Keratinocyte Growth Factor Promotes Melanosome Transfer to Keratinocytes  Giorgia Cardinali, Simona Ceccarelli, Daniela Kovacs, Nicaela Aspite, Lavinia Vittoria Lotti, Maria Rosaria Torrisi, Mauro Picardo  Journal of Investigative Dermatology  Volume 125, Issue 6, Pages 1190-1199 (December 2005) DOI: 10.1111/j.0022-202X.2005.23929.x Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 KGF induces melanosome transfer in co-cultures of HaCaT (K) and melanoma (M) cells. (A–D) Double immunolabeling with anti-tyrosinase antibody (green) and anti-cytokeratin antibody (red) shows that, different from untreated cultures, in both keratinocyte growth factor (KGF) (20 ng per mL for 6 h) (B) or ultraviolet B (UVB)-treated cultures (40 mJ per cm2) (C) the keratinocytes, identified by the positive staining for cytokeratin, appear frequently to contain intracytoplasmic dots positive for tyrosinase, indicating melanosome transfer. Parallel experiments using melanocyte-stimulating hormone (α-MSH) (160 ng per mL for 6 h) (D) also show an increased uptake of melanosomes by keratinocytes. Scale bar=10 μm. (E) Percentage of tyrosinase-positive HaCaT cells co-cultured with melanoma cells treated for 6 h with KGF (20 ng per mL) or α-MSH (160 ng per mL) and exposed to UVB (40 mJ per cm2) compared with untreated cells and to cells treated with heat-denatured KGF (20 ng per mL) or with bovine serum albumin (0.7 nM). Immunolabeling with anti-tyrosinase antibody shows that both KGF and UVB treatments clearly increase melanosome transfer to HaCaT cells. Results represent the mean value±SE. For each treatment, a total of 500 cells, randomly observed from 20 microscopic fields in two different experiments, were analyzed. Journal of Investigative Dermatology 2005 125, 1190-1199DOI: (10.1111/j.0022-202X.2005.23929.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 KGF induces melanosome transfer in co-cultures of human primary keratinocytes and melanocytes. (A–H) Immunofluorescence and electron microscopy analysis following keratinocyte growth factor (KGF) treatment (20 ng per mL for 6 h) or exposure to different doses of ultraviolet B (UVB) (40–50–60 mJ per cm2). Immunolabeling with anti-tyrosinase antibody to identify melanosomes shows an increase in fluorescent spotted signal in keratinocytes, revealing melanosome transfer from melanocytes (M) to the neighboring keratinocytes (K). In UVB-exposed cells, the melanosomes appear to be transferred to the keratinocytes from the tips of long and narrow melanocyte dendrites (arrows in C, F, G), whereas KGF treatment promotes melanosome transfer to keratinocytes from large extensions of the melanocyte body and through cell–cell contacts (B, E). Nuclei are stained with 4′,6-diamido-2-phenylindole dihydrochloride. Ultrastructural analysis shows intact melanosomes inside phagosomal vacuoles after KGF treatment (F, arrow). Scale bars: (A–E, G, H)=10 and F=1 μm. (I) Percentage of tyrosinase-positive human keratinocytes in co-culture of human primary keratinocytes and melanocytes treated with KGF (20 ng per mL for 6 h) or melanocyte-stimulating hormone (α-MSH) (160 ng per mL for 6 h) or exposed to UVB (40–50–60 mJ per cm2) compared to untreated cells. The melanosome transfer process appears to be increased by KGF and α-MSH treatments with respect to untreated cells and the UVB dose of 40 mJ per cm2 is sufficient for the maximal transfer. Results represent the mean value±SE. For each treatment, a total of 500 cells, randomly observed from 20 microscopic fields in two different experiments, were analyzed. Journal of Investigative Dermatology 2005 125, 1190-1199DOI: (10.1111/j.0022-202X.2005.23929.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 KGF treatment promotes phagocytosis of 0.1 μm (green) and 1 μm (red) beads in keratinocytes. (B) Keratinocyte growth factor (KGF) treatment (20 ng per mL for 6 h). Exposure to ultraviolet B (UVB) (40 mJ per cm2) (C). Nuclei are stained with 4′,6-diamido-2-phenylindole dihydrochloride. Scale bar=10 μm. (D) Quantitative analysis of the fluorescence intensity resulting from the intracellular green and red beads shows an increased uptake, in particular of the 0.1 μm green beads, induced by KGF (20 ng per mL for 6 h) or UVB exposure (40 mJ per cm2) compared with untreated cells. Analysis was performed evaluating 150 cells for each condition, randomly taken from 10 different microscopic fields, and values are expressed as the average of measurements±SE. Journal of Investigative Dermatology 2005 125, 1190-1199DOI: (10.1111/j.0022-202X.2005.23929.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 KGF produces the internalization of 0.5 μm beads in human primary keratinocytes and HaCaT cells. (A–P) Immunofluorescence analysis and parallel phase contrast microscopy of human primary keratinocytes (A–H) and human immortalized keratinocytes (HaCaT) cells (I–P) incubated with 0.5 μm fluorescent microspheres (red) in the presence or absence of keratinocyte growth factor (KGF) treatment (20 ng per mL). In KGF-treated cells (C, G, K, O), an increase in the number of internalized beads compared to untreated cells (A, E, I, M) is evident after both 1 and 4 h of incubation. In addition, at the 4 h time point, the beads appear preferentially localized in the perinuclear area, indicating intracellular transport of the beads inside phagocytic structures. Nuclei are stained with 4′,6-diamido-2-phenylindole dihydrochloride. Scale bar=10 μm. (Q) Quantitative analysis of the 0.5 μm beads in normal human keratinocytes (NHK), NHK of light and dark skin individuals, and human immortalized keratinocytes (HaCaT) cells demonstrates that KGF (20 ng per mL) induces an increased uptake of the beads (red and green lines) compared to untreated cells (blue and brown lines). Analysis was performed by counting the number of internalized beads in 150 cells for each condition, randomly taken from 10 microscopic fields in three different experiments, and values are expressed as the mean value±SE. Journal of Investigative Dermatology 2005 125, 1190-1199DOI: (10.1111/j.0022-202X.2005.23929.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Inhibitors of PAR-2 and of Src-family tyrosine kinases significantly reduce KGF-induced phagocytosis. (A–C) Ultrastructural analysis of normal human keratinocytes (NHK) incubated with 0.5 μm microspheres for 30 min (A, B) or 4 h (C) in the presence of keratinocyte growth factor (KGF) (20 ng per mL). After 30 min of incubation, membrane protrusions, which extend around the beads to be ingested (arrows in A and B) are visible on the cell surface, whereas after 4 h the internalized beads are surrounded by the phagosomal membranes (arrow in C). PM, plasma membrane; er, endoplasmic reticulum; k, keratin filaments; NM, nuclear membrane; Nu, nucleus. Scale bars=0.5 μm. (D) Quantitative immunofluorescence analysis of NHK incubated with 0.5 μm fluorescent microspheres, treated with soybean trypsin inhibitor (STI, 0.01%, 0.1%, and 1%) and 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo-D-3,4-pyrimidine (PP1) (10 and 20 μM) in the presence or absence of KGF (20 ng per mL) for 4 h. Both inhibitors appear to block the KGF effect on the stimulation of beads uptake. The analysis was performed by counting the number of internalized beads in 150 cells for each condition, randomly taken from 10 microscopic fields in three different experiments, and values are expressed as the mean value±SE. Journal of Investigative Dermatology 2005 125, 1190-1199DOI: (10.1111/j.0022-202X.2005.23929.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 KGF promotes phagocytosis in HaCaT cells transfected with KGFR. Cells untreated (A and B) or treated with KGF (20 ng per mL for 2 h) (C–F) were incubated with 0.5 μm fluorescent microspheres (red): the uptake induced by KGF is enhanced in cells overexpressing KGFR by transfection and identified by staining with anti-Bek antibody (green) (C, D). Confocal analysis reveals the presence of KGFR in phagocytic structures: co-localization of the two signals (KGFR: green; 0.5 μm beads: red) either at the cell surface (arrow and inset in E) or in intracellular compartment after bead internalization (arrow and inset in F) is shown in yellow. Arrowhead in E points to an extracellular red bead. Scale bars: (A–D)=10 and (E, F)=5 μm. (G) Quantitative analysis of the 0.5 μm beads shows that the uptake of beads induced by KGF is greater in HaCaT cells highly positive for KGFR. Analysis was performed by counting the number of internalized beads in 150 cells for each condition, randomly taken from 10 microscopic fields in three different experiments, and values are expressed as the mean value±SE. (H) Quantitative analysis of normal human keratinocytes (NHK) incubated with 0.5 μm fluorescent microspheres, treated with genistein (100 μM) in the presence or absence of KGF (20 ng per mL) for 6 h: the tyrosine kinase inhibitor appear to block the KGF effect on the stimulation of beads uptake. Parallel quantitative analysis of NHK treated for 6 h with KGF (20 ng per mL) and epidermal growth factor (EGF) (20 ng per mL) shows that, unlike KGF, EGF does not affect the beads uptake. The analysis was performed by counting the number of internalized beads in 150 cells for each condition, randomly taken from 10 microscopic fields in three different experiments, and values are expressed as the mean value±SE. Journal of Investigative Dermatology 2005 125, 1190-1199DOI: (10.1111/j.0022-202X.2005.23929.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions