Role of regenerating islet-derived proteins in inflammatory bowel disease.

Inflammatory bowel disease (IBD) is an inflammatory disorder of the gastrointestinal tract that affects millions of patients worldwide. It has a complex and multifactorial etiology leading to excessive exposure of intestinal epithelium to microbial antigens, inappropriate activation of the immune system and ultimately to the damage of intestinal tissues. Although numerous efforts have been made to improve the disease management, IBD remains persistently recurring and beyond cure. This is due largely to the gaps in our understanding of the pathogenesis of IBD that hamper the development of timely diagnoses and effective treatment. However, some recent discoveries, including the beneficial effects of interleukin-22 (IL-22) on the inflamed intestine, have shed light on a self-protective mechanism in IBD. Regenerating islet-derived (REG/Reg) proteins are small secretory proteins which function as IL-22's downstream effectors. Mounting studies have demonstrated that IBD patients have significantly increased REG expressions in the injured intestine, but with undefined mechanisms and roles. The reported functions of REG/Reg proteins in intestinal homeostasis, such as those of antibacterial, anti-inflammatory and tissue repair, lead us to discuss their potential mechanisms and clinical relevance in IBD in order to advance IBD research and management.

Airway Resistance Caused by Sphingomyelin Synthase 2 Insufficiency in Response to Cigarette Smoke.

Sphingomyelin synthase is responsible for the production of sphingomyelin (SGM), the second most abundant phospholipid in mammalian plasma, from ceramide, a major sphingolipid. Knowledge of the effects of cigarette smoke on SGM production is limited. In the present study, we examined the effect of chronic cigarette smoke on sphingomyelin synthase (SGMS) activity and evaluated how the deficiency of Sgms2, one of the two isoforms of mammalian SGMS, impacts pulmonary function. Sgms2-knockout and wild-type control mice were exposed to cigarette smoke for 6 months, and pulmonary function testing was performed. SGMS2-dependent signaling was investigated in these mice and in human monocyte-derived macrophages of nonsmokers and human bronchial epithelial (HBE) cells isolated from healthy nonsmokers and subjects with chronic obstructive pulmonary disease (COPD). Chronic cigarette smoke reduces SGMS activity and Sgms2 gene expression in mouse lungs. Sgms2-deficient mice exhibited enhanced airway and tissue resistance after chronic cigarette smoke exposure, but had similar degrees of emphysema, compared with smoke-exposed wild-type mice. Sgms2-/- mice had greater AKT phosphorylation, peribronchial collagen deposition, and protease activity in their lungs after smoke inhalation. Similarly, we identified reduced SGMS2 expression and enhanced phosphorylation of AKT and protease production in HBE cells isolated from subjects with COPD. Selective inhibition of AKT activity or overexpression of SGMS2 reduced the production of several matrix metalloproteinases in HBE cells and monocyte-derived macrophages. Our study demonstrates that smoke-regulated Sgms2 gene expression influences key COPD features in mice, including airway resistance, AKT signaling, and protease production.

Liver serine palmitoyltransferase activity deficiency in early life impairs adherens junctions and promotes tumorigenesis.

Serine palmitoyltransferase is the key enzyme in sphingolipid biosynthesis. Mice lacking serine palmitoyltransferase are embryonic lethal. We prepared liver-specific mice deficient in the serine palmitoyltransferase long chain base subunit 2 gene using an albumin-cyclization recombination approach and found that the deficient mice have severe jaundice. Moreover, the deficiency impairs hepatocyte polarity, attenuates liver regeneration after hepatectomy, and promotes tumorigenesis. Importantly, we show that the deficiency significantly reduces sphingomyelin but not other sphingolipids in hepatocyte plasma membrane; greatly reduces cadherin, the major protein in adherens junctions, on the membrane; and greatly induces cadherin phosphorylation, an indication of its degradation. The deficiency affects cellular distribution of ß-catenin, the central component of the canonical Wnt pathway. Furthermore, such a defect can be partially corrected by sphingomyelin supplementation in vivo and in vitro. CONCLUSION: The plasma membrane sphingomyelin level is one of the key factors in regulating hepatocyte polarity and tumorigenesis. (Hepatology 2016;64:2089-2102).

Myeloid cell-specific serine palmitoyltransferase subunit 2 haploinsufficiency reduces murine atherosclerosis.

Serine palmitoyltransferase (SPT) is the first and rate-limiting enzyme of the de novo biosynthetic pathway of sphingomyelin (SM). Both SPT and SM have been implicated in the pathogenesis of atherosclerosis, the development of which is driven by macrophages; however, the role of SPT in macrophage-mediated atherogenesis is unknown. To address this issue, we have analyzed macrophage inflammatory responses and reverse cholesterol transport, 2 key mediators of atherogenesis, in SPT subunit 2-haploinsufficient (Sptlc2(+/-)) macrophages. We found that Sptlc2(+/-) macrophages have significantly lower SM levels in plasma membrane and lipid rafts. This reduction not only impaired inflammatory responses triggered by TLR4 and its downstream NF-κB and MAPK pathways, but also enhanced reverse cholesterol transport mediated by ABC transporters. LDL receptor-deficient (Ldlr(-/-)) mice transplanted with Sptlc2(+/-) bone marrow cells exhibited significantly fewer atherosclerotic lesions after high-fat and high-cholesterol diet feeding. Additionally, Ldlr(-/-) mice with myeloid cell-specific Sptlc2 haploinsufficiency exhibited significantly less atherosclerosis than controls. These findings suggest that SPT could be a novel therapeutic target in atherosclerosis.

Acute pancreatitis in aging animals: loss of pancreatitis-associated protein protection?

AIM: To investigate the effect of age on severity of acute pancreatitis (AP) using biochemical markers, histology and expression of the protective pancreatitis-associated proteins (PAPs). METHODS: AP was induced via intraductal injection of 4% sodium taurocholate in young and old rats. Sera and pancreata were assayed at 24 h for the parameters listed above; we also employed a novel molecular technique to assess bacterial infiltration using polymerase chain reaction to measure bacterial genomic ribosomal RNA. RESULTS: At 24 h after induction of AP, the pancreata of older animals had less edema (mean ± SE histologic score of young vs old: 3.11 ± 0.16 vs 2.50 ± -0.11, P < 0.05), decreased local inflammatory response (histologic score of stromal infiltrate: 3.11 ± 0.27 vs 2.00 ± 0.17, P < 0.05) and increased bacterial infiltration (174% ± 52% increase from sham vs 377% ± 4%, P < 0.05). A decreased expression of PAP1 and PAP2 was demonstrated by Western blotting analysis and immunohistochemical staining. There were no differences in serum amylase and lipase activity, or tissue myeloperoxidase or monocyte chemotactic protein-1 levels. However, in the most-aged group, serum C-reactive protein levels were higher (young vs old: 0.249 ± 0.04 mg/dL vs 2.45 ± 0.68 mg/dL, P < 0.05). CONCLUSION: In older animals, there is depressed PAP expression related to a blunted inflammatory response in AP which is associated with worsened bacterial infiltration and higher C-reactive protein level; this may explain the more aggressive clinical course.

Impact of sphingomyelin synthase 1 deficiency on sphingolipid metabolism and atherosclerosis in mice.

OBJECTIVE: Sphingomyelin synthase (SMS) catalyzes the conversion of ceramide to sphingomyelin and sits at the crossroads of sphingolipid biosynthesis. SMS has 2 isoforms: SMS1 and SMS2. Although they have the same SMS activity, they are different enzymes with distinguishable subcellular localizations and cell expression patterns. It is conceivable that these differences could yield different consequences, in terms of sphingolipid metabolism and its related atherogenesis. METHODS AND RESULTS: We created Sms1 gene knockout mice and found that Sms1 deficiency significantly decreased plasma, liver, and macrophage sphingomyelin (59%, 45%, and 54%, respectively), but only had a marginal effect on ceramide levels. Surprisingly, we found that Sms1 deficiency dramatically increased glucosylceramide and GM3 levels in plasma, liver, and macrophages (4- to 12-fold), whereas Sms2 deficiency had no such effect. We evaluated the total SMS activity in tissues and found that Sms1 deficiency causes 77% reduction in SMS activity in macrophages, indicating SMS1 is the major SMS in macrophages. Moreover, Sms1-deficient macrophages have a significantly higher glucosylceramide synthase activity. We also found that Sms1 deficiency significantly attenuated toll-like 4 receptor-mediated nuclear factor-κB and mitogen-activated protein kinase activation after lipopolysaccharide treatment. To evaluate atherogenicity, we transplanted Sms1 knockout mouse bone marrow into low-density lipoprotein receptor knockout mice (Sms1(-/-)→Ldlr(-/-)). After 3 months on a western diet, these animals showed a significant decrease of atherosclerotic lesions in the root and the entire aorta (35% and 44%, P<0.01, respectively) and macrophage content in lesions (51%, P<0.05), compared with wild-type→Ldlr(-/-) mice. CONCLUSIONS: Sms1 deficiency decreases sphingomyelin, but dramatically increases the levels of glycosphingolipids. Atherosclerosis in Sms1(-/-)→Ldlr(-/-) mice is significantly decreased.

Macrophage sphingomyelin synthase 2 deficiency decreases atherosclerosis in mice.

RATIONALE: Sphingomyelin synthase (SMS)2 contributes to de novo sphingomyelin (SM) biosynthesis and plasma membrane SM levels. SMS2 deficiency in macrophages diminishes nuclear factor kappaB and mitogen-activated protein kinase activation induced by inflammatory stimuli. OBJECTIVE: The effects of SMS2 deficiency on the development of atherosclerosis are investigated. METHODS AND RESULTS: We measured cholesterol efflux from macrophages of wild-type (WT) and SMS2 knockout (KO) mice. We transplanted SMS2 KO mouse bone marrow into low-density lipoprotein (LDL) receptor (LDLr) knockout mice (SMS2(-/-)-->LDLr(-/-)), creating a mouse model of SMS2 deficiency in the macrophages. We found that SMS2 deficiency caused significant induction of cholesterol efflux in vitro and in vivo. Moreover, we found that SMS2 KO mice had less interleukin-6 and tumor necrosis factor alpha in the circulation before and after endotoxin stimulation, compared with controls. More importantly, after 3 months on a western-type diet, SMS2(-/-)-->LDLr(-/-) mice showed decreased atherosclerotic lesions in the aortic arch, root (57%, P<0.001), and the entire aorta (42%, P<0.01), compared with WT-->LDLr(-/-) mice. Analysis of plaque morphology revealed that SMS2(-/-)-->LDLr(-/-) mice had significantly less necrotic core area (71%, P<0.001), less macrophage content (37%, P<0.01), and more collagen content (35%, P<0.05) in atherosclerotic lesions. We also found that SMS2(-/-)-->LDLr(-/-) mice had significantly lower free cholesterol and cholesteryl ester levels in the brachiocephalic artery than WT-->LDLr(-/-) mice (33 and 52%, P<0.01 and P<0.001, respectively). CONCLUSIONS: SMS2 deficiency in the macrophages reduces atherosclerosis in mice. Macrophage SMS2 is thus a potential therapeutic target for treatment of this disease.

Sphingomyelin synthase 2 deficiency attenuates NFkappaB activation.

BACKGROUND: NFkappaB has long been regarded as a proatherogenic factor, mainly because of its regulation of many of the proinflammatory genes linked to atherosclerosis. Metabolism of sphingomyelin (SM) has been suggested to affect NFkappaB activation, but the mechanism is largely unknown. SMS2 regulates SM levels in cell plasma membrane and lipid rafts and has a potential to regulate NFkappaB activation. METHODS AND RESULTS: To investigate the role of SMS2 in NFkappaB activation we used macrophages from SMS2 knockout (KO) mice and SMS2 siRNA-treated HEK 293 cells. We found that NFkappaB activation and its target gene expression are attenuated in macrophages from SMS2 KO mice in response to lipopolysaccharide (LPS) stimulation and in SMS2 siRNA- treated HEK 293 cells after tumor necrosis factor (TNF)-alpha simulation. In line with attenuated NFkappaB activation, we found that SMS2 deficiency substantially diminished the abundance of toll like receptor 4 (TLR4)-MD2 complex levels on the surface of macrophages after LPS stimulation, and SMS2 siRNA treatment reduced TNF-alpha-stimulated lipid raft recruitment of TNF receptor-1 (TNFR1) in HEK293 cells. SMS2 deficiency decreased the relative amounts of SM and diacylglycerol (DAG) and increased ceramide, suggesting multiple mechanisms for the decrease in NFkappaB activation. CONCLUSIONS: SMS2 is a modulator of NFkappaB activation, and thus it could play an important role in NFkappaB-mediated proatherogenic process.

Transcription factors TFE3 and TFEB are critical for CD40 ligand expression and thymus-dependent humoral immunity.

TFE3 and TFEB are broadly expressed transcription factors related to the transcription factor Mitf. Although they have been linked to cytokine signaling pathways in nonlymphoid cells, their function in T cells is unknown. TFE3-deficient mice are phenotypically normal, whereas TFEB deficiency causes early embryonic death. We now show that combined inactivation of TFE3 and TFEB in T cells resulted in a hyper-immunoglobulin M syndrome due to impaired expression of CD40 ligand by CD4(+) T cells. Native TFE3 and TFEB bound to multiple cognate sites in the promoter of the gene encoding CD40 ligand (Cd40lg), and maximum Cd40lg promoter activity and gene expression required TFE3 or TFEB. Thus, TFE3 and TFEB are direct, physiological and mutually redundant activators of Cd40lg expression in activated CD4(+) T cells critical for T cell-dependent antibody responses.

Renal carcinoma-associated transcription factors TFE3 and TFEB are leukemia inhibitory factor-responsive transcription activators of E-cadherin.

Translocations of the genes encoding the related transcription factors TFE3 and TFEB are almost exclusively associated with a rare juvenile subset of renal cell carcinoma and lead to overexpression of TFE3 or TFEB protein sequences. A better understanding of how deregulated TFE3 and TFEB contribute to the transformation process requires elucidating more of the normal cellular processes in which they participate. Here we identify TFE3 and TFEB as cell type-specific leukemia inhibitory factor-responsive activators of E-cadherin. Overexpression of TFE3 or TFEB in 3T3 cells activated endogenous and reporter E-cadherin expression. Conversely, endogenous TFE3 and/or TFEB was required for endogenous E-cadherin expression in primary mouse embryonic fibroblasts and human embryonic kidney cells. Chromatin precipitation analyses and E-cadherin promoter reporter gene assays revealed that E-cadherin induction by TFE3 or TFEB was primarily or exclusively direct and mitogen-activated protein kinase-dependent in those cell types. In mouse embryonic fibroblasts, TFE3 and TFEB activation of E-cadherin was responsive to leukemia inhibitory factor. In 3T3 cells, TFE3 and TFEB expression also induced expression of Wilms' tumor-1, another E-cadherin activator. In contrast, E-cadherin expression in model mouse and canine renal epithelial cell lines was indifferent to inhibition of endogenous TFE3 and/or TFEB and was reduced by TFE3 or TFEB overexpression. These results reveal new cell type-specific activities of TFE3 and TFEB which may be affected by their mutation.