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Yan J. Jiang Yoshikazu Uchida Biao Lu Peggy Kim Cungui Mao Masashi Akiyama Peter M. Elias Walter M. Holleran Carl Grunfeld Kenneth R. Feingold 《The Journal of biological chemistry》2009,284(28):18942-18952
ABCA12 (ATP binding cassette transporter, family 12) is a cellular membrane transporter that facilitates the delivery of glucosylceramides to epidermal lamellar bodies in keratinocytes, a process that is critical for permeability barrier formation. Following secretion of lamellar bodies into the stratum corneum, glucosylceramides are metabolized to ceramides, which comprise ∼50% of the lipid in stratum corneum. Gene mutations of ABCA12 underlie harlequin ichthyosis, a devastating skin disorder characterized by abnormal lamellar bodies and a severe barrier abnormality. Recently we reported that peroxisome proliferator-activated receptor (PPAR) and liver X receptor activators increase ABCA12 expression in human keratinocytes. Here we demonstrate that ceramide (C2-Cer and C6-Cer), but not C8-glucosylceramides, sphingosine, or ceramide 1-phosphate, increases ABCA12 mRNA expression in a dose- and time-dependent manner. Inhibitors of glucosylceramide synthase, sphingomyelin synthase, and ceramidase and small interfering RNA knockdown of human alkaline ceramidase, which all increase endogenous ceramide levels, also increased ABCA12 mRNA levels. Moreover, simultaneous treatment with C6-Cer and each of these same inhibitors additively increased ABCA12 expression, indicating that ceramide is an important inducer of ABCA12 expression and that the conversion of ceramide to other sphingolipids or metabolites is not required. Finally, both exogenous and endogenous ceramides preferentially stimulate PPARδ expression (but not other PPARs or liver X receptors), whereas PPARδ knockdown by siRNA transfection specifically diminished the ceramide-induced increase in ABCA12 mRNA levels, indicating that PPARδ is a mediator of the ceramide effect. Together, these results show that ceramide, an important lipid component of epidermis, up-regulates ABCA12 expression via the PPARδ-mediated signaling pathway, providing a substrate-driven, feed-forward mechanism for regulating this key lipid transporter.The outermost layer of mammalian epidermis, the stratum corneum, is essential for permeability barrier function and critical for terrestrial life. The stratum corneum consists of terminally differentiated, anucleate keratinocytes, or corneocytes, surrounded by lipid-enriched lamellar membranes composed of three major lipids, ceramides, cholesterol, and free fatty acids (1). These lipids are delivered to the extracellular spaces of the stratum corneum through exocytosis of lamellar body contents from outermost stratum granulosum cells (2). Mature lamellar bodies contain primarily cholesterol, phospholipids, and glucosylceramides (3). Following lamellar body secretion, the secreted phospholipids and glucosylceramides are converted to free fatty acids and ceramides by phospholipases and β-glucocerebrosidase, respectively (1, 4). ABCA12 (ATP binding cassette transporter, family 12), a lipid transporter predominantly expressed in epidermis, has been shown to play a vital role in the formation of mature lamellar bodies (5, 6), although how this transporter is regulated remains unresolved.ABCA12 is a member of the ABCA subfamily of transporters, which are involved in the transport of a variety of lipids (7). Mutations in ABCA1 cause Tangier disease, which is due to a defect in transporting cholesterol and phospholipids from intracellular lipid stores to apolipoproteins, particularly apolipoprotein A-I (8–11). Mutations in ABCA3 cause neonatal respiratory failure due to a defect in surfactant transport from alveolar type II cells into the alveolar space (12). Mutations in ABCA4 cause Stargardt''s macular degeneration, with visual loss due to a defect in transporting phosphatidylethanolamine-retinylidene out of retinal pigment cells (13).Recently, mutations in ABCA12 have been shown to cause harlequin ichthyosis and a subgroup of lamellar ichthyosis, two disorders of keratinization (5, 14, 15). ABCA12 mutations lead to an abnormality in lamellar body formation, a decrease in lamellar membranes in the extracellular spaces of the stratum corneum, an accumulation of glucosylceramide in the epidermis with a reduction in ceramide (16), and ultimately loss of permeability barrier function (17), which in harlequin ichthyosis can result in neonatal lethality (5, 15). Strikingly, genetic correction of ABCA12 deficiency in patients'' keratinocytes by gene transfer normalized loading of glucosylceramides into lamellar bodies (5). These studies demonstrate a critical role for ABCA12 in epidermal physiology, specifically in the formation of mature lamellar bodies and subsequent permeability barrier homeostasis. Hence, it is crucial to understand how ABCA12 is regulated.Our laboratory recently demonstrated that activation of peroxisome proliferator-activated receptor (PPARδ and PPARγ) or liver X receptor (LXR) stimulates ABCA12 expression in cultured human keratinocytes (18). Both PPARs and LXR are important lipid sensors that stimulate keratinocyte differentiation and enhance permeability barrier function (19). Additionally, PPARα and -δ as well as LXR activators stimulate ceramide synthesis in keratinocytes (20, 21). Likewise, ceramide synthesis increases in keratinocytes during differentiation, foreshadowing the formation of lamellar bodies (22, 23).In addition to serving as structural membrane components, ceramides are also important signaling molecules that can induce growth arrest, differentiation, and apoptosis in various cells, including keratinocytes (24–26). Moreover, distal ceramide metabolites, sphingosine and sphingosine-1-phosphate (Fig. 1), are also important signaling molecules (27).Open in a separate windowFIGURE 1.The central role of ceramide in sphingolipid metabolism in keratinocytes. C1P, ceramide 1-phosphate; Sph, sphingosine; S1P, sphingosine-1-phosphate; GlcCer, glucosylceramide; SM, sphingomyelin.It is well established that the expression of ABCA1 is regulated by cellular cholesterol levels in many cell types, including keratinocytes (28). Cholesterol, if metabolized to certain oxysterols, can activate LXR, which then stimulates ABCA1 expression and the transport of cholesterol out of cells (29). This example of feed-forward regulation leads us to hypothesize that either ceramide or a metabolite of ceramide might stimulate ABCA12 expression, thereby leading to an increase in the transport of glucosylceramides into maturing lamellar bodies. Here, we provide evidence that ceramide stimulates ABCA12 expression in keratinocytes via a mechanism involving PPARδ signaling. 相似文献
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Ting Wang Yatrik M. Shah Tsutomu Matsubara Yueying Zhen Tomotaka Tanabe Tomokazu Nagano Serge Fotso Kristopher W. Krausz T. Mark Zabriskie Jeffrey R. Idle Frank J. Gonzalez 《The Journal of biological chemistry》2010,285(10):7670-7685
A previous study identified the peroxisome proliferator-activated receptor α (PPARα) activation biomarkers 21-steroid carboxylic acids 11β-hydroxy-3,20-dioxopregn-4-en-21-oic acid (HDOPA) and 11β,20-dihydroxy-3-oxo-pregn-4-en-21-oic acid (DHOPA). In the present study, the molecular mechanism and the metabolic pathway of their production were determined. The PPARα-specific time-dependent increases in HDOPA and 20α-DHOPA paralleled the development of adrenal cortex hyperplasia, hypercortisolism, and spleen atrophy, which was attenuated in adrenalectomized mice. Wy-14,643 activation of PPARα induced hepatic FGF21, which caused increased neuropeptide Y and agouti-related protein mRNAs in the hypothalamus, stimulation of the agouti-related protein/neuropeptide Y neurons, and activation of the hypothalamic-pituitary-adrenal (HPA) axis, resulting in increased adrenal cortex hyperplasia and corticosterone production, revealing a link between PPARα and the HPA axis in controlling energy homeostasis and immune regulation. Corticosterone was demonstrated as the precursor of 21-carboxylic acids both in vivo and in vitro. Under PPARα activation, the classic reductive metabolic pathway of corticosterone was suppressed, whereas an alternative oxidative pathway was uncovered that leads to the sequential oxidation on carbon 21 resulting in HDOPA. The latter was then reduced to the end product 20α-DHOPA. Hepatic cytochromes P450, aldehyde dehydrogenase (ALDH3A2), and 21-hydroxysteroid dehydrogenase (AKR1C18) were found to be involved in this pathway. Activation of PPARα resulted in the induction of Aldh3a2 and Akr1c18, both of which were confirmed as target genes through introduction of promoter luciferase reporter constructs into mouse livers in vivo. This study underscores the power of mass spectrometry-based metabolomics combined with genomic and physiologic analyses in identifying downstream metabolic biomarkers and the corresponding upstream molecular mechanisms. 相似文献
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Francisco J. Schopfer Marsha P. Cole Alison L. Groeger Chen-Shan Chen Nicholas K. H. Khoo Steven R. Woodcock Franca Golin-Bisello U. Nkiru Motanya Yong Li Jifeng Zhang Minerva T. Garcia-Barrio Tanja K. Rudolph Volker Rudolph Gustavo Bonacci Paul R. S. Baker H. Eric Xu Carlos I. Batthyany Y. Eugene Chen Tina M. Hallis Bruce A. Freeman 《The Journal of biological chemistry》2010,285(16):12321-12333
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Norihisa Nishimichi Fumiko Higashikawa Hiromi H. Kinoh Yoshiko Tateishi Haruo Matsuda Yasuyuki Yokosaki 《The Journal of biological chemistry》2009,284(22):14769-14776
Osteopontin (OPN) is a cytokine and ligand for multiple members of the
integrin family. OPN undergoes the in vivo polymerization catalyzed
by cross-linking enzyme transglutaminase 2, which consequently increases the
bioactivity through enhanced interaction with integrins. The integrin
α9β1, highly expressed on neutrophils, binds to the sequence
SVVYGLR only after intact OPN is cleaved by thrombin. The SVVYGLR sequence
appears to be cryptic in intact OPN because α9β1 does not recognize
intact OPN. Because transglutaminase 2-catalyzed polymers change their
physical and chemical properties, we hypothesized that the SVVYGLR site might
also be exposed on polymeric OPN. As expected, α9β1 turned into a
receptor for polymeric OPN, a result obtained by cell adhesion and migration
assays with α9-transfected cells and by detection of direct binding of
recombinant soluble α9β1 with colorimetry and surface plasmon
resonance analysis. Because the N-terminal fragment of thrombin-cleaved OPN, a
ligand for α9β1, has been reported to attract neutrophils, we next
examined migration of neutrophils to polymeric OPN using time-lapse
microscopy. Polymeric OPN showed potent neutrophil chemotactic activity, which
was clearly inhibited by anti-α9β1 antibody. Unexpectedly,
mutagenesis studies showed that α9β1 bound to polymeric OPN
independently of the SVVYGLR sequence, and further, SVVYGLR sequence of
polymeric OPN was cryptic because SVVYGLR-specific antibody did not recognize
polymeric OPN. These results demonstrate that polymerization of OPN generates
a novel α9β1-binding site and that the interaction of this site
with the α9β1 integrin is critical to the neutrophil chemotaxis
induced by polymeric OPN.Acidic phosphorylated secreted glycoprotein osteopontin
(OPN),4 known as a
cytokine, has multiple functions, including roles in tissue remodeling,
fibrosis, mineralization, immunomodulation, inflammation, and tumor metastasis
(1–3).
OPN is also an integrin ligand. At least nine integrins can function as OPN
receptors. α5β1, α8β1, αvβ1, αvβ3,
αvβ5 (1), and
αvβ6 (4) recognize
the linear tripeptide RGD, and α9β1, α4β1, and
α4β7 recognize the sequence, SVVYGLR
(5), adjacent to RGD but only
after OPN has been cleaved by the protease, thrombin
(Fig. 1).Open in a separate windowFIGURE 1.Schematic diagram of OPN. Two integrin-binding sites
(boxed), a thrombin cleavage site (arrow), and a putative
transglutamination site (circled) are shown. The term
thrombin-cleaved nOPN is defined as in the figure.The overlap of receptors for OPN does not necessarily mean that these
integrins play redundant roles in cellular responses to OPN because the
patterns of integrin expression and utilization vary widely among cell types.
In addition, interactions of different integrins with a single ligand can
exert distinct effects on cell behavior in a single cell type. For example, we
have previously reported that signals by ligation of αvβ3,
αvβ6, or α9β1 to a single ligand, tenascin-C,
differently affected cell adhesion, spreading, and proliferation of the colon
cancer cell line, SW480 (6).
Furthermore, intact OPN or thrombin- or matrix metalloproteinase-cleaved OPN
interact with distinct subsets of integrins and exhibit distinct effects on
cell behavior (4,
7,
8). Collectively, some of the
functional diversity of OPN could be attributed to this multiplicity of
receptors and responses. We have recently shown that polymerization of OPN
results in enhanced biological activity
(9). We thus set out to
determine whether polymerized OPN exerts its effects through unique
interactions with integrins.OPN is polymerized by transglutaminase 2 (TG2, EC 2.3.2.13)
(10) that catalyzes formation
of isopeptide cross-links between glutamine and lysine residues in substrate
proteins (11) including OPN.
Polymeric OPN has been identified in vivo in bone
(12) and calcified aorta
(13). We have previously
reported that upon polymerization, OPN displays increased integrin binding
accompanied by enhanced cell adhesion, spreading, migration, and focal contact
formation (9). However, very
little is known about how polymeric OPN induces its biological effects.Integrin α9β1, highly expressed on neutrophils
(14), does not act as a
receptor for intact OPN but does bind to an N-terminal fragment of OPN (nOPN)
that is generated by thrombin cleavage
(15) through the new
C-terminal sequence, SVVYGLR. Protein polymerization can expose otherwise
cryptic domains (16), so we
hypothesized that the SVVYGLR site might be exposed upon polymerization and
serve as a binding site for α9β1. In the present study, we
demonstrate that α9β1 is indeed a receptor for polymeric OPN and
that neutrophil migration induced by polymeric OPN is largely mediated by this
interaction. However, mutational analysis and antibody studies demonstrate
that this interaction does not involve the SVVYGLR site, suggesting the
presence of de novo binding site in polymeric OPN. 相似文献
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Benoit Pourcet In��s Pineda-Torra Bruno Derudas Bart Staels Corine Glineur 《The Journal of biological chemistry》2010,285(9):5983-5992
The nuclear receptor peroxisome proliferator-activated receptor α (PPARα) is a key regulator of genes implicated in lipid homeostasis and inflammation. PPARα trans-activity is enhanced by recruitment of coactivators such as SRC1 and CBP/p300 and is inhibited by binding of corepressors such as NCoR and SMRT. In addition to ligand binding, PPARα activity is regulated by post-translational modifications such as phosphorylation and ubiquitination. In this report, we demonstrate that hPPARα is SUMOylated by SUMO-1 on lysine 185 in the hinge region. The E2-conjugating enzyme Ubc9 and the SUMO E3- ligase PIASy are implicated in this process. In addition, ligand treatment decreases the SUMOylation rate of hPPARα. Finally, our results demonstrate that SUMO-1 modification of hPPARα down-regulates its trans-activity through the specific recruitment of corepressor NCoR but not SMRT leading to the differential expression of a subset of PPARα target genes. In conclusion, hPPARα SUMOylation on lysine 185 down-regulates its trans-activity through the selective recruitment of NCoR. 相似文献
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Haipeng Cheng Kulandaivelu S. Vetrivel Renaldo C. Drisdel Xavier Meckler Ping Gong Jae Yoon Leem Tong Li Meghan Carter Ying Chen Phuong Nguyen Takeshi Iwatsubo Taisuke Tomita Philip C. Wong William N. Green Maria Z. Kounnas Gopal Thinakaran 《The Journal of biological chemistry》2009,284(3):1373-1384
Proteolytic processing of amyloid precursor protein (APP) by β- and
γ-secretases generates β-amyloid (Aβ) peptides, which
accumulate in the brains of individuals affected by Alzheimer disease.
Detergent-resistant membrane microdomains (DRM) rich in cholesterol and
sphingolipid, termed lipid rafts, have been implicated in Aβ production.
Previously, we and others reported that the four integral subunits of the
γ-secretase associate with DRM. In this study we investigated the
mechanisms underlying DRM association of γ-secretase subunits. We report
that in cultured cells and in brain the γ-secretase subunits nicastrin
and APH-1 undergo S-palmitoylation, the post-translational covalent
attachment of the long chain fatty acid palmitate common in lipid
raft-associated proteins. By mutagenesis we show that nicastrin is
S-palmitoylated at Cys689, and APH-1 is
S-palmitoylated at Cys182 and Cys245.
S-Palmitoylation-defective nicastrin and APH-1 form stable
γ-secretase complexes when expressed in knock-out fibroblasts lacking
wild type subunits, suggesting that S-palmitoylation is not essential
for γ-secretase assembly. Nevertheless, fractionation studies show that
S-palmitoylation contributes to DRM association of nicastrin and
APH-1. Moreover, pulse-chase analyses reveal that S-palmitoylation is
important for nascent polypeptide stability of both proteins. Co-expression of
S-palmitoylation-deficient nicastrin and APH-1 in cultured cells
neither affects Aβ40, Aβ42, and AICD production, nor intramembrane
processing of Notch and N-cadherin. Our findings suggest that
S-palmitoylation plays a role in stability and raft localization of
nicastrin and APH-1, but does not directly modulate γ-secretase
processing of APP and other substrates.Alzheimer disease is the most common among neurodegenerative diseases that
cause dementia. This debilitating disorder is pathologically characterized by
the cerebral deposition of 39–42 amino acid peptides termed Aβ,
which are generated by proteolytic processing of amyloid precursor protein
(APP)2 by β- and
γ-secretases (1,
2). The β-site APP
cleavage enzyme 1 cleaves full-length APP within its luminal domain to
generate a secreted ectodomain leaving behind a C-terminal fragment
(β-CTF). γ-Secretase cleaves β-CTF within the transmembrane
domain to release Aβ and APP intracellular
C-terminal domain (AICD). γ-Secretase is a
multiprotein complex, comprising at least four subunits: presenilins (PS1 and
PS2), nicastrin, APH-1, and PEN-2 for its activity
(3). PS1 is synthesized as a
42–43-kDa polypeptide and undergoes highly regulated endoproteolytic
processing within the large cytoplasmic loop domain connecting putative
transmembrane segments 6 and 7 to generate stable N-terminal (NTF) and
C-terminal fragments (CTF) by an uncharacterized proteolytic activity
(4). This endoproteolytic event
has been identified as the activation step in the process of PS1 maturation as
it assembles with other γ-secretase subunits
(3). Nicastrin is a heavily
glycosylated type I membrane protein with a large ectodomain that has been
proposed to function in substrate recognition and binding
(5), but this putative function
has not been confirmed by others
(6). APH-1 is a
seven-transmembrane protein encoded by two human or three rodent genes that
are alternatively spliced (7).
Although PS1 (or PS2), nicastrin, APH-1, and PEN-2 are sufficient for
γ-secretase processing of APP, a type I membrane protein, termed p23
(also referred toTMP21), was recently identified as a γ-secretase
component that modulates γ-secretase activity and regulates secretory
trafficking of APP (8,
9).A growing number of type I integral membrane proteins has been identified
as γ-secretase substrates within the last few years, including Notch1
homologues, Notch ligands, Delta and Jagged, cell adhesion receptors N- and
E-cadherins, low density lipoprotein receptor-related protein, ErbB-4, netrin
receptor DCC, and others (10).
Mounting evidence suggests that APP processing occurs within cholesterol- and
sphingolipid-enriched lipid rafts, which are biochemically defined as
detergentresistant membrane microdomains (DRM)
(11,
12). Previously we reported
that each of the γ-secretase subunits localizes in lipid rafts in
post-Golgi and endosome membranes enriched in syntaxin 6
(13). Moreover, loss of
γ-secretase activity by gene deletion or exposure to γ-secretase
inhibitors results in the accumulation of APP CTFs in lipid rafts indicating
that cleavage of APP CTFs likely occurs in raft microdomains
(14). In contrast, CTFs
derived from Notch1, Jagged2, N-cadherin, and DCC are processed by
γ-secretase in non-raft membranes
(14). The mechanisms
underlying association of γ-secretase subunits with lipid rafts need
further clarification to elucidate spatial segregation of amyloidogenic
processing of APP in membrane microdomains.Post-translational S-palmitoylation is increasingly recognized as
a potential mechanism for regulating raft association, stability,
intracellular trafficking, and function of several cytosolic and transmembrane
proteins
(15–17).
S-palmitoylation refers to the addition of 16-carbon palmitoyl moiety
to certain cysteine residues through thioester linkage. Cysteines close to
transmembrane domains or membrane-associated domains in non-integral membrane
proteins are preferred S-palmitoylation sites, although no conserved
motif has been identified
(18). Palmitoylation modifies
numerous neuronal proteins, including postsynaptic density protein PSD-95
(19),
a-amino-3-hydroxyl-5-methyl-4-isoxazole propionic acid receptors
(20), nicotinic α7
receptors (21), neuronal
t-SNAREs SNAP-25, synaptobrevin 2 and synaptogagmin
(22,
23), neuronal
growth-associated protein GAP-43
(24), protein kinase CLICK-III
(CL3)/CaMKIγ (25),
β-secretase (26), and
Huntingtin (27). Although
palmitoylation can occur in vitro without the involvement of an
enzyme, a family of palmitoyltransferases that specifically catalyze
S-palmitoylation has been identified
(28,
29).In this study, we have identified S-palmitoylation of
γ-secretase subunits nicastrin and APH-1, and characterized its role on
DRM association, protein stability, and γ-secretase enzyme activities.
We show that nicastrin is S-palmitoylated at Cys689, and
APH-1 at Cys182 and Cys245. Mutagenesis of
palmitoylation sites results in increased degradation of nascent nicastrin and
APH-1 polypeptides and reduced association with DRM. Nevertheless, in cultured
cells overexpression of S-palmitoylation-deficient nicastrin and
APH-1 does not modulate γ-secretase processing of APP or other
substrates. 相似文献
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Maria L. R. Hughes Bonan Liu Michelle L. Halls Kylie M. Wagstaff Rahul Patil Tony Velkov David A. Jans Nigel W. Bunnett Martin J. Scanlon Christopher J. H. Porter 《The Journal of biological chemistry》2015,290(22):13895-13906
Nuclear hormone receptors (NHRs) regulate the expression of proteins that control aspects of reproduction, development and metabolism, and are major therapeutic targets. However, NHRs are ubiquitous and participate in multiple physiological processes. Drugs that act at NHRs are therefore commonly restricted by toxicity, often at nontarget organs. For endogenous NHR ligands, intracellular lipid-binding proteins, including the fatty acid-binding proteins (FABPs), can chaperone ligands to the nucleus and promote NHR activation. Drugs also bind FABPs, raising the possibility that FABPs similarly regulate drug activity at the NHRs. Here, we investigate the ability of FABP1 and FABP2 (intracellular lipid-binding proteins that are highly expressed in tissues involved in lipid metabolism, including the liver and intestine) to influence drug-mediated activation of the lipid regulator peroxisome proliferator-activated receptor (PPAR) α. We show by quantitative fluorescence imaging and gene reporter assays that drug binding to FABP1 and FABP2 promotes nuclear localization and PPARα activation in a drug- and FABP-dependent manner. We further show that nuclear accumulation of FABP1 and FABP2 is dependent on the presence of PPARα. Nuclear accumulation of FABP on drug binding is driven largely by reduced nuclear egress rather than an increased rate of nuclear entry. Importin binding assays indicate that nuclear access occurs via an importin-independent mechanism. Together, the data suggest that specific drug-FABP complexes can interact with PPARα to effect nuclear accumulation of FABP and NHR activation. Because FABPs are expressed in a regionally selective manner, this may provide a means to tailor the patterns of NHR drug activation in a tissue-specific manner. 相似文献
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