p38 MAPK regulates Bax activity and apoptosis in enterocytes at baseline and after intestinal resection

Increased apoptosis in crypt enterocytes is a key feature of intestinal adaptation following massive small bowel resection (SBR). Expression of the proapoptotic factor Bax has been shown to be required for resection-induced apoptosis. It has also been demonstrated that p38-α MAPK (p38) is necessary for Bax activation and apoptosis in vitro. The present studies were designed to test the hypothesis that p38 is a key regulator of Bax activation during adaptation after SBR in vivo. Enterocyte expression of p38 was deleted by tamoxifen administration to activate villin-Cre in adult mice with a floxed Mapk14 (p38-α) gene. Proximal 50% SBR or sham operations were performed on wild-type (WT) and p38 intestinal knockout (p38-IKO) mice under isoflurane anesthesia. Mice were killed 3 or 7 days after operation, and adaptation was analyzed by measuring intestinal morphology, proliferation, and apoptosis. Bax activity was quantified by immunoprecipitation, followed by Western blotting. After SBR, p38-IKO mice had deeper crypts, longer villi, and accelerated proliferation compared with WT controls. Rates of crypt apoptosis were significantly lower in p38-IKO mice, both at baseline and after SBR. Levels of activated Bax were twofold higher in WT mice after SBR relative to sham. In contrast, activated Bax levels were reduced by 67% in mice after p38 MAPK deletion. Deleted p38 expression within the intestinal epithelium leads to enhanced adaptation and reduced levels of enterocyte apoptosis after massive intestinal resection. p38-regulated Bax activation appears to be an important mechanism underlying resection-induced apoptosis.     

Roles for p21waf1/cip1 and p27kip1 during the adaptation response to massive intestinal resection

The magnitude of gut adaptation is a decisive factor in determining whether patients are able to live independent of parenteral nutrition after massive small bowel loss. We previously established that the cyclin-dependent kinase inhibitor (CDKI) p21(waf1/cip1) is necessary for enterocyte proliferation and a normal adaptation response. In the present study, we have further elucidated the role of this CDKI in the context of p27(kip1), another member of the Cip/Kip CDKI family. Small bowel resections (SBRs) or sham operations were performed in control (C57/BL6), p21(waf1/cip1)-null, p27(kip1)-null, and p21(waf1/cip1)/p27(kip1) double-null mice. Morphological (villus height/crypt depth) alterations in the mucosa, the kinetics of enterocyte turnover (rates of enterocyte proliferation and apoptosis), and the protein expression of various cell cycle-regulatory proteins were recorded at various postoperative times. Enterocyte compartment-specific mRNA expression was investigated using laser capture microdissection. Resection-induced adaptation in control mice coincided with increased protein expression of p21(waf1/cip1) and decreased p27(kip1) within 3 days postoperatively. Identical changes in mRNA expression were detected in crypt but not in villus enterocytes. Adaptation occurred normally in control and p27(kip1)-null mice; however, mice deficient in both p21(waf1/cip1) and p27(kip1) failed to increase baseline rates of enterocyte proliferation and adaptation. The expression of p21(waf1/cip1) protein and mRNA in the proliferative crypt compartment is necessary for resection-induced enterocyte proliferation and adaptation. The finding that deficient expression of p27(kip1) does not affect adaptation suggests that these similar CDKI family members display distinctive cellular functions during the complex process of intestinal adaptation.     

Ret heterozygous mice have enhanced intestinal adaptation after massive small bowel resection

Intestinal adaptation is an important compensatory response to massive small bowel resection (SBR) and occurs because of a proliferative stimulus to crypt enterocytes by poorly understood mechanisms. Recent studies suggest the enteric nervous system (ENS) influences enterocyte proliferation. We, therefore, sought to determine whether ENS dysfunction alters resection-induced adaptation responses. Ret+/- mice with abnormal ENS function and wild-type (WT) littermates underwent sham surgery or 50% SBR. After 7 days, ileal morphology, enterocyte proliferation, apoptosis, and selected signaling proteins were characterized. Crypt depth and villus height were equivalent at baseline in WT and Ret+/- mice. In contrast after SBR, Ret+/- mice had longer villi (Ret+/- 426.7 ± 46.0 μm vs. WT 306.5 ± 7.7 μm, P < 0.001) and deeper crypts (Ret+/- 119 ± 3.4 μm vs. WT 82.4 ± 3.1 μm, P < 0.001) than WT. Crypt enterocyte proliferation was higher in Ret+/- (48.8 ± 1.3%) than WT (39.9 ± 2.1%; P < 0.001) after resection, but apoptosis rates were similar. Remnant bowel of Ret+/- mice also had higher levels of glucagon-like peptide 2 (6.2-fold, P = 0.005) and amphiregulin (4.6-fold, P < 0.001) mRNA after SBR, but serum glucagon-like peptide 2 protein levels were equal in WT and Ret+/- mice, and there was no evidence of increased c-Fos nuclear localization in submucosal neurons. Western blot confirmed higher crypt epidermal growth factor receptor (EGFR) protein levels (1.44-fold; P < 0.001) and more phosphorylated EGFR (2-fold; P = 0.003) in Ret+/- than WT mice after SBR. These data suggest that Ret heterozygosity enhances intestinal adaptation after massive SBR, likely via enhanced EGFR signaling. Reducing Ret activity or altering ENS function may provide a novel strategy to enhance adaptation attenuating morbidity in patients with short bowel syndrome.     

Delineation of the cell-extrinsic apoptosis pathway in the zebrafish

The mammalian extrinsic apoptosis pathway is triggered by Fas ligand (FasL) and Apo2 ligand/tumor necrosis factor (TNF)-related apoptosis-inducing ligand (Apo2L/TRAIL). Ligand binding to cognate receptors activates initiator caspases directly in a death-inducing signaling complex. In Drosophila, TNF ligand binding activates initiator caspases indirectly, through JNK. We characterized the extrinsic pathway in zebrafish to determine how it operates in a nonmammalian vertebrate. We identified homologs of FasL and Apo2L/TRAIL, their receptors, and other components of the cell death machinery. Studies with three Apo2L/TRAIL homologs demonstrated that they bind the receptors zHDR (previously linked to hematopoiesis) and ovarian TNFR (zOTR). Ectopic expression of these ligands during embryogenesis induced apoptosis in erythroblasts and notochord cells. Inhibition of zHDR, zOTR, the adaptor zFADD, or caspase-8-like proteases blocked ligand-induced apoptosis, as did antiapoptotic Bcl-2 family members. Thus, the extrinsic apoptosis pathway in zebrafish closely resembles its mammalian counterpart and cooperates with the intrinsic pathway to trigger tissue-specific apoptosis during embryogenesis in response to ectopic Apo2L/TRAIL expression.     

p53-dependent Fas expression is critical for Ginsenoside Rh2 triggered caspase-8 activation in HeLa cells

We have recently reported that Ginsenoside Rh2 (G-Rh2) induces the activation of two initiator caspases, caspase-8 and caspase-9 in human cancer cells. However, the molecular mechanism of its death-inducing function remains unclear. Here we show that G-Rh2 stimulated the activation of both caspase-8 and caspase-9 simultaneously in HeLa cells. Under G-Rh2 treatment, membrane death receptors Fas and TNFR1 are remarkably upregulated. However, the induced expression of Fas but not TNFR1 was contributed to the apoptosis process. Moreover, significant increases in Fas expression and caspase-8 activity temporally coincided with an increase in p53 expression in p53-nonmutated HeLa and SK-HEP-1 cells upon G-Rh2 treatment. In contrast, Fas expression and caspase-8 activity remained constant with G-Rh2 treatment in p53-mutated SW480 and PC-3 cells. In addition, siRNA-mediated knockdown of p53 diminished G-Rh2-induced Fas expression and caspase-8 activation. These results indicated that G-Rh2-triggered extrinsic apoptosis relies on p53-mediated Fas over-expression. In the intrinsic apoptotic pathway, G-Rh2 induced strong and immediate translocation of cytosolic BAK and BAX to the mitochondria, mitochondrial cytochrome c release, and subsequent caspase-9 activation both in HeLa and in SW480 cells. p53-mediated Fas expression and subsequent downstream caspase-8 activation as well as p53-independent caspase-9 activation all contribute to the activation of the downstream effector caspase-3/-7, leading to tumor cell death. Taken together, we suggest that G-Rh2 induces cancer cell apoptosis in a multi-path manner and is therefore a promising candidate for antitumor drug development.     

Regulation of Fas ligand-induced apoptosis by TNF.

Fas ligand (FasL, CD95L) expression helps control inflammatory reactions in immune privileged sites such as the eye. Cellular activation is normally required to render lymphoid cells sensitive to FasL-induced death; however, both activated and freshly isolated Fas(+) lymphoid cells are efficiently killed in the eye. Thus, we examined factors that might regulate cell death in the eye. TNF levels rapidly increased in the eye after the injection of lymphoid cells, and these cells underwent apoptosis within 24 h. Coinjection of anti-TNF Ab with the lymphoid cells blocked this cell death. Furthermore, TNFR2(-/-) T cells did not undergo apoptosis in the eyes of normal mice, while normal and TNFR1(-/-) T cells were killed by apoptosis. In vitro, TNF enhanced the Fas-mediated apoptosis of unactivated T cells through decreased intracellular levels of FLIP and increased production of the pro-apoptotic molecule Bax. This effect was mediated through the TNFR2 receptor. In vivo, intracameral injection of normal or TNFR1(-/-) 2,4,6-trinitrophenyl-coupled T cells into normal mice induced immune deviation, but TNFR2(-/-) 2,4,6-trinitrophenyl-coupled T cells were ineffective. Collectively, our results provide evidence of a role for the p75 TNFR in cell death in that TNF signaling through TNFR2 sensitizes lymphoid cells for Fas-mediated apoptosis. We conclude that there is complicity between apoptosis and elements of the inflammatory response in controlling lymphocyte function in immune privileged sites.     

Salivary epidermal growth factor and intestinal adaptation in male and female mice

Salivary epidermal growth factor (sEGF) levels are increased in male mice after small bowel resection (SBR) and may be important during intestinal adaptation. Since males have greater sEGF than females, the influence of sex on postresection adaptation was tested. Females had lower sEGF; however, sEGF substantially increased in both sexes after a massive (50%) SBR. Adaptive increases in DNA and protein content, villus height, and crypt depth, as well as crypt cell proliferation rates in the remnant ileum, were not different between males and females. Although significant postresection increases in sEGF were identified, EGF mRNA and protein did not change within the submandibular gland. Glandular kallikrein-13 and ileal EGF receptor expression were greater after SBR in female mice. Intestinal adaptation is equivalent in female and male mice after SBR. Despite lower sEGF, females demonstrated increased expression of a kallikrein responsible for sEGF precursor cleavage as well as amplified ileal EGF receptor expression. These results endorse an important differential response between sexes regarding sEGF mobilization and intestinal receptor availability during adaptation.     

Caspase-8, c-FLIP, and caspase-9 in c-Myc-induced apoptosis of fibroblasts

c-Myc is known to induce or potentiate apoptotic processes predominantly by triggering or enhancing the activity of caspases, but the activation mechanisms of caspases by c-Myc remain still poorly understood. Here we found that in MycER™ rat fibroblasts the activation of c-Myc led to an early activation and cleavage of the initiator caspase-8, and concurrent processing and activation of the effector caspases 3 and 7. Interestingly, the expression of cellular FLICE inhibitory protein (c-FLIP) mRNA and the encoded protein, c-FLIP_L, a catalytically inactive homologue of caspase-8, were down-regulated prior to or coincidently with the activation of caspase-8. Of the other known initiators, caspase-9, involved in the mitochondrial pathway, was activated/processed surprisingly late, only after the effector caspases 3/7. Further, we studied the potential involvement of the Fas- and tumor necrosis factor receptor (TNFR)-mediated signaling in the activation of caspase-8 by c-Myc. Blocking of the function of these death receptors by neutralizing antibodies against Fas ligand and TNF-α did not prevent the processing of caspase-8 or cell death. c-Myc was neither found to induce any changes in the expression of TNF-related apoptosis inducing ligand (TRAIL) or its receptor. These data suggest that caspase-8 does not become activated through an extrinsic but an “intrinsic/intracellular” apoptotic pathway unleashed by the down-regulation of c-FLIP by c-Myc. Moreover, ectopic expression of c-FLIP_L inhibited the c-Myc-induced apoptosis.     

Deciphering the pathways of death of Histoplasma capsulatum-infected macrophages: implications for the immunopathogenesis of early infection

Apoptosis of leukocytes is known to strongly influence the immunopathogenesis of infection. In this study, we dissected the death pathways of murine macrophages (MΦs) infected with the intracellular pathogen Histoplasma capsulatum. Yeast cells caused apoptosis of MΦs at a wide range of multiplicity of infection, but smaller inocula resulted in delayed detection of apoptosis. Upon infection, caspases 3 and 1 were activated, and both contributed to cell death; however, only the former was involved in apoptosis. The principal driving force for apoptosis involved the extrinsic pathway via engagement of TNFR1 by TNF-α. Infected MΦs produced IL-10 that dampened apoptosis. The chronology of TNF-α and IL-10 release differed in vitro. The former was detected by 2 h postinfection, and the latter was not detected until 8 h postinfection. In vivo, the lungs of TNFR1(-/-) mice infected for 1 d contained fewer apoptotic MΦs than wild-type mice, whereas the lungs of IL-10(-/-) mice exhibited more. Blockade of apoptosis by a pan-caspase inhibitor or by simvastatin sharply reduced the release of TNF-α but enhanced IL-10. However, these treatments did not modify the fungal burden in vitro over 72 h. Thus, suppressing cell death modulated cytokine release but did not alter the fungal burden. These findings provide a framework for the early pathogenesis of histoplasmosis in which yeast cell invasion of lung MΦs engenders apoptosis, triggered in part in an autocrine TNF-α-dependent manner, followed by release of IL-10 that likely prevents apoptosis of newly infected neighboring phagocytes.     

Epidermal growth factor receptor signaling modulates apoptosis via p38alpha MAPK-dependent activation of Bax in intestinal epithelial cells

Previous studies have demonstrated that the proapoptotic protein Bax plays an important role in the elevated enterocyte apoptosis that occurs during the intestinal adaptation response to massive small bowel resection (SBR). Additionally, epidermal growth factor receptor (EGFR) activation prevents SBR-induced enterocyte apoptosis. The present study aims to delineate the relationship between EGFR activity and intestinal epithelial cell apoptosis. Treatment of model intestinal epithelial cells (RIEC-18) with both a selective EGFR inhibitor (ZD1839) and EGFR small interfering RNA knockdown resulted in a dramatic increase in apoptosis, accompanied by rapid phosphorylation of p38alpha. Concurrently, Bax underwent conformational changes consistent with activation and translocated to mitochondria. In contrast, EGF stimulation enhanced cell survival by attenuating p38alpha phosphorylation, Bax conformational change, mitochondrial trafficking, and apoptosis. These results demonstrate that that diminished EGFR activity initiates the intrinsic pathway of apoptosis through p38alpha-dependent Bax activation in intestinal epithelial cells. These finding provide mechanistic insight into the role that EGFR signaling plays in the regulation of enterocyte apoptosis following massive intestinal loss.     

DR3 regulates negative selection during thymocyte development

DR3 (Ws1, Apo3, LARD, TRAMP, TNFSFR12) is a member of the death domain-containing tumor necrosis factor receptor (TNFR) superfamily, members of which mediate a variety of developmental events including the regulation of cell proliferation, differentiation, and apoptosis. We have investigated the in vivo role(s) of DR3 by generating mice congenitally deficient in the expression of the DR3 gene. We show that negative selection and anti-CD3-induced apoptosis are significantly impaired in DR3-null mice. In contrast, both superantigen-induced negative selection and positive selection are normal. The pre-T-cell receptor-mediated checkpoint, which is dependent on TNFR signaling, is also unaffected in DR3-deficient mice. These data reveal a nonredundant in vivo role for this TNF receptor family member in the removal of self-reactive T cells in the thymus.     

Inhibition of Fas-mediated fulminant hepatitis in CrmA gene-transfected mice

Hyperimmune response via Fas/Fas-ligand and perforin/granzyme pathways may be essential in pathogenesis of virus-induced fulminant hepatitis. CrmA inhibits activation of caspases and granzyme B, suggesting it may block these pathways. We investigated whether CrmA expression would inhibit Fas-associated lethal hepatitis in mice. We successfully generated AxCALNLCrmA, a recombinant adenovirus expressing CrmA gene with a Cre-mediated switching cassette. We increased CrmA expression level in the liver transfected with AxCALNLCrmA (10(9) pfu) by increasing administration dose (10(7)-10(9) pfu) of AxCANCre, a recombinant, adenovirus-expressing Cre gene. Injection of anti-Fas antibody into the control mice rapidly led to animal death due to massive liver apoptosis, while the apoptosis was dramatically reduced in the CrmA-expressed mice. The animal survival increased with an increase of CrmA expression. The formation of active caspase-3 was markedly inhibited in the crmA-transfected hepatocytes in vitro. These results suggest that crmA is an effective gene that can inhibit immune-related liver apoptosis.     

Mechanisms of p75-mediated death of hippocampal neurons. Role of caspases

Neurotrophins support neuronal survival and differentiation via Trk receptors, yet can also induce cell death via the p75 receptor. In these studies, we investigated signaling mechanisms governing p75-mediated death of hippocampal neurons, specifically the role of caspases. Although p75 is structurally a member of the Fas/TNFR1 receptor family, caspase-8 was not required for p75-mediated death, unlike other members of this receptor family. In contrast, p75-mediated neuronal death was associated with mitochondrial loss of cytochrome c and required Apaf-1 and caspase-9, -6, and -3. In particular, caspase-6 plays a central role in mediating neurotrophin-induced death, illuminating a novel role for this caspase. Inhibition of DIABLO/Smac, which blocks inhibitor of apoptosis proteins, protected cells from death, whereas simultaneous inhibition of both DIABLO/Smac and MIAP3 allowed trophin-induced death to proceed. In vivo, pilocarpine-induced seizures, previously shown to up-regulate p75 expression and increase neurotrophin production, caused activation of caspase-6 and -3 and cleavage of poly(ADP-ribose) polymerase in p75-expressing hippocampal neurons. In p75(-/-) mice, no activated caspase-3 was detected, and there was a marked reduction in the number of dying neurons after pilocarpine treatment compared with wild type mice. Neurotrophin-induced p75-mediated death is likely to play an important role in mediating neuronal loss consequent to brain injury.     

Apoptosis control by death and decoy receptors

The death receptors Fas and tumor necrosis factor receptor 1 (TNFR1) trigger apoptosis upon engagement by their cognate death ligands. Recently, researchers have discovered several novel homologues of Fas and TNFR1: DR 3, 4, 5, and 6 function as death receptors that signal apoptosis, whereas DcR 1, 2, and 3 act as decoys that compete with specific death receptors for ligand binding. Further, mouse gene knockout studies have enabled researchers to delineate some of the signaling pathways that connect death receptors to the cell's apoptotic machinery.     

Competitive control of independent programs of tumor necrosis factor receptor-induced cell death by TRADD and RIP1

Stimulation of tumor necrosis factor receptor 1 (TNFR1) can initiate several cellular responses, including apoptosis, which relies on caspases, necrotic cell death, which depends on receptor-interacting protein kinase 1 (RIP1), and NF-kappaB activation, which induces survival and inflammatory responses. The TNFR-associated death domain (TRADD) protein has been suggested to be a crucial signal adaptor that mediates all intracellular responses from TNFR1. However, cells with a genetic deficiency of TRADD are unavailable, precluding analysis with mature immune cell types. We circumvented this problem by silencing TRADD expression with small interfering RNA. We found that TRADD is required for TNFR1 to induce NF-kappaB activation and caspase-8-dependent apoptosis but is dispensable for TNFR1-initiated, RIP1-dependent necrosis. Our data also show that TRADD and RIP1 compete for recruitment to the TNFR1 signaling complex and the distinct programs of cell death. Thus, TNFR1-initiated intracellular signals diverge at a very proximal level by the independent association of two death domain-containing proteins, RIP1 and TRADD. These single transducers determine cell fate by triggering NF-kappaB activation, apoptosis, and nonapoptotic death signals through separate and competing signaling pathways.     

Smooth muscle overexpression of IGF-I induces a novel adaptive response to small bowel resection

Prior studies of intestinal adaptation after massive small bowel resection (SBR) have focused on growth factors and their effects on amplification of the gut mucosa. Because adaptive changes have also been described in intestinal smooth muscle, we sought to determine the effect of targeted smooth muscle growth factor overexpression on resection-induced intestinal adaptation. Male transgenic mice with smooth muscle cell overexpression of insulin-like growth factor I (IGF-I) by virtue of an alpha-smooth muscle actin promoter were obtained. SMP8 IGF-I transgenic (IGF-I TG) and nontransgenic (NT) littermates underwent 50% proximal SBR or sham operation and were then killed after 3 or 28 days. NT mice showed the expected alterations in mucosal adaptive parameters after SBR, such as increased wet weight and villus height. The IGF-I TG mice had inherently taller villi, which did not increase significantly after SBR. In addition, IGF-I TG mice had a 50% postresection persistent increase in remnant intestinal length, which was associated with an early decline and later increase in relative mucosal surface area. These results indicate that growth factor overexpression within the muscularis layer of the bowel wall induces significant postresection adaptive intestinal lengthening and a unique mucosal response. IGF-I signaling within the muscle wall may play an important role in the pathogenesis of resection-induced adaptation.     

CD47 augments Fas/CD95-mediated apoptosis

Fas (CD95) mediates apoptosis of many cell types, but the susceptibility of cells to killing by Fas ligand and anti-Fas antibodies is highly variable. Jurkat T cells lacking CD47 (integrin-associated protein) are relatively resistant to Fas-mediated death but are efficiently killed by Fas ligand or anti-Fas IgM (CH11) upon expression of CD47. Lack of CD47 impairs events downstream of Fas activation including caspase activation, poly-(ADP-ribose) polymerase cleavage, cytochrome c release from mitochondria, loss of mitochondrial membrane potential, and DNA cleavage. Neither CD47 signaling nor raft association of CD47 is required to enable Fas apoptosis. CH11 induces association of Fas and CD47. Primary T cells from CD47-null mice are also protected from Fas-mediated killing relative to wild type T cells. Thus CD47 associates with Fas upon its activation and augments Fas-mediated apoptosis.     

Bid activation during induction of extrinsic and intrinsic apoptosis in eosinophils

Eosinophils readily undergo apoptosis when removed from a physiological environment via activation of the intrinsic cell death pathway. This can be further enhanced by certain chemicals (for example, glucocorticoid), or by extrinsic means (that is, Fas receptor engagement). In this investigation, we examined the relative importance of these pathways in cultured human peripheral blood eosinophils in the context of expression and activation of the BH3-only Bcl2 homologue Bid. Bid activation was examined under conditions where programmed cell death was either stimulated (via Fas engagement or glucocorticoid treatment) or inhibited (interleukin-5 (IL-5)) relative to control. Full-length Bid was found to be highly expressed in eosinophils, and processed to a similar extent during either agonist anti-Fas or glucocorticoid treatment. IL-5 blocked intrinsic Bid activation during factor withdrawal or glucocorticoid treatment, and partially attenuated that caused by Fas activation. Caspase 8 (but not caspase 9) antagonism partly but significantly affected receptor-mediated Bid activation and cell death; these processes were not altered by either caspase inhibitor during simple factor withdrawal or glucocorticoid treatment. Bid processing appears to be central to both intrinsic and extrinsic pathways of cell death in eosinophils. The role of IL-5 in blocking the intrinsic pathway of eosinophil apoptosis is underscored. Results of specific inhibition support the existence of Bid activation pathways in eosinophils other than those mediated by the classic initiator caspases.     

Altering the sphingolipid acyl chain composition prevents LPS/GLN-mediated hepatic failure in mice by disrupting TNFR1 internalization

The involvement of ceramide in death receptor-mediated apoptosis has been widely examined with most studies focusing on the role of ceramide generated from sphingomyelin hydrolysis. We now analyze the effect of the ceramide acyl chain length by studying tumor necrosis factor α receptor-1 (TNFR1)-mediated apoptosis in a ceramide synthase 2 (CerS2) null mouse, which cannot synthesize very-long acyl chain ceramides. CerS2 null mice were resistant to lipopolysaccharide/galactosamine-mediated fulminant hepatic failure even though TNFα secretion from macrophages was unaffected. Cultured hepatocytes were also insensitive to TNFα-mediated apoptosis. In addition, in both liver and in hepatocytes, caspase activities were not elevated, consistent with inhibition of TNFR1 pro-apoptotic signaling. In contrast, Fas receptor activation resulted in the death of CerS2 null mice. Caspase activation was blocked because of the inability of CerS2 null mice to internalize the TNFR1; whereas Fc-TNFα was internalized to a perinuclear region in hepatocytes from wild-type mice, no internalization was detected in CerS2 null mice. Our results indicate that altering the acyl chain composition of sphingolipids inhibits TNFR1 internalization and inhibits selective pro-apoptotic downstream signaling for apoptosis.