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Besides its classical function of bone metabolism regulation, 1alpha, 25-dihydroxyvitamin D3 (1,25(OH) 2 D 3 ), acts on a variety of tissues including the nervous system, where the hormone plays an important role as neuroprotective, antiproliferating and differentiating agent. Sphingolipids are bioactive lipids that play critical and complex roles in regulating cell fate. In the present paper we have investigated whether sphingolipids are involved in the protective action of 1,25(OH) 2 D 3. We have found that 1,25(OH) 2 D 3 prevents amyloid-β peptide (Aβ(1−42)) cytotoxicity both in differentiated SH-SY5Y human neuroblastoma cells and in vivo . In differentiated SH-SY5Y cells, Aβ(1−42) strongly reduces the sphingosine-1-phosphate (S1P)/ceramide (Cer) ratio while 1,25(OH) 2 D 3 partially reverts this effect. 1,25(OH) 2 D 3 reverts also the Aβ(1−42)-induced reduction of sphingosine kinase activity. We have also studied the crosstalk between 1,25(OH) 2 D 3 and S1P signaling pathways downstream to the activation of S1P receptor subtype S1P1. Notably, we found that 1,25(OH) 2 D 3 prevents the reduction of S1P1 expression promoted by Aβ(1−42) and thereby it modulates the downstream signaling leading to ER stress damage (p38MAPK/ATF4). Similar effects were observed by using ZK191784. In addition, chronic treatment with 1,25(OH) 2 D 3 protects from aggregated Aβ(1−42)-induced damage in the CA1 region of the rat hippocampus and promotes cell proliferation in the hippocampal dentate gyrus of adult mice. In conclusion, these results represent the first evidence of the role of 1,25(OH) 2 D 3 and its structural analogue ZK191784 in counteracting the Aβ(1−42) peptide-induced toxicity through the modulation of S1P/S1P1/p38MAPK/ATF4 pathway in differentiated SH-SY5Y cells.

1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), a crucial regulator of calcium/phosphorus homeostasis, has important physiological effects on growth and differentiation in a variety of malignant and non-malignant cells. Synthetic structural hormone analogues, with lower hypercalcemic side effects, are currently under clinical investigation. Sphingolipids appear to be crucial bioactive factors in the control of the cell fate: the phosphorylated forms, sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P), are mitogenic factors, whereas sphingosine and ceramide (Cer) usually act as pro-apoptotic agents. Although many studies correlate S1P function to impaired cell growth, the relevance of C1P/Cer system and its involvement in neuroblastoma cells remain to be clarified. Here, we demonstrated the anti-proliferative effect of 1,25(OH)2D3 as well as of its structural analogues, ZK156979 and ZK191784, in human SH-SY5Y cells, as judged by [3H]thymidine incorporation, cell growth and evaluation of active ERK1/2 levels. The inhibition of ceramide kinase (CerK), the enzyme responsible for C1P synthesis, by specific gene silencing or pharmacological inhibition, drastically reduced cell proliferation. 1,25(OH)2D3 and ZK191784 treatment induced a significant decrease in CerK expression and C1P content, and an increase of Cer. Notably, the treatment of SH-SY5Y cells with ZK159222, antagonist of 1,25(OH)2D3 receptor, trichostatin A, inhibitor of histone deacetylases, and COUP-TFI-siRNA prevented the decrease of CerK expression elicited by 1,25(OH)2D3 supporting the involvement of VDR/COUP-TFI/histone deacetylase complex in CerK regulation. Altogether, these findings provide the first evidence that CerK/C1P axis acts as molecular effector of the anti-proliferative action of 1,25(OH)2D3 and its analogues, thereby representing a new possible target for anti-cancer therapy of human neuroblastoma.

Transient receptor potential canonical (TRPC) channels provide cation and Ca2+ entry pathways, which have important regulatory roles in many physio-pathological processes, including muscle dystrophy. However, the mechanisms of activation of these channels remain poorly understood. Using siRNA, we provide the first experimental evidence that TRPC channel 1 (TRPC1), besides acting as a store-operated channel, represents an essential component of stretch-activated channels in C2C12 skeletal myoblasts, as assayed by whole-cell patch-clamp and atomic force microscopic pulling. The channel's activity and stretch-induced Ca2+ influx were modulated by sphingosine 1-phosphate (S1P), a bioactive lipid involved in satellite cell biology and tissue regeneration. We also found that TRPC1 was functionally assembled in lipid rafts, as shown by the fact that cholesterol depletion resulted in the reduction of transmembrane ion current and conductance. Association between TRPC1 and lipid rafts was increased by formation of stress fibres, which was elicited by S1P and abolished by treatment with the actin-disrupting dihydrocytochalasin B, suggesting a role for cytoskeleton in TRPC1 membrane recruitment. Moreover, TRPC1 expression was significantly upregulated during myogenesis, especially in the presence of S1P, implicating a crucial role for TRPC1 in myoblast differentiation. Collectively, these findings may offer new tools for understanding the role of TRPC1 and sphingolipid signalling in skeletal muscle regeneration and provide new therapeutic approaches for skeletal muscle disorders.

Although sphingosine 1-phosphate (S1P) has been considered a potent regulator of skeletal muscle biology, acting as a physiological anti-mitogenic and prodifferentiating agent, its downstream effectors are poorly known. In the present study, we provide experimental evidence for a novel mechanism by which S1P regulates skeletal muscle differentiation through the regulation of gap junctional protein connexin (Cx) 43. Indeed, the treatment with S1P greatly enhanced Cx43 expression and gap junctional intercellular communication during the early phases of myoblast differentiation, whereas the down-regulation of Cx43 by transfection with short interfering RNA blocked myogenesis elicited by S1P. Moreover, calcium and p38 MAPK-dependent pathways were required for S1P-induced increase in Cx43 expression. Interestingly, enforced expression of mutated Cx43Δ130–136reduced gap junction communication and totally inhibited S1P-induced expression of the myogenic markers, myogenin, myosin heavy chain, caveolin-3, and myotube formation. Notably, in S1P-stimulated myoblasts, endogenous or wild-type Cx43 protein, but not the mutated form, coimmunoprecipitated and colocalized with F-actin and cortactin in a p38 MAPK-dependent manner. These data, together with the known role of actin remodeling in cell differentiation, strongly support the important contribution of gap junctional communication, Cx43 expression and Cx43/cytoskeleton interaction in skeletal myogenesis elicited by S1P.

Infliximab treatment demonstrated clinical and endoscopic benefits in active refractory and fistulizing Crohn's disease. The aim of this research was to investigate the proliferative response of peripheral blood mononuclear cells (PBMC) obtained from patients with active and fistulizing Crohn's disease treated with infliximab therapy. PBMC proliferation and VDR protein levels were also studied when 1,25(OH)2D3 or its analogues (EB 1089, KH 1060) were added to cells cultures. At day 5 of culture, the proliferation of PBMC obtained from patients responsive to the therapy showed a remarkable decrease (about 60%) at T6 (after two infusions) with respect to T0 (before the first infusion). On the contrary, in the unresponsive patient, the proliferative response was four times higher at T6 in comparison with T0. Vitamin D derivatives induced a decrease in cell proliferation higher in responsive patients than in the unresponsive one. Increased VDR levels during therapy were registered only in the unresponsive patient. Our results indicate that PBMC proliferation and VDR expression may be useful indicators to predict the response of patients with Crohn's disease to the infliximab therapy.

We recently demonstrated that skeletal muscle differentiation induced by sphingosine 1-phosphate (S1P) requires gap junctions and transient receptor potential canonical 1 (TRPC1) channels. Here, we searched for the signaling pathway linking the channel activity with Cx43 expression/function, investigating the involvement of the Ca(2+)-sensitive protease, m-calpain, and its targets in S1P-induced C2C12 myoblast differentiation. Gene silencing and pharmacological inhibition of TRPC1 significantly reduced Cx43 up-regulation and Cx43/cytoskeletal interaction elicited by S1P. TRPC1-dependent functions were also required for the transient increase of m-calpain activity/expression and the subsequent decrease of PKCα levels. Remarkably, Cx43 expression in S1P-treated myoblasts was reduced by m-calpain-siRNA and enhanced by pharmacological inhibition of classical PKCs, stressing the relevance for calpain/PKCα axis in Cx43 protein remodeling. The contribution of this pathway in myogenesis was also investigated. In conclusion, these findings provide novel mechanisms by which S1P regulates myoblast differentiation and offer interesting therapeutic options to improve skeletal muscle regeneration.

Crohn's disease (CD) is an inflammatory disease characterized by the activation of the immune system in the gut. Since tumor necrosis factor (TNF-alpha) plays an important role in the initiation and perpetuation of intestinal inflammation in CD, we investigated whether TX 527 [19-nor-14,20-bisepi-23-yne-1,25(OH)(2)D(3)], a Vitamin D analogue, could affect peripheral blood mononuclear cells (PBMC) proliferation and exert an immunosuppressive effect on TNF-alpha production in CD patients, and whether this immunosuppressive action could be mediated by NF-kappaB down-regulation. TX 527 significantly decreased cell proliferation and TNF-alpha levels. On activation, NF-kappaB, rapidly released from its cytoplasmatic inhibitor (IKB-alpha), transmigrates into the nucleus and binds to DNA response elements in gene promoter regions. The activation of NF-kappaB, stimulated by TNF-alpha, and its nuclear translocation together with the degradation of IKB-alpha were blocked by TX 527. At the same time, NF-kappaB protein levels present in cytoplasmic extracts decreased in the presence of TNF-alpha and increased when PBMC were incubated with TX 527. The results of our studies indicate that TX 527 inhibits TNF-alpha mediated effects on PBMC and the activation of NF-kappaB and that its action is mediated by Vitamin D receptor (VDR), which is activated when the cells are stimulated with TX 527.

Background The active form of vitamin D, 1,25(OH) 2 D 3 , exerts important effects on proliferation and differentiation of many cell types, and immunoregulatory activities in particular on T cell-mediated immunity. Aim The aim of this study was to investigate whether KH 1060, a vitamin D analogue, could decrease tumor necrosis factor-alpha (TNF-α) levels in patients with inflammatory bowel disease (IBD). Methods PBMC proliferation was determined by [ 3 H]thymidine incorporation. TNF-α levels were measured by ELISA kit; VDR, Bcl-2 and Bax protein levels with Western blot analysis. Results KH 1060 inhibited PBMC proliferation and decreased TNF-α levels in IBD patients and this effect was synergistic with anti-TNF-α. VDR protein levels were significantly increased by PBMC treatment with KH 1060 or anti-TNF-α or their combination in ulcerative colitis (UC) patients, and decreased in Crohn's disease (CD) patients, treating the cells with KH 1060. In UC patients an increase in Bcl-2 and Bax levels was observed incubating, PBMC with KH 1060 or anti-TNF-α or their combination. In CD patients a slight decrease in Bcl-2 levels was registered when anti-TNF alone or in association with KH 1060 was used. Bax protein levels were slightly increased in the presence of KH 1060 alone or in combination with anti-TNF. Conclusion This study shows that KH 1060 acts as an immunomodulator on PBMC, acting as TNF-α inhibitor. This finding provides strong evidence that vitamin D status could be an important regulator of immunity IBD.

The aim of this study was to investigate whether the vitamin D analogue KH 1060 could exert a suppressive action on Tumor necrosis factor-alpha (TNF-alpha). The chimeric anti-TNF-alpha monoclonal antibody (anti-TNF), alone or in combination with KH 1060, was also used. KH 1060 (0.01, 0.1, 1 nM) significantly inhibited cell proliferation, determined after 5 days by [3H]thymidine incorporation, when peripheral blood mononuclear cells (PBMC), obtained from healthy subjects, were stimulated with phytohaemagglutinin (PHA) and incubated for 24 h in the absence and in the presence of lipopolysaccharide (LPS). In the same experimental conditions, anti-TNF exerted a significant inhibition on PBMC proliferation, at the lowest doses (0.001, 0.01 microg/ml) in the absence of LPS, and at 0.001, 1, 10 microg/ml in its presence. A synergistic inhibition was registered combining KH 1060 and anti-TNF, at well-defined concentrations. 0.1 nM KH 1060 produced a significant decrease in TNF-alpha levels, determined by ELISA, although less remarkable than in the presence of anti-TNF. This decrease was synergistic, associating 0.1 nM KH 1060 and 0.1 microg/ml anti-TNF. VDR protein levels were increased by 0.1 nM KH 1060, 0.1 microg/ml anti-TNF or their combination. The protein levels of two oncogenes, Bax and Bcl-2, remained unchanged, when PBMC were incubated with KH 1060, anti-TNF or their combination in the absence of LPS, while, in its presence, an increase was registered. The demonstrated anti-TNF-alpha effect of KH 1060 may suggest for this compound an immunosuppressive action and the possibility to synergistically act with other drugs.