WNK1 is a chloride-stimulated scaffold that regulates mTORC2 activity and ion transport.

Mammalian (or mechanistic) target of rapamycin complex 2 (mTORC2) is a kinase complex that targets predominantly Akt family proteins, SGK1 and protein kinase C (PKC), and has well-characterized roles in mediating hormone and growth factor effects on a wide array of cellular processes. Recent evidence suggests that mTORC2 is also directly stimulated in renal tubule cells by increased extracellular K+ concentration, leading to activation of the Na+ channel, ENaC, and increasing the electrical driving force for K+ secretion. We identify here a signaling mechanism for this local effect of K+. We show that an increase in extracellular [K+] leads to a rise in intracellular chloride (Cl-), which stimulates a previously unknown scaffolding activity of the protein 'with no lysine-1' (WNK1) kinase. WNK1 interacts selectively with SGK1 and recruits it to mTORC2, resulting in enhanced SGK1 phosphorylation and SGK1-dependent activation of ENaC. This scaffolding effect of WNK1 is independent of its own kinase activity and does not cause a generalized stimulation of mTORC2 kinase activity. These findings establish a novel WNK1-dependent regulatory mechanism that harnesses mTORC2 kinase activity selectively toward SGK1 to control epithelial ion transport and electrolyte homeostasis.

Aldosterone-independent regulation of K+ secretion in the distal nephron.

PURPOSE OF REVIEW: Maintenance of plasma K+ concentration within a narrow range is critical to all cellular functions. The kidneys are the central organ for K+ excretion, and robust renal excretory responses to dietary K+ loads are essential for survival. Recent advances in the field have challenged the view that aldosterone is at the center of K+ regulation. This review will examine recent findings and propose a new mechanism for regulating K+ secretion. RECENT FINDINGS: Local aldosterone-independent response systems in the distal nephron are increasingly recognized as key components of the rapid response to an acute K+ load, as well as playing an essential role in sustained responses to increased dietary K+. The master kinase mTOR, best known for its role in mediating the effects of growth factors and insulin on growth and cellular metabolism, is central to these aldosterone-independent responses. Recent studies have shown that mTOR, particularly in the context of the "type 2" complex (mTORC2), is regulated by K+ in a cell-autonomous fashion. SUMMARY: New concepts related to cell-autonomous K+ signaling and how it interfaces with aldosterone-dependent regulation are emerging. The underlying signaling pathways and effectors of regulated K+ secretion, as well as implications for the aldosterone paradox and disease pathogenesis are discussed.

Potassium Activates mTORC2-dependent SGK1 Phosphorylation to Stimulate Epithelial Sodium Channel: Role in Rapid Renal Responses to Dietary Potassium.

SIGNIFICANCE STATEMENT: Rapid renal responses to ingested potassium are essential to prevent hyperkalemia and also play a central role in blood pressure regulation. Although local extracellular K + concentration in kidney tissue is increasingly recognized as an important regulator of K + secretion, the underlying mechanisms that are relevant in vivo remain controversial. To assess the role of the signaling kinase mTOR complex-2 (mTORC2), the authors compared the effects of K + administered by gavage in wild-type mice and knockout mice with kidney tubule-specific inactivation of mTORC2. They found that mTORC2 is rapidly activated to trigger K + secretion and maintain electrolyte homeostasis. Downstream targets of mTORC2 implicated in epithelial sodium channel regulation (SGK1 and Nedd4-2) were concomitantly phosphorylated in wild-type, but not knockout, mice. These findings offer insight into electrolyte physiologic and regulatory mechanisms. BACKGROUND: Increasing evidence implicates the signaling kinase mTOR complex-2 (mTORC2) in rapid renal responses to changes in plasma potassium concentration [K + ]. However, the underlying cellular and molecular mechanisms that are relevant in vivo for these responses remain controversial. METHODS: We used Cre-Lox-mediated knockout of rapamycin-insensitive companion of TOR (Rictor) to inactivate mTORC2 in kidney tubule cells of mice. In a series of time-course experiments in wild-type and knockout mice, we assessed urinary and blood parameters and renal expression and activity of signaling molecules and transport proteins after a K + load by gavage. RESULTS: A K + load rapidly stimulated epithelial sodium channel (ENaC) processing, plasma membrane localization, and activity in wild-type, but not in knockout, mice. Downstream targets of mTORC2 implicated in ENaC regulation (SGK1 and Nedd4-2) were concomitantly phosphorylated in wild-type, but not knockout, mice. We observed differences in urine electrolytes within 60 minutes, and plasma [K + ] was greater in knockout mice within 3 hours of gavage. Renal outer medullary potassium (ROMK) channels were not acutely stimulated in wild-type or knockout mice, nor were phosphorylation of other mTORC2 substrates (PKC and Akt). CONCLUSIONS: The mTORC2-SGK1-Nedd4-2-ENaC signaling axis is a key mediator of rapid tubule cell responses to increased plasma [K + ] in vivo . The effects of K + on this signaling module are specific, in that other downstream mTORC2 targets, such as PKC and Akt, are not acutely affected, and ROMK and Large-conductance K + (BK) channels are not activated. These findings provide new insight into the signaling network and ion transport systems that underlie renal responses to K +in vivo .

Interactions between mTORC2 core subunits Rictor and mSin1 dictate selective and context-dependent phosphorylation of substrate kinases SGK1 and Akt.

Mechanistic target of rapamycin complex 2 (mTORC2) is a multi-subunit kinase complex, central to multiple essential signaling pathways. Two core subunits, Rictor and mSin1, distinguish it from the related mTORC1 and support context-dependent phosphorylation of its substrates. mTORC2 structures have been determined previously; however, important questions remain, particularly regarding the structural determinants mediating substrate specificity and context-dependent activity. Here, we used cryo-EM to obtain high-resolution structures of the human mTORC2 apo-complex in the presence of substrates Akt and SGK1. Using functional assays, we then tested predictions suggested by substrate-induced structural changes in mTORC2. For the first time, we visualized in the apo-state the side chain interactions between Rictor and mTOR that sterically occlude recruitment of mTORC1 substrates and confer resistance to the mTORC1 inhibitor rapamycin. Also in the apo-state, we observed that mSin1 formed extensive contacts with Rictor via a pair of short α-helices nestled between two Rictor helical repeat clusters, as well as by an extended strand that makes multiple weak contacts with Rictor helical cluster 1. In co-complex structures, we found that SGK1, but not Akt, markedly altered the conformation of the mSin1 N-terminal extended strand, disrupting multiple weak interactions while inducing a large rotation of mSin1 residue Arg-83, which then interacts with a patch of negatively charged residues within Rictor. Finally, we demonstrate mutation of Arg-83 to Ala selectively disrupts mTORC2-dependent phosphorylation of SGK1, but not of Akt, supporting context-dependent substrate selection. These findings provide new structural and functional insights into mTORC2 specificity and context-dependent activity.

Role of mTORC2 in biphasic regulation of brown fat metabolism in response to mild and severe cold.

Nonshivering thermogenesis is essential for mammals to maintain body temperature. According to the canonical view, temperature is sensed by cutaneous thermoreceptors and nerve impulses transmitted to the hypothalamus, which generates sympathetic signals to ß-adrenergic receptors in brown adipocytes. The energy for heat generation is primarily provided by the oxidation of fatty acids derived from triglyceride hydrolysis and cellular uptake. Fatty acids also activate the uncoupling protein, UCP1, which creates a proton leak that uncouples mitochondrial oxidative phosphorylation from ATP production, resulting in energy dissipation as heat. Recent evidence supports the idea that in response to mild cold, ß-adrenergic signals stimulate not only lipolysis and fatty acid oxidation, but also act through the mTORC2-Akt signaling module to stimulate de novo lipogenesis. This opposing anabolic effect is thought to maintain lipid fuel stores during increased catabolism. We show here, using brown fat-specific Gs-alpha knockout mice and cultured adipocytes that, unlike mild cold, severe cold directly cools brown fat and bypasses ß-adrenergic signaling to inhibit mTORC2. This cell-autonomous effect both inhibits lipogenesis and augments UCP1 expression to enhance thermogenesis. These findings suggest a novel mechanism for overriding ß-adrenergic-stimulated anabolic activities while augmenting catabolic activities to resolve the homeostatic crisis presented by severe cold.

Phosphorylation at distinct subcellular locations underlies specificity in mTORC2-mediated activation of SGK1 and Akt.

mTORC2 lies at the intersection of signaling pathways that control metabolism and ion transport through phosphorylation of the AGC-family kinases, the Akt and SGK1 proteins. How mTORC2 targets these functionally distinct downstream effectors in a context-specific manner is not known. Here, we show that the salt- and blood pressure-regulatory hormone, angiotensin II (AngII) stimulates selective mTORC2-dependent phosphorylation of SGK1 (S422) but not Akt (S473 and equivalent sites). Conventional PKC (cPKC), a critical mediator of the angiotensin type I receptor (AT1R, also known as AGTR1) signaling, regulates the subcellular localization of SIN1 (also known as MAPKAP1) and SGK1. Inhibition of cPKC catalytic activity disturbs SIN1 and SGK1 subcellular localization, re-localizing them from the nucleus and a perinuclear compartment to the plasma membrane in advance of hormonal stimulation. Surprisingly, pre-targeting of SIN1 and SGK1 to the plasma membrane prevents SGK1 S422 but not Akt S473 phosphorylation. Additionally, we identify three sites on SIN1 (S128, S315 and S356) that are phosphorylated in response to cPKC activation. Collectively, these data demonstrate that SGK1 activation occurs at a distinct subcellular compartment from that of Akt and suggests a mechanism for the selective activation of these functionally distinct mTORC2 targets through subcellular partitioning of mTORC2 activity.

POLD1 Germline Mutations in Patients Initially Diagnosed with Werner Syndrome.

Segmental progeroid syndromes are rare, heterogeneous disorders characterized by signs of premature aging affecting more than one tissue or organ. A prototypic example is the Werner syndrome (WS), caused by biallelic germline mutations in the Werner helicase gene (WRN). While heterozygous lamin A/C (LMNA) mutations are found in a few nonclassical cases of WS, another 10%-15% of patients initially diagnosed with WS do not have mutations in WRN or LMNA. Germline POLD1 mutations were recently reported in five patients with another segmental progeroid disorder: mandibular hypoplasia, deafness, progeroid features syndrome. Here, we describe eight additional patients with heterozygous POLD1 mutations, thereby substantially expanding the characterization of this new example of segmental progeroid disorders. First, we identified POLD1 mutations in patients initially diagnosed with WS. Second, we describe POLD1 mutation carriers without clinically relevant hearing impairment or mandibular underdevelopment, both previously thought to represent obligate diagnostic features. These patients also exhibit a lower incidence of metabolic abnormalities and joint contractures. Third, we document postnatal short stature and premature greying/loss of hair in POLD1 mutation carriers. We conclude that POLD1 germline mutations can result in a variably expressed and probably underdiagnosed segmental progeroid syndrome.

Atypical Aicardi-Goutieres syndrome: is the WRN locus a modifier?

We describe a 28-year-old Turkish man with consanguineous parents who presented with an aged appearance with prematurely gray hair and scleroderma-like skin, spastic paraplegia, and apparent disability. The proband and each of his parents were heterozygous for a mutation in WRN, which could not explain his symptoms. Exome sequencing of the proband's blood DNA showed a homozygous c.626-1G > C mutation in intron 5 of the SAMHD1 gene, which encodes a triphosphohydrolase involved in the regulation of intracellular dNTP pools and which is mutated in Aicardi-Goutieres syndrome. The RNA studies confirmed aberrant splicing of exon 6, and family studies showed that both parents are heterozygous for this mutation. We conclude that mutations in SAMHD1 - in addition to causing an early-onset form of encephalopathy in Aicardi-Goutieres syndrome - may present with modest signs of accelerated aging similar to Werner syndrome. The extent to which heterozygosity at the WRN locus may modify the effect of biallelic SAMHD1 mutations is unknown. It is conceivable that synergistic effects of these two mutations might be responsible for the unusual phenotype.

Coordinated Regulation of Renal Glucose Reabsorption and Gluconeogenesis by mTORC2 and Potassium.

Background: The kidney proximal tubule is uniquely responsible for reabsorption of filtered glucose and gluconeogenesis (GNG). Insulin stimulates glucose transport and suppresses GNG in the proximal tubule, however, the signaling mechanisms and coordinated regulation of these processes remain poorly understood. The kinase complex mTORC2 is critical for regulation of growth, metabolism, solute transport, and electrolyte homeostasis in response to a wide array of inputs. Here we examined its role in the regulation of renal glucose reabsorption and GNG. Methods: Rictor, an essential component of mTORC2, was knocked out using the Pax8-LC1 system to generate inducible tubule specific Rictor knockout (TRKO) mice. These animals were subjected to fasting, refeeding, and variation in dietary K + . Metabolic parameters including glucose homeostasis and renal function were assessed in balance cages. Kidneys and livers were also harvested for molecular analysis of gluconeogenic enzymes, mTORC2-regulated targets, and plasma membrane glucose transporters. Results: On a normal chow diet, TRKO mice had marked glycosuria despite indistinguishable blood glucose relative to WT controls. Kidney plasma membrane showed lower SGLT2 and SGLT1 in the fed state, supporting reduced renal glucose reabsorption. Additional metabolic testing provided evidence for renal insulin resistance with elevated fasting insulin, impaired pyruvate tolerance, elevated hemoglobin A1c, and increased renal gluconeogenic enzymes in the fasted and fed states. These effects were correlated with reduced downstream phosphorylation of Akt and the transcription factor FOXO4, identifying a novel role of FOXO4 in the kidney. Interestingly, high dietary K + prevented glycosuria and excessive GNG in TRKO mice, despite persistent reduction in mTORC2 substrate phosphorylation. Conclusion: Renal tubule mTORC2 is critical for coordinated regulation of sodium-glucose cotransport by SGLT2 and SGLT1 as well as renal GNG. Dietary K + promotes glucose reabsorption and suppresses GNG independently of insulin signaling and mTORC2, potentially providing an alternative signaling mechanism in states of insulin resistance. SIGNIFICANCE STATEMENT: The kidney contributes to regulation of blood glucose through reabsorption of filtered glucose and gluconeogenesis. This study shows that mTORC2 and dietary potassium coordinate the regulation of sodium-glucose cotransport and glucose production in the kidney via independent mechanisms. New insights into the regulation of these processes in the kidney offer promising implications for diabetes mellitus management and treatment.

Coronary artery disease in a Werner syndrome-like form of progeria characterized by low levels of progerin, a splice variant of lamin A.

Classical Hutchinson-Gilford progeria syndrome (HGPS) is caused by LMNA mutations that generate an alternatively spliced form of lamin A, termed progerin. HGPS patients present in early childhood with atherosclerosis and striking features of accelerated aging. We report on two pedigrees of adult-onset coronary artery disease with progeroid features, who were referred to our International Registry of Werner Syndrome (WS) because of clinical features consistent with the diagnosis. No mutations were identified in the WRN gene that is responsible for WS, among these patients. Instead, we found two novel heterozygous mutations at the junction of exon 10 and intron 11 of the LMNA gene. These mutations resulted in the production of progerin at a level substantially lower than that of HGPS. Our findings indicate that LMNA mutations may result in coronary artery disease presenting in the fourth to sixth decades along with short stature and a progeroid appearance resembling WS. The absence of early-onset cataracts in this setting should suggest the diagnosis of progeroid laminopathy. This study illustrates the evolving genotype-phenotype relationship between the amount of progerin produced and the age of onset among the spectrum of restrictive dermopathy, HGPS, and atypical forms of WS.

WRN mutations in Werner syndrome patients: genomic rearrangements, unusual intronic mutations and ethnic-specific alterations.

Werner syndrome (WS) is an autosomal recessive segmental progeroid syndrome caused by null mutations at the WRN locus, which codes for a member of the RecQ family of DNA helicases. Since 1988, the International Registry of Werner syndrome had enrolled 130 molecularly confirmed WS cases from among 110 worldwide pedigrees. We now report 18 new mutations, including two genomic rearrangements, a deep intronic mutation resulting in a novel exon, a splice consensus mutation leading to utilization of the nearby splice site, and two rare missense mutations. We also review evidence for founder mutations among various ethnic/geographic groups. Founder WRN mutations had been previously reported in Japan and Northern Sardinia. Our Registry now suggests characteristic mutations originated in Morocco, Turkey, The Netherlands and elsewhere.

Potassium acts through mTOR to regulate its own secretion.

Potassium (K+) secretion by kidney tubule cells is central to electrolyte homeostasis in mammals. In the K+ secretory "principal" cells of the distal nephron, electrogenic Na+ transport by the epithelial sodium channel (ENaC) generates the electrical driving force for K+ transport across the apical membrane. Regulation of this process is attributable in part to aldosterone, which stimulates the gene transcription of the ENaC-regulatory kinase, SGK1. However, a wide range of evidence supports the conclusion that an unidentified aldosterone-independent pathway exists. We show here that in principal cells, K+ itself acts through the type 2 mTOR complex (mTORC2) to activate SGK1, which stimulates ENaC to enhance K+ excretion. The effect depends on changes in K+ concentration on the blood side of the cells, and requires basolateral membrane K+-channel activity. However, it does not depend on changes in aldosterone, or on enhanced distal delivery of Na+ from upstream nephron segments. These data strongly support the idea that K+ is sensed directly by principal cells to stimulate its own secretion by activating the mTORC2-SGK1 signaling module, and stimulate ENaC. We propose that this local effect acts in concert with aldosterone and increased Na+ delivery from upstream nephron segments to sustain K+ homeostasis.

Dysfunction of the MDM2/p53 axis is linked to premature aging.

The tumor suppressor p53, a master regulator of the cellular response to stress, is tightly regulated by the E3 ubiquitin ligase MDM2 via an autoregulatory feedback loop. In addition to its well-established role in tumorigenesis, p53 has also been associated with aging in mice. Several mouse models with aberrantly increased p53 activity display signs of premature aging. However, the relationship between dysfunction of the MDM2/p53 axis and human aging remains elusive. Here, we have identified an antiterminating homozygous germline mutation in MDM2 in a patient affected by a segmental progeroid syndrome. We show that this mutation abrogates MDM2 activity, thereby resulting in enhanced levels and stability of p53. Analysis of the patient's primary cells, genome-edited cells, and in vitro and in vivo analyses confirmed the MDM2 mutation's aberrant regulation of p53 activity. Functional data from a zebrafish model further demonstrated that mutant Mdm2 was unable to rescue a p53-induced apoptotic phenotype. Altogether, our findings indicate that mutant MDM2 is a likely driver of the observed segmental form of progeria.

Rapamycin decreases DNA damage accumulation and enhances cell growth of WRN-deficient human fibroblasts.

Werner syndrome (WS), caused by mutations at the WRN helicase gene, is a progeroid syndrome characterized by multiple features consistent with accelerated aging. Aberrant double-strand DNA damage repair leads to genomic instability and reduced replicative lifespan of somatic cells. We observed increased autophagy in WRN knockdown cells; this was further increased by short-term rapamycin treatment. Long-term rapamycin treatment resulted in improved growth rate, reduced accumulation of DNA damage foci and improved nuclear morphology; autophagy markers were reduced to near-normal levels, possibly due to clearance of damaged proteins. These data suggest that protein aggregation plays a role in the development of WS phenotypes and that the mammalian target of rapamycin complex 1 pathway is a potential therapeutic target of WS.

DNA damage accumulation and TRF2 degradation in atypical Werner syndrome fibroblasts with LMNA mutations.

Segmental progeroid syndromes are groups of disorders with multiple features suggestive of accelerated aging. One subset of adult-onset progeroid syndromes, referred to as atypical Werner syndrome, is caused by mutations in the LMNA gene, which encodes a class of nuclear intermediate filaments, lamin A/C. We previously described rapid telomere attrition and accelerated replicative senescence in cultured fibroblasts overexpressing mutant lamin A. In this study, we investigated the cellular phenotypes associated with accelerated telomere shortening in LMNA mutant primary fibroblasts. In early passage primary fibroblasts with R133L or L140R LMNA mutations, shelterin protein components were already reduced while cells still retained telomere lengths comparable to those of controls. There was a significant inverse correlation between the degree of abnormal nuclear morphology and the level of TRF2, a shelterin subunit, suggesting a potential causal relationship. Stabilization of the telomeres via the introduction of the catalytic subunit of human telomerase, hTERT (human telomerase reverse transcriptase), did not prevent degradation of shelterin components, indicating that reduced TRF2 in LMNA mutants is not mediated by short telomeres. Interestingly, γ-H2AX foci (reflecting double strand DNA damage) in early passage LMNA mutant primary fibroblasts and LMNA mutant hTERT fibroblasts were markedly increased in non-telomeric regions of DNA. Our results raise the possibility that mutant lamin A/C causes global genomic instability with accumulation of non-telomeric DNA damage as an early event, followed by TRF2 degradation and telomere shortening.

Ethnic-Specific WRN Mutations in South Asian Werner Syndrome Patients: Potential Founder Effect in Patients with Indian or Pakistani Ancestry.

Werner syndrome is a rare autosomal recessive disorder characterized by multiple features consistent with accelerated aging. It is caused by mutations in the WRN gene, which encodes a RecQ type helicase. To date, more than 70 disease-causing mutations have been reported. While founder mutations and a corresponding relatively high incidence of WS have been reported in Japan and Sardinia, such mutations have not been previously described among patients of South Asian descent. Here we report two novel WRN mutations in three pedigrees. A homozygous c.561A>G mutation in exon 6 was identified both in a pedigree from Kerala, India and in a British patient of Pakistani ancestry. Although c.561A>G does not alter the corresponding amino acid (p.K187K), it creates a cryptic splice site resulting in a 98bp deletion at the mRNA level (r.557-654del98) followed by a frameshift (p.K187fs). These two cases shared the same haplotype across the WRN gene, and were distinct from another Indian Werner patient with a homozygous stop codon mutation, c.2855 C>A (p.S952*) in exon 24. As the Indian population increases and the awareness of Werner syndrome grows, we anticipate that more cases will be identified with these founder mutations among South Asian Werner syndrome patients.

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.

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.

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.