Suman Kumar Singh

Melanin transfer in human skin cells is mediated by filopodia—a model for homotypic and heterotypic lysosome-related organelle transfer

Transfer of the melanocyte‐specific and lysosome‐related organelle, the melanosome, from melanocytes to keratinocytes is crucial for the protection of the skin against harmful ultraviolet radiation (UVR)—our main physiological cutaneous stressor. However, this commonplace event remains a most enigmatic process despite several early hypotheses. Recently, we and others have proposed a role for filopodia in melanin transfer, although conclusive experimental proof remained elusive. Using known filopodial markers (MyoX/Cdc42) and the filopodial disrupter, low‐dose cytochalasin‐B, we demonstrate here a requirement for filopodia in melanosome transfer from melanocytes to keratinocytes and also, unexpectedly, between keratinocytes. Melanin distribution throughout the skin represents the key phenotypic event in skin pigmentation. Melanocyte filopodia were also necessary for UVR‐stimulated melanosome transfer, as this was also inhibited by MyoX knockdown and low‐dose cytochalasin‐B. Knockdown of keratinocyte MyoX protein, in its capacity as a phagocytosis effector, resulted in the inhibition of melanin uptake by keratinocytes. This indicates a central role for phagocytosis by keratinocytes of melanocyte filopodia. In summary, we propose a new model for the regulation of pigmentation in human skin cells under both constitutive and facultative (post‐UVR) conditions, which we call the “filopodial‐phagocytosis model.” This model also provides a unique and highly accessible way to study lysosome‐related organelle movement between mammalian cells.—Singh, S. K., Kurfurst, R., Nizard, C., Schnebert, S., Perrier, E., Tobin, D. J. Melanin transfer in human skin cells is mediated by filopodia—a model for homotypic and heterotypic lysosome‐related organelle transfer. FASEB J. 24, 3756–3769 (2010). www.fasebj.org

The silver locus product (Silv⁄gp100⁄Pmel17) as a new tool for the analysis of melanosome transfer in human melanocyte-keratinocyte co-culture

Abstract:  Melanosomes are melanocyte‐specific lysosome‐related organelles that are transferred to keratinocytes of the epidermis and anagen hair bulb. Transferred melanin forms supra‐nuclear caps that protect epidermal keratinocytes against UV irradiation. The mechanism(s) responsible for melanosome transfer into keratinocytes and their subsequent intra‐keratinocyte distribution has long remained one of the most enigmatic of heterotypic cell interactions. Although there have been many attempts to study this process, significant progress has been hindered by the absence of an adequate in vitro model. During our ongoing study of melanocyte–keratinocyte interactions in skin and hair follicle, we have developed a novel in vitro assay that exploits the specificity of Silv/Pmel17/gp100 expression for melanosome/melanin granules. Using matched cultures of keratinocytes and melanocytes isolated from normal healthy epidermis together with double immunofluorescence, we have determined that gp100 is a surprisingly useful tracker of transferred melanin. Moreover, transferred gp100 stained melanin granules emit a bright fluorescence signal, facilitating ready quantification of melanin transfer levels between melanocytes and keratinocytes. This quantitative approach was validated using known inducers and inhibitors of the melanocyte phenotype. This assay further confirmed that cytophagocytosis of melanocyte components (e.g. dendrite tips) by keratinocytes is one route for melanin incorporation into keratinocytes. Lastly, a role for the recently proposed filopodium as a direct conduit for melanin transfer was substantiated using this novel approach. In conclusion, this assay promises to significantly aid our investigations of the molecular basis of melanosome transfer and offers a new tool for the clinical evaluation of melanocyte modulators.

Bone morphogenetic proteins differentially regulate pigmentation in human skin cells

Summary Bone morphogenetic proteins (BMPs) are a large family of multi-functional secreted signalling molecules. Previously BMP2/4 were shown to inhibit skin pigmentation by downregulating tyrosinase expression and activity in epidermal melanocytes. However, a possible role for other BMP family members and their antagonists in melanogenesis has not yet been explored. In this study we show that BMP4 and BMP6, from two different BMP subclasses, and their antagonists noggin and sclerostin were variably expressed in melanocytes and keratinocytes in human skin. We further examined their involvement in melanogenesis and melanin transfer using fully matched primary cultures of adult human melanocytes and keratinocytes. BMP6 markedly stimulated melanogenesis by upregulating tyrosinase expression and activity, and also stimulated the formation of filopodia and Myosin-X expression in melanocytes, which was associated with increased melanosome transfer from melanocytes to keratinocytes. BMP4, by contrast, inhibited melanin synthesis and transfer to below baseline levels. These findings were confirmed using siRNA knockdown of BMP receptors BMPR1A/1B or of Myosin-X, as well as by incubating cells with the antagonists noggin and sclerostin. While BMP6 was found to use the p38MAPK pathway to regulate melanogenesis in human melanocytes independently of the Smad pathway, p38MAPK, PI3-K and Smad pathways were all involved in BMP6-mediated melanin transfer. This suggests that pigment formation may be regulated independently of pigment transfer. These data reveal a complex involvement of regulation of different members of the BMP family, their antagonists and inhibitory Smads, in melanocytes behaviour.

Topobiology of human pigmentation: P-cadherin selectively stimulates hair follicle melanogenesis.

P-cadherin serves as a major topobiological cue in mammalian epithelium. In human hair follicles (HFs), it is prominently expressed in the inner hair matrix that harbors the HF pigmentary unit. However, the role of P-cadherin in normal human pigmentation remains unknown. As patients with mutations in the gene that encodes P-cadherin show hypotrichosis and fair hair, we explored the hypothesis that P-cadherin may control HF pigmentation. When P-cadherin was silenced in melanogenically active organ-cultured human scalp HFs, this significantly reduced HF melanogenesis and tyrosinase activity as well as gene and/or protein expression of gp100, stem cell factor, c-Kit, and microphthalmia-associated transcription factor (MITF), both in situ and in isolated human HF melanocytes. Instead, epidermal pigmentation was unaffected by P-cadherin knockdown in organ-cultured human skin. In hair matrix keratinocytes, P-cadherin silencing reduced plasma membrane β-catenin, whereas glycogen synthase kinase 3 beta (GSK3β) and phospho-β-catenin expression were significantly upregulated. This suggests that P-cadherin-GSK3β/Wnt signaling is required for maintaining the expression of MITF to sustain intrafollicular melanogenesis. Thus, P-cadherin-mediated signaling is a melanocyte subtype-specific topobiological regulator of normal human pigmentation, possibly via GSK3β-mediated canonical Wnt signaling.

Sphingolipid-mediated restoration of Mitf expression and repigmentation in vivo in a mouse model of hair graying.

Recent advances in the identification and characterisation of stem cell populations has led to substantial interest in understanding the precise triggers that would operate to induce activation of quiescent stem cells. Melanocyte stem cells (MSCs) reside in the bulge region of the hair follicles and are characterised by reduced expression of the microphthalmia‐associated transcription factor (Mitf) and its target genes implicated in differentiation. Vitiligo is characterised by progressive destruction of differentiated melanocytes. However, therapies using UV irradiation therapy can induce a degree of repigmentation, suggesting that MSCs may be activated. As Mitf is implicated in control of proliferation, we have explored the possibility that inducing Mitf expression via lipid‐mediated activation of the p38 stress‐signalling pathway may represent a re‐pigmentation strategy. Here we have isolated from placental extract a C18:0 sphingolipid able to induce Mitf and tyrosinase expression via activation of the p38 stress‐signalling pathway. Strikingly, in age‐onset gray‐haired C57BL/6J mice that exhibit decaying Mitf expression, topical application of placental sphingolipid leads to increased Mitf in follicular melanocytes and fresh dense black hair growth. The results raise the possibility that lipid‐mediated activation of the p38 pathway may represent a novel approach to an effective vitiligo therapy.

Transcriptional activation of tyrosinase gene by human placental sphingolipid

The sphingolipids, a class of complex bioactive lipids, are involved in diverse cellular functions such as proliferation, differentiation, and apoptosis as well as growth inhibition. Recently sphingosylphosphorylcholine (SPC), sphingosine-1-phosphate (S1P), and C2-ceramide (C2-Cer), sphingolipid containing acetic acid are emerging as melanogenic regulators. A bioactive sphingolipid (PSL) was isolated from hydroalcoholic extract of fresh term human placenta and it induced melanogenesis in an in vitro culture of mouse melanoma B16F10 cells. Tyrosinase, the rate-limiting enzyme for melanogenesis, is required to be upregulated for the increased melanin production. The expression of tyrosinase, both at protein as well as mRNA level, was higher in the PSL treated B16F10 cells as evidenced by Western blot and RT-PCR analysis. Actinomycin D and cycloheximide, inhibitors of transcription and translation, respectively, inhibited PSL-induced tyrosinase activity and its protein expression showing decrease in melanogenesis, correspondingly. The activity of GFP coupled tyrosinase promoter was upregulated in transfected B16F10 cells after treating with PSL as determined by fluorescence microscopy, fluorometric analysis, and Western blot. These results, thus, suggested that PSL upregulated tyrosinase gene expression at transcription level through promoter activation to show increased melanogenesis. Therefore, PSL as an inducer of melanogenesis might account for the recovery of pigment in depigmentation disorder.

Attenuated Leishmanial sphingolipid induces apoptosis in A375 human melanoma cell via both caspase-dependent and -independent pathways

A fraction of attenuated Leishmanial lipid (ALL) rich in sphingolipids, previously shown to have apoptosis inducing activity in mouse melanoma (B16F10) and human melanoma (A375) cells, was resolved to isolate the bioactive sphingolipid. The mechanism of apoptosis induction by this bioactive attenuated Leishmanial sphingolipid (ALSL) was studied in A375 cells. Apoptosis induced by ALSL in A375 cells was found to be dose and time-dependent. Exposure of cells to ALSL resulted in a rapid increase in reactive oxygen species generation. Pretreatment of cells with the antioxidant N-acetyl-cystein reduced ROS generation and attenuated apoptosis induced by ALSL. Again, ALSL sensitization resulted in the activation of caspase-3 and -9 but not caspase-8. However, inhibitors of these caspases could not protect the cells completely from ALSL-induced apoptosis. N-acetyl-cystein pretreatment was again found to attenuate the activation of caspase-3 and -9. ALSL treatment also resulted in the alteration of mitochondrial membrane potential, and release of pro-apoptotic factors such as cytochrome c and apoptosis inducing factor (AIF) from mitochondria. Furthermore, c-Jun N-terminal kinase was activated that resulted in apoptosis of A375 cells, whereas p38 MAPK was activated to counteract the stress generated in cells in response to ALSL treatment. Taken together, our results indicate that ALSL-induced apoptosis of A375 cells is mediated by both mitochondrial caspase-dependent and -independent pathways and it involves ROS and JNK activation in the mitogen-activated protein kinase cascade.

The effects of Sophora angustifolia and other natural plant extracts on melanogenesis and melanin transfer in human skin cells

Skin pigmentation is a multistep process of melanin synthesis by melanocytes, its transfer to recipient keratinocytes and its degradation. As dyspigmentation is a prominent marker of skin ageing, novel effective agents that modulate pigmentation safely are being sought for both clinical and cosmetic use. Here, a number of plant extracts were examined for their effect on melanogenesis (by melanin assay and Western blotting) and melanin transfer (by confocal immunomicroscopy of gp100‐positive melanin granules in cocultures and by SEM analysis of filopodia), in human melanocytes and in cocultures with phototype‐matched normal adult epidermal keratinocytes. Mulberry, Kiwi and Sophora extracts were assessed against isobutylmethylxanthine, hydroquinone, vitamin C and niacinamide. Compared with unstimulated control, all extracts significantly reduced melanogenesis in human melanoma cells and normal adult epidermal melanocytes. These extracts also reduced melanin transfer and reduced filopodia expression on melanocytes, similar to hydroquinone and niacinamide, indicating their effectiveness as multimode pigmentation actives.

Human placental protein/peptides stimulate melanin synthesis by enhancing tyrosinase gene expression

Placental protein/peptides as biological response modifier are well documented, but not much known about melanogenesis. We possibly for the first time, demonstrated melanogenesis in B16F10 mouse melanoma by a placental protein/peptide fraction (PPPF) prepared from a hydroalcoholic extract of fresh term human placenta. This study described the effect of PPPF on the induction of tyrosinase; the key enzyme of melanogenesis to investigate the basis of PPPF induced pigmentation in primary melanocyte and B16F10 melanoma. Tyrosinase induction by PPPF in B16F10 cells was found dose- and time dependent at the level of activity. Tyrosinase, at the level of transcription and protein expression when assessed by RT-PCR and Western blot analyses found to have considerable induction over untreated control. PPPF led to enhanced activation of tyrosinase promoter resulting higher transcription thus substantiating the role of PPPF as a stimulator of melanogenesis. Actinomycin D, the transcriptional inhibitor of protein synthesis, blocked the stimulatory action of PPPF since the induction of tyrosinase and melanin was markedly reduced in presence of this inhibitor. Thus the results suggested that PPPF mediated increase in tyrosinase expression occurred through transcriptional upregulation to stimulate melanogenesis in B16F10 cells and in primary melanocyte also. (Mol Cell Biochem xxx: 1–10, 2004)

E‐cadherin mediates ultraviolet radiation‐ and calcium‐induced melanin transfer in human skin cells

Skin pigmentation is directed by epidermal melanin units, characterized by long‐lived and dendritic epidermal melanocytes (MC) that interact with viable keratinocytes (KC) to contribute melanin to the epidermis. Previously, we reported that MC:KC contact is required for melanosome transfer that can be enhanced by filopodi, and by UVR/UVA irradiation, which can upregulate melanosome transfer via Myosin X‐mediated control of MC filopodia. Both MC and KC express Ca2+‐dependent E‐cadherins. These homophilic adhesion contacts induce transient increases in intra‐KC Ca2+, while ultraviolet radiation (UVR) raises intra‐MC Ca2+ via calcium‐selective ORAI1 ion channels; both are associated with regulating melanogenesis. However, how Ca2+ triggers melanin transfer remains unclear. Here we evaluated the role of E‐cadherin in UVR‐mediated melanin transfer in human skin cells. MC and KC in human epidermis variably express filopodia‐associated E‐cadherin, Cdc42, VASP and β‐catenin, all of which were upregulated by UVR in human MC in vitro. Knockdown of E‐cadherin revealed that this cadherin is essential for UVR‐induced MC filopodia formation and melanin transfer. Moreover, Ca2+ induced a dose‐dependent increase in filopodia formation and melanin transfer, as well as increased β‐catenin, Cdc42, Myosin X and E‐cadherin expression in these skin cells. Together, these data suggest that filopodial proteins and E‐cadherin, which are upregulated by intracellular (UVR‐stimulated) and extracellular Ca2+ availability, are required for filopodia formation and melanin transfer. This may open new avenues to explore how Ca2+ signalling influences human pigmentation.