Knockout of Ceramide Kinase Aggravates Pathological and Lethal Responses in Mice with Experimental Colitis

Abstract

Sphingolipids and their metabolic enzymes are implicated in ulcerative colitis. Ceramide kinase (CerK) catalyzes the phosphorylation of ceramide to ceramide-1-phosphate (C1P). Previous studies showed the activation of CerK by the pro-inflammatory cytokine interleukin-1β, the C1P-induced up-regulation of prostanoids exerting protective effects against colitis, and the C1P-induced down-regulation of the pro-inflammatory cytokine tumor necrosis factor-α. In order to elucidate CerK/C1P functions in colitis, we examined the severity of dextran sodium sulfate (DSS)-induced colitis in wild-type (WT) and CerK deletion (CerK^(−/−)) mice. Lethal responses were observed in C57BL/6 mice treated with DSS in dose- and time-dependent manners. The depletion of CerK enhanced DSS-induced lethal responses without affecting the onset of these responses. In colons from mice treated with 2.5% DSS for 10 d, epithelial damage was significantly enhanced by the depletion of CerK by day 5, whereas decreases in occluding and E-cadherin levels were similar in both mice. On day 5, the DSS treatment increased spleen weights and colonic levels of cyclooxygenase-2, but not cytosolic phospholipase A₂α, and induced a contractile dysfunction in the colons of both mice. The DSS-induced increase in the damage activity index score between days 5 and 10 was slightly enhanced and the decrease in this score from day 10 was slower in CerK^(−/−) mice than in WT mice. On day 7 after the DSS treatment, spleen weights slightly decreased and increased in WT and CerK^(−/−) mice, respectively. These results indicate that the depletion of CerK enhances the pathology of colitis and lethal responses in DSS-treated mice.

Inflammatory bowel disease (IBD), which includes ulcerative colitis (UC) and Crohn’s disease, is a chronic inflammatory disorder of gastrointestinal tissues. Symptoms include abdominal pain, diarrhea, rectal bleeding, and weight loss, and the number of patients with IBD is increasing worldwide. The etiology of IBD remains unclear, but involves a complex interaction between genetic factors and environmental risk factors including an altered microbiota, infection, and smoking.^1,2) These factors appear to trigger abnormal immune responses, which result in impaired intestinal barrier function.^1,2) Sphingolipids including ceramide, sphingosine, and sphingosine-1-phosphate (S1P) regulate various immunological responses such as lymphocyte activation and cytokine formation,³⁾ and these lipids have emerged as targets for IBD including UC.^2,4) The inhibition of type 1 S1P receptors and the pharmacological and genetic inhibition of sphingosine kinase-1 (SphK-1) were shown to reduce the severity of colitis in animal UC models,^5–7) whereas deletion of S1P degradative enzymes including S1P phosphatase-1 exacerbated its severity.^8,9) Thus, the SphK-1/S1P/S1P receptor pathway appears to enhance the severity of colitis. In contrast, the deletion of S1P phosphatase-2⁹⁾ and sphingomyelin synthase-2¹⁰⁾ suppressed dextran sodium sulfate (DSS)-induced colitis in mice. A ceramide synthase-2 deficiency was shown to exacerbate DSS-induced colitis.^11,12) Thus, sphingolipids and/or their metabolic enzymes may have distinct roles in colitis that depend on their molecules, enzyme subtypes, and cell/tissue types. To the best of our knowledge, the role of ceramide kinase (CerK), an enzyme involved in the formation of ceramide-1-phosphate (C1P) from ceramide, in experimental colitis has not yet been elucidated.

The existence of C1P and ceramide phosphorylating activity has long been recognized, and CerK was finally cloned by Kohama and Spiegel’s group.¹³⁾ Various roles for CerK/C1P have been demonstrated in the regulation of enzymes and cellular/biological functions.^14,15) Many studies including ours^16,17) revealed that C1P directly binds to and activates phospholipase group IVA (PLA2G4A, α-type cytosolic phospholipase A₂, cPLA₂α). Arachidonic acid (AA) produced by the activation of cPLA₂α is metabolized to prostaglandins (PGs) including PGE₂ in a cyclooxygenase (COX)-mediated manner, and regulates inflammatory responses. We previously reported that the up-regulation of inducible COX2 and resulting abnormal contractile responses occurred in the colon and rectum of DSS-treated mice, an experimental UC mouse model.¹⁸⁾ In the present study, we examined the role of the depletion of CerK in the induction, progression, and relapse of UC using wild-type (WT) and CerK deletion (CerK^(−/−)) mice administered DSS.

MATERIALS AND METHODS

Animals

CerK^(+/−) mice (heterozygous C57BL/6J) were kindly gifted from Dr. Igarashi (Hokkadio University, Sapporo, Japan) and Dr. Kohama (Daiichi-Sankyo RD Novare, Tokyo, Japan)’s group. Seven- to eight-week-old mice (WT (CerK^(+/+)) and CerK^(−/−)) were generated and used in experiments. Animals were housed in the Animal Resource Facility of Chiba University under pathogen-free conditions and cared for in accordance with the animal care guidelines of Chiba University. At the end of the experiment, the DSS-treated mice were euthanized by cervical dislocation. Experiments were performed according to an animal protocol approved by the Animal Welfare Committee of Chiba University.

Induction and Evaluation of Experimental Colitis in Vivo

DSS is a sulfated molecule and the powerful electro-negativity of its poly-anions appeared to have cytotoxic actions including apoptotic effect on the colonic epithelial cells.^19,20) Several treatments with DSS (average molecular weight 5000, Wako Pure Chemical Industries, Ltd., Osaka, Japan) were performed in the present study. DSS was dissolved in sterile water and provided ad libitum for the indicated days. In order to evaluate the severity of colitis, damage activity index (DAI) scores were monitored as described previously.²¹⁾ Briefly, weight loss (0=normal; 1=1–5% loss; 2=5–10% loss; 3=10–15% loss; 4=>15% loss), stool consistency (0=normal; 2=loose stools, pasty stools that do not stick to the anus; 4=diarrhea, liquid stools that stick to the anus), and the degree of blood in stools (0=normal; 2=guaiac; 4=gross bleeding) were evaluated, and DAI was their combined score/3. The length of the colon from the anus to the appendix in male mice was measured using a ruler. The weight of the spleen from both sexes was adjusted by body weight and expressed as a percentage of the body weight of individual mice.

Histological Assessments of the Severity of Colitis

On day 5 after the administration of DSS, mice were killed and the colon was excised, opened longitudinally, and fixed with 4% paraformaldehyde. Preparations were embedded in optimum cutting temperature (OCT) compound after dehydration, and sections (thickness of 10 µm) were prepared and stained with hematoxylin and eosin. Analyses of histopathological/morphological changes were performed by experimenters “blinded” to sample identity, and conducted based on methods described previously.²²⁾ Briefly, epithelial damage was assessed as follows: 0=normal; 1=irregular crypts and goblet cell loss; 2=10–50% crypt loss; 3=50–90% crypt loss; 4=complete crypt loss and an intact surface epithelium; 5=small ulcer (<10 crypt widths); and 6=large ulcer (≥10 crypt widths). Infiltration with inflammatory cells was assigned scores separately for the mucosa (0=normal, 1=mild, 2=modest, 3=severe), submucosa (0=normal, 1=mild/modest, 2=severe), and muscle/serosa (0=normal, 1=moderate/severe). Scores for epithelial damage and inflammatory cell infiltration were added, resulting in a total scoring range of 0–12.

Protein Extraction and Western Blot Analyses

Immunoblotting to detect colonic proteins was performed as described previously¹⁸⁾ with minor modifications. Briefly, the whole distal colon was cut and homogenized in buffer (250 mM sucrose, 10 mM ethylene glycol bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, 2 mM ethylenediaminetetraacetic acid, 1% Triton-X 100, 20 mM Tris–HCl, and protease inhibitors, pH 7.4) at 4°C with 20 strokes of a glass-teflon homogenizer. The supernatant after centrifugation (17400×g, 4°C, 30 min) was used as a protein extract (50 µg of protein per lane). The following antibodies were used (1 : 500–1 : 1000): an anti-occludin antibody (6HCLC, ThermoFisher, Waltham, MA, U.S.A.), anti-E-cadherin antibody (#610182, BD Sciences, San Jose, CA, U.S.A.), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (5A12, Wako). An anti-cPLA₂α antibody (sc-454) and anti-COX2 antibody (sc-376861) were from Santa Cruz Biotech (Santa Cruz, CA, U.S.A.). An anti-caspase-3 antibody (#9662) and anti-β-catenin antibody (#9562) were from Cell Signaling (Beverly, MA). Anti-rabbit and anti-mouse immunoglobulin G (IgG) horseradish peroxidase antibodies were from Amersham (Buckinghamshire, U.K.). The ratio of the respective protein to GAPDH was calculated, and data were expressed as fold differences from the control value from a WT mouse without the DSS treatment. Individual variations were observed in the expression levels of cPLA₂α and E-cadherin.

Preparation and Measurement of Contractile Responses

The contractile responses of the distal colon were measured as described previously¹⁸⁾ with minor modifications. Briefly, whole preparations including mucosal layers (1 cm in length) were suspended in a longitudinal direction under 0.5 gf in a 5-mL organ bath containing Krebs–Henseleit buffer (112 mM NaCl, 5.9 mM KCl, 2.0 mM CaCl₂, 1.2 mM MgCl₂, 1.2 mM NaH₂PO₄, 25.0 mM NaHCO₃, and 11.5 mM glucose, pH 7.4). The bath was maintained at 37°C and continuously bubbled with a mixture of 95% O₂ and 5% CO₂. A phasic contraction was defined as the maximal difference between the peak and lowest values, and evaluated based on dimension. Dimension indicates the area showing contractile or relaxant responses in the measurement period, and was expressed as a percentage of the standard dimension, which is a continuous contraction for 1 min with a 0.1-gf peak (100%). A stimulator (Sen-7203, Nihon Koden, Tokyo, Japan) was used for electrical stimulation (ES) of the colon, and the precise conditions of ES were as described previously¹⁸⁾: 10V, 0.1-ms for a contraction and 0.2-msec for relaxation, a 5-ms interval, and 10 trains. The amplitude (gf) of an ES-induced contraction was the mean of the top three values for the tested period. The maximal contraction (peak amplitude, gf) induced by 3–10 µM acetylcholine (ACh) and the pEC₅₀ values of ACh were examined. ACh was obtained from Sigma (St. Louis, MO, U.S.A.). A treatment with 2.5% DSS for 7 d markedly reduced contractile responses (approximately 80%) in WT and CerK^(−/−) mice (C57BL/6J mice), similar to those in ddY mice.¹⁸⁾ In the present study, we examined the effects of treatment with 1.5% DSS for 7 d, which causes moderate changes in contractile responses. Contractile data from male and female mice were combined and analyzed.

Statistical Analysis

Survival curves (Kaplan–Meier plots) were analyzed using the Log-rank test. Other results were expressed as the mean±standard deviation (S.D.) for the indicated number (n) of independent experiments. Each experiment was conducted using different animals and different times. Differences between experimental groups were analyzed by an ANOVA with Bonferroni post hoc comparisons. Significance was assumed at p<0.05.

RESULTS

Enhanced Lethal Responses in CerK^(−/−) Mice with Experimental Colitis

Survival rates after the administration of DSS were examined. The application of 4.0% DSS-containing water caused lethal responses: WT mice died on days 9, 10, and 13, and CerK^(−/−) mice died on day 11 (Exp. I in Table 1). In 2.5% DSS-treated mice, WT mice died on days 8, 14, and 15, and CerK^(−/−) mice died on days 8, 10, and 14 (Exp. II). The treatment with 1.5% DSS caused lethal responses from day 10, and more than 80% of both mice died before day 30 (Exp. III). The onset and degree of DSS-induced lethal responses did not appear to differ by sex or the depletion of CerK. We then examined the effects of a short-term treatment with DSS. Initially, 2.5% DSS was administrated for 7 d, and mice were then housed with normal water (Exp. IV). Lethal responses induced by the DSS treatment in CerK^(−/−) mice appeared to be greater than those in WT mice. Therefore, we analyzed the significance of survival curves (Kaplan–Meier plots) between WT and CerK^(−/−) mice using the Log-rank test. The survival of CerK^(−/−) mice (n=12) was slightly shorter (p value of 0.22) than that of WT mice (n=16). We then administered 2.5% DSS for 10 d (Exp. V and Fig. 1A). A small number of WT mice died from days 7–8 after the DSS treatment, and several mice died from days 10–15 when consuming normal water. The survival of CerK^(−/−) mice was significantly shorter (p<0.01) than that of WT mice, whereas the time dependency of mortality after the DSS treatment in the two groups appeared to be similar. In both male and female mice, the lethal responses in CerK^(−/−) mice were greater than those in WT mice (p values of 0.06 and 0.07 in male and female, respectively). The DAI score was slightly higher in CerK^(−/−) mice than in WT mice after the DSS treatment (Fig. 1A-2); days 8 and 10 showed maximal scores in CerK^(−/−) and WT mice, respectively, and the maximal value in CerK^(−/−) mice, approximately 3.5, was slightly higher than that in WT mice of 3.2. The recovery of scores after day 10 with normal water was slower in CerK^(−/−) mice than in WT mice. The depletion of CerK slightly accelerated decreases in body weights and decelerated the recovery of body weights after the removal of DSS from those in WT mice (Fig. 1A-3). These results suggest that CerK^(−/−) mice are affected more by DSS than WT mice.

Table 1. Survival Rates of DSS-Treated WT and CerK^(−/−) Mice

Number of surviving mice
Exp. I: on day 11 after 4% DSS every day
WT	1/3	(2 males and 1 female)
CerK(−/−)	0/3	(2 males and 1 female)
Exp. II: on day 15 after 2.5% DSS every day
WT	0/3	(3 males)
CerK(−/−)	0/3	(2 males and 1 female)
Exp. III: on day 30 after 1.5% DSS every day
WT	2/12 (16.6)	Male: 0/7 (0)
		Female: 2/5 (40.0)
CerK(−/−)	1/12 (8.3)	Male: 1/10 (10.0)
		Female: 0/2 (0)
Exp. IV: on day 30 after 2.5% DSS for 7 d
WT	14/16 (87.5)	Male: 7/7 (100)
		Female: 7/9 (77.7)
CerK(−/−)	8/12 (66.6)	Male: 5/6 (83.3)
		Female: 3/6 (50.0)
Exp. V: on day 30 after 2.5% DSS for 10 d
WT	17/29 (58.6)	Male: 6/10 (60.0)
		Female: 11/19 (57.8)
CerK(−/−)	4/18 (22.2)	Male: 2/10 (20.0)
		Female: 2/8 (25.0)
Exp. VI: on day 30 after the DSS treatment (2.5%, 7 d, twice)
WT	22/24 (91.6)	Male: 11/13 (84.6)
		Female: 11/11 (100)
CerK(−/−)	9/13 (69.2)	Male: 3/5 (60.0)
		Female: 6/8 (75.0)

In Exps. I–III, water containing the indicated concentrations of DSS was administrated for successive days. In Exps. IV and V, 2.5% DSS was administrated for 7 and 10 d, respectively, and DSS-free water was then applied. In VI, the DSS treatment (2.5%, for 7 d) was repeated twice with an interval of 7 d with DSS-free water. Survival rates were assessed on the respective days, and the percentages of surviving mice (surviving mice/all mice, %) were shown in parentheses.

Fig. 1. Survival Rates of DSS-Treated WT and CerK^(−/−) Mice

In A, 2.5% DSS was administrated for 10 d, and mice were then housed with normal water. In B, 2.5% DSS was administrated for 7 d, and mice were then re-treated with 2.5% DSS for 7 d after an interval of 7 d with normal water. Survival curves (A-1 and B-1), DAI scores (A-2 and B-2), and body weight losses (A-3 and B-3) were examined. Survival curves (Kaplan–Meier plots) in A-1 and B-1 were analyzed using the Log-rank test. The numbers of surviving mice on days 10 (A-2) and 20 (A-2 and B-2) were shown. The data shown were combined data from males and females. Data for sex-dependent analyses were described in the text.

The effects of the repeated application of DSS on survival were investigated. Mice were treated with 2.5% DSS for 7 d and were then re-treated with 2.5% DSS for 7 d after an interval of 7 d with normal water (Experiment VI). Under these conditions, the onset times of increases in DAI scores and mortality induced by the second DSS treatment were not accelerated by the depletion of CerK (Fig. 1B). Although a few mice died by day 14 after the initial DSS treatment among WT and CerK^(−/−) mice independent of sex, the majority of surviving mice did not die after the secondary DSS treatment by day 30. In WT and CerK^(−/−) mice, the maximal DAI score, approximately 2.5, was observed on day 7 after the DSS treatment, and a subsequent score, 2.3–2.5, was noted on day 21, the last day of the repeated treatment. The depletion of CerK did not enhance the toxicity induced by the repeated DSS treatment.

Changes in Histological Scores, Colon Lengths, and Spleen Weights by the DSS Treatment

A previous study reported that CerK^(−/−) mice were healthy including their life span.²³⁾ In our studies, the survival rate of CerK^(−/−) mice was similar to that of WT mice in the absence of DSS. The depletion of CerK did not modify body weights, colon lengths, or spleen weights. WT and CerK^(−/−) mice did not exhibit any histological abnormalities in the distal colon: histopathological scores were approximately 1–2 in WT and CerK^(−/−) mice (Figs. 2A, 2B). The treatment with 2.5% DSS for 5 d induced epithelial damage and infiltration with inflammatory cells to colon layers namely, the mucosa, submucosa, and muscle/serosa, in WT mice (Fig. 2A). The response to DSS widely varied depending on individual mice and was similar in males and females; therefore, data were combined without the distinction of sex in this assay. Abnormal histological changes by DSS were also observed in CerK^(−/−) mice, and scores were significantly higher in CerK^(−/−) mice than in WT mice (Fig. 2B). Scores for epithelial damage were 2.5–3.5 and 4.0–5.8 in WT and CerK^(−/−) mice, respectively, and those for inflammatory cell infiltration were slightly higher in CerK^(−/−) mice. Abnormal histological changes in the colon were prominent on day 7 after the DSS treatment, and scores were higher than 8–9 in WT and CerK^(−/−) mice (data not shown). We then examined colon lengths and spleen weights as indicators of DSS-induced colitis.^7,24–26) DSS treatments for 5 and 7 d significantly decreased colon lengths in mice with and without the depletion of CerK (Table 2, Exp. I). Spleen weights in mice treated with vehicle for 5–7 d were 0.35±0.06% in WT (n=8) and 0.31±0.04% in CerK^(−/−) mice (n=5). The DSS treatment for 5 d significantly increased spleen weights, which were adjusted by body weights in both sexes, in WT and CerK^(−/−) mice in a similar manner (Fig. 2C). On day 7 after the DSS treatment, spleen weights slightly decreased in WT mice, but slightly increased in CerK^(−/−) mice from data obtained on day 5.

Fig. 2. Histological Analyses of Colon Sections from DSS-Treated WT and CerK^(−/−) Mice

In A and B, colonic sections were prepared from WT and CerK^(−/−) mice treated with 2.5% DSS for 5 d, and were stained with hematoxylin and eosin. Images in A are from typical experiments, and quantitative data for histopathological scores were shown in B. In C, spleens were prepared from WT and CerK^(−/−) mice treated with 2.5% DSS for 5 and 7 d. Data are presented as a box plot.

Table 2. Colon Lengths and Inflammation- and Tight Junction-Related Protein Levels in Colons of DSS-Treated Mice

	WT		CerK(−/−)
	Control	DSS	Control	DSS
Exp. I. Colon length (cm)
5 d	7.64±0.21 (9)	6.99±0.08a) (16)	7.79±0.16 (7)	6.93±0.12a) (15)
7 d	8.11±0.21 (4)	6.01±0.06a) (8)	8.01±0.22 (3)	6.34±0.12a) (7)
Exp. II. Ratio (fold)
cPLA2α (3)	0.62±0.23	0.63±0.18	0.84±0.18	0.79±0.23
COX2 (3)	1.26±0.21	2.23±0.32a)	1.05±0.19	1.98±0.34
Caspase-3 (3)	0.81±0.06	0.43±0.12a)	0.80±0.08	0.44±0.07a)
Occludin (9)	0.91±0.34	0.48±0.10a)	1.09±0.27	0.36±0.11a)
E-Cadherin (9)	0.69±0.22	0.26±0.14a)	0.91±0.37	0.21±0.08a)
β-Catenin (9)	1.01±0.22	0.73±0.19	1.23±0.25	0.74±0.21

In Exp. I, male WT and CerK^(−/−) mice were treated with vehicle or 2.5% DSS for 5 or 7 d. Colon lengths (cm) were measured. In Exp. II, WT and CerK^(−/−) mice (both the sexes) were treated with 2.5% DSS for 5 d. The levels of the indicated proteins were analyzed by Western blotting using total protein extracts of the distal colon. Image data in a typical experiment were shown in Fig. 3. The numbers of animals used are given in parentheses. a) p<0.05, significantly different from the control.

Levels of Inflammatory- and Tight Junction-Related Proteins in Distal Colons of DSS-Treated CerK^(−/−) Mice

cPLA₂α, COX2, and caspase-3 play key roles in colonic inflammation and damage. cPLA₂α levels appeared to be similar in the distal colons of WT and CerK^(−/−) mice, and were not changed by the DSS treatment (Fig. 3 and Table 2, Exp. II). The up-regulation of COX2 in the colonic epithelium of UC patients and model animals is well documented. The treatment of WT and CerK^(−/−) mice with DSS increased COX2 levels to a similar extent. Caspase-3 (CPP-32) is cleaved by proteolysis and its fragments exhibit caspase activity in apoptotic cells. Caspase-3 levels were not modified by the depletion of CerK, and the DSS treatment reduced these levels by 50% in both mice. In order to confirm the role of CerK at epithelial tight junctions, junction-associated proteins levels were measured. In the colons of DSS-treated WT mice, occludin and epithelial (E)-cadherin levels were significantly decreased, while those of β-catenin were slightly decreased, as previously reported.^11,27) The levels of these three adhesion proteins in the colon were not affected by the depletion of CerK, and the DSS treatment reduced their levels to a similar extent in both mice under the experimental conditions employed in the present study.

Fig. 3. DSS-Induced Changes in Inflammatory- and Tight Junction-Related Proteins in the Colon

Images of Western blotting are from typical experiments. Quantitative data are shown in Experiment II in Table 2.

Contractile Responses in Colons of DSS-Treated CerK^(−/−) Mice

A ceramide treatment was previously reported to regulate contractility in the cat esophagus,²⁸⁾ while the contractility of gastric smooth muscle was modified in ceramide synthase-2 null mice.²⁹⁾ The depletion of CerK did not change phasic contractions or ES-induced responses in the distal colon from those in WT mice (Table 3). The DSS treatment slightly decreased phasic and ES-induced contractions and significantly decreased ES-induced relaxation in WT mice, and similar responses were observed in CerK^(−/−) mice. The DSS treatment did not change ACh responses including half maximal effective concentration (EC₅₀) values in WT (C57BL/6J) mice, similar to ddY mice.¹⁸⁾ The depletion of CerK with and without the DSS treatment did not change ACh responses.

Table 3. Contractile Responses in Colons of WT and CerK^(−/−) Mice

Treatment	Control	DSS	Control	DSS
Exp. I: Phasic contraction (dimension)
WT	70.9±11.8	57.2±15.4
CerK(−/−)	90.7±8.9	45.2±19.0
Exp. II: ES-induced contraction
	(dimension)		(peak, gf)
WT	130±16	120±15	0.39±0.06	0.41±0.05
CerK(−/−)	138±15	113±19	0.44±0.05	0.41±0.06
Exp. III: ES-induced relaxation
	(dimension)		(peak, gf)
WT	6.17±0.99	1.95±0.61a)	0.06±0.01	0.03±0.01
CerK(−/−)	4.56±0.65	1.62±0.66a)	0.05±0.01	0.02±0.01
Exp. IV: ACh-induced contraction
	(peak, gf)		(pEC50 values of ACh)
WT	2.01±0.05	1.94±0.14	−6.04±0.26	−6.23±0.38
CerK(−/−)	2.04±0.06	1.73±0.12	−6.13±0.34	−6.38±0.41

Preparations of the distal colons of WT and CerK^(−/−) mice (male and female) treated with 1.5% DSS for 7 d were isolated. Phasic, ES-, and ACh-induced contractile activities were recorded. The numbers of mice used were as follows: WT/vehicle, 14; WT/DSS, 16; CerK^(−/−)/vehicle, 24; CerK^(−/−)/DSS, 10. a) p<0.05, significantly different from the control.

DISCUSSION

In the present study, we demonstrated that lethal responses induced by the 2.5% DSS treatment for 10 d were more prominent in CerK^(−/−) mice than in WT mice. Lethal responses in the late and succeeding stages (days 8–15) after the DSS treatment were enhanced by the depletion of CerK, and the onset times of these responses were not changed. Histopathological scores in the colon on day 5 were significantly higher in DSS-treated CerK^(−/−) mice than in WT mice, however, DAI scores and decreases in colonic adhesion protein levels were similar in both mice. The recovery of increased DAI scores after day 10 was decelerated in CerK^(−/−) mice.

Characteristics of Enhanced Lethal Responses to the DSS Treatment by the Depletion of CerK

Lethal responses induced by the repeated application of DSS were prominent in WT mice (C57BL/6 mice). DSS-induced mortality appeared to be dependent on DSS doses, and the effects of the depletion of CerK on mortality were not observed under the conditions tested (Table 1, Exps. I–III). We then investigated the effects of a short-term treatment with DSS. Lethal responses were significantly enhanced in CerK^(−/−) mice treated with 2.5% DSS for 10 d (Exp. V and Fig. 1). Enhanced lethality induced by the depletion of CerK was observed in mice treated with 2.5% DSS for 7 d (Exp. IV). The onset and rate of increases in DAI scores and the onset of mortality were not accelerated by the depletion of CerK in DSS-treated mice, although mortality after day 10 was significantly enhanced. In WT mice treated 2.5% DSS for 10 d, lethal responses began from days 7–9 and continued to days 16–18 in spite of the consumption of DSS-free water. In surviving (and dying) WT mice after the DSS treatment, increased DAI scores and decreased body weights returned to control levels, and 50% of mice were alive by day 30. However, recovery from increased DAI scores on days 14–16 after the DSS treatment was delayed in CerK^(−/−) mice. The prolongation of abnormal stages in CerK^(−/−) mice appears to be supported by the prolonged increase in the spleen weights of DSS-treated CerK^(−/−) mice (Fig. 2C): spleen weights on day 5 were similar in WT and CerK^(−/−) mice, while those on day 7 slightly decreased and increased in WT and CerK^(−/−) mice, respectively. Thus, the CerK depletion-induced aggravation of colitis and mortality may be explained by impaired protective mechanisms and/or enhanced inflammation within and after the DSS treatment. A previous study reported that CerK^(−/−) mice developed more impaired defenses against Streptococcus pneumonia infection.³⁰⁾ Wang et al. showed that the depletion of alkaline ceramidase-3 enhanced DSS-induced mortality without affecting the onset or rate of increases in colitis activity scores in mice.²⁶⁾ In contrast, the early onset of DSS-induced colitis and mortality without changes in maximal lethal responses were reported in mice with genetic mutations in Toll-like receptors³¹⁾ and endoplasmic reticulum stress transducers.³²⁾ DSS-treated WT mice (C57BL/6 mice) are known to be resistant to secondary DSS treatments.³³⁾ In the present study, the lethal responses of WT mice induced by the secondary DSS treatment was less prominent than those of the first treatment (Fig. 1C), and the depletion of CerK did not affect this phenomenon. Thus, the depletion of CerK did not appear to modify the relapse/remission of DSS-treated colitis.

Possible Roles for Prostanoid and Cytokine Levels in DSS-Induced Colitis in CerK^(−/−) Mice

The incidence of UC appears to be mediated by T-helper 2 (Th2)-dominant mechanisms, and gastrointestinal tissues including the colon show a Th1/Th2 imbalance.^34,35) The up-regulation of pro-inflammatory cytokines such as tumor necrosis factor α (TNFα) and interleukin (IL)-1β and down-regulation of anti-inflammatory cytokines have been reported in the colons of DSS-treated mice.^35,36) C1P directly inhibits a processing enzyme of TNFα,³⁷⁾ and an intraperitoneal (i.p.) injection of a C1P analog reduced serum levels of pro-inflammatory cytokines including TNFα in lipopolysaccharide-treated mice.³⁸⁾ Enhanced DSS-induced lethal responses by the depletion of CerK may couple with the up-regulation of TNFα. The early onset of colitis was previously shown to be dependent on TNFα in DSS-treated rats.³⁹⁾ However, the onset of DSS-induced colitis was not affected by the depletion of CerK, and, thus, this possibility may be excluded. DSS-induced colitis in mice has been associated with an increase in the inflammatory cytokine IL-1β in the colon.^9,26,27) A treatment with IL-1β increased C1P levels through the activation of CerK in lung cancer cells,⁴⁰⁾ and C1P is a direct activator of cPLA₂α.^16,17) In gastrointestinal tissues including the colon, PGs, specifically PGE₂, prevented colitis by enhancing mucosal repair.^41,42) PGE₂ levels in bronchoalveolar lavage fluid in asthma model mice were significantly reduced by the depletion of CerK.⁴³⁾ In our studies, damage to the epithelium on day 5 (Figs. 2A, 2B) and mortality from days 7–8 (Fig. 1A) after the DSS treatment were enhanced by the depletion of CerK, and the recovery of increased DAI scores in the late and succeeding stages (days 7–16) was decelerated by the depletion of CerK (Fig. 1B). Thus, the depletion of CerK may aggravate colitis by disturbing the AA/PGs-mediated repair process. Several anti-inflammatory cytokines including IL-4 were found to be reduced by the depletion of CerK in asthma model mice.⁴³⁾ In native CerK^(−/−) mice, a defect in neutrophil/leucocyte/lymphocyte homeostasis was reported in blood and the spleen.³⁰⁾ A treatment with DSS changed immune cell homeostasis and inflammation-related gene expression in tissues and individuals, and these changes were different in mice showing modified sphingolipid metabolism.^{7,10,12,14,25,44)} C1P and/or C1P-regulated cytokines modify the migration of various cells.¹⁴⁾ Changes in immune cell homeostasis, the cytokine balance, and cell migration in DSS-treated CerK^(−/−) mice need to be examined in more detail in future studies. A similar dysfunction in colon contractility induced by DSS treatment was observed in both mice (Table 3); thus, colon contractility did not appear to be involved in the CerK depletion-induced enhancement of lethal responses.

Roles of Sphingolipid Metabolism and/or Metabolites in UC

Ceramide plays a central role in sphingolipid metabolism, and interacts with various lipids including S1P and C1P. The depletion of CerK was shown to increase serum ceramide levels,⁴⁵⁾ and a treatment with DSS increased ceramide levels in intestinal epithelial cells by approximately 1.7-fold that of control levels.⁴⁶⁾ Previous studies revealed that sphingolipids are involved in IBD including UC. The depletion of SphK-1,^7,25) S1P phosphatase-2 mainly expressed in gut tissues,⁹⁾ and sphingomyelin synthase-2¹⁰⁾ prevented DSS-induced colitis in mice, whereas colitis was enhanced in gut-specific conditional S1P lyase knockout mice.⁴⁷⁾ The loss of S1P phosphatase-1,⁹⁾ ceramide synthase-2,¹¹⁾ alkaline ceramidase-3,²⁶⁾ and neutral ceramidase⁴⁸⁾ exacerbated DSS-induced colitis in mice. The oral application of S1P receptor modulators, which functionally act as S1P receptor antagonists, prevented the development of colitis.^5,44) Thus, the role of sphingolipids in colitis remains controversial. In the present study, we showed for the first time that the global depletion of CerK aggravated DSS-induced colitis and reduced survival rates. There are several inhibitors of ceramide kinase,⁴⁹⁾ and activation of peroxisome proliferator-activated receptor β enhanced the mRNA expression of ceramide kinase.⁵⁰⁾ It is interesting to examine the effects of chemical inhibitors and activators of ceramide kinase on colitis. Also, the precise roles of CerK/C1P in colitis and mechanisms including effects of C1P molecules with different acyl chains, the C1P balance with other ceramide metabolites, and tissue/cell specificity, have yet to be elucidated.

Acknowledgments

We thank Dr. Yasuyuki Igarashi (Hokkaido University, Sapporo, Japan) and Dr. Takafumi Kohama (Daiichi-Sankyo, Tokyo, Japan) for providing ceramide kinase knockout mice. These studies were partially supported by Grants-in-Aid (26460060 to H.N. and 26460093 to T.M.) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan.

Conflict of Interest

The authors declare no conflict of interest.

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