L-4-thiazolylalanine (Protinol), a novel non-proteinogenic amino acid, demonstrates epidermal and dermal efficacy with clinically observable benefits.

OBJECTIVE: Facial skin undergoes major structural and functional changes as a result of intrinsic and extrinsic factors. The goal of the current work is to demonstrate L-4-thiazolylalaine (L4, Protinol), a non-proteinogenic amino acid shown to stimulate the production of dermal proteins by fibroblasts, is an alternative efficacious topical ingredient for visible signs of ageing. METHODS: In vitro studies using 3D human skin tissue models were performed to show changes in protein and gene expression of key dermal markers in samples treated with 0.3% L4 compared to vehicle control. In vivo evaluation of skin turnover was measured in volunteers after treatment with L4 compared to retinol. Skin biopsies (n = 30) were taken to investigate epidermal and dermal changes in cases treated with L4 and compared to retinol. Finally, a clinical evaluation (n = 28) was conducted to assess the efficacy of L4 over a base formulation using various ageing parameters within a population of women 46-66 years old with mild-to-moderate wrinkles. RESULTS: In vitro studies on 3D tissues displayed significant changes in the dermal matrix via an increase in HA and pro-collagen I production and a decrease in the expression of inflammatory genes. In vivo biopsy studies demonstrated that L4 and retinol independently increased epidermal thickness and collagen remodelling significantly more compared with the base formula. Clinical evaluation showed firmer and smoother skin at day 28 post-treatment with L4 over the vehicle control without causing side effects such as redness or irritation. CONCLUSION: L4 is a novel, multi-functional ingredient which offers a superior alternative to currently available technologies for improving epidermal and dermal parameters that change during ageing and photodamage.

OBJECTIF: La peau du visage est sujet à des changements majeurs structuraux et fonctionnels dus à des facteurs intrinsèques et extrinsèques. Dans cette étude, nous montrons que l'acide aminé non-protéinogène L-4-thiazolylalanine (L4, Protinol) est une alternative intéressante pour une application topique. MÉTHODES: Des modèles 3D de peaux ont été utilisés pour mesurer les changements d'expressions géniques et protéiques de marqueurs clés du derme à partir d'échantillons traités avec L4 comparés à une condition contrôle. In vivo, après un traitement L4, le renouvellement cutané a été mesuré chez les volontaires et comparé à un traitement au rétinol. Des biopsies de peaux (n = 30) traitées soit à L4 soit au rétinol ont été isolées afin d'évaluer les changements au niveau du derme et de l'épiderme. Pour finir, une étude clinique (n = 28) a été menée pour évaluer l'efficacité de L4 par rapport à une formulation de base en utilisant différents paramètres de vieillissement au sein d'une population de femmes de 46 à 66 ans présentant des rides légères à modérées. RÉSULTATS: Les études in vitro sur tissues 3D ont montré des changements dans la matrice du derme avec une augmentation de la production d'acide hyaluronique et de procollagène I et une diminution d'expression de gènes pro-inflammatoires. Les études menées in vivo sur biopsies ont démontré que L4 et rétinol augmentaient indépendamment tous deux significativement l'épaisseur de l'épiderme et le remodelage du collagène par rapport à leur base seule. Pour finir, une peau plus ferme et plus lisse a été mesurée cliniquement après 28 jours de traitement L4 par rapport au véhicule et cela sans effets indésirables tels que rougeur et irritation. CONCLUSION: L4 est un ingrédient, innovant et multifonctionnel. Il offre une sérieuse alternative aux technologies actuellement disponibles dans les traitements contre le vieillissement de la peau ou le photodommage.

Transcriptomic and histological analysis of exposed facial skin areas wrinkled or not and unexposed skin.

BACKGROUND: Skin aging involves genetic, environmental and hormonal factors. Facial wrinkles also depend on muscular activity. Gene expression investigation may be useful for new anti-aging products. METHODS AND RESULTS: To evaluate structure and gene expression differences among exposed and unexposed skin in menopausal women. Cross-sectional study, including 15 menopausal women, 55-65 years, phototype III; photo-exposed, periorbital wrinkles (A1), preauricular, not wrinkled (A2), and unexposed gluteal (A3) areas were described and compared by non-invasive measures, histology, immunohistochemistry and gene expression (RNASeq); participants mean age was 61yo, presenting moderate periorbital wrinkles and light facial photodamage. Higher roughness, wrinkles number and echogenicity were observed in A1 and A2 versus A3. Decreased epidermal thickness and dermal collagen IV were demonstrated in A1 versus A2 and A3. Exposed areas impacted different pathways compared to unexposed. Exposed wrinkled skin (A1) showed impact on cell movement with decreased inflammatory activation state. Pathways related to lipid and aminoacids metabolism were modulated in non-wrinkled exposed (A2) compared to unexposed (A3) skin. CONCLUSIONS: Expected histological findings and gene expression differences among areas were observed. Photoaging in menopausal women may modulate lipid and aminoacids metabolism and decrease inflammatory and keratinization pathways, cellular homeostasis, immune response, fibrogenesis and filament formation. These findings may help development of new therapies for skin health and aging control.

Novel Rotational Combination Regimen of Skin Topicals Improves Facial Photoaging: Efficacy Demonstrated in Double-Blinded Clinical Trials and Laboratory Validation.

Topical antiaging products are often a first-line intervention to counter visible signs of facial photoaging, aiming for sustained cosmetic improvement. However, prolonged application of a single active topical compound was observed clinically to lead to a plateau effect in improving facial photoaging. In view of this, we set out to reduce this effect systematically using a multi-tiered approach with laboratory evidence and clinical trials. The objective of the study was to evaluate the effects of active topical ingredients applied either alone, in combination, or in a rotational manner on modulation of facial photoaging. The study methodology included in vitro, organotypic, and ex vivo skin explants; in vivo biopsy study; as well as clinical trials. We demonstrate for the first time that a pair of known antiaging ingredients applied rotationally, on human dermal fibroblasts, maximized pro-collagen I production. Indeed, rotational treatment with retinol and phytol/glycolic acid (PGA) resulted in better efficacy than application of each active ingredient alone as shown by explants and in vivo biopsy study, with penetration of active ingredients confirmed by Raman spectroscopy. Furthermore, two split-face, randomized, double-blinded clinical trials were conducted, one for 12 months to compare treated vs. untreated and the other for 6 months followed by a 2-month regression to compare treated vs. commercially marketed products. In both studies, rotational regimen showed superior results to its matching comparison as assessed by clinical grading and image analysis of crow's feet wrinkles. In conclusion, rotational regimen using retinol and PGA is effective in treating facial photoaging signs with long-lasting benefits.

Growth differentiation factor 11 (GDF11) has pronounced effects on skin biology.

Growth differentiation factor 11 (GDF11) belongs to the TGF-ß superfamily of proteins and is closely related to myostatin. Recent findings show that GDF11 has rejuvenating properties with pronounced effects on the cardiovascular system, brain, skeletal muscle, and skeleton in mice. Several human studies were also conducted, some implicating decreasing levels of circulating GDF11 with age. To date, however, there have not been any reports on its role in human skin. This study examined the impact of GDF11 on human skin, specifically related to skin aging. The effect of recombinant GDF11 on the function of various skin cells was examined in human epidermal keratinocytes, dermal fibroblasts, melanocytes, dermal microvascular endothelial cells and 3D skin equivalents, as well as in ex vivo human skin explants. GDF11 had significant effects on the production of dermal matrix components in multiple skin models in vitro and ex vivo. In addition, it had a pronounced effect on expression of multiple skin related genes in full thickness 3D skin equivalents. This work, for the first time, demonstrates an important role for GDF11 in skin biology and a potential impact on skin health and aging.

Sustained PKCßII activity confers oncogenic properties in a phospholipase D- and mTOR-dependent manner.

Protein kinase C (PKC) is a family of serine/threonine kinases implicated in a variety of physiological processes. We have shown previously that sustained activation of the classical PKCα and PKCßII induces their phospholipase D (PLD)-dependent internalization and translocation to a subset of the recycling endosomes defined by the presence of PKC and PLD (the pericentrion), which results in significant differences in phosphorylation of PKC substrates. Here, we have investigated the biological consequences of sustained PKC activity and the involvement of PLD in this process. We find that sustained activation of PKC results in activation of the mammalian target of rapamycin (mTOR)/S6 kinase pathway in a PLD- and endocytosis-dependent manner, with both pharmacologic inhibitors and siRNA implicating the PLD2 isoform. Notably, dysregulated overexpression of PKCßII in A549 lung cancer cells was necessary for the enhanced proliferation and migration of these cancer cells. Inhibition of PKCßII with enzastaurin reduced A549 cell proliferation by >60% (48 h) and migration by >50%. These biological effects also required both PLD activity and mTOR function, with both the PLD inhibitor FIPI and rapamycin reducing cell growth by >50%. Reciprocally, forced overexpression of wild-type PKCßII, but not an F666D mutant that cannot interact with PLD, was sufficient to enhance cell growth and increase migration of noncancerous HEK cells; indeed, both properties were almost doubled when compared to vector control and PKC-F666D-overexpressing cells. Notably, this condition was also dependent on both PLD and mTOR activity. In summary, these data define a PKC-driven oncogenic signaling pathway that requires both PLD and mTOR, and suggest that inhibitors of PLD or mTOR would be beneficial in cancers where PKC overexpression is a contributing or driving factor.

The protein kinase Sch9 is a key regulator of sphingolipid metabolism in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae protein kinase Sch9 is an in vitro and in vivo effector of sphingolipid signaling. This study examines the link between Sch9 and sphingolipid metabolism in S. cerevisiae in vivo based on the observation that the sch9Δ mutant displays altered sensitivity to different inhibitors of sphingolipid metabolism, namely myriocin and aureobasidin A. Sphingolipid profiling indicates that sch9Δ cells have increased levels of long-chain bases and long-chain base-1 phosphates, decreased levels of several species of (phyto)ceramides, and altered ratios of complex sphingolipids. We show that the target of rapamycin complex 1-Sch9 signaling pathway functions to repress the expression of the ceramidase genes YDC1 and YPC1, thereby revealing, for the first time in yeast, a nutrient-dependent transcriptional mechanism involved in the regulation of sphingolipid metabolism. In addition, we establish that Sch9 affects the activity of the inositol phosphosphingolipid phospholipase C, Isc1, which is required for ceramide production by hydrolysis of complex sphingolipids. Given that sphingolipid metabolites play a crucial role in the regulation of stress tolerance and longevity of yeast cells, our data provide a model in which Sch9 regulates the latter phenotypes by acting not only as an effector but also as a regulator of sphingolipid metabolism.

Sustained activation of protein kinase C induces delayed phosphorylation and regulates the fate of epidermal growth factor receptor.

It is well established that acute activation of members of the protein kinase C (PKC) family induced by activation of cellular receptors can transduce extracellular stimuli to intracellular signaling. However, the functions of sustained activation of PKC are not well studied. We have previously shown that sustained activation of classical PKC isoforms over 15-60 min induced the formation of the pericentrion, a subset of recycling endosomes that are sequestered perinuclearly in a PKC- and phospholipase D (PLD)-dependent manner. In this study, we investigated the role of this process in the phosphorylation of EGFR on threonine 654 (Thr-654) and in the regulation of intracellular trafficking and fate of epidermal growth factor receptor (EGFR). Sustained stimulation of the angiotensin II receptor induced translocation of the EGFR to the pericentrion, which in turn prevents full access of EGF to the EGFR. These effects required PKC and PLD activities, and direct stimulation of PKC with phorbol esters was sufficient to reproduce these effects. Furthermore, activation of PKC induced delayed phosphorylation of EGFR on Thr-654 that coincided with the formation of the pericentrion and which was dependent on PLD and endocytosis of EGFR. Thus, Thr-654 phosphorylation required the formation of the pericentrion. On the other hand, using a T654A mutant of EGFR, we find that the phosphorylation on Thr-654 was not required for translocation of EGFR to the pericentrion but was required for protection of EGFR from degradation in response to EGF. Taken together, these results demonstrate a novel role for the pericentrion in the regulation of EGFR phosphorylation, which in turn is important for the fates of EGFR.

Dihydroceramide accumulation and reactive oxygen species are distinct and nonessential events in 4-HPR-mediated leukemia cell death.

4-(Hydroxyphenyl)retinamide (4-HPR) is a synthetic retinoid with a strong apoptotic effect towards different cancer cell lines in vitro, and it is currently tested in clinical trials. Increases of reactive oxygen species (ROS) and modulation of endogenous sphingolipid levels are well-described events observed upon 4-HPR treatment, but there is still a lack of understanding of their relationship and their contribution to cell death. LC-MS analysis of sphingolipids revealed that in human leukemia CCRF-CEM and Jurkat cells, 4-HPR induced dihydroceramide but not ceramide accumulation even at sublethal concentrations. Myriocin prevented the 4-HPR-induced dihydroceramide accumulation, but it did not prevent the loss of viability and increase of intracellular ROS production. On the other hand, ascorbic acid, Trolox, and vitamin E reversed 4-HPR effects on cell death but not dihydroceramide accumulation. NDGA, described as a lipoxygenase inhibitor, exerted a significantly higher antioxidant activity than vitamin E and abrogated 4-HPR-mediated ROS. It did not however rescue cellular viability. Taken together, this study demonstrates that early changes observed upon 4-HPR treatment, i.e., sphingolipid modulation and ROS production, are mechanistically independent events. Furthermore, the results indicate that 4-HPR-driven cell death may occur even in the absence of dihydroceramide or ROS accumulation. These observations should be taken into account for an improved design of drug combinations.

The plant defensin RsAFP2 induces cell wall stress, septin mislocalization and accumulation of ceramides in Candida albicans.

The antifungal plant defensin RsAFP2 isolated from radish interacts with fungal glucosylceramides and induces apoptosis in Candida albicans. To further unravel the mechanism of RsAFP2 antifungal action and tolerance mechanisms, we screened a library of 2868 heterozygous C. albicans deletion mutants and identified 30 RsAFP2-hypersensitive mutants. The most prominent group of RsAFP2 tolerance genes was involved in cell wall integrity and hyphal growth/septin ring formation. Consistent with these genetic data, we demonstrated that RsAFP2 interacts with the cell wall of C. albicans, which also contains glucosylceramides, and activates the cell wall integrity pathway. Moreover, we found that RsAFP2 induces mislocalization of septins and blocks the yeast-to-hypha transition in C. albicans. Increased ceramide levels have previously been shown to result in apoptosis and septin mislocalization. Therefore, ceramide levels in C. albicans membranes were analysed following RsAFP2 treatment and, as expected, increased accumulation of phytoC24-ceramides in membranes of RsAFP2-treated C. albicans cells was detected. This is the first report on the interaction of a plant defensin with glucosylceramides in the fungal cell wall, causing cell wall stress, and on the effects of a defensin on septin localization and ceramide accumulation.

Evaluation of bioactive sphingolipids in 4-HPR-resistant leukemia cells.

BACKGROUND: N-(4-hydroxyphenyl)retinamide (4-HPR, fenretinide) is a synthetic retinoid with potent pro-apoptotic activity against several types of cancer, but little is known regarding mechanisms leading to chemoresistance. Ceramide and, more recently, other sphingolipid species (e.g., dihydroceramide and dihydrosphingosine) have been implicated in 4-HPR-mediated tumor cell death. Because sphingolipid metabolism has been reported to be altered in drug-resistant tumor cells, we studied the implication of sphingolipids in acquired resistance to 4-HPR based on an acute lymphoblastic leukemia model. METHODS: CCRF-CEM cell lines resistant to 4-HPR were obtained by gradual selection. Endogenous sphingolipid profiles and in situ enzymatic activities were determined by LC/MS, and resistance to 4-HPR or to alternative treatments was measured using the XTT viability assay and annexin V-FITC/propidium iodide labeling. RESULTS: No major crossresistance was observed against other antitumoral compounds (i.e. paclitaxel, cisplatin, doxorubicin hydrochloride) or agents (i.e. ultra violet C, hydrogen peroxide) also described as sphingolipid modulators. CCRF-CEM cell lines resistant to 4-HPR exhibited a distinctive endogenous sphingolipid profile that correlated with inhibition of dihydroceramide desaturase. Cells maintained acquired resistance to 4-HPR after the removal of 4-HPR though the sphingolipid profile returned to control levels. On the other hand, combined treatment with sphingosine kinase inhibitors (unnatural (dihydro)sphingosines ((dh)Sph)) and glucosylceramide synthase inhibitor (PPMP) in the presence or absence of 4-HPR increased cellular (dh)Sph (but not ceramide) levels and were highly toxic for both parental and resistant cells. CONCLUSIONS: In the leukemia model, acquired resistance to 4-HPR is selective and persists in the absence of sphingolipid profile alteration. Therapeutically, the data demonstrate that alternative sphingolipid-modulating antitumoral strategies are suitable for both 4-HPR-resistant and sensitive leukemia cells. Thus, whereas sphingolipids may not be critical for maintaining resistance to 4-HPR, manipulation of cytotoxic sphingolipids should be considered a viable approach for overcoming resistance.

A novel mechanism of lysosomal acid sphingomyelinase maturation: requirement for carboxyl-terminal proteolytic processing.

Acid sphingomyelinase (aSMase) catalyzes the hydrolysis of sphingomyelin (SM) to form the bioactive lipid ceramide (Cer). Notably, aSMase exists in two forms: a zinc (Zn(2+))-independent lysosomal aSMase (L-SMase) and a Zn(2+)-dependent secreted aSMase (S-SMase) that arise from alternative trafficking of a single protein precursor. Despite extensive investigation into the maturation and trafficking of aSMase, the exact identity of mature L-SMase has remained unclear. Here, we describe a novel mechanism of aSMase maturation involving C-terminal proteolytic processing within, or in close proximity to, endolysosomes. Using two different C-terminal-tagged constructs of aSMase (V5, DsRed), we demonstrate that aSMase is processed from a 75-kDa, Zn(2+)-activated proenzyme to a mature 65 kDa, Zn(2+)-independent L-SMase. L-SMase is recognized by a polyclonal Ab to aSMase, but not by anti-V5 or anti-DsRed antibodies, suggesting that the C-terminal tag is lost during maturation. Furthermore, indirect immunofluorescence staining demonstrated that mature L-SMase colocalized with the lysosomal marker LAMP1, whereas V5-aSMase localized to the Golgi secretory pathway. Moreover, V5-aSMase possessed Zn(2+)-dependent activity suggesting it may represent the common protein precursor of S-SMase and L-SMase. Importantly, the 65-kDa L-SMase, but not V5-aSMase, was sensitive to the lysosomotropic inhibitor desipramine, co-fractionated with lysosomes, and migrated at the same M(r) as partially purified human aSMase. Finally, three aSMase mutants containing C-terminal Niemann-Pick mutations (R600H, R600P, ΔR608) exhibited defective proteolytic maturation. Taken together, these results demonstrate that mature L-SMase arises from C-terminal proteolytic processing of pro-aSMase and suggest that impaired C-terminal proteolysis may lead to severe defects in L-SMase function.

Regulated secretion of acid sphingomyelinase: implications for selectivity of ceramide formation.

The acid sphingomyelinase (aSMase) gene gives rise to two distinct enzymes, lysosomal sphingomyelinase (L-SMase) and secretory sphingomyelinase (S-SMase), via differential trafficking of a common protein precursor. However, the regulation of S-SMase and its role in cytokine-induced ceramide formation remain ill defined. To determine the role of S-SMase in cellular sphingolipid metabolism, MCF7 breast carcinoma cells stably transfected with V5-aSMase(WT) were treated with inflammatory cytokines. Interleukin-1ß and tumor necrosis factor-α induced a time- and dose-dependent increase in S-SMase secretion and activity, coincident with selective elevations in cellular C(16)-ceramide. To establish a role for S-SMase, we utilized a mutant of aSMase (S508A) that is shown to retain L-SMase activity, but is defective in secretion. MCF7 expressing V5-aSMase(WT) exhibited increased S-SMase and L-SMase activity, as well as elevated cellular levels of specific long-chain and very long-chain ceramide species relative to vector control MCF7. Interestingly, elevated levels of only certain very long-chain ceramides were evident in V5-aSMase(S508A) MCF7. Secretion of the S508A mutant was also defective in response to IL-1ß, as was the regulated generation of C(16)-ceramide. Taken together, these data support a crucial role for Ser(508) in the regulation of S-SMase secretion, and they suggest distinct metabolic roles for S-SMase and L-SMase.

Anterograde and retrograde transport of neutral sphingomyelinase-2 between the Golgi and the plasma membrane.

The activation of neutral sphingomyelinase-2 (nSMase2) and consequent ceramide production are implicated in many stress-induced signaling pathways. Trafficking of nSMase2 from the Golgi compartment to the plasma membrane (PM) in response to signaling stimuli has been described. However, the precise mechanisms of transport remain unknown. This study aimed to investigate the trafficking of nSMase2 between the Golgi and the PM. We show here that V5-nSMase2 localizes at the PM and Golgi in MCF-7 cells and confirm relocalization of nSMase2 to the PM at confluence. Although cycloheximide (CHX) treatment partially inhibited the Golgi localization of GFP-nSMase2, recovery of GFP-nSMase2 to an intracellular compartment was still observed after photobleaching. Moreover, in the presence of CHX, GFP- and V5-nSMase2 co-localized with endosomal/recycling markers. In HEK293 cells, activation of either protein kinase C-alpha or betaII, with the phorbol ester PMA led to relocalization of both wild-type and inactive nSMase2 to the pericentrion, a PKC-dependent subset of recycling endosomes. Finally, inhibition of nSMase2 endocytosis by K+depletion reduced the intracellular pool of nSMase2 and increased nSMase2 activity resulting in elevated ceramide levels. Altogether, these results suggest that nSMase2 traffics from the Golgi to the PM as a membrane protein en route to the cell surface and recycles back to the Golgi through the endosomal/recycling compartment. Moreover, the recycling of nSMase2 from the PM is important for its catalytic regulation.

Dihydroceramide desaturase activity is modulated by oxidative stress.

Oxidative stress has been implicated previously in the regulation of ceramide metabolism. In the present study, its effects on dihydroceramide desaturase were investigated. To stimulate oxidative stress, HEK (human embyronic kidney)-293, MCF7, A549 and SMS-KCNR cells were treated with H2O2, menadione or tert-butylhydroperoxide. In all cell lines, an increase in dihydroceramide was observed upon oxidative stress as measured by LC (liquid chromatography)/MS. In contrast, total ceramide levels were relatively unchanged. Mechanistically, dihydroceramide desaturase activity was measured by an in situ assay and decreased in a time- and dose-dependent fashion. Interestingly, no detectable changes in the protein levels were observed, suggesting that oxidative stress does not induce degradation of dihydroceramide desaturase. In summary, oxidative stress leads to potent inhibition of dihydroceramide desaturase resulting in significant elevation in dihydroceramide levels in vivo.

Skn1 and Ipt1 negatively regulate autophagy in Saccharomyces cerevisiae.

We demonstrated that a yeast deletion mutant in IPT1 and SKN1, encoding proteins involved in the biosynthesis of mannosyldiinositolphosphoryl ceramides, is characterized by increased autophagy and DNA fragmentation upon nitrogen (N) starvation as compared with the single deletion mutants or wild type (WT). Apoptotic features were not significantly different between single and double deletion mutants upon N starvation, pointing to increased autophagy in the double Deltaipt1 Deltaskn1 deletion mutant independent of apoptosis. We observed increased basal levels of phytosphingosine in membranes of the double Deltaipt1 Deltaskn1 deletion mutant as compared with the single deletion mutants or WT. These data point to a negative regulation of autophagy by both Ipt1 and Skn1 in yeast, with a putative involvement of phytosphingosine in this process.

Sustained receptor stimulation leads to sequestration of recycling endosomes in a classical protein kinase C- and phospholipase D-dependent manner.

Considerable insight has been garnered on initial mechanisms of endocytosis of plasma membrane proteins and their subsequent trafficking through the endosomal compartment. It is also well established that ligand stimulation of many plasma membrane receptors leads to their internalization. However, stimulus-induced regulation of endosomal trafficking has not received much attention. In previous studies, we showed that sustained stimulation of protein kinase C (PKC) with phorbol esters led to sequestration of recycling endosomes in a juxtanuclear region. In this study, we investigated whether G-protein-coupled receptors that activate PKC exerted effects on endosomal trafficking. Stimulation of cells with serotonin (5-hydroxytryptamine (5-HT)) led to sequestration of the 5-HT receptor (5-HT2AR) into a Rab11-positive juxtanuclear compartment. This sequestration coincided with translocation of PKC as shown by confocal microscopy. Mechanistically the observed sequestration of 5-HT2AR was shown to require continuous PKC activity because it was inhibited by pretreatment with classical PKC inhibitor Gö6976 and could be reversed by posttreatment with this inhibitor. In addition, classical PKC autophosphorylation was necessary for receptor sequestration. Moreover inhibition of phospholipase D (PLD) activity and inhibition of PLD1 and PLD2 using dominant negative constructs also prevented this process. Functionally this sequestration did not affect receptor desensitization or resensitization as measured by intracellular calcium increase. However, the PKC- and PLD-dependent sequestration of receptors resulted in co-sequestration of other plasma membrane proteins and receptors as shown for epidermal growth factor receptor and protease activated receptor-1. This led to heterologous desensitization of those receptors and diverted their cellular fate by protecting them from agonist-induced degradation. Taken together, these results demonstrate a novel role for sustained receptor stimulation in regulation of intracellular trafficking, and this process requires sustained stimulation of PKC and PLD.

The sphingolipid salvage pathway in ceramide metabolism and signaling.

Sphingolipids are important components of eukaryotic cells, many of which function as bioactive signaling molecules. Of these, ceramide is a central metabolite and plays key roles in a variety of cellular responses, including regulation of cell growth, viability, differentiation, and senescence. Ceramide is composed of the long-chain sphingoid base, sphingosine, in N-linkage to a variety of acyl groups. Sphingosine serves as the product of sphingolipid catabolism, and it is mostly salvaged through reacylation, resulting in the generation of ceramide or its derivatives. This recycling of sphingosine is termed the "salvage pathway", and recent evidence points to important roles for this pathway in ceramide metabolism and function. A number of enzymes are involved in the salvage pathway, and these include sphingomyelinases, cerebrosidases, ceramidases, and ceramide synthases. Recent studies suggest that the salvage pathway is not only subject to regulation, but it also modulates the formation of ceramide and subsequent ceramide-dependent cellular signals. This review focuses on the salvage pathway in ceramide metabolism, its regulation, its experimental analysis, and emerging biological functions.

Mechanism of inhibition of sequestration of protein kinase C alpha/betaII by ceramide. Roles of ceramide-activated protein phosphatases and phosphorylation/dephosphorylation of protein kinase C alpha/betaII on threonine 638/641.

Sustained activation of protein kinase C (PKC) isoenzymes alpha and betaII leads to their translocation to a perinuclear region and to the formation of the pericentrion, a PKC-dependent subset of recycling endosomes. In MCF-7 human breast cancer cells, the action of the PKC activator 4beta-phorbol-12-myristate-13-acetate (PMA) evokes ceramide formation, which in turn prevents PKCalpha/betaII translocation to the pericentrion. In this study we investigated the mechanisms by which ceramide negatively regulates this translocation of PKCalpha/betaII. Upon PMA treatment, HEK-293 cells displayed dual phosphorylation of PKCalpha/betaII at carboxyl-terminal sites (Thr-638/641 and Ser-657/660), whereas in MCF-7 cells PKCalpha/betaII were phosphorylated at Ser-657/660 but not Thr-638/641. Inhibition of ceramide synthesis by fumonisin B1 overcame the defect in PKC phosphorylation and restored translocation of PKCalpha/betaII to the pericentrion. To determine the involvement of ceramide-activated protein phosphatases in PKC regulation, we employed small interference RNA to silence individual Ser/Thr protein phosphatases. Knockdown of isoforms alpha or beta of the catalytic subunits of protein phosphatase 1 not only increased phosphorylation of PKCalpha/betaII at Thr-638/641 but also restored PKCbetaII translocation to the pericentrion. Mutagenesis approaches in HEK-293 cells revealed that mutation of either Thr-641 or Ser-660 to Ala in PKCbetaII abolished sequestration of PKC, implying the indispensable roles of phosphorylation of PKCalpha/betaII at those sites for their translocation to the pericentrion. Reciprocally, a point mutation of Thr-641 to Glu, which mimics phosphorylation, in PKCbetaII overcame the inhibitory effects of ceramide on PKC translocation in PMA-stimulated MCF-7 cells. Therefore, the results demonstrate a novel role for carboxyl-terminal phosphorylation of PKCalpha/betaII in the translocation of PKC to the pericentrion, and they disclose specific regulation of PKC autophosphorylation by ceramide through the activation of specific isoforms of protein phosphatase 1.

Protein kinase C-induced activation of a ceramide/protein phosphatase 1 pathway leading to dephosphorylation of p38 MAPK.

Recently we showed that, in human breast cancer cells, activation of protein kinase C by 4beta-phorbol 12-myristate 13-acetate (PMA) produced ceramide formed from the salvage pathway (Becker, K. P., Kitatani, K., Idkowiak-Baldys, J., Bielawski, J., and Hannun, Y. A. (2005) J. Biol. Chem. 280, 2606-2612). In this study, we investigated intracellular signaling events mediated by this novel activated pathway of ceramide generation. PMA treatment resulted in transient activation of mitogen-activated protein kinases (ERK1/2, JNK1/2, and p38) followed by dephosphorylation/inactivation. Interestingly, fumonisin B1 (FB1), an inhibitor of the salvage pathway, attenuated loss of phosphorylation of p38, suggesting a role for ceramide in p38 dephosphorylation. This was confirmed by knock-down of longevity-assurance homologue 5, which partially suppressed the formation of C(16)-ceramide induced by PMA and increased the phosphorylation of p38. These results demonstrate a role for the salvage pathway in feedback inhibition of p38. To determine which protein phosphatases act in this pathway, specific knock-down of serine/threonine protein phosphatases was performed, and it was observed that knock-down of protein phosphatase 1 (PP1) catalytic subunits significantly increased p38 phosphorylation, suggesting activation of PP1 results in an inhibitory effect on p38. Moreover, PMA recruited PP1 catalytic subunits to mitochondria, and this was significantly suppressed by FB1. In addition, phospho-p38 resided in PMA-stimulated mitochondria. Upon PMA treatment, a mitochondria-enriched/purified fraction exhibited significant increases in C(16)-ceramide, a major ceramide specie, which was suppressed by FB1. Taken together, these data suggest that accumulation of C(16)-ceramide in mitochondria formed from the protein kinase C-dependent salvage pathway results at least in part from the action of longevity-assurance homologue 5, and the generated ceramide modulates the p38 cascade via PP1.

Dynamic sequestration of the recycling compartment by classical protein kinase C.

It has been previously shown that upon sustained stimulation (30-60 min) with phorbol esters, protein kinase C (PKC) alpha and betaII become sequestered in a juxtanuclear region, the pericentrion. The activation of PKC also results in sequestration of transferrin, suggesting a role for PKC in regulating endocytosis and sequestration of recycling components. In this work we characterize the pericentrion as a PKC-dependent subset of the recycling compartment. We demonstrate that upon sustained stimulation of PKC, both protein (CD59, caveolin) and possibly also lipid (Bodipy-GM1) cargo become sequestered in a PKC-dependent manner. This sequestration displayed a strict temperature requirement and was inhibited below 32 degrees C. Treatment of cells with phorbol myristate acetate for 60 min led to the formation of a distinct membrane structure. PKC sequestration and pericentrion formation were blocked by hypertonic sucrose as well as by potassium depletion (inhibitors of clathrin-dependent endocytosis) but not by nystatin or filipin, which inhibit clathrin-independent pathways. Interestingly, it was also observed that some molecules that internalize through clathrin-independent pathways (CD59, Bodipy-GM1, caveolin) also sequestered to the pericentrion upon sustained PKC activation, suggesting that PKC acted distal to the site of internalization of endocytic cargo. Together these results suggest that PKC regulates sequestration of recycling molecules into this compartment, the pericentrion.