts.