AP-1 binding transcriptionally regulates human neutral ceramidase
Highlights
► Serum increases nCDase mRNA expression. ► Serum-induced nCDase mRNA is mediated through an AP-1 binding site in the promoter. ► Knockdown of the AP-1 subunit, c-jun, inhibits nCDase transcription. ► Overexpression of c-jun increases nCDase expression and protects from apoptosis.
Introduction
Mounting evidence has demonstrated that sphingolipid metabolites, including ceramide (Cer)1, sphingosine (Sph), and sphingosine-1-phosphate (So1P), are critical modulators of cellular function. Cer and Sph are second messengers shown to induce cell growth arrest or apoptosis [1], [2]. In contrast, So1P typically promotes cell growth and differentiation [3], [4] and can suppress the apoptotic effects of Cer [5]. It is postulated that overall balance between sphingolipid metabolites, in part, determines cellular responses.
Sph, the precursor for So1P, is formed through the catalytic activity of ceramidases (CDases) and reverted back to its unphosphorylated state via So1P phosphatases. CDases cleave the N-acyl linkage of Cer, yielding Sph and free fatty acids as products [6]. CDases are classified into three subtypes according to their pH optima: acidic, neutral, and alkaline. Evidence suggests that neutral CDases (nCDases) are critical physiological enzymes in regulating the balance of sphingolipid metabolites. Human, rat, and mouse nCDases have all been defined as integral membrane proteins mainly localizing to the plasma membrane [7], [8]. Furthermore, rat kidney nCDase was shown to be enriched in lipid rafts, which are structured membrane microdomains [9]. Not only has nCDase been shown to localize to these lipid rafts, but exogenously delivered Cer [10] as well as endogenous ceramide, sphingosine and the enzyme sphingosine kinase 1 [11]. We and others have previously shown that Cer recruits and activates a downstream target, PKCζ, within these lipid rafts [12], [13]. The abilities of nCDase to modulate lipid mediated signaling in response to various cytokine and growth factor stimuli are supported by the co-localization of enzyme, substrate, and targets within lipid rafts.
The physiological consequences of altering Cer mass are becoming more apparent. Many forms of cellular stress, including, but not limited to, heat shock, ionizing radiation, ultraviolet light, Fas ligand, growth factor removal, and oxidative stress cause an elevation of endogenous Cer levels [3], [14]. Similarly, many anticancer drugs mediate apoptosis through elevating levels of Cer [15], [16]. Evidence has accumulated demonstrating the importance of nCDase in regulating Cer concentration in response to cytokine- and growth factor-mediated signaling. We have previously shown that platelet derived growth factor increased nCDase activity in rat mesangial cells [17]. In addition, nCDase activity was regulated by the cytokine interleukin-1β (IL-1β) in a bimodal manner in rat hepatocytes [18]. Furthermore, after an initial release of Cer, long-term exposure of mesangial cells to IL-1β results in cytoprotective up-regulation of nCDase mRNA expression and protein synthesis [19]. Degradation of nCDase induced by nitric oxide (NO) results in apoptosis from increased Cer levels [20]. Moreover, nCDase was shown to protect against TNFα-induced Cer accumulation and apoptosis in primary hepatocytes and against TNFα-induced hepatotoxicity in a rat in vivo model [21]. Despite the increasing evidence of the critical role of nCDase in cellular function, little is known concerning the transcriptional regulation of this enzyme. Putative transcriptional response elements (TRE) have been identified, but not confirmed, in the mouse nCDase promoter [22]. In concurrent work in this issue, we identified the proximal promoter region of the human nCDase gene and also, identified several important TRE within this proximal promoter region [23]. In the present study, we investigated the physiological transcriptional regulation of human nCDase promoter in human embryonic kidney (HEK 293) and coronary artery vascular smooth muscle cell models, focusing on the serum-responsive AP-1 TRE. Identifying the physiological mechanisms underlying transcriptional regulation of nCDase may identify new targets in diseases of dysfunctional ceramide metabolism, including vascular diseases, diabetes and cancer.
Section snippets
Cell culture
Human embryonic kidney 293 (HEK 293) cells were obtained from American Type Culture Collection (Rockville, MD). Passages 5–20 were maintained in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS). Human coronary artery smooth muscle cells (HCASMC) were obtained from Cascade Biologics, Inc. (Portland, Oregon). Passages 3–10 were maintained in Medium-231(Cascade Biologics, Inc.) supplemented with Smooth Muscle Cell Growth Supplement (Cascade Biologics, Inc.).
Sequence analysis
Putative
Serum induces human nCDase mRNA expression through proximal regulatory sequences
As Cer metabolism can produce the promitogenic phosphorylated metabolite SoP through the initial production of sphingosine, we investigated if serum treatment induced nCDase expression. We initially chose human coronary artery smooth muscle cells (HCASMC) as a model system, since we have previously reported that balloon angioplasty induced smooth muscle cell migration and proliferation, which correlated with increased promitogenic signaling cascades and metabolism of Cer [29], [30]. Treatment
Discussion
The present study characterizes the physiological regulation of human nCDase. EMSA analyses revealed a putative cis-element for AP-1 within the proximal promoter region of nCDase gene that is regulated by serum in two cell models. We chose to focus on the transcriptional factors that regulate this overlapping AP-1/CCAAT element because the activation of AP-1 by growth factors and serum and the role of AP-1 in proliferation and transformation is well documented [33]. AP-1 trans-activators are
Acknowledgments
NIH Grants HL66371 and HL76789 to M.K. supported this work. We would like to acknowledge the Molecular Genetics Core Facility at the Penn State College of Medicine for their assistance in DNA sequencing analyses. Core Facility services and instruments used in this project were funded, in part, under a grant with the Pennsylvania Department of Health using Tobacco Settlement Funds. The Department specifically disclaims responsibility for any analyses, interpretations or conclusions. We would
References (45)
Biochim. Biophys. Acta
(2002)- et al.
Curr. Opin. Cell Biol.
(1996) - et al.
Biochem. Biophys. Res. Commun.
(2005) - et al.
J. Biol. Chem.
(2003) - et al.
J. Biol. Chem.
(2001) - et al.
Arch. Biochem. Biophys.
(2009) - et al.
J. Biol. Chem.
(2007) - et al.
J. Biol. Chem.
(1995) - et al.
J. Biol. Chem.
(1997) - et al.
J. Biol. Chem.
(2001)
J. Biol. Chem
J. Biol. Chem.
J. Biol. Chem.
Arch. Biochem. Biophys.
Methods
J. Lipid Res.
Gene
Biochim. Biophys. Acta
Cell
J. Biol. Chem.
Int. J. Biochem. Cell Biol.
FEBS Lett.
Cited by (16)
Essential roles of neutral ceramidase and sphingosine in mitochondrial dysfunction due to traumatic brain injury
2014, Journal of Biological ChemistryCitation Excerpt :Evidence has accumulated demonstrating the importance of NCDase in regulating ceramide levels in response to cytokine and growth factor-mediated signaling. Cytokines, including TNF-α, IL-1β, and interferon-γ, could up-regulate the mRNA and protein expression of NCDase as a part of the mechanism protecting the cell against cytokine-induced ceramide toxicity (56–58). Our studies are the first demonstration of the pathophysiological role of NCDase in promoting mitochondrial dysfunction and brain damage after TBI.
Sphingolipid abnormalities in cancer multidrug resistance: Chicken or egg?
2017, Cellular SignallingCitation Excerpt :While transformational changes associated with cancer often involve genetic mutations, sphingolipid metabolizing enzymes are rarely mutated but rather are regulated by aberrant upstream signaling. Transcriptional [190–195] or post-transcriptional [196] regulation of sphingolipid metabolizing enzymes has been poorly investigated. Despite this, two general lines of evidence support altered sphingolipid intermediary metabolism at an early stage of oncogenesis: 1) sphingolipid metabolizing enzymes often serve as targets for or are modifiers of oncoproteins and 2) sphingolipids often regulate growth factor receptor signaling [197,198].
Regulation of alkaline ceramidase activity by the c-Src-mediated pathway
2014, Archives of Biochemistry and BiophysicsCitation Excerpt :Thus, ceramidase activity at pH 8.8 may reflect neutral ceramidase activity. It has been reported that the activity of neutral ceramidase was generally insensitive to Ca2+ [24,25,27,28], and that serum starvation downregulated the expression of the enzyme in cells [29,30]. In the lysates of A549 cells, however, CaCl2 addition significantly enhanced ceramidase activity at pH 8.8, but not the activity at pH 7.2.
New insight into the structure, reaction mechanism, and biological functions of neutral ceramidase
2014, Biochimica et Biophysica Acta - Molecular and Cell Biology of LipidsCitation Excerpt :In addition, the overexpression of c-Jun increased CDase mRNA expression by increasing the AP-1 promoter activity, resulting in a decrease in ceramide and protection against caspase 3/7-dependent apoptosis. This work indicates that c-Jun/AP-1 signaling may be involved in the regulation of serum-induced human neutral CDase [62,63]. In inflammatory bowel disease, S1P generation was increased, possibly through the activation of sphingosine kinase; therefore, S1P plays an important role in the disease [64].
Transcriptional regulation of the human neutral ceramidase gene
2011, Archives of Biochemistry and BiophysicsCitation Excerpt :As briefly aforementioned, the CCAAT box in the nCDase promoter overlaps a functional AP-1 binding site. Our concurrent manuscript in this issue investigates the cooperatively between NF-Y and AP-1 more thoroughly [38]. Here, we will focus on potential interactions of NF-Y with transcription factors that may bind other cis-elements identified in the nCDase promoter.
Lysophospholipids and Their G-Coupled Protein Signaling in Alzheimer’s Disease: From Physiological Performance to Pathological Impairment
2020, Frontiers in Molecular Neuroscience