Ceramide in Stress Response (Sphingolipids) Part 1

Abstract

Evidence has consistently indicated that activation of sphingomyelinases and/or ceramide synthases and the resulting accumulation of ceramide mediate cellular responses to stressors such as lipopolysaccharide, interleukin 1P, tumor necrosis factor a, serum deprivation, irradiation and various antitumor treatments. Recent studies had identified the genes encoding most of the enzymes responsible for the generation of ceramide and ongoing research is aimed at characterizing their individual functions in cellular response to stress. This topic discusses the seminal and more recent discoveries in regards to the pathways responsible for the accumulation of ceramide during stress and the mechanisms by which ceramide affects cell functions. The former group includes the roles of neutral sphingomyelinase 2, serine palmitoyltransferase, ceramide synthases, as well as the secretory and endosomal/lysosomal forms of acid sphingomyelinase. The latter summarizes the mechanisms by which ceramide activate its direct targets, PKC^, PP2A and cathepsin D. The ability of ceramide to affect membrane organization is discussed in the light of its relevance to cell signaling. Emerging evidence to support the previously assumed notion that ceramide acts in a strictly structure-specific manner are also included. These findings are described in the context of several physiological and pathophysiological conditions, namely septic shock, obesity-induced insulin resistance, aging and apoptosis of tumor cells in response to radiation and chemotherapy.

Introduction

Cells and organisms have developed various strategies to deal with adverse changes in their environment. Cellular insult by infectious agents, toxins, nutrient deprivation, or genotoxic stress produces a coordinated systemic response (generally referred to as inflammation), which is aimed at neutralization ofthe insult and initiation oftissue repair. The first line of defense at systemic level is stimulation of the innate immune response, which consists of regulated production of inflammatory mediators, including cytokines like IL-1P, TNFa and IL-6. The typical cellular response to environmental stressors includes the induction of cellular apoptosis (in response to either genotoxic stress or cytokines like TNFa), growth arrest (during nutrient deprivation), increased eicosanoid production, cell migration and adhesion (in the presence of infectious agent). While acute inflammation is protective for the organisms, excessive and long-standing inflammation is harmful and underlies diseases like septic shock, atherosclerosis, asthma, rheumatoid arthritis and inflammatory bowel disease.

Diverse signaling pathways mediate cellular response to stress. The sphingolipid second messengers ceramide, ceramide-1-phosphate, sphingosine and sphingosine-1-phosphate play important role as mediators in many of these pathways. This topic is focused on the role of ceramide in cellular stress response and the mechanisms of its generation and action.

Figure 1. Structure of ceramide.

Chemical Structure and Biophysical Properties of Ceramide

Ceramides (Fig. 1) form the hydrophobic backbone of all complex sphingolipids1 and consist of a long chain sphingoid base and amid linked fatty acid which is either saturated or unsaturated and vary in length from two to 28 carbon atoms. In mammalian cells, the most commonly found ceramides have D-erythro-sphingosine and a saturated fatty acyl chain of 16 carbon atoms and are among the most hydrophobic lipids in the membrane. Free ceramide has a very low critical micellar concentration (cmc < 10~10M) and cannot exist in aqueous solutions.2 Nevertheless, ceramides are still considered as amphiphiles because the hydroxyl group at the first carbon and the amide bond are hydrophilic moieties. Dihydroceramide differs from ceramide inasmuch as the latter contains a trans 4, 5 double bond, which is essential for some of the bioactive roles of ceramide.

The structural features in the ceramide molecule that are required for its biological properties are not well understood, however the two hydroxyl groups, amid group protons and the trans-double bond seems to be involved.3 A network of intramolecular hydrogen bonds involving the OH and NH groups establishes a unique conformation arrangement of ceramide molecule.

Changes in Ceramide Mass during Stress

The basal ceramide concentrations in the cells are low and may change during cellular differentiation or progression through the cell cycle. Various inducers of cellular stress however lead to an accumulation of ceramide that promotes apoptotic, inflammatory and growth inhibitory signals and also mediates the onset of a specific response. The data in Table 1 illustrate what is the magnitude of changes in ceramide levels under various conditions of stress. These reported differences however, are likely to be an underestimation since they are measured in total cell preparations and not in the specific membrane fractions where ceramide was generated. Furthermore, with the exception of mass spectrometry—based assays, the most commonly used methods of ceramide quantification do not separate the individual ceramide species.4 This might be important as the impact ceramide has on cell functions seemingly depends on the type of fatty acid attached to the sphingoid base. Recent data indeed support the notion that the different biological effects caused by ceramide may be mediated by distinct molecular species of the lipid, underscoring the necessity to evaluate changes in ceramide content in a structure-specific manner.

A comparison of the magnitudes of ceramide accumulation observed under various treatments reveals that the changes are more robust in response to chemotherapeutics or irradiation. As the outcome of these treatments has been the induction of cell death via apoptosis, some investigators suggest that ceramide plays the role of a gauge that senses the level of cell injury and depending on downstream factors determines specific biological outcome.5

Table 1. Changes in ceramide content in response to stress and during some pathophysiological conditions

	Control	Treatment	Assay	Tissue/Cell Type	Ref.
Hypoxia/	4.5 pmol/nmol LP	8.5 pmol/nmol LP	Mass Spec	NT-2	6
Re-	100%	200% of control	DAGK	Cardiac myocytes	7
oxygenation	22 pmol/nmol LP#	55 pmol/nmol LP	DAGK	NRK-52E	8
	250 pmol/mg Pr	900 pmol/mg Pr	DAGK	PC-12	9
	100%	300% of control	DAGK	HUVEC	10
	100%	50% of control	DAGK	A7r5	11
Ischemia/	110% of sham	175% of sham	Mass Spec	Brain	12
Reperfusion	100%	200% of control	DAGK	Rat cardiomyocytes	13
	50 pmol/nmol	90 pmol/nmol LP	DAGK	Renal cortex	14
	190 pmol/mg Pr	290 pmol/mg Pr	DAGK	Liver	15
	100%	390% of control	DAGK	Rat astrocytes	16
	100%	170% of control	DAGK	CG-4	17
	18 pmol/nmol LP	45 pmol/nmol LP	DAGK	Rat myocytes	18
	75 cpm/106cells	160 cpm/106 cells	Labeling	Rat mesangial cells	19
	1 nmol/mg Pr	1.5 nmol/mg Pr	HPLC	Rat hepatocytes	20
	1.1 nmol/mg Pr	1.48 nmol/mg Pr	HPLC	Rat hepatocytes	21
	250 pmol/106cells	500 pmol/106 cells	DAGK	HUVEC	22
	450 pmol/106cells	800 pmol/106 cells	DAGK	Dendritic cells	23
	100%	170% of control	Labeling	Thyroid cells	24
	100%	150% of control	HPLC	Cortical neurons	25
	100%	330% of control	DAGK	Rat astrocytes	16
	200 pmol/mg Pr	850 pmol/mg Pr	DAGK	U-87 MG cells	26
	22 pmol/106 cells	37 pmol/106cell	DAGK	Mesangial cells	27
	100%	300% of control	HPLC	Hepatocytes	28
	0.8 nmol/mg Pr	1.4 nmol/mg Pr	DAGK	Lung microsomes	29
	5.0 nmol/mg Pr	10.9 nmol/mg Pr	HPLC	Alveolar lavage	30
	0.5 nmol/nmol LP	1.1 nmol/nmol LP	Mass Spec	Endothelial cells	31
	100%	460% of control	DAGK	MIN6 cells	32
	100%	700% of control	DAGK	MCF-7 cells	33
	100%	175% of control	DAGK	MCF-7 cells	34
	100%	250% of control	DAGK	L929	35
	5 pmol/nmol LP	13 pmol/nmol LP	DAGK	U937 cells	36
	5 pmol/nmol LP	12 pmol/nmol LP	DAGK	SK2	36
Serum starvation	100%	200% of control	DAGK	Mouse keratinocytes	37
LPS	2.8 pmol/nmol LP	4.3 pmol/nmol LP	HPLC	Brain	38
	4 nmol/ml	8 nmol/ml	HPLC	Plasma	38
	3.2 nmol/ml	4.8 nmol/ml	HPLC	Mouse serum,	39
	100%	200-500% of contr.	HPLC	Human plasma	39
	3.5 nM/mg Pr	16 nM/mg Pr	HPLC	THP-1 cells	40
	258 pmol/mg Pr	1634 pmol/mg Pr	HPLC	Mouse Mo	41
	8 nM/mg Pr	22 nM/mg Pr	HPLC	Human alveolar Mo	42
	1.1 nmol/mg Pr	2.1 nmol/mg Pr	DAGK	Intestinal mucosa	43

Table 1.

Table 1.

In some cases numerical values were taken from graphically represented data and were rounded to the nearest whole number. Abbreviations: General: CSF, cerebrospinal fluid; DAGK, diacylglycerol Kinase; LP, lipid phosphate; M0, primary macrophages; OLG, oligodendrocytes; Pr, Protein. Cells lines: A7r5, rat embryonic thoracic aorta smooth muscle; APRE-19, human retinal pigment epithelium; BAEC, bovine aortic endothelium; BT-20—human breast carcinoma; CG4, oligodendrocyte progenitor; CHK, Chinese hamster kidney; CHLA-90, human neuroblastoma; HAEC, human airway epithelial; HDF, human diploid fibroblasts; HEK 293, human embryonic kidney; HeLa, cervical carcinoma; HL60/VCR, drug resistant human leukemia; Hs-578T, breast carcinoma; HUVEC, human umbilical vein endothelium; INS-1, rat insulinoma; JHU-022, squamous cell carcinoma; Jurkat, T-lymphocytes; L929, mouse fibroblasts; MDA-MB 231, human Caucasian breast adenocarcinoma; MDA-MB 468, human Black breast adenocarcinoma; MCF-7, human breast adenocarcinoma; MIN6, murine beta cells; Molt-4; human acute lymphoblastic leukemia; LnCaP, human prostate carcinoma; NRK-52E, rat kidney epithelium; NT-2, human neuronal precursor; PC3, human prostate carcinoma, PC12, rat adrenal medulla carcinoma; SK2, hybridoma; SP-1, mammary intraductal adenocarcinoma; T47D, human ductal breast epithelial tumor; TNP-1, human acute monocytic leukemia; U87, human astrocytoma; U937, human leukemic monocyte lymphoma.