Melatonin reduces endothelin-1 expression and secretion in colon cancer cells through the inactivation of FoxO-1 and NF-κß.

Melatonin is an indoleamine that is synthesised from tryptophan under the control of the enzymes arylalkylamine N-acetyltransferase (AA-NAT) and acetylserotonin methyltransferase (ASMT). Melatonin inhibits colon cancer growth in both in vivo and in vitro models; however, a precise mechanism responsible for inhibiting tumour growth has not been clearly described. Endothelin-1 (ET-1) is a peptide that acts as a survival factor in colon cancer, inducing cell proliferation, protecting carcinoma cells from apoptosis and promoting angiogenesis. The data presented show that melatonin inhibits edn-1 mRNA expression (the first step in ET-1 synthesis), ECE-1 protein expression and the release of ET-1 from colorectal cancer cells in vitro. ET-1 levels in cultured media present a similar inhibition pattern to that of edn-1 mRNA expression despite the inhibition of ECE-1 protein after melatonin treatment, which suggests that an endopeptidase other than ECE-1 could be mainly responsible for ET-1 synthesis. The inhibition of edn-1 expression is due to an inactivation of FoxO1 and NF-κß transcription factors. FoxO1 inactivation is associated with an increased Src phosphorylation, due to elevated cAMP content and PKA activity, whereas NF-κß inactivation is associated with the blockade of Akt and ERK phosphorylation due to the inhibition of PKC activity after melatonin treatment. Melatonin also inhibits edn-1 promoter activity regulated by FoxO1 and NF-κß. Finally, a significant correlation was observed between AA-NAT and edn-1 expression downregulation in human colorectal cancer tissues. In conclusion, melatonin may be useful in treating colon carcinoma in which the activation of ET-1 plays a role in tumour growth and progression.

Increased CD38 expression in T cells and circulating anti-CD38 IgG autoantibodies differentially correlate with distinct cytokine profiles and disease activity in systemic lupus erythematosus patients.

CD38 is a multifunctional protein possessing ADP-ribosyl cyclase activity responsible for both the synthesis and the degradation of several Ca(2+)-mobilizing second messengers. In mammals, CD38 also functions as a receptor. In this study CD38 expression in CD4(+), CD8(+), or CD25(+) T cells was significantly higher in systemic lupus erythematosus (SLE) patients than in Normal controls. Increased CD38 expression in SLE T cells correlated with plasma levels of Th2 (IL-4, IL-10, IL-13) and Th1 (IL-1ß, IL-12, IFN-γ, TNF-α) cytokines, and was more prevalent in clinically active SLE patients than in Normal controls. In contrast, elevated anti-CD38 IgG autoantibodies were more frequent in clinically quiescent SLE patients (SLEDAI=0) than in Normal controls, and correlated with moderate increased plasma levels of IL-10 and IFN-γ. However, clinically active SLE patients were mainly discriminated from quiescent SLE patients by increased levels of IL-10 and anti-dsDNA antibodies, with odds ratios (ORs) of 3.7 and 4.8, respectively. Increased frequency of anti-CD38 autoantibodies showed an inverse relationship with clinical activity (OR=0.43), and in particular with the frequency of anti-dsDNA autoantibodies (OR=0.21). Increased cell death occurred in CD38(+) Jurkat T cells treated with anti-CD38(+) SLE plasmas, and not in these cells treated with anti-CD38(-) SLE plasmas, or Normal plasmas. This effect did not occur in CD38-negative Jurkat T cells, suggesting that it could be attributed to anti-CD38 autoantibodies. These results support the hypothesis that anti-CD38 IgG autoantibodies or their associated plasma factors may dampen immune activation by affecting the viability of CD38(+) effector T cells and may provide protection from certain clinical SLE features.

Increased expression and phosphorylation of the two S100A9 isoforms in mononuclear cells from patients with systemic lupus erythematosus: a proteomic signature for circulating low-density granulocytes.

Proteins differentially expressed in peripheral blood mononuclear cells (PBMCs) from systemic lupus erythematosus (SLE) patients versus Normal controls were identified by 2-DE and MALDI-MS. Thus, S100A9 expression was significantly increased in SLE PBMCs relative to Normal PBMCs at both mRNA and protein levels. Increased S100A9 levels in SLE PBMCs correlated positively with the abnormal presence of low-density granulocytes (LDGs) detected by flow-cytometry in the mononuclear cell fractions. Another set of proteins that were differentially expressed in SLE PBMCs formed S100A9-independent clusters, suggesting that these differences in protein expression are in fact reflecting changes in the abundance of specific cell types. In SLE PBMCs spots of the two S100A9 isoforms, S100A9-l and S100A9-s, and their phosphorylated counterparts were identified and confirmed to be phosphorylated at Thr(113) by MS/MS analyses. In addition, the phorbol ester PMA alone or in combination with ionomycin induced a stronger increase in threonine phosphorylation of S100A9 in SLE than in Normal PBMCs, while the same stimuli caused the opposite effect on phosphorylation and activation of Erk1/2, suggesting the existence of an abnormal S100A9 signaling in SLE PBMCs. Therefore, the expansion and activation of LDGs in SLE seems to underlie this prominent S100A9 signature.

Exosomes from human lymphoblastoid B cells express enzymatically active CD38 that is associated with signaling complexes containing CD81, Hsc-70 and Lyn.

Exosome vesicles of endocytic origin are involved in communication between tumor and immune cells. In addition, membrane rafts (MR) may support the sorting of proteins associated with exosomes. CD38 is found at the plasma membrane and in recycling endosomes, which are both redistributed toward the immunological synapse (IS) upon T cell antigen receptor (TCR) engagement. The data of this study provide evidence that CD38 is expressed on the surface of secreted exosomes derived from lymphoblastoid B cells. Exosomic CD38 is associated with the signaling molecules CD81, Hsc-70 and Lyn. Likewise, in MR, CD38 is associated with CD81, CD19, Lyn, Galphai-2, Hsc-70 and actin. Therefore, a high degree of overlap in the pattern of signaling proteins associated with CD38 in exosomes and MR exists. Exosomic and MR CD38, by virtue of these interactions, have signaling potential. Indeed, CD38 is enzymatically active in both exosomes and MR, and CD38 ligation induces Akt/PKB and Erk activation, which is accompanied by increased translocation of CD38 into MR. In conclusion, the present study indicates that CD38 localizes to MR, where it promotes cell signaling, and it is exported out of the cells through the exosome-mediated exocytic pathway, where it may act as an intercellular messenger.

Proteomic analysis of plasma from patients with systemic lupus erythematosus: increased presence of haptoglobin alpha2 polypeptide chains over the alpha1 isoforms.

In the present study plasma samples from 15 systemic lupus erythematosus (SLE) patients and 16 healthy controls of initially unknown haptoglobin (Hp) phenotype were separated by 2-DE, and tryptic digests of the excised Hpalpha polypeptide chain spots were analyzed by MALDI-TOF-MS. Selected tryptic peptides were sequenced by nano-(n)ESI-IT MS/MS. The six major Hp phenotypes were present, although with distinct frequencies in controls and SLE patients. Thus, there were an increased proportion of SLE patients with Hp 2-2, or Hp 2-1S phenotypes. The Hp phenotype distribution resulted in allele frequencies of 0 625 (Hp(2)), 0.281 (Hp(1S)), and 0.093 (Hp(1F)) in healthy controls, correlating fairly well with the allele frequencies of European populations. In contrast, the Hp allele frequencies of the SLE patients were 0.733 (Hp(2)), 0.233 (Hp(1S)), and 0.033 (Hp1(1F)), which clearly indicated an increased frequency of Hp(2), a similar proportion of Hp(1S) and a diminished proportion of Hp(1F) in SLE patients compared with that in healthy controls. Preferential Hpalpha2 expression in SLE patients may contribute to some of the clinical manifestations of the disease such as hypergammaglobulinemia, systemic vasculitis, and cardiovascular disorders.

Increased association of CD38 with lipid rafts in T cells from patients with systemic lupus erythematosus and in activated normal T cells.

In this study we have determined whether there is a relationship between CD38 expression on T cells, its distribution in different membrane microdomains, and T cell activation in SLE patients. The data show that CD38 expression is augmented in ex vivo CD3+, CD4+, CD8+, and CD25+ SLE T cells, which correlates with its increased insolubility in Brij 98 detergent, and its translocation into lipid rafts. Moreover, SLE T cells show an altered CD4:CD8 ratio, which is due to a decreased proportion of CD4+ T cells and a concomitant increase in the proportion of CD8+ T cells. These data are consistent with the increased CD38 expression and lipid raft formation, and the significant reduction in the CD4:CD8 ratio observed in mitogen-stimulated normal T cells as compared with that in ex vivo untouched normal T cells. Increased expression of CD38 in floating rafts from SLE T cells, or from activated normal T cells may modulate TCR signaling by providing or sequestering signaling molecules to the engaged TCR.

CD38 signaling in T cells is initiated within a subset of membrane rafts containing Lck and the CD3-zeta subunit of the T cell antigen receptor.

In this study we present data supporting that most CD38 is pre-assembled in a subset of Brij 98-resistant raft vesicles, which were stable at 37 degrees C, and have relatively high levels of Lck and the CD3-zeta subunit of T cell antigen receptor-CD3 complex in contrast with a Brij 98-soluble pool, where CD38 is associated with CD3-zeta, and Lck is not detected. Our data further indicate that following CD38 engagement, LAT and Lck are tyrosine phosphorylated exclusively in Brij 98-resistant rafts, and some key signaling components translocate into rafts (i.e. Sos and p85-phosphatidylinositol 3-kinase). Moreover, N-Ras results activated within rafts immediately upon CD38 ligation, whereas activated Erk was mainly found in soluble fractions with delayed kinetics respective to Ras activation. Furthermore, full phosphorylation of CD3-zeta and CD3-epsilon only occurs in rafts, whereas partial CD3-zeta tyrosine phosphorylation occurs exclusively in the soluble pool, which correlated with increased levels of c-Cbl tyrosine phosphorylation in the non-raft fractions. Taken together, these results suggest that, unlike the non-raft pool, CD38 in rafts is able to initiate and propagate several activating signaling pathways, possibly by facilitating critical associations within other raft subsets, for example, LAT rafts via its capacity to interact with Lck and CD3-zeta. Overall, these findings provide the first evidence that CD38 operates in two functionally distinct microdomains of the plasma membrane.