Alkaline Ceramidase 3 (ACER3) Hydrolyzes Unsaturated Long-chain Ceramides, and Its Down-regulation Inhibits Both Cell Proliferation and Apoptosis

Abstract

Ceramides with different fatty acyl chains may vary in their physiological or pathological roles; however, it remains unclear how cellular levels of individual ceramide species are regulated. Here, we demonstrate that our previously cloned human alkaline ceramidase 3 (ACER3) specifically controls the hydrolysis of ceramides carrying unsaturated long acyl chains, unsaturated long-chain (ULC) ceramides. In vitro, ACER3 only hydrolyzed C 18:1 -, C 20:1 -, C 20:4 -ceramides, dihydroceramides, and phytoceramides. In cells, ACER3 overexpression decreased C 18:1 -and C 20:1 -ceramides and dihydroceramides, whereas ACER3 knockdown by RNA interference had the opposite effect, suggesting that ACER3 controls the catabolism of ULC ceramides and dihydroceramides. ACER3 knockdown inhibited cell proliferation and up-regulated the cyclin-dependent kinase inhibitor p21 CIP1/WAF1 . Blocking p21 CIP1/WAF1 up-regulation attenuated the inhibitory effect of ACER3 knockdown on cell proliferation, suggesting that ACER3 knockdown inhibits cell proliferation because of p21 CIP1/WAF1 up-regulation. ACER3 knockdown inhibited cell apoptosis in response to serum deprivation. ACER3 knockdown up-regulated the expression of the alkaline ceramidase 2 (ACER2), and the ACER2 up-regulation decreased non-ULC ceramide species while increasing both sphingosine and its phosphate. Collectively, these data suggest that ACER3 catalyzes the hydrolysis of ULC ceramides and dihydroceramides and that ACER3 coordinates with ACER2 to regulate cell proliferation and survival.Ceramidases catalyze the hydrolysis of ceramides to form free fatty acids and sphingosine (SPH) 3 (1), and as such they are important intermediates of complex sphingolipids that play an important role in the integrity and function of cell membranes. Ceramides also act as bioactive molecules to mediate various cellular responses such as cell growth arrest, differentiation, and apoptosis in response to a variety of stress stimuli, including the cytokine tumor necrosis factor-␣ (2), cancer chemotherapeutic agents (3), UV (4), and ionizing radiation (5). Similar to ceramides, SPH also potently induces cell growth arrest, differentiation, or apoptosis (6, 7). SPH is phosphorylated by sphingosine kinases to generate sphingosine 1-phosphate (S1P) (8, 9). In contrast to ceramides and SPH, S1P mainly promotes cell proliferation and survival (10). Therefore, ceramidases may play an important role in regulating the levels of ceramides, SPH, and S1P as well as cellular responses mediated by these bioactive lipids.Ceramidases, according to their pH optima for in vitro activity, are classified into acid, neutral, and alkaline types (1, 11). Five human ceramidases encoded by five distinct genes have been identified as follows: the acid ceramidase (ASAH1) (12); neutral ceramidase (ASAH2) (13); alkaline ceramidase 1 (ACER1/ASAH3 (14); alkaline ceramidase 2 (ACER2/ASAH3L) (15); and alkaline ceramidase 3 (ACER3/ APHC/PHCA) (16).Ceramidases have different cellular localizations and substrate specificities. AS...

Activity of neutral and alkaline ceramidases on fluorogenic N-acylated coumarin-containing aminodiols

“…The lack of hydrolysis of RBM14 compounds by ACER1 and ACER2 could be explained by their reported preference for very long chain ceramides ( 1,2,17,27 ), while ACER3 hydrolyzes long chain, but not very long chain, unsaturated ceramides ( 4 ). Considering that RBM14 bears a modifi cation in the sphingoid base moiety, another plausible explanation is that ACER3 tolerates this modifi cation, while ACER1 and ACER2 do not.…”

Acid ceramidase as a therapeutic target in metastatic prostate cancer

“…A similar scenario might lead to the slight increase of sphingosine found in PC-3/Mc cells knocked down for AC. In support of this hypothesis, Hu et al ( 24 ) demonstrated that knockdown of the alkaline ceramidase 3 upregulated the expression of the alkaline ceramidase 2 with increases of both sphingosine and its phosphate.…”

“…Unexpectedly, sphingosine was increased in both knockdown clones ( Fig. 2F ), which suggests that other ceramidases are upregulated upon chronic knockdown of ASAH1 ( 24 ).…”

Plasma Sphingolipids Associated with Chronic Obstructive Pulmonary Disease Phenotypes

“…Although the significance of this finding remains to be tested, the extracellular matrix protein CCN1/Cyr61 is activated by S1P via receptor 2 (32), which also activates rho kinase to cause lung damage during cigarette smoke exposure (33,34). Other disrupted gene-metabolite associations of interest in those with severe COPD exacerbations were between ceramide and alkaline ceramidase, which degrades unsaturated long chain ceramides (35); ORMDL1, which regulates de novo ceramide biosynthesis, recently involved in asthma (36); and 3-ketodihydrosphingosine reductase, which reduces 3-ketodihydrosphingosine to dihydrosphingosine (37). Another intriguing association was that between circulating ceramide and COL4A3BP, also known as CERTL, a gene encoding for ceramide transporter protein Goodpasture antigen-binding protein, which mediates the nonvesicular intracellular transport of ceramides (38).…”