Keratinocyte Growth Factor Promotes Melanosome Transfer to Keratinocytes
2005; Elsevier BV; Volume: 125; Issue: 6 Linguagem: Inglês
10.1111/j.0022-202x.2005.23929.x
ISSN1523-1747
AutoresGiorgia Cardinali, Simona Ceccarelli, Daniela Kovacs, Nicaela Aspite, Lavinia Vittoria Lotti, Maria Rosaria Torrisi, Mauro Picardo,
Tópico(s)Mast cells and histamine
ResumoMelanogenesis and melanosome transfer from the melanocytes to the neighboring keratinocytes are induced by ultraviolet radiation and modulated by autocrine and paracrine factors. Keratinocyte growth factor (KGF/fibroblast growth factor (FGF)7) is a paracrine mediator of human keratinocyte growth and differentiation. We evaluated the influence of KGF on melanosome transfer in co-cultures of keratinocytes and melanocytes. Immunofluorescence analysis using anti-tyrosinase and anti-human cytokeratin antibodies, phagocytic assays using fluorescent latex beads, and ultrastructural analysis indicated that KGF is able to induce melanosome transfer acting only on the recipient keratinocytes and as a consequence of a general role of KGF in the promotion of the phagocytic process. Inhibition of proteinase-activated receptor-2, to block the Rho-dependent phagocytic pathway, or of the Src family tyrosine kinases, to inhibit the Rac-dependent pathway, showed that KGF promotes phagocytosis through both mechanisms. Increased expression of the KGF receptor (KGFR) on the keratinocytes by transfection led to increased phagocytosis of latex beads following KGF treatment, suggesting that the KGF effect is directly mediated by KGFR expression and activation. Moreover, confocal microscopic analysis revealed that KGFR localize in phagosomes during KGF-induced phagocytosis, suggesting a direct role of the receptor in regulating both the early steps of uptake and the intracellular traffic of the phagosomes. Melanogenesis and melanosome transfer from the melanocytes to the neighboring keratinocytes are induced by ultraviolet radiation and modulated by autocrine and paracrine factors. Keratinocyte growth factor (KGF/fibroblast growth factor (FGF)7) is a paracrine mediator of human keratinocyte growth and differentiation. We evaluated the influence of KGF on melanosome transfer in co-cultures of keratinocytes and melanocytes. Immunofluorescence analysis using anti-tyrosinase and anti-human cytokeratin antibodies, phagocytic assays using fluorescent latex beads, and ultrastructural analysis indicated that KGF is able to induce melanosome transfer acting only on the recipient keratinocytes and as a consequence of a general role of KGF in the promotion of the phagocytic process. Inhibition of proteinase-activated receptor-2, to block the Rho-dependent phagocytic pathway, or of the Src family tyrosine kinases, to inhibit the Rac-dependent pathway, showed that KGF promotes phagocytosis through both mechanisms. Increased expression of the KGF receptor (KGFR) on the keratinocytes by transfection led to increased phagocytosis of latex beads following KGF treatment, suggesting that the KGF effect is directly mediated by KGFR expression and activation. Moreover, confocal microscopic analysis revealed that KGFR localize in phagosomes during KGF-induced phagocytosis, suggesting a direct role of the receptor in regulating both the early steps of uptake and the intracellular traffic of the phagosomes. epidermal growth factor fibroblast growth factor keratinocyte growth factor keratinocyte growth factor receptor melanocyte-stimulating hormone normal human keratinocytes Pigmentation of the skin and protection from ultraviolet (UV) irradiation are known to be strictly dependent on the type and quantity of melanin synthesized by the melanocytes and its subsequent transfer to the neighboring keratinocytes. Melanin is synthesized and packaged in organelles containing melanogenic enzymes (tyrosinase gene family of proteins), the melanosomes, which are translocated down to the tips of the melanocyte dendrites and then transferred to the neighboring keratinocytes, where they form melanin cap over the nuclei to protect DNA from UV damage (for recent reviews, seeBoissy, 2003Boissy R.E. Melanosome transfer to and translocation in the keratinocytes.Exp Dermatol. 2003; 12: 5-12Crossref PubMed Scopus (193) Google Scholar; Imokawa, 2004Imokawa G. Autocrine and paracrine regulation of melanocytes in human skin and in pigmentary disorders.Pigment Cell Res. 2004; 17: 96-110Crossref PubMed Scopus (286) Google Scholar). The biological processes involved in melanosome transfer are not completely defined and different mechanisms that may coexist have been proposed: (i) exocytosis of the melanosomes into the extracellular space and their subsequent endocytosis by keratinocytes; (ii) cytophagocytosis of the dendritic tips of the melanocytes by keratinocytes; (iii) direct injection of melanosomes into the keratinocytes. In support of the model involving phagocytosis, there are several studies which analyzed the epidermal keratinocyte phagocytic ability in vitro through internalization of latex microspheres (Wolff and Konrad, 1972Wolff K. Konrad K. Phagocytosis of latex beads by epidermal keratinocytes in vivo.J Ultrastruct Res. 1972; 39: 262-280Crossref PubMed Scopus (66) Google Scholar; Virador et al., 2002Virador V.M. Muller J. Wu X. et al.Influence of α-melanocyte-stimulating hormone and of ultraviolet radiation on the transfer of melanosomes to keratinocytes.FASEB J. 2002; 16: 105-107PubMed Google Scholar; Desjardins and Griffiths, 2003Desjardins M. Griffiths G. Phagocytosis: Latex leads the way.Curr Opin Cell Biol. 2003; 15: 498-503Crossref PubMed Scopus (116) Google Scholar). It is well known that both melanosome release by melanocytes and melanosome endocytosis and phagocytosis by keratinocytes are induced by UV radiation and are modulated by autocrine and paracrine factors (Imokawa, 2004Imokawa G. Autocrine and paracrine regulation of melanocytes in human skin and in pigmentary disorders.Pigment Cell Res. 2004; 17: 96-110Crossref PubMed Scopus (286) Google Scholar) such as the melanocyte-stimulating hormone (α-MSH) (Virador et al., 2002Virador V.M. Muller J. Wu X. et al.Influence of α-melanocyte-stimulating hormone and of ultraviolet radiation on the transfer of melanosomes to keratinocytes.FASEB J. 2002; 16: 105-107PubMed Google Scholar). Ultraviolet B (UVB) irradiation of keratinocytes stimulates pigmentation in human skin through different mechanisms, including upregulation of melanogenic enzymes and increase of melanocyte dendricity and melanosome transfer to keratinocytes. More recently it has been demonstrated that one of the mechanisms responsible for pigmentation induced by UVB is an increase in the phagocytic activity of keratinocytes by upregulation in the expression and activation of the proteinase-activated receptor-2 (PAR-2) (Seiberg et al., 2000aSeiberg M. Paine C. Sharlow E. Andrade-Gordon P. Costanzo M. Eisinger M. Shapiro S.S. The protease-activated receptor-2 regulates pigmentation via keratinocyte–melanocyte interactions.Exp Cell Res. 2000; 254: 25-32Crossref PubMed Scopus (196) Google Scholar; Sharlow et al., 2000Sharlow E.R. Paine C. Babiarz L. Eisinger M. Shapiro S.S. Seiberg M. The protease activated receptor-2 upregulates keratinocyte phagocytosis.J Cell Sci. 2000; 113: 3093-3101PubMed Google Scholar). In fact, the expression of PAR-2 is increased in human skin and in cultured keratinocytes after ultraviolet R (UVR) (Scott et al., 2001Scott G. Deng A. Rodriguez-Burford C. et al.Protease-activated receptor 2, a receptor involved in melanosome transfer, is upregulated in human skin by ultraviolet irradiation.J Invest Dermatol. 2001; 117: 1412-1420Crossref PubMed Scopus (88) Google Scholar), and the inhibition of its activation prevents UVB-induced pigmentation in vivo and melanosome transfer in vitro (Seiberg et al., 2000bSeiberg M. Paine C. Sharlow E. Andrade-Gordon P. Costanzo M. Eisinger M. Shapiro S.S. Inhibition of melanosome transfer results in skin lightening.J Invest Dermatol. 2000; 115: 162-167Crossref PubMed Scopus (184) Google Scholar; Paine et al., 2001Paine C. Sharlow E. Liebel F. Eisinger M. Shapiro S. Seiberg M. An alternative approch to depigmentation by soybean extracts via inhibition of the PAR-2 pathway.J Invest Dermatol. 2001; 116: 587-595Crossref PubMed Scopus (134) Google Scholar), reducing the phagocytic capability in keratinocytes. Finally, it has been shown that the activation of PAR-2 results in activation of Rho (Scott et al., 2003Scott G. Leopardi S. Parker L. Babiarz L. Seiberg M. Han R. The Proteinase-Activated Receptor-2 mediates phagocytosis in a Rho-dependent manner in human keratinocytes.J Invest Dermatol. 2003; 121: 529-541Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), one of the Rho family GTPase, which are known to play critical roles in cytoskeletal remodeling during phagocytosis (Conner and Schmid, 2003Conner S.D. Schmid S.L. Regulated portals of entry into cell.Nature. 2003; 422: 37-44Crossref PubMed Scopus (2850) Google Scholar; Niedergang and Chayrier, 2004Niedergang F. Chayrier P. Signaling and membrane dynamics during phagocytosis: Many roads lead to the phagos(R)ome.Curr Opin Cell Biol. 2004; 16: 422-428Crossref PubMed Scopus (113) Google Scholar). The earliest step in the cellular response to UV involves ligand-independent phosphorylation and activation of growth factor receptors (Coffer et al., 1995Coffer P.J. Burgering B.M. Peppelenbosch M.P. Bos J.L. Kruijer W. UV activation of receptor tyrosine kinase activity.Oncogene. 1995; 11: 561-569PubMed Google Scholar; Huang et al., 1996Huang R.P. Wu J.X. Fan Y. Adamson E.D. UV activates growth factor receptors via reactive oxygen intermediates.J Cell Biol. 1996; 133: 211-220Crossref PubMed Scopus (246) Google Scholar), subsequent triggering of signal transduction pathways (Rosette and Karin, 1996Rosette C. Karin M. Ultraviolet light and osmotic stress: Activation of the JNK cascade through multiple growth factor and cytokine receptors.Science. 1996; 15: 1194-1197Crossref Scopus (926) Google Scholar), and rapid expression of early growth response genes (Egr-1) (Huang and Adamson, 1995Huang R.P. Adamson E.D. A biological role for Egr-1 in cell survival following ultra-violet irradiation.Oncogene. 1995; 2: 467-475Google Scholar). We have recently demonstrated that exposure to UVB is able to trigger activation and internalization of the receptor for the keratinocyte growth factor (KGF/fibroblast growth factor (FGF)7) (Marchese et al., 2003Marchese C. Maresca V. Cardinali G. et al.UVB-induced activation and internalization of keratinocyte growth factor receptor.Oncogene. 2003; 22: 2422-2431Crossref PubMed Scopus (53) Google Scholar), which is a member of the FGF family and represents a key mediator of epithelial growth and differentiation. Secreted from mesenchymal cells, KGF elicits its activity specifically on epithelial cells (Finch et al., 1989Finch P.W. Rubin J.S. Miki T. Ron D. Aaronson S.A. Human KGF is FGF-related with properties of a paracrine effector of epithelial cell growth.Science. 1989; 245: 752-755Crossref PubMed Scopus (795) Google Scholar; Rubin et al., 1989Rubin J.S. Osada H. Finch P.W. Taylor W.G. Rudikoff S. Aaronson S.A. Purification and characterization of a newly identified growth factor specific for epithelial cells.Proc Natl Acad Sci USA. 1989; 86: 802-806Crossref PubMed Scopus (719) Google Scholar) through binding to the keratinocyte growth factor receptor (KGFR), a splicing transcript variant of the FGFR2 (Miki et al., 1991Miki T. Fleming T.P. Bottaro D.P. Rubin J.S. Ron D. Aaronson S.A. Expression cDNA cloning of the KGF receptor by creation of a transforming autocrine loop.Science. 1991; 251: 72-75Crossref PubMed Scopus (357) Google Scholar,Miki et al., 1992Miki T. Bottaro D.P. Fleming T.P. Smith C.L. Burgess W.H. Chan A.M. Aaronson S.A. Determination of ligand-binding specificity by alternative splicing: Two distinct growth factor receptors encoded by a single gene.Proc Natl Acad Sci USA. 1992; 89: 246-250Crossref PubMed Scopus (633) Google Scholar). On cultured human keratinocytes, KGF acts as a potent mitogen, promotes their early differentiation (Marchese et al., 1990Marchese C. Rubin J. Ron D. et al.Human keratinocyte growth factor activity on proliferation and differentiation of human keratinocyte: Differentiation response distinguishes KGF from EGF family.J Cell Physiol. 1990; 144: 326-332Crossref PubMed Scopus (196) Google Scholar) and inhibits their terminal differentiation and apoptosis (Hines and Allen-Hoffman, 1996Hines M.D. Allen-Hoffman B.L. Keratinocyte growth factor inhibits cross-linked envelope formation and nucleosomal fragmentation in cultured human keratinocytes.J Biol Chem. 1996; 271: 6245-6251Crossref PubMed Scopus (69) Google Scholar). In vivo, KGF appears to play a role in experimental and human wound healing (Werner et al., 1992Werner S. Peters K.G. Longaker M.T. Fuller-Pace F. Banda M.J. Williams L.T. Large induction of keratinocyte growth factor expression in the dermis during wound healing.Proc Natl Acad Sci USA. 1992; 89: 6896-6900Crossref PubMed Scopus (514) Google Scholar; Marchese et al., 1995Marchese C. Chedid M. Dirsch O.R. et al.Modulation of keratinocyte growth factor and its receptor in reepithelializing human skin.J Exp Med. 1995; 182: 1369-1376Crossref PubMed Scopus (142) Google Scholar). In addition, KGF induces cell migration and reorganization of the actin cytoskeleton (Putnins et al., 1999Putnins E.E. Firth J.D. Lohachitranont A. Uitto V.J. Larjava H. Keratinocyte growth factor (KGF) promotes keratinocyte cell attachment and migration on collagen and fibronectin.Cell Adhes Commun. 1999; 7: 211-221Crossref PubMed Scopus (41) Google Scholar; Mehta et al., 2001Mehta P.B. Robson C.N. Neal D.E. Leung H.Y. Keratinocyte growth factor activates p38 MAPK to induce stress fibre formation in human prostate DU145 cells.Oncogene. 2001; 20: 5359-5365Crossref PubMed Scopus (39) Google Scholar; Galiacy et al., 2003Galiacy S. Planus E. Lepetit H. et al.Keratinocyte growth factor promotes cell motility during alveolar epithelial repair in vitro.Exp Cell Res. 2003; 283: 215-229Crossref PubMed Scopus (42) Google Scholar). Since both UVB and KGF may activate KGFR, inducing a similar signal transduction, the aim of this study was to evaluate if KGF, similarly to UVB, could promote melanosome transfer through direct action on the keratinocytes, providing also a model for selective analysis of the contribution of the recipient keratinocytes to the transfer process. The process of melanosome transfer from melanocytes to keratinocytes has been widely studied in vitro using co-culture models of the two cell types (Seiberg et al., 2000aSeiberg M. Paine C. Sharlow E. Andrade-Gordon P. Costanzo M. Eisinger M. Shapiro S.S. The protease-activated receptor-2 regulates pigmentation via keratinocyte–melanocyte interactions.Exp Cell Res. 2000; 254: 25-32Crossref PubMed Scopus (196) Google Scholar; Duval et al., 2001Duval C. Regnier M. Schmidt R. Distinct melanogenic response of human melanocytes in mono-culture, in co-culture with keratinocytes and in reconstructed epidermis, to UV exposure.Pigment Cell Res. 2001; 14: 348-355Crossref PubMed Scopus (78) Google Scholar; Scott, 2002Scott G. Rac and Rho: The story behind melanocyte dendrite formation.Pigment Cell Res. 2002; 15: 322-330Crossref PubMed Scopus (66) Google Scholar; Boissy, 2003Boissy R.E. Melanosome transfer to and translocation in the keratinocytes.Exp Dermatol. 2003; 12: 5-12Crossref PubMed Scopus (193) Google Scholar). Therefore, to evaluate in vitro the possible role of KGF on melanosome transfer, we used either primary co-cultures of human keratinocytes and melanocytes (40:1 seeding ratio) or co-cultures of the human immortalized keratinocytes HaCaT with a melanoma cell line (20:1 seeding ratio). Co-cultures of the two cell lines were incubated with human recombinant KGF (5–50 ng per mL) for different times ranging from 30 min to 6 h, or they were exposed to different doses of UVB irradiation (20–60 mJ per cm2) or treated with α-MSH (160 ng per mL) as positive controls. To evaluate the melanosome transfer, we performed immunofluorescence analysis with anti-tyrosinase polyclonal antibodies to identify melanosomes and to unequivocally identify the keratinocytes in the co-cultures, double immunolabeling with anti-cytokeratin monoclonal antibody was performed. Immunostaining of untreated co-cultures showed tyrosinase-positive dots almost exclusively in the cytoplasm of melanocytes (Figure 1a), whereas in co-cultures treated with either KGF (Figure 1b) or α-MSH (Figure 1d) as well as in those exposed to UVB (Figure 1c) the fluorescence spots became visible also in the cytoplasm of cytokeratin-positive HaCaT cells surrounding melanocytes, which indicates increased melanosome transfer to the keratinocytes (p<0.001, p<0.001, and p<0.001, respectively). Quantitative analysis of the melanosome transfer in co-cultures of HaCaT keratinocytes and melanocytes exposed to UVB, KGF, or α-MSH, compared to unexposed cells or to cells treated with heat-denatured KGF or with an unrelated protein (bovine serum albumin (BSA)) was performed by counting for each treatment the percentage of tyrosinase-positive keratinocytes (containing at least 25 dots) on a total of 500 cells, randomly observed from 10 fields in two different experiments and expressed as mean value±SE: the results showed increased melanosome transfer at the UVB dose of 40 mJ per cm2 and at the KGF dose of 20 ng per mL (Figure 1e), which correspond to the plateau doses (data not shown). Similar results were obtained using primary cell co-cultures of human keratinocytes and melanocytes: in fact, as described above, double immunofluorescence staining with anti-tyrosinase antibody revealed that both KGF treatment and UVB exposure induced an increase of intracytoplasmic tyrosinase-positive dots corresponding to transferred melanosomes in keratinocytes (p<0.05 and p<0.01, respectively) (Figure 2a–e and g, i). Parallel ultrastructural analysis showed intact melanosomes inside phagosomal structures in the keratinocytes following KGF treatment (Figure 2f, arrow), confirming the in vitro transfer of melanosomes. As expected, however (Hara et al., 1995Hara M. Yaar M. Gilchrest B.A. Endothelin-1 of keratinocyte origin is a mediator of melanocyte dendricity.J Invest Dermatol. 1995; 105: 744-748Crossref PubMed Scopus (116) Google Scholar; Hearing, 2000Hearing V.J. The melanosome: The perfect model for cellular responses to the environment.Pigment Cell Res. 2000; 13: 23-34Crossref PubMed Scopus (102) Google Scholar; Duval et al., 2001Duval C. Regnier M. Schmidt R. Distinct melanogenic response of human melanocytes in mono-culture, in co-culture with keratinocytes and in reconstructed epidermis, to UV exposure.Pigment Cell Res. 2001; 14: 348-355Crossref PubMed Scopus (78) Google Scholar; Virador et al., 2002Virador V.M. Muller J. Wu X. et al.Influence of α-melanocyte-stimulating hormone and of ultraviolet radiation on the transfer of melanosomes to keratinocytes.FASEB J. 2002; 16: 105-107PubMed Google Scholar), whereas exposure of melanocytes co-cultured with keratinocytes to α-MSH or UVB irradiation triggered dendrite formation and transport of melanosomes to the dendritic tips (arrows in Figure 2c, g, h), KGF treatment appeared to promote melanosome transfer to keratinocytes without affecting melanocyte morphology (Figure 2b and e); in addition, melanosome transfer in KGF-treated co-cultures appeared to occur from extensions of both melanocyte and keratinocyte bodies and through large cell–cell contacts (Figure 2b and e). Thus, differently from UVB and α-MSH, KGF appears to induce melanosome transfer acting primarily on the recipient keratinocytes. Since our observations demonstrated that the melanosome transfer could be increased by signaling induced by KGF, we wondered if this effect could be a consequence of a possible role of KGF in promoting the phagocytic process, which seems to represent a crucial step for melanosome uptake by keratinocytes. To evaluate the possible modulation of keratinocyte phagocytic activity induced by KGF we performed immunofluorescence analysis using inert latex beads. Differently sized fluorescent beads, which could correspond to individual small or large melanosomes as well as to clusters of small melanosomes, were added to human primary keratinocytes and a quantitative analysis of the intracellular fluorescence intensity was performed to estimate the quantity of beads internalized. We incubated the keratinocytes for 6 h with fluorescent microspheres of 0.1 μm (green) and 1 μm (red) in diameter in presence or absence of KGF (20 ng per mL) or after UVB irradiation (40 mJ per cm2). Compared to untreated cells (Figure 3a), KGF treatment induced an increased uptake of fluorescent latex beads as a consequence of stimulation of the phagocytic activity (Figure 3b and d) (green beads: p<0.0001; red beads: p<0.001). Cells exposed to UVB, as positive controls, show a clear increase of intracellular fluorescent beads (Figure 3c and d) (green beads: p<0.0001; red beads: p<0.001). Quantitative analysis of the fluorescence intensity in KGF treated or UVB exposed keratinocytes, compared to control cells, confirmed that both treatments were able to increase the phagocytic activity of the keratinocytes (Figure 3d), although the effect of all treatments was more evident for the uptake of the smaller 0.1 μm beads, as expected (Virador et al., 2002Virador V.M. Muller J. Wu X. et al.Influence of α-melanocyte-stimulating hormone and of ultraviolet radiation on the transfer of melanosomes to keratinocytes.FASEB J. 2002; 16: 105-107PubMed Google Scholar). Since the 0.1 and 1 μm beads are respectively smaller and larger than melanosomes, we next assessed the phagocytic process using beads of 0.5 μm in diameter, more similar to the melanosome size. First we performed dose–response experiments evaluating the uptake of the beads after incubation with KGF at different doses (5–50 ng per mL). We observed that the number of internalized beads/cell reaches a plateau using KGF at the dose of 20 ng per mL (data not shown). In addition, we performed, on primary keratinocytes and HaCaT cells incubated with 0.5 μm beads in presence or absence of KGF (20 ng per mL), a time course (30 min, 1, 2, 4, and 6 h) to evaluate the uptake kinetic of fluorescent latex beads. Quantitative analysis performed counting the number of internalized beads/cell demonstrated that the KGF induces at every time point analyzed an increase of beads uptake compared to untreated cells (Figure 4q) (normal human keratinocytes (NHK): p<0.05 at 30 min, 1 h; p<0.01 at 2, 3 h; p<0.001 at 4 h; p<0.01 at 6 h) (HaCaT: p<0.05 at 30 min, 1, 2 h; p<0.01 at 4, 6 h). Examination of the cells by fluorescence microscopy and parallel phase contrast (Figure 4a–p) revealed that the efficiency of beads uptake is KGF dependent. The amount of internalized beads 1 h after addition was reduced with respect to the accumulation of beads observed at 4 h, and furthermore, NHK revealed a higher phagocytic activity compared to the HaCaT cells. Moreover, the internalized beads were localized largely beneath the plasma membrane after 1 h of beads incubation (Figure 4a–d, i–l), whereas after 4 h were distributed throughout the cells and preferentially accumulated near the perinuclear region of the cells (Figure 4e–h, m–p). To search for possible expected differences (Minwalla et al., 2001Minwalla L. Zhao Y. Le Poole I.C. Wickett R.R. Boissy R.E. Keratinocytes play a role in regulating distribution patterns of recipient melanosomes in vitro.J Invest Dermatol. 2001; 117: 341-347Crossref PubMed Google Scholar) in the phagocytic behavior of keratinocytes from light or dark skin, we compared the effect of KGF on the uptake kinetics of fluorescent latex beads in primary keratinocytes derived from light and dark skin individuals. Although preliminary, the results showed no differences, at least up to 6 h of incubation, in the phagocytic uptake of the keratinocytes; however, both cell cultures respond similarly to the KGF treatment with a significant increase in the uptake of the beads (Figure 4q). In order to analyze in more detail the phagocytic mechanism induced by KGF, we performed conventional thin section electron microscopy on human primary keratinocytes incubated with 0.5 μm beads in the presence or absence of KGF at different time points (30 min and 4 h). Ultrastructural examination revealed typical features of human keratinocytes such as bundles of cytoplasmic keratin tonofilaments (Figure 5a–c). As shown in Figure 5a and b, treatment with KGF for 30 min triggered the formation of membrane protrusions extending around the bead to be ingested (arrows in 5A and B), features characteristic of the zippering mechanism in the early phase of the phagocytic process regulated by activation of Cdc42 and Rac (Conner and Schmid, 2003Conner S.D. Schmid S.L. Regulated portals of entry into cell.Nature. 2003; 422: 37-44Crossref PubMed Scopus (2850) Google Scholar; DeMali and Burridge, 2003DeMali K. Burridge K. Coupling membrane protrusion and cell adhesion.J Cell Sci. 2003; 116: 2389-2397Crossref PubMed Scopus (145) Google Scholar). Furthermore, ultrastructural analysis of KGF-treated cells after 4 h of incubation showed the presence of internalized beads surrounded by phagosomal membranes localized in the perinuclear area (arrow in Figure 5c), suggesting intracellular transport in phagocytic structures from the cell periphery to central locations as occurring for melanosomes after transfer to keratinocytes (Boissy, 2003Boissy R.E. Melanosome transfer to and translocation in the keratinocytes.Exp Dermatol. 2003; 12: 5-12Crossref PubMed Scopus (193) Google Scholar). Since there are multiple phagocytic pathways, that are known to be associated with different signaling events, we were interested to evaluate whether the cytoskeletal remodeling occurring in the KGF-induced phagocytic process would involve activation of Rho or of Cdc42 and Rac. In fact, Rho is required for actin assembly in the phagocytosis mediated by activation of the C3 complement receptor (Conner and Schmid, 2003Conner S.D. Schmid S.L. Regulated portals of entry into cell.Nature. 2003; 422: 37-44Crossref PubMed Scopus (2850) Google Scholar; DeMali and Burridge, 2003DeMali K. Burridge K. Coupling membrane protrusion and cell adhesion.J Cell Sci. 2003; 116: 2389-2397Crossref PubMed Scopus (145) Google Scholar), and in human keratinocytes it is involved in the mechanism of melanosome transfer mediated by the PAR-2 (Scott et al., 2001Scott G. Deng A. Rodriguez-Burford C. et al.Protease-activated receptor 2, a receptor involved in melanosome transfer, is upregulated in human skin by ultraviolet irradiation.J Invest Dermatol. 2001; 117: 1412-1420Crossref PubMed Scopus (88) Google Scholar,Scott et al., 2003Scott G. Leopardi S. Parker L. Babiarz L. Seiberg M. Han R. The Proteinase-Activated Receptor-2 mediates phagocytosis in a Rho-dependent manner in human keratinocytes.J Invest Dermatol. 2003; 121: 529-541Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), whereas Cdc42 and Rac are required for actin recruitment to the phagocytic cup during FcγR-mediated phagocytosis (Conner and Schmid, 2003Conner S.D. Schmid S.L. Regulated portals of entry into cell.Nature. 2003; 422: 37-44Crossref PubMed Scopus (2850) Google Scholar; DeMali and Burridge, 2003DeMali K. Burridge K. Coupling membrane protrusion and cell adhesion.J Cell Sci. 2003; 116: 2389-2397Crossref PubMed Scopus (145) Google Scholar). Therefore, we investigated the possibility to affect keratinocyte cytoskeletal reorganization and to reduce the KGF-induced phagocytosis using specific inhibitors of PAR-2 to block the Rho-dependent pathway, and inhibitors of the Src-family tyrosine kinases to inhibit the Rac-dependent pathway. NHK were treated with soybean trypsin inhibitor (STI, 1%, 0.1%, 0.01%) for inhibition of PAR-2 (Paine et al., 2001Paine C. Sharlow E. Liebel F. Eisinger M. Shapiro S. Seiberg M. An alternative approch to depigmentation by soybean extracts via inhibition of the PAR-2 pathway.J Invest Dermatol. 2001; 116: 587-595Crossref PubMed Scopus (134) Google Scholar), or with PP1 [4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo-D-3,4-pyrimidine] (10, 20 μM) for inhibition of Src-family protein tyrosine kinases (Kwiatkowska et al., 2002Kwiatkowska K. Frey J. Sobota A. Phosphorylation of FcγRIIA is required for the receptor-induced actin rearrangement and capping: The role of membrane rafts.J Cell Sci. 2002; 116: 537-550Crossref Scopus (89) Google Scholar), in the presence or absence of KGF (20 ng per mL) and incubated with 0.5 μm fluorescent microspheres for 4 h. As shown in Figure 5d, both inhibitors appeared to decrease the uptake of the beads induced by KGF, interfering with the keratinocyte phagocytic ability. Thus, KGF seems to promote phagocytosis through both Rho-dependent and Cdc42/Rac-dependent mechanisms. To evaluate if the KGF effect on keratinocytes in inducing phagocytosis and melanosome transfer would be mediated by KGFR expression and activation, we used HaCaT cells transfected with KGFR and we performed the phagocytic assay with 0.5 μm beads in presence or absence of KGF. Immunofluorescence staining with an anti-Bek antibody (Figure 6a–f), which recognizes the extracellular portion of the two splicing variants FGFR2 and KGFR, followed by quantitative analysis of the internalized beads (Figure 6g) showed that in HaCaT cells overexpressing KGFR by transfection, compared to cells with endogenous levels of the receptor, the ability to ingest the fluorescent beads after KGF treatment is drastically increased (p<0.01). Furthemore, the observation that the internalized beads appeared yellow instead of red as the extracellular ones (Figure 6a–d) prompted us to investigate the possible localization of KGFR in the phagocytic structures. Subsequent confocal analysis confirmed that the two signals (KGFR: green and beads: red) colocalized in yellow dots located either beneath the cell surface (Figure 6e) or in intracellular spots (Figure 6f). These results sh
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