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.

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.

Activation of the Mitf promoter by lipid-stimulated activation of p38-stress signalling to CREB.

The microphthalmia-associated transcription factor Mitf plays a critical role in regulating many aspects of melanocyte biology. It is required for melanoblast and postnatal melanocyte survival, regulates proliferation, and activates genes associated with differentiation such as tyrosinase and related genes involved in melanogenesis. Identifying the signals that regulate Mitf expression is crucial if we are to understand how cells of the melanocyte lineage respond to environmental cues. Here we show that the Mitf promoter is induced by lipid signalling via the p38 stress-activated kinase pathway that is also activated by a wide range of receptors as well as UV irradiation. Signalling via p38 leads to increased phosphorylation and activation of cyclic adenosine monophosphate response element-binding (CREB) that binds and activates the Mitf promoter via the cyclic adenosine monophosphate (cAMP) response element. Moreover, we also show that activation of p38 mediated by lipids is potentiated by inhibition of the PI3kinase pathway but not by inhibition of protein kinase A (PKA). The results identify a mechanism in which stress signalling via p38 leads to activation of CREB, enhanced Mitf expression and consequently increased tyrosinase expression. The results are relevant for the regulation of melanocytes by Mitf, but also raise the possibility that lipid mediated activation of p38 signalling may represent a potential therapy for vitiligo.

Immunobiological activities of a new nontoxic lipopolysaccharide from Acidiphilium GS18h/ATCC55963, a soil isolate from an Indian copper mine.

A novel nontoxic lipopolysaccharide (LPS) was purified from Acidiphilium strain GS18h/ATCC55963. The chemical composition of the lipid A part of this LPS is distinctly different from that of known lipid A molecules. The LPS was investigated to determine its capacity to provide protection against toxic LPS or endotoxic shock, as has been reported for other nontoxic LPSs (Rhodobacter sphaeroides and Rhodobacter capsulatus), and also the extent and type of immunomodulatory response in terms of tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-beta), and IL-6 release as well as NO secretion by stimulated monocyte-macrophage systems. This study demonstrates clearly that mice immunized or primed with this LPS are fully protected against challenge with toxic Escherichia coli LPS. Unlike most of the extensively studied nontoxic LPSs, this LPS induced reactive nitrogen intermediates and released TNF-alpha, IL-beta and IL-6 in both mouse and human monocyte-macrophage systems. However, the extent of the cytokine and lymphokine releasing response was well below the range of the toxic LPS, for example that of E. coli. Owing to its capacity to provide immunostimulation of the host without causing any lethality to ensure protection against endotoxic shock, this LPS appears to have potential therapeutic value.

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.

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.

Human placental lipid induces melanogenesis through p38 MAPK in B16F10 mouse melanoma.

Melanogenesis is one of the characteristic functional activities of melanocyte/melanoma and is regulated via mitogen-activated protein kinase (MAPK) and Akt/protein kinase B (PKB) pathways. Placental total lipid fraction (PTLF), prepared from a hydroalcoholic extract of fresh term human placenta contains sphingolipids and was recently shown to stimulate melanogenesis via up-regulation of the key enzyme tyrosinase in B16F10 mouse melanoma cells. How such lipids mediate their effects on pigmentation and tyrosinase expression is a particularly important aspect of melanogenesis. To study the signaling that leads to tyrosinase expression, we have investigated the roles of the MAPK and Akt/PKB pathways in B16F10 melanoma cells in melanogenesis in response to PTLF. Treatment of cells with PTLF led to the time dependent phosphorylation of p38 MAPK. SB203580, a p38 MAPK inhibitor, completely blocked the PTLF-induced melanogenesis by inhibiting promoter activity and subsequent expression of tyrosinase. Phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002 a blocker of the Akt signaling pathway, or an inhibitor of MEK (MAPK/ERK Kinase), PD98059 when included along with PTLF was found to potentiate PTLF-induced phosphorylation of p38 MAPK together with tyrosinase expression and melanogenesis. The results suggest that the activation of p38 MAPK plays a crucial role in PTLF-induced B16F10 melanogenesis by up-regulating tyrosinase expression.

Human placental lipid induces melanogenesis by increasing the expression of tyrosinase and its related proteins in vitro.

Lipids, particularly sphingolipids, are emerging as novel regulators of cellular activity. A placental total lipid fraction (PTLF), the total lipid prepared from an hydroalcoholic extract of fresh term human placenta, was previously shown to have a pigment-inducing activity in an animal model. The PTLF contains sphingolipids which stimulate DNA synthesis and melanin formation with marked morphological changes in B16F10 melanoma cells. In order to identify the mechanism underlying the increased melanin synthesis, B16F10 cells were treated with PTLF to assess the catalytic activities of tyrosinase (i.e. tyrosine hydroxylase and DOPA oxidase), the key regulatory enzyme of melanin synthesis. Tyrosine hydroxylase (estimated by the release of (3)H(2)O) as well as DOPA oxidase (measured spectrophotometrically and also in non-denaturing gels), was stimulated significantly by PTLF. Western blot analysis demonstrated an increase in the expression of tyrosinase, tyrosinase related proteins 1 and 2 (TRP1 and TRP2) at the protein level and RT-PCR analysis revealed stimulated transcription of tyrosinase, TRP1 and TRP2 mRNAs in PTLF-treated B16F10 cells. Actinomycin D and cycloheximide, inhibitors of transcription and translation, respectively, inhibited PTLF-induction of tyrosinase activity with a corresponding decrease in melanogenesis. In all cases, the response to PTLF was similar to that induced by alpha-melanocyte stimulating hormone, a well-known stimulator of melanogenesis. Thus, these results provide the basis of action of PTLF stimulated melanogenesis in B16F10 cells showing that this placental extract is a strong inducer of pigmentation at the transcriptional and translational levels.