Effect of Recent Antirheumatic Drug on Features of Rheumatoid Arthritis-Associated Lymphoproliferative Disorders.

OBJECTIVE: In this study, we examine how advancements in novel antirheumatic drugs affect the clinicopathologic features of lymphoproliferative disorder (LPD) in patients with rheumatoid arthritis (RA). METHODS: In this multicenter study across 53 hospitals in Japan, we characterized patients with RA who developed LPDs and visited the hospitals between January 1999 and March 2021. The statistical tools used included Fisher's exact test, the Mann-Whitney U-test, the log-rank test, logistic regression analysis, and Cox proportional hazards models. RESULTS: Overall, 752 patients with RA-associated LPD (RA-LPD) and 770 with sporadic LPD were included in the study. We observed significant differences in the clinicopathologic features between patients with RA-LPD and those with sporadic LPD. Histopathological analysis revealed a high frequency of LPD-associated immunosuppressive conditions. Furthermore, patients with RA-LPD were evaluated based on the antirheumatic drugs administered. The methotrexate (MTX) plus tacrolimus and MTX plus tumor necrosis factor inhibitor (TNFi) groups had different affected site frequencies and histologic subtypes than the MTX-only group. Moreover, MTX and TNFi may synergistically affect susceptibility to Epstein-Barr virus infection. In case of antirheumatic drugs administered after LPD onset, tocilizumab (TCZ)-only therapy was associated with lower frequency of regrowth after spontaneous regression than other regimens. CONCLUSION: Antirheumatic drugs administered before LPD onset may influence the clinicopathologic features of RA-LPD, with patterns changing over time. Furthermore, TCZ-only regimens are recommended after LPD onset.

Eliglustat exerts anti-fibrotic effects by activating SREBP2 in TGF-ß1-treated myofibroblasts derived from patients with idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive chronic lung disease. Myofibroblasts play a critical role in fibrosis. These cells produce the extracellular matrix (ECM), which contributes to tissue regeneration; however, excess ECM production can cause fibrosis. Transforming growth factor-ß (TGF-ß)/Smad signaling induces ECM production by myofibroblasts; therefore, the inhibition of TGF-ß/Smad signaling may be an effective strategy for IPF treatment. We recently reported that miglustat, an inhibitor of glucosylceramide synthase (GCS), ameliorates pulmonary fibrosis by inhibiting the nuclear translocation of Smad2/3. In the present study, we examined the anti-fibrotic effects of another GCS inhibitor, eliglustat, a clinically approved drug for treating Gaucher disease type 1, in myofibroblasts derived from patient with IPF (IPF-MyoFs). We found that eliglustat exerted anti-fibrotic effects independent of GCS inhibition, and inhibited TGF-ß1-induced expression of α-smooth muscle actin, a marker of fibrosis, without suppressing the phosphorylation and nuclear translocation of Smad2/3. RNA sequencing analysis of eliglustat-treated human lung fibroblasts identified sterol regulatory element-binding protein 2 (SREBP2) activation. Transient overexpression of SREBP2 attenuated the TGF-ß1-induced increase in the expression of Smad target genes in IPF-MyoFs, and SREBP2 knockdown nullified the inhibitory effect of eliglustat on TGF-ß1-induced expression of α-SMA. These results suggested that eliglustat exerts its anti-fibrotic effects through SREBP2 activation. The findings of this study may contribute to the development of novel therapeutic strategies for IPF treatment.

N-butyldeoxynojirimycin (miglustat) ameliorates pulmonary fibrosis through inhibition of nuclear translocation of Smad2/3.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease. The disease involves excessive accumulation of fibroblasts and myofibroblasts, and myofibroblasts differentiated by pro-fibrotic factors promote the deposition of extracellular matrix proteins such as collagen and fibronectin. Transforming growth factor-ß1 is a pro-fibrotic factor that promotes fibroblast-to-myofibroblast differentiation (FMD). Therefore, inhibition of FMD may be an effective strategy for IPF treatment. In this study, we screened the anti-FMD effects of various iminosugars and showed that some compounds, including N-butyldeoxynojirimycin (NB-DNJ, miglustat, an inhibitor of glucosylceramide synthase (GCS)), a clinically approved drug for treating Niemann-Pick disease type C and Gaucher disease type 1, inhibited TGF-ß1-induced FMD by inhibiting the nuclear translocation of Smad2/3. N-butyldeoxygalactonojirimycin having GCS inhibitory effect did not attenuate the TGF-ß1-induced FMD, suggesting that NB-DNJ exerts the anti-FMD effects by GCS inhibitory effect independent manner. N-butyldeoxynojirimycin did not inhibit TGF-ß1-induced Smad2/3 phosphorylation. In a mouse model of bleomycin (BLM)-induced pulmonary fibrosis, intratracheal or oral administration of NB-DNJ at an early fibrotic stage markedly ameliorated lung injury and deterioration of respiratory functions, such as specific airway resistance, tidal volume, and peak expiratory flow. Furthermore, the anti-fibrotic effects of NB-DNJ in the BLM-induced lung injury model were similar to those of pirfenidone and nintedanib, which are clinically approved drugs for the treatment of IPF. These results suggest that NB-DNJ may be effective for IPF treatment.

Conversion Surgery After Atezolizumab Plus Bevacizumab for Primary and Peritoneal Metastasis After Hepatocellular Carcinoma Rupture.

BACKGROUND/AIM: Conversion surgery (CS) following atezolizumab plus bevacizumab (Atez+Bev) is a treatment strategy for unresectable hepatocellular carcinoma (UR-HCC). Herein, we report a case of CS after transcatheter arterial embolization (TAE) and Atez+Bev for primary HCC with peritoneal metastases and multiple liver metastasis after HCC rupture. CASE REPORT: A 75-year-old man with a suspected ruptured HCC in segment 4b was referred to the National Hospital Organization Kumamoto Medical Center. TAE was performed to stop the bleeding. Subsequently, 15 courses of Atez+Bev were administered for UR-HCC with primary tumor, peritoneal metastasis, and multiple liver metastases. Multiple liver metastases and peritoneal metastasis resolved 7 months after initiation of Atez+Bev. The primary HCC had shrunk, but the patient decided not to continue treatment because of severe numbness in his fingers. Six months after stopping Atez+Bev, CS was performed because no new lesions were observed, and the patient wished to become cancer-free by resection of the remaining tumor. HCC was successfully resected, and he was discharged without any complications. The pathological findings demonstrated that there was no remnant viable HCC. CONCLUSION: We herein present a case of CS following TAE and Atez+Bev for unresectable and ruptured HCC. The patient did not require chemotherapy after CS and is alive and recurrence-free for 7 months.

The case of T-ALL presenting with NK phenotype after COVID-19 vaccination.

NK-lymphoblastic leukemia/lymphoma (NK-LL) is an extremely rare hematopoietic tumor consisting of natural killer (NK) precursor cells, and their lineage overlaps with T-cells, making it challenging to diagnose. COVID-19 vaccination is recommended for people with a risk of aggravation such as cancer-bearing patients, including hematopoietic tumors. We present a 55-year-old man who had cervical lymph node swelling post vaccination for COVID-19. Hematological malignancy was suspected due to the presence of atypical lymphoid cells with an elevated IL-2R in laboratory data. Tumor cells were positive for CD7, CD56, cyCD3, and terminal deoxynucleotidyl transferase (TdT) evidenced through flow cytometry of the bone marrow and the lymph node. The histopathological findings showed monotonous tumor cell proliferation, the cells being positive for CD3 and TdT in the bone marrow and they were positive for CD3, TdT, and CD56 in lymph node. Even though these findings suggested NK-LL, clonal T-cell receptor (TCR) ß gene rearrangement by Southern blot hybridization was observed in the bone marrow. TCRß rearrangement led to the final diagnosis of T-cell lymphoblastic leukemia (T-ALL). The causal relationship between COVID-19 vaccination and carcinogenesis is not clear, and more cases need to be studied in order to elucidate the relationship between the two factors.

Effects of ceramide kinase knockout on lipopolysaccharide-treated sepsis-model mice: Changes in serum cytokine/chemokine levels and increased lethality.

Ceramide, a central molecule of sphingolipid metabolism, is phosphorylated to ceramide-1-phosphate (C1P) by ceramide kinase (CerK). The CerK/C1P pathway regulates many cellular functions, but its roles in immune/inflammation-related (IIR) diseases in vivo are not well known. Sepsis is an acute systemic inflammatory disease accompanied by damage/dysfunction in multiple organs. In the present study, we investigated the effects of CerK knockout on the onset/progression of sepsis-related events in lipopolysaccharide (LPS)-treated sepsis-model mice. In CerK-null mice, the lethality at 48 h after i.v. injection of LPS was significantly increased compared with that in wild-type (WT) mice. The increased lethality by CerK knockout was reproduced in mice treated with i.p. injections of LPS. Changes in serum levels of 23 IIR molecules, including cytokines and chemokines, were measured. In WT mice, levels of these molecules increased 4 and/or 20 h after i.v. injection of LPS. Although the basal levels of IIR molecules were not affected, LPS-induced increases in interleukin-17 (IL-17), C-C motif chemokine ligands (CCL-2 and CCL-11), and tumor necrosis factor-α were significantly up-regulated, whereas IL-2 levels were slightly down-regulated by CerK knockout. Putative mechanisms for the CerK/C1P pathway-mediated regulation of IIR molecules and increased lethality in LPS-treated mice are discussed.

Tankyrase Regulates Neurite Outgrowth through Poly(ADP-ribosyl)ation-Dependent Activation of ß-Catenin Signaling.

Poly(ADP-ribosyl)ation is a post-translational modification of proteins by transferring poly(ADP-ribose) (PAR) to acceptor proteins by the action of poly(ADP-ribose) polymerase (PARP). Two tankyrase (TNKS) isoforms, TNK1 and TNK2 (TNKS1/2), are ubiquitously expressed in mammalian cells and participate in diverse cellular functions, including wnt/ß-catenin signaling, telomere maintenance, glucose metabolism and mitosis regulation. For wnt/ß-catenin signaling, TNKS1/2 catalyze poly(ADP-ribosyl)ation of Axin, a key component of the ß-catenin degradation complex, which allows Axin's ubiquitination and subsequent degradation, thereby activating ß-catenin signaling. In the present study, we focused on the functions of TNKS1/2 in neuronal development. In primary hippocampal neurons, TNKS1/2 were detected in the soma and neurites, where they co-localized with PAR signals. Treatment with XAV939, a selective TNKS1/2 inhibitor, suppressed neurite outgrowth and synapse formation. In addition, XAV939 also suppressed norepinephrine uptake in PC12 cells, a rat pheochromocytoma cell line. These effects likely resulted from the inhibition of ß-catenin signaling through the stabilization of Axin, which suggests TNKS1/2 enhance Axin degradation by modifying its poly(ADP-ribosyl)ation, thereby stabilizing wnt/ß-catenin signaling and, in turn, promoting neurite outgrowth and synapse formation.

Endometrial stromal sarcoma of the sigmoid colon: a case report and literature review.

Endometrial stromal sarcoma (ESS) is a rare mesenchymal tumor of the uterus that accounts for 7-25% of uterine sarcomas and < 1% of uterine tumors. Previously reported sites include the ovary, bowel wall, abdomen, peritoneum, pelvis, and vagina; however, ESS in the extrauterine area is rare. We report a rare case of endometrial stromal sarcoma that developed in the sigmoid colon along the gonadal vasculature, which was difficult to distinguish from colon cancer. A large polyp was found in the sigmoid colon of a 74-year-old woman during a routine medical examination and was diagnosed as tubular adenoma. On colonoscopy 7 months later, the tumor had grown and blocked the lumen, causing stenosis. She was referred to our hospital for surgery. Although detailed examination at our hospital did not yield a definitive diagnosis, bowel obstruction was considered likely and we performed laparoscopic low anterior resection under a preoperative diagnosis of sigmoid colon cancer. The tumor protruded into the sigmoid colon from the stump of the ovarian arteries and veins outside the intestinal tract. As the left ovarian artery and vein were involved in the tumor, we extracted them as a lump. The tumor was diagnosed as low-grade ESS (LG-ESS). She had a history of hysterectomy and left salpingo-oophorectomy for uterine myoma 25 years ago, and radiation therapy was performed after surgery for an unknown reason. The postoperative course was uneventful, and follow-up was continued at the request of the patient. We report a rare case of ESS infiltrating the sigmoid colon, which was probably a lesion derived from endometriosis of the ovarian arteriovenous stump remaining after surgery 25 years ago.

The first case of methotrexate-associated lymphoproliferative disorder presenting as follicular T-cell lymphoma.

This is the first case of follicular T-cell lymphoma (FTCL) presenting as methotrexate-associated lymphoproliferative disorders (MTX-LPDs). A 69-year-old man treated rheumatoid arthritis with methotrexate presented with cervical swelling, hoarseness and fever. Imaging studies revealed multiple lymphadenopathy and lymphoma was suspected. Lymph node biopsy was performed to confirm the diagnosis. Pathologically, the lymph node was composed of atypical lymphocytes with a follicular growth pattern and area of necrosis. Immunohistochemical examination showed the atypical lymphocytes were positive for CD3, CD4, programmed cell death protein 1, and inducible T-cell co-stimulator. These findings are consistent with FTCL. During hospitalization, the patient's fever subsided and cervical lymphadenopathy improved, probably due to discontinuation of MTX. Here we presented the first case of FTCL presenting as MTX-LPDs. The T-cell phenotype MTX-LPDs are relatively rare and accounts for only 3.4%-6.3% of all MTX-LPD cases. Therefore, detailed clinicopathological features have not been clarified sufficiently. It is hoped that similar cases should be accumulated and studied to better understand the clinical and pathological features of this condition.

The differential functional coupling of phosphodiesterase 4 to human DP and EP2 prostanoid receptors stimulated with PGD2 or PGE2.

BACKGROUND: Human DP and EP2 receptors are two of the most homologically related receptors coupling with Gαs-protein, which stimulate adenylyl cyclase to produce cAMP. Indeed, both receptors are considered to be generated by tandem duplication. It has been reported that other highly homologous and closely related ß1- and ß2-adrenergic receptors interact distinctly with and differentially regulate cAMP-specific phosphodiesterase (PDE) 4 recruitment. METHODS: First, we focused on the cAMP degradation pathways of DP and EP2 receptors stimulated by prostaglandin (PG) D2 or PGE2 using HEK cells stably expressing either human DP receptors or EP2 receptors. Then, distances between ligands and amino acids of the receptors were evaluated by molecular dynamics (MD) analysis. RESULTS: We found that PGD2/EP2 receptors exerted a greater effect on PDE4 activity than PGE2/EP2 receptors. Moreover, by MD analysis, either the PGD2 or EP2 receptor was moved and the distance was shortened between them. According to the results, DP receptors retain reactivity for PGE2, but EP2 receptors may be activated only by PGE2, at least in terms of cAMP formation, through the differential functional coupling of PDE4 probably with ß-arrestin. CONCLUSION: Since DP receptors and EP2 receptors are considered to be duplicated genes, DP receptors may still be in a rapid evolutionary stage as a duplicated copy of EP2 receptors and have not yet sufficient selectivity for their cognate ligand, PGD2.

Py3-FITC: a new fluorescent probe for live cell imaging of collagen-rich tissues and ionocytes.

Polypyrrole-based polyamides are used as sequence-specific DNA probes. However, their cellular uptake and distribution are affected by several factors and have not been extensively studied in vivo. Here, we generated a series of fluorescence-conjugated polypyrrole compounds and examined their cellular distribution using live zebrafish and cultured human cells. Among the evaluated compounds, Py3-FITC was able to visualize collagen-rich tissues, such as the jaw cartilage, opercle and bulbus arteriosus, in early-stage living zebrafish embryos. Then, we stained cultured human cells with Py3-FITC and found that the staining became more intense as the amount of collagen was increased. In addition, Py3-FITC-stained HR cells, which represent a type of ionocyte on the body surface of living zebrafish embryos. Py3-FITC has low toxicity, and collagen-rich tissues and ionocytes can be visualized when soaked in Py3-FITC solution. Therefore, Py3-FITC may be a useful live imaging tool for detecting changes in collagen-rich tissue and ionocytes, including their mammalian analogues, during both normal development and disease progression.

Topical Application of BMS-509744, a Selective Inhibitor of Interleukin-2-Inducible T Cell Kinase, Ameliorates Imiquimod-Induced Skin Inflammation in Mice.

Psoriasis is an immune disorder-related inflammatory skin disease. Recent studies have suggested a contribution of T cell activation in the pathogenesis of psoriasis. Interleukin-2 (IL-2)-inducible T cell kinase (ITK) regulates T cell activation, including proliferation, and cytokine production. In this study, we investigated the effect of the topically administered selective ITK inhibitor BMS-509744 on imiquimod (IMQ)-induced psoriasis-like skin inflammation in mice. Topically administered BMS-509744 ameliorated IMQ-induced psoriasis-like skin inflammation as shown by decreased skin lesions, epidermal thickening, and cell infiltration into the dermis. These suppressive effects occurred with lower numbers of cluster of differentiation antigen-3+ (CD3+) T cells and T helper subset 17 (Th17)-related cytokine expression in IMQ-treated skin. IMQ-induced upregulation of proinflammatory cytokine expression was also inhibited by topical application of BMS-509744 in IMQ-treated skin. Our report showed for the first time that topical application of BMS-509744 ameliorated psoriasis-like skin inflammation in mice, which is likely mediated by the inhibition of T cell activation in the skin lesions.

Tyrosine-phosphorylation and activation of glucosylceramide synthase by v-Src: Its role in survival of HeLa cells against ceramide.

Sphingolipids represent a family of cellular lipid-molecules that regulate physiological and pathophysiological processes. Glucosylceramide (GlcCer), the simplest glycosphingolipid (GSL), is synthesized from ceramide and UDP-glucose by GlcCer synthase (GCS). Both GlcCer (and resulting GSLs) and ceramide regulate various cellular functions including cell death and multiple drug resistance. Src family tyrosine kinases are up-regulated in various human cancer cells. We examined the effect of v-Src expression on GCS activity, the formation of 4-nitrobenzo-2-oxa-1,3-diazole (NBD)-labeled GlcCer from NBD-ceramide, and the effect of tyrosine132 mutation in GCS on ceramide-induced cytotoxicity in HeLa cells. Expression of v-Src increased the formation of NBD-GlcCer in both intact cells without marked changes in other sphingolipid metabolites and cell homogenates without changing affinities of NBD-ceramide and UDP-glucose. Expression of v-Src also increased tyrosine-phosphorylated levels in GCS proteins in HeLa and HEK293T cells. In HEK293T cells transiently expressing the GCS mutant, GCS-Y132F-HA, showing replacement of the tyrosine132 residue with phenylalanine, tyrosine-phosphorylated levels in GCS proteins were significantly lower than those in control cells expressing the GCS-wild-type-HA. The formation of NBD-GlcCer in HeLa cells stably expressing GCS-Y132F-HA was significantly lower than that in the control. Ceramide-induced cytotoxicity in HeLa-GCS-Y132F-HA cells was significantly greater than in the control. In this study, we showed for the first time that expression of v-Src up-regulated GCS activity via tyrosine phosphorylation of the enzyme in a post-translational manner. Mechanisms of Src-induced resistance to ceramide-induced cytotoxicity are discussed in relation to the Src-induced up-regulation of GCS activity.

The first reported case of primary extranodal counterpart of follicular T-cell lymphoma of submandibular gland.

This is the first reported case of follicular T-cell lymphoma (FTCL) that primarily developed in the extranodal site of the right submandibular gland. An 86-year-old man was detected with a right cervical mass suspected to be malignant lymphoma during his physical examination. Imaging studies revealed that the mass was a submandibular gland tumor. The tumor was excised for diagnosis and treatment. Pathologically, the tumor was composed of densely aggregated lymphocytes with a follicular growth pattern. The immunohistochemical investigation showed that the lymphoma cells expressed CD3, CD4, programmed cell death protein 1, BCL6, chemokine (C-X-C motif) ligand 13, and BCL2. Staining of the follicular dendritic cell revealed its meshwork structure limited in the germinal center. Monoclonal rearrangement of the T-cell receptor was detected using polymerase chain reaction. These findings are consistent with the characteristics of FTCL. Here, we describe the first reported case of extranodal counterpart of FTCL of the submandibular gland. Accumulation and investigation of such extranodal cases is essential.

Down-regulation of ceramide kinase via proteasome and lysosome pathways in PC12 cells by serum withdrawal: Its protection by nerve growth factor and role in exocytosis.

Ceramide kinase (CerK) phosphorylates ceramide to ceramide-1-phosphate (C1P). CerK is highly expressed in the brain, and its association with the neuronal function has been reported. Previous reports showed that the activity of CerK is regulated by post-translational modifications including phosphorylation, whereas the cellular fate of CerK protein and its role in neuronal functions have not been clearly elucidated. Therefore, we investigated these issues in PC12 cells. Treatment with nerve growth factor (NGF) for 6 h increased the formation of C1P but not CerK mRNA. Knockdown of CerK and overexpression of HA-tagged CerK down- and up-regulated the formation of C1P, respectively. In PC12-CerK-HA cells, serum withdrawal caused ubiquitination of CerK-HA protein and down-regulated both CerK-HA protein and C1P formation within 6 h, and these down-regulations were abolished by co-treatments with NGF or proteasome inhibitors such as MG132 and clasto-lactacystin. Microscopic analysis showed that treatment with the proteasome inhibitors increased CerK-HA in puncture structures, possibly endosomes and/or vesicles, in cells. Treatment with the lysosome inhibitors reduced serum withdrawal-induced down-regulation of CerK-HA protein but not C1P formation. When knockdown or overexpression of CerK was performed, Ca2+-induced release of [3H] noradrenaline was reduced or enhanced, respectively, but neurite extension was not modified. There was a positive correlation between noradrenaline release and formation of C1P and/or CerK-HA levels in NGF- and clasto-lactacystin-treated cells. These results suggest that levels of CerK were down-regulated by the ubiquitin/proteasome and lysosome pathways and the former pathway-sensitive pool of CerK was suggested to be linked with exocytosis in PC12 cells.

Inhibitory effects of ceramide kinase on Rac1 activation, lamellipodium formation, cell migration, and metastasis of A549 lung cancer cells.

Ceramide kinase (CerK) phosphorylates ceramide to ceramide-1-phosphate (C1P), a bioactive sphingolipid. Since the mechanisms responsible for regulating the proliferation and migration/metastasis of cancer cells by the CerK/C1P pathway remain unclear, we conducted the present study. The knockdown of CerK in A549 lung and MCF-7 breast cancer cells (shCerK cells) increased the formation of lamellipodia, which are membrane protrusions coupled with cell migration. Mouse embryonic fibroblasts prepared from CerK-null mice also showed an enhanced formation of lamellipodia. The overexpression of CerK inhibited lamellipodium formation in A549 cells. The knockdown of CerK increased the number of cells having lamellipodia with Rac1 and the levels of active Rac1-GTP form, whereas the overexpression of CerK decreased them. CerK was located in lamellipodia after the epidermal growth factor treatment, indicating that CerK functioned there to inhibit Rac1. The migration of A549 cells was negatively regulated by CerK. An intravenous injection of A549-shCerK cells into nude mice resulted in markedly stronger metastatic responses in the lungs than an injection of control cells. The in vitro growth of A549 cells and in vivo expansion after the injection into mouse flanks were not affected by the CerK knockdown. These results suggest that the activation of CerK/C1P pathway has inhibitory roles on lamellipodium formation, migration, and metastasis of A549 lung cancer cells.

Short chain fatty acid butyrate uptake reduces expressions of prostanoid EP4 receptors and their mediation of cyclooxygenase-2 induction in HCA-7 human colon cancer cells.

Microbiota produce short chain fatty acids (SCFAs), which are known to maintain gut homeostasis, by the fermentation of dietary fiber in the human colon. Among SCFAs, butyrate has been considered as the most physiologically effective SCFA in colorectal epithelial cells for growth and differentiation. Here we show that the E-type prostanoid 4 (EP4) receptor expression level is regulated by different concentrations of butyrate, but not by other SCFAs, in human colon cancer HCA-7 cells, through sodium-coupled monocarboxylate transporter-1 (SMCT-1)-mediated uptake followed by the activation of histone acetyltransferase: cAMP response element binding protein-binding protein/p300. Of particular interest, the prostanoid EP4 receptors are known to be expressed in normal colorectal crypt epithelial cells and maintain intestinal homeostasis by preserving mucosal integrity, while they are also known to be involved in the early stage of carcinogenesis. Thus, the links between butyrate and the expression of prostanoid EP4 receptors are both important factors for maintaining homeostasis. Based on in silico analysis, almost half of colorectal cancer tissues have lost the expression of SMCT-1 mRNA when compared with healthy corresponding tissues. Therefore, with the collapse of homeostasis systems such as a decrease in the concentration of butyrate in colorectal tissues, or reduced butyrate uptake, there is a possibility of early stage colorectal cancer development; the transformation of normal cells to the cancerous phenotype may be due to the overexpression of prostanoid EP4 receptors followed by excessive cyclooxygenase-2 induction, which are caused by a reduced amount of butyrate and/or its uptake, in/around colorectal epithelial cells.

Translocation and activation of sphingosine kinase 1 by ceramide-1-phosphate.

Sphingosine kinases (SphKs) and ceramide kinase (CerK) phosphorylate sphingosine to sphingosine-1-phosphate (S1P) and ceramide to ceramide-1-phosphate (C1P), respectively. S1P and C1P are bioactive lipids that regulate cell fate/function and human health/diseases. The translocation and activity of SphK1 are regulated by its phosphorylation of Ser 225 and by anionic lipids such as phosphatidic acid and phosphatidylserine. However, the roles of another anionic lipid C1P on SphK1 functions have not yet been elucidated, thus, we here investigated the regulation of SphK1 by CerK/C1P. C1P concentration dependently bound with and activated recombinant human SphK1. The inhibition of CerK reduced the phorbol 12-myristate 13-acetate-induced translocation of SphK1 to the plasma membrane (PM) and activation of the enzyme in membrane fractions of cells. A treatment with C1P translocated wild-type SphK1, but not the SphK1-S225A mutant, to the PM without affecting phosphorylation signaling. A cationic RxRH sequence is proposed to be a C1P-binding motif in α-type cytosolic phospholipase A 2 and tumor necrosis factor α-converting enzyme. The mutation of four cationic amino acids to Ala in the 56-RRNHAR-61 domain in SphK1 reduced the phorbol 12-myristate 13-acetate- and C1P-induced translocation of SphK1 to the PM, however, the capacity of C1P to bind with and activate SphK1 was not affected by this mutation. In conclusion, C1P modulates SphK1 functions by interacting with multiple sites in SphK1.

Cellular density-dependent increases in HIF-1α compete with c-Myc to down-regulate human EP4 receptor promoter activity through Sp-1-binding region.

The up-regulated expression of E-type prostanoid (EP) 4 receptors has been implicated in carcinogenesis; however, the expression of EP4 receptors has also been reported to be weaker in tumor tissues than in normal tissues. Indeed, EP4 receptors have been suggested to play a role in the maintenance of colorectal homeostasis. This study aimed to examine the underlying mechanisms/reasons for why inconsistent findings have been reported regarding EP4 receptor expression levels in homeostasis and carcinogenesis by focusing on cellular densities. Thus, the human colon cancer HCA-7 cells, which retain some functional features of normal epithelia, and luciferase reporter genes containing wild-type or mutated EP4 receptor promoters were used for elucidating the cellular density-dependent mechanisms about the regulation of EP4 receptor expression. In silico analysis was also utilized for confirming the relevance of the findings with respect to colon cancer development. We here demonstrated that the expression of EP4 receptors was up-regulated by c-Myc by binding to Sp-1 under low cellular density conditions, but was down-regulated under high cellular density conditions via the increase in the expression levels of HIF-1α protein, which may pull out c-Myc and Sp-1 from DNA-binding. The tightly regulated EP4 receptor expression mechanism may be a critical system for maintaining homeostasis in normal colorectal epithelial cells. Therefore, once the system is altered, possibly due to the transient overexpression of EP4 receptors, it may result in aberrant cellular proliferation and transformation to cancerous phenotypes. However, at the point, EP4 receptors themselves and their mediated homeostasis would be no longer required.

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

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-1ß, 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.