Protoporphyrin IX Stimulates Melanogenesis, Melanocyte Dendricity, and Melanosome Transport Through the cGMP/PKG Pathway

Abstract

Protoporphyrin IX (PPIX) is a heterocyclic organic compound that is the last intermediate in the heme biosynthetic pathway. PPIX, due to its photodynamic effects, is utilized in the treatment of skin diseases. Furthermore, PPIX has been utilized as a melanogenesis-stimulating agent in various studies. However, the exact function and mechanism underlying PPIX action in melanocytes remain to be elucidated. In the present study, we sought to further investigate how PPIX affects melanocyte melanogenesis, and whether PPIX is involved in melanin transport. Our findings demonstrated that PPIX increased melanocyte dendricity and melanosome transport, in addition to increasing melanogenesis. PPIX functions primarily by activating the guanylate cyclase (GC) and cyclic guanosine 3', 5'-monophosphate/protein kinase G (cGMP/PKG) signaling pathways. Once activated, these pathways increase tyrosinase activity and the expression of microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein-1 and -2 (TRP-1 and TRP-2), myosin Va, melanophinin, Ras-related protein Rab-27A (Rab27a), and cell division cycle 42 (Cdc42), promoting melanogenesis, melanocyte dendricity, and melanosome transport. Furthermore, the melanogenic effects of PPIX were confirmed in vivo in a zebrafish model system. Our results indicate that PPIX is not cytotoxic and may, thus, be utilized as a pigmentation enhancer.

Keywords: 5’-monophosphate/protein kinase G; cyclic guanosine 3’; guanylate cyclase; melanogenesis; melanosome transport; protoporphyrin IX.

Figure 1

Protoporphyrin IX (PPIX) induced hyperpigmentation in melanocytes. (A) The chemical structure of PPIX. (B) After incubation of with various concentrations (10-40μM) of PPIX for 48 h, cell viability was determined using MTT assay. (C) SK-MEL-2 and HNM were treated with PPIX (10, 20, and 30 μM) for 48 h, and melanin contents were measured. (D) SK-MEL-2 cells were treated with PPIX for 48 h and then stained with Masson-Fontana ammoniacal silver stain. Bar = 20 μm. Total length of dendrites per cell was measured on the pictures using ruler. Data are expressed as the mean ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 versus non-treated cells.

Figure 2

PPIX increased the tyrosinase activity and expression of tyrosinase and MITF. (A) Cellular tyrosinase activity and (B) mushroom tyrosinase activity in a cell-free assay system were measured after PPIX-treated as described in methods. SK-MEL-2 and HNM cells were treated with PPIX (10, 20, and 30 μM) for 12 h, and RT-qPCR was then applied to detect tyrosinase (C, G), TRP-1 (D, H), TRP-2 (E, I), and MITF (F, J) gene expression. (K) SK-MEL-2 cells and (L) HNM were treated with PPIX (10, 20, and 30 μM) for 48 h, and western blot was then applied to detect the protein levels of tyrosinase, TRP-1, TRP-2, and MITF. Data are expressed as the mean ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 versus non-treated cells.

Figure 3

PPIX induced melanosome transport in melanocytes. (A) In cocultured SK-MEL-2 and HaCaT cells, melanosome with yellow signal (arrow) were evident in cytokeratin-positive HaCaT cells. Melanosome labeling with GP100 (green). HaCaT cells labeling with cytokeratin (red). Bar = 20 μm. Quantification of melanosomes transferred to HaCaT cells. Twenty cells per condition were assessed in each of three independent experiments. SK-MEL-2 cells and HNM were treated with PPIX (10, 20, and 30 μM) for 12 hours, and RT-qPCR was then applied to detect myosin Va (B, G), KIF5b (C, H), melanophinin (D, I), Rab27a (E, J), and Cdc42 (F, K) gene expression. (L) SK-MEL-2 and (M) HNM cells were treated with PPIX (10, 20, and 30 μM). After 48-h treatment, the expression levels of myosin Va, KIF5b, melanophinin, Rab27a, and Cdc42 were examined. The results were shown as relative values to the control. Data are expressed as the mean ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 versus non-treated cells.

Figure 4

The effects of PPIX on the activity of GC/cGMP/PKG signaling pathways in melanocytes. (A) Guanylate cyclase (GC) activity in melanocytes treated with PPIX was measured. Effect of PPIX on cellular cGMP levels (B) and PKG activity (C) in melanocytes was examined after cells were treated with PPIX. (D) Quantification of cAMP-response element-binding protein (CREB) and phosphorylation of CREB (p-CREB) protein expression levels were evaluated by western blotting. Data are expressed as the mean ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 versus non-treated cells.

Figure 5

Inhibitory effects of LY83583 and KT5823 on PPIX-induced pigmentation in SK-MEL-2 cells. SK-MEL-2 was pretreated or not with 10 μM LY83583 (or 1 μM KT5823) for 1 h before PPIX was applied for 48 h at 30μM. (A) Melanin contents were measured, and (B) SK-MEL-2 cells were stained with Masson-Fontana ammoniacal silver stain. Bar = 20 μm. Total length of dendrites per cell was measured on the pictures using ruler. (C) Western blotting for the protein expression relating to melanogenesis and melanosome transport were measured. Data are expressed as the mean ± SEM (n = 3). *p < 0.05, **p < 0.01 versus non-treated cells. ^#p < 0.05, ^##p < 0.01 versus PPIX-treated cells.

Figure 6

Effect of PPIX on pigmentation in zebrafish. (A) Representative photographs of zebrafish. Zebrafish embryos were treated with PPIX from 35 to 60 h. The effects on the pigmentation of zebrafish were observed under the stereomicroscope. (B) Tyrosinase activity. For measurement of tyrosinase activity, 100 μg of total protein was incubated with L-DOPA (final, 0.5 mM) and then quantified using a spectrometer. The results were shown as relative values to the control. Data are expressed as the mean ± SEM (n = 3). *p < 0.05, **p < 0.01 versus non-treated zebrafish.

Figure 7

Schematic description of changes in pigmentation upon PPIX treatment.