AP-1 binding transcriptionally regulates human neutral ceramidase

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Abstract

Many forms of cellular stress cause an elevation of endogenous ceramide levels leading to growth arrest or apoptosis. Ceramidases (CDase) play a critical role in regulating apoptosis by hydrolyzing ceramide into sphingosine, a precursor for promitogenic sphingosine-1-phosphate. Growth factor induction of neutral CDase (nCDase) has been shown to have a cytoprotective effect against cytokine-induced increases in ceramide levels. To further define the physiological regulation of nCDase, we identified a 200 bp promoter region and demonstrated that serum activated this proximal promoter, which correlated with a serum-induced increase in human nCDase mRNA expression. Computational analysis revealed a putative cis-element for AP-1, a transcription factor activated by serum. Electrophoretic mobility shift assays demonstrated that the identified transcriptional response element binds to AP-1 transcription factors. RNA interference-mediated knockdown of the AP-1 subunit, c-Jun, inhibited the activity of the human nCDase proximal promoter, whereas, c-Jun overexpression increased promoter activity, which directly correlated with human nCDase mRNA transcription, decreased ceramide mass, and protection against caspase 3/7-dependent apoptosis. Taken together, our findings suggest that c-Jun/AP-1 signaling may, in part, regulate serum-induced human nCDase gene transcription.

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

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