Keratinocyte-derived Laminin-332 Protein Promotes Melanin Synthesis via Regulation of Tyrosine Uptake*

Background: Laminin-332 derived from keratinocytes plays a critical role in adhesion-related cell functions in melanocytes. Results: Keratinocyte-derived laminin-332 promotes the uptake of extracellular tyrosine and subsequent melanin synthesis in melanoma cells and melanocytes. Conclusion: Keratinocyte-derived laminin-332 promotes melanogenesis by controlling the uptake of tyrosine into melanocytes. Significance: Our finding reports novel means for regulating melanogenesis by the insoluble extracellular protein laminin-332. Melanocytes, which produce the pigment melanin, are known to be closely regulated by neighboring keratinocytes. However, how keratinocytes regulate melanin production is unclear. Here we report that melanin production in melanoma cells (B16F10 and MNT-1) was increased markedly on a keratinocyte-derived extracellular matrix compared with a melanoma cell-derived extracellular matrix. siRNA-mediated reduction of keratinocyte-derived laminin-332 expression decreased melanin synthesis in melanoma cells, and laminin-332, but not fibronectin, enhanced melanin content and α-melanocyte-stimulating hormone-regulated melanin production in melanoma cells. Similar effects were observed in human melanocytes. Interestingly, however, laminin-332 did not affect the expression or activity of tyrosinase. Instead, laminin-332 promoted the uptake of extracellular tyrosine and, subsequently, increased intracellular levels of tyrosine in both melanocytes and melanoma cells. Taken together, these data strongly suggest that keratinocyte-derived laminin-332 contributes to melanin production by regulating tyrosine uptake.

Melanocytes, which are present in the skin, hair, eyes, and ears, synthesize melanin via a process known as melanogenesis in the melanosome, a specialized organelle of melanocytes (1). Because melanin is an essential component of the pigmentary system of the skin, melanogenesis is a crucial and special step for the regulation of melanocyte functions such as photoprotection (2). Therefore, many pigmentary skin diseases are closely correlated with failure of the regulation of melanin synthesis. Albinism is induced by the complete or partial absence of pigmentation in the skin, hair, and eyes (3). On the other hand, vitiligo is caused by depigmentation in the skin when melanocytes die or loss of function because of autoimmune, genetic, oxidative stress, or viral causes (4). Genetic disorder in melanocytes also causes Waardenburg syndrome, which has various symptoms, including pale or blue eyes, white hair, and white patches on the skin (5).
To serve a photoprotective role, melanin produced in melanocytes needs to transfer to neighbor keratinocytes, the predominant cell type in the skin epidermis, to protect keratinocytes from UV light (7). Therefore, keratinocytes are actively involved in the regulation of melanin synthesis. They produce various regulatory soluble factors such as ␣-melanocyte-stimulating hormone (␣-MSH), adrenocorticotropic hormone, stem cell factor, and endothelin 1 (8). ␣-MSH, for instance, binds to melanocortin receptor 1 (MC1R) (9) and increases the cAMP level by stimulating adenylyl cyclase. cAMP subsequently activates PKA and cAMP-responsive element-binding protein. cAMP-responsive element-binding protein works as a transcription factor for microphthalmia-related transcription factor (MITF), a critical regulator of tyrosinase, TRP-1, and TRP-2 (10,11).
In addition to soluble factors, keratinocytes also produce extracellular proteins to regulate the cellular behaviors of melanocytes. It has been known that keratinocytes produce various extracellular matrix (ECM) proteins, such as fibronectin, laminin, and collagen (12,13). These ECM proteins have been commonly reported to regulate cell adhesion, proliferation, and migration (14 -16). One of the ECM proteins produced by keratinocytes is laminin-332. Laminin-332 is involved in the initiation of hemidesmosome formation and in the stable structure of the epidermis (17). In addition, laminin-332 is known to stimulate the migration of keratinocytes in wound healing (18), tumor growth and invasion of melanoma cells (19), and lamellipodia formation of keratinocytes (20).
Recently, we have shown that keratinocyte-derived laminin-332 plays a crucial role in adhesion-related cell functions of melanocytes and melanoma cells (21). This suggests that laminin-332 might be involved in the regulation of melanin synthesis. Here we examined whether keratinocyte-derived laminin-332 regulates melanogenesis in melanoma and melanocytes cells.
Immunofluorescence Analysis-Cells were plated onto 12well plates containing coverslips and fixed with 3.5% paraformaldehyde for 10 min. After being washed with PBS, cells were blocked with 0.5% BSA and incubated overnight with an antilaminin ␥2 antibody at 4°C. After being washed with PBS, cells were incubated with Texas Red-conjugated goat anti-rabbit antibody (Invitrogen) for 1 h at 25°C. For F-actin staining, cells were fixed with 3.5% paraformaldehyde and permeabilized with 0.5% Triton X-100. After blocking with 0.5% BSA, cells were incubated with FITC-conjugated phalloidin antibody for 1 h at 25°C. Coverslips were then mounted with mounting solution containing DAPI on glass slides and observed by fluorescence microscopy.
Preparation of Tissue Culture Plates Coated with ECM Substrate-ECM proteins were diluted in serum-free medium (laminin-332, 1 g/cm 2 ; fibronectin, 0.5 g/cm 2 ) added to the plates and incubated at 25°C for 1 h to allow adsorption onto the plates. After being washed with PBS, plates were blocked with 0.2% heat-inactivated BSA in PBS for 1 h and then washed three times with PBS. For preparing cells, the cells were detached with 0.05% trypsin and 1 mM EDTA, suspended in medium containing 0.5% FBS, harvested, resuspended in medium containing 0.5% FBS, plated onto ECM-coated plates, and incubated for 24 h at 37°C and 5% CO 2 .
Preparation of Keratinocyte-derived ECM-The keratinocyte-derived ECM was prepared according to the method of Rodeck et al. (22). Briefly, HaCaT cells (90ϳ100% confluent) grown on culture plates were detached with 0.05% trypsin and 1 mM EDTA in PBS. The detached cells were removed, and the adherent ECM on the culture plate was washed with PBS and treated with 0.1 mg/ml soybean trypsin inhibitor (Invitrogen). The plates were then washed with PBS, blocked with 0.2% heatinactivated BSA for 1 h, and washed with PBS. Alternatively, HaCaT cells grown on tissue culture plates were removed by sequential extraction with 1% Triton X-100 in PBS, 2 M urea in 1 M NaCl, and 8 M urea in 1 M NaCl (23). After removing cells, ECM-deposited plates were washed and blocked with the same method used to remove cells with 0.05% trypsin and 1 mM EDTA. Melanoma cells were plated on HaCaT ECM for 24 h at 37°C in medium containing 1% FBS.
Melanin Determination-Cells were plated on an ECMcoated 6-well tissue culture dish for 24 h at 37°C. Cells were detached using 0.05% trypsin and 1 mM EDTA in PBS at 37°C in 5% CO 2 . Detached cells were harvested into a 1.5-ml tube and centrifuged at 1000 ϫ g for 3 min. After removing the supernatant, cell pellets were suspended with PBS. Cells were counted using a hemocytometer. Equal numbers of cells (B16F10 and MNT-1 cells, 3.5 ϫ 10 5 cells; melanocytes, 2.0 ϫ 10 5 cells) were centrifuged at 1000 ϫ g for 3 min and solubilized in 50 l of 1 N NaOH and 10% dimethyl sulfoxide for 2 h at 80°C. The dissolved melanin was assessed by absorbance at 405 nm, and the melanin content was determined using a standard curve generated with synthetic melanin (Sigma). The results were analyzed in percentage terms.
Tyrosinase Activity Assay-Tyrosinase activity was assayed using a modified version of the method described by Ando et al. (24). After incubation on ECM or ␣-MSH for 24 h, cells were lysed in tyrosinase assay buffer (50 mM sodium phosphate (pH 6.8), 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1 g/ml aprotinin, and 10 g/ml leupeptin) The lysates were clarified by centrifugation at 13,000 ϫ g for 15 min at 4°C and denatured with SDS sample buffer without mercaptoethanol and heating. The proteins (20 g) were resolved by SDS-PAGE, and the gels were rinsed with 50 mM sodium phosphate buffer (pH 6.8) and equilibrated at room temperature. After 30 min, each gel was reacted with staining solution (10 mM L-DOPA in 50 mM sodium phosphate buffer) and incubated in the dark for 2 h at 37°C. Tyrosinase activity was visualized in the gels as dark bands containing L-DOPA-melanin. Alternatively, clarified lysates were reacted with L-DOPA (5 mM) at 37°C, and the absorbance was measured at 470 nm (25).
Statistical Analysis-Data are represented as the mean Ϯ S.D. from three independent experiments. Statistical analysis was performed using one-way analysis of variance. p Ͻ 0.01 or 0.05 was considered to be statistically significant.

RESULTS
Keratinocyte-derived Laminin-332 Promotes Melanin Synthesis of Melanoma Cells-To investigate the effect of keratinocyte-derived ECM on melanin synthesis, melanoma cells were detached and replated on ECM derived from HaCaT keratinocytes (HaCaT ECM) or rat embryonic fibroblasts (REF ECM), and the amount of melanin was compared with that of cells on melanoma ECM. Interestingly, B16F10 mouse melanoma cells on HaCaT ECM produced more melanin than cells on B16F10 ECM (F10 ECM) or REF ECM (Fig. 1A). Similarly, MNT-1 human melanoma cells on HaCaT ECM produced more melanin than cells on MNT-1 ECM or REF ECM (Fig. 1A). HaCaT ECM increased the amount of melanin from 98.23 pg/cell to 163.2 pg/cell in B16F10 cells and from 153.86 pg/cell to 598.81 pg/cell in MNT-1 cells. We obtained similar results in B16F10 cells on HaCaT ECM that was prepared by removing cells using either trypsin/EDTA or 1% Triton X-100 (Fig. 1B), suggesting that ECM produced specifically by keratinocytes regulates melanin synthesis in melanoma cells.
Because laminin-332 is known to regulate adhesion-dependent signaling in melanoma and melanocytes (21), we investi-gated whether laminin-332 regulates melanin synthesis of melanoma cells. We found that both B16F10 and MNT-1 cells grown on laminin-332 showed increased production of melanin compared with cells grown on either BSA or fibronectin (Fig. 1C). In addition, laminin-332 enhanced melanin synthesis more strongly than other laminins (Fig. 1D).
To further investigate the potential involvement of laminin-332 in the regulation of melanin synthesis, we used unique 21-bp siRNA sequences targeted against the laminin ␥2 chain (si-LAMC2) to knock down the expression levels of laminin-332. HaCaT transfected with the siRNA constructs showed decreased mRNA (Fig. 1E, top panel) and protein expression of the targeted proteins (Fig. 1E, center panel). Consistently, our results revealed that B16F10 cells grown on HaCaT ECM derived from laminin-332 knockdown cells showed reduced melanin synthesis (Fig. 1E, bottom panel). Integrins are important cell surface receptors binding to ECM proteins (27), and integrin ␣6␤1, ␣6␤4, and ␣3␤1 are known receptors for laminin-332 (28). Interestingly, knockdown of integrin ␣6 using unique siRNA sequences targeted against integrin ␣6 (si-ITGA6) significantly reduced melanin synthesis in B16F10 cells grown on HaCaT ECM (Fig. 1F). However, those effects were not seen in B16F10 cells transfected with siRNA of integrin ␣3 (si-ITGA3), suggesting that integrin ␣6 participates in the laminin-332-mediated melanin synthesis of melanoma cells to keratinocyte-derived ECM. Together, these data strongly suggest that laminin-332 enhances melanin synthesis in melanoma cells.
Laminin-332 Potentiates the Melanogenic Response to ␣-MSH in Melanoma Cells-Because ␣-MSH is known to be a key regulator of melanin synthesis in melanocytes (9), we next investigated whether keratinocyte-derived laminin-332 was involved in the regulation of ␣-MSH-induced melanin synthesis. We found that growth on HaCaT ECM enhanced ␣-MSHinduced melanin synthesis in B16F10 cells ( Fig. 2A), whereas growth on HaCaT ECM derived from laminin-332 knockdown cells did not (Fig. 2B). Consistent with these results, growth on laminin-332 directly enhanced ␣-MSH-induced melanin synthesis in both B16F10 (Fig. 2C) and MNT-1 cells (Fig. 2D). These findings indicate that laminin-332 potentiates ␣-MSHinduced melanin synthesis in melanoma cells and melanocytes.
Laminin-332-mediated Melanin Synthesis Is Independent of Tyrosinase-Various enzymes and transcription factors are involved in melanogenesis, including tyrosinase, TRP-1, TRP-2, and MITF (29 -31). Therefore, we investigated whether laminin-332 regulates melanin synthesis by modulating the expression levels of these regulators (Fig. 3). However, the mRNA levels of tyrosinase and MITF in B16F10 cells did not differ significantly in cells grown on B16F10 ECM and those grown on HaCaT ECM (Fig. 3A). In addition, growth on HaCaT ECM failed to affect tyrosinase activity (Fig. 3B). Similarly, growth on laminin-332 did not affect the protein expression level of tyrosinase (Fig. 3C) and the activity of tyrosinase (Fig. 3, D and E). These results suggest that laminin-332 stimulates melanin synthesis through a tyrosinase-independent pathway.

Laminin-332 Promotes Tyrosine Uptake into Melanoma
Cells-Tyrosine is a precursor of melanin (32,33), so its availability is a critical regulator of melanogenesis. Because laminin-332 enhanced melanin synthesis without affecting the expression or activity of tyrosinase, we next investigated whether laminin-332 was involved in the regulation of tyrosine uptake. We found that B16F10 cells grown on high-tyrosine medium showed enhanced intracellular tyrosine levels and melanin synthesis compared with cells grown on low-tyrosine medium (Fig.  4A), suggesting the possible involvement of intracellular tyrosine levels in the regulation of melanin synthesis. As expected, in culture media containing tyrosine, B16F10 cells grown on HaCaT ECM showed increased intracellular tyrosine levels in parallel with increased melanin synthesis (Fig. 4B). Consistent with the observation of B16F10 cells grown on HaCaT ECM, growth on laminin-332 directly enhanced both intracellular levels of tyrosine (Fig. 4C) and uptake of extracellular tyrosine (Fig. 4D). Although fibronectin also showed increased tyrosine uptake, laminin-332 showed a much better effect. Interestingly, the expression levels of these transporters, including larger amino acid transporter (LAT1), membrane-associated transporter protein, and P-protein, did not appear to be altered in cells grown on HaCaT ECM (data not shown). In addition, although B16F10 cells growth on laminin-332 had a more cylindrical morphology compared with those incubated on fibronectin, tyrosine did not cause significant morphological changes in both cells (Fig. 4E). Collectively, our data suggest  that laminin-332 regulates melanin synthesis by promoting tyrosine uptake.
Laminin-332 Promotes Tyrosine Uptake into Melanoma Cells via Adhesion-dependent MAP Kinase Activation-Cell adhesion to the ECM is known to activate diverse intracellular signaling molecules, including the MAP kinases, which are involved in melanogenesis. Among the MAP kinases, ERK regulates MITF expression and stability (34), and p38 MAPK has been reported to play a role in pigmentation (35). To investigate the effect of the HaCaT ECM on MAP kinase activation, B16F10 cells and MNT-1 cells were incubated on either their own ECM or HaCaT ECM, and MAP kinase activation was monitored by immunoblotting with the indicated MAP kinase antibodies (Fig. 5). As expected, lysates from B16F10 cells incubated on HaCaT ECM showed a clear increase in ERK activation, with a measurable increase in ERK phosphorylation as early as 15 min after replating (data not shown), and HaCaT ECM-mediated ERK activation remained high for up to 24 h when we observed increased melanin production in B16F10 cells on laminin-332 (Fig. 5A). Similarly, lysates from MNT-1 cells incubated on HaCaT ECM showed a clear increase in ERK activation (Fig. 5A). Consistent with these observations, laminin-332 enhanced HaCaT ECM-mediated ERK phosphorylation in both B16F10 and MNT-1 cells (Fig. 5B). These findings indicate that laminin-332-induced ERK activation is involved in melanin synthesis. Indeed, PD98059-mediated inhibition of ERK reduced laminin-332-mediated melanin synthesis (Fig. 5C). Because cell adhesion and spreading is dependent on ERK activation (36), it is possible that laminin-332mediated ERK activation is involved indirectly in the regulation of melanin synthesis through enhanced cell adhesion. Under our experimental conditions, however, inhibition of ERK activity did not affect the laminin-332-mediated spreading of melanoma cells (data not shown), suggesting that laminin-332-mediated ERK activation regulates melanin synthesis. Consistent with these results, laminin-332-mediated tyrosine uptake was reduced significantly when ERK activity was inhibited using the MEK inhibitor PD98059 (Fig. 5D). Therefore, laminin-332 seems to regulate tyrosine uptake into melanoma cells via adhesion-dependent MAP kinase activation.
Laminin-332 Regulates Melanin Synthesis in Human Primary Melanocytes-Finally, we used similar experiments to investigate whether laminin-332 regulates melanin synthesis in human melanocytes (Fig. 6). Consistent with our observations in melanoma cells, laminin-332 stimulated melanin synthesis in melanocytes (Fig. 6A), and its effect were synergistic with that  After 24 h, tyrosine uptake was measured as described in Figure 4D. Error bars indicate mean Ϯ S.D.

DISCUSSION
We reported previously that keratinocyte-derived laminin-332 plays important roles in the adhesion, spreading, and migration of melanoma cells and melanocytes (21). Here we further investigated the role of laminin-332 in melanin synthesis. As expected, growth on HaCaT ECM enhanced melanin synthesis in both melanoma cells and melanocytes (Figs. 1 and  6). Keratinocyte-derived laminin-332 stimulated melanin synthesis in melanoma cells (Fig. 1) and had a synergic effect with ␣-MSH on melanin synthesis (Fig. 2). Therefore, keratinocytederived laminin-332 appears to promote melanin synthesis in melanocyte-derived cells.
One of the major regulatory pathways in melanogenesis is the regulation of tyrosinase expression. Even though we observed increased melanin synthesis in cells grown on laminin-332, we failed to detect any altered expression of tyrosinase (Fig. 3, A and C). Tyrosinase activity was not regulated by keratinocyte-derived laminin-332 (Fig. 3, B, D, and E), suggesting that laminin-332 promotes melanin synthesis through a tyrosinase-independent pathway. Because melanin is produced from tyrosine, changes in the intracellular concentration of tyrosine form another important regulatory mechanism with increases in tyrosine triggering increases in cellular melanin content (37). Interestingly, increased intracellular tyrosine levels correlated with enhanced melanin synthesis (Fig. 4A), keratinocyte-derived ECM and laminin-332 caused an increase of intracellular tyrosine levels (Fig. 4, B and C), and laminin-332 enhanced the uptake of extracellular tyrosine (Fig. 4D). Together, these results indicate that laminin-332 regulates melanin synthesis by regulating tyrosine uptake. Notably, our results revealed that laminin-332 stimulates melanin synthesis not only in melanoma cells but also in human primary melanocytes (Fig. 6). As in melanoma cells, melanin synthesis in melanocytes was promoted by laminin-332 (Fig. 6, A and B), as was tyrosine uptake (Fig. 6, C-G). Therefore, laminin-332 appears to enhance melanin synthesis via the same mechanism in melanocytes and melanoma cells. Several transporters are involved in tyrosine uptake, including LAT1 in the plasma membrane (38) and membrane-associated transporter protein and P-protein in the membrane of the melanosome (39). It appears likely that laminin-332 regulates tyrosine uptake through a still  FN(ϩ). C, human primary melanocytes were cultured as described in B. Cell lysates were analyzed by immunoblotting with anti-tyrosinase antibody (␣-TYR). D, total cell lysates (5 g) were reacted with L-DOPA for 20 min at 37°C, and tyrosinase activity was measured at 470 nm. Error bars indicate mean Ϯ S.D. *, p Ͻ 0.05; ns, non-significant. E, cell lysates from human melanocytes on the indicated ECM were analyzed by tyrosinase activity assay (L-DOPA reaction product). F, human primary melanocytes were treated with L-tyrosine (500 M). After 24 h, cells were harvested, and melanin content was determined. Error bars indicate mean Ϯ S.D. *, p Ͻ 0.05 versus control. G, melanocytes were incubated on the laminin-332, and tyrosine uptake was measured as described in Fig. 4D. BSA was used as a control. The results were normalized with total cell numbers and analyzed in percentage terms. Error bars indicate mean Ϯ S.D. **, p Ͻ 0.01 versus BSA. unknown mechanism rather than via the regulation of transporter expression.
We further found that adhesion-mediated MAPK activation was crucial to melanin synthesis. It has been known that the integrin family, the major receptors involved in mediating cellular response to ECM, can activate the focal adhesion kinase and/or Src kinases to promote integrin-mediated intracellular signaling (40). Indeed, we found that siRNA-mediated knockdown of integrin ␣6 diminished the ability of laminin-332 to enhance melanin synthesis (Fig. 1F). Therefore, it is likely that integrin ␣6␤1 mediates the laminin-332-mediated melanin synthesis in melanoma cells. Another key regulatory kinase family is the MAP kinase family. Integrin-mediated MAP kinase activation of the Ras pathway can activate cell proliferation (41), and the integrin ␤1 subunit can activate a MAPK signaling pathway in neural stem cells, contributing to their maintenance (42). The integrin ␤3 subunit stimulates actin polymerization and migration by activating ERK in human mesangial cells (43), whereas the integrin ␣v subunit induces phosphorylation of p38 for invasion of breast cancer cells (44). Interestingly, the MAPK pathway is crucial to melanin synthesis. Activated ERK regulates the activity or stability of MITF, a critical regulator of tyrosinase (45,34), whereas ERK phosphorylates MITF at serine 73 or serine 407, increasing its transcriptional activity or ubiquitination, respectively (46). Furthermore, p38 MAPK is required for pigmentation (35) and the degradation of tyrosinase (47). Therefore, it is likely that ERK activation plays a role in melanin synthesis. Consistent with this notion, we found that keratinocyte-derived laminin-332 specifically increased the phosphorylation of ERK (Fig. 5, A and B), whereas PD98059-mediated inhibition of ERK decreased melanin synthesis (Fig. 5C) and laminin-332-mediated tyrosine uptake (Fig. 5D). These results indicate that keratinocyte-derived laminin-332 stimulates melanin synthesis via ERK activation.
Keratinocytes produce various soluble regulators for melanin synthesis, including ␣-MSH, adrenocorticotropic hormone, agouti signaling protein, stem cell factor, and endothelin 1, all of which are involved in mediating the expression of tyrosinase and TRPs. Interestingly, however, our results indicate that laminin-332 is an insoluble protein that regulates melanin synthesis via a tyrosinase-independent pathway. Instead, laminin-332 enhances the uptake of tyrosine, a substrate of tyrosinase and a precursor of melanin (Figs. 4 -6). These findings suggest that keratinocytes cooperatively regulate melanin synthesis by producing both soluble and insoluble regulators. Our data showing a synergistic effect on melanin synthesis by ␣-MSH and laminin-332 (Fig. 2) strongly support this idea of cooperative regulation.
In sum, here we show the role of keratinocyte-derived laminin-332 in melanin synthesis. Laminin-332 promotes melanin synthesis by a tyrosinase-independent pathway. Interestingly, laminin-332 stimulates the uptake of tyrosine, which is a precursor of melanin. Although future studies will be required to fully elucidate the mechanism underlying laminin-332-induced melanin synthesis in melanocytes, our present findings provide important new insights into the cooperative regulation of melanogenesis by keratinocytes.