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 Cx43⌬130 -136 reduced 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.

This study tested the hypothesis that 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] plays a role in human umbilical vein endothelial cells (HUVEC) cultures. HUVEC were incubated with 10 or 100 nM 1,25(OH)(2)D(3) for 24 h, in the absence or presence of 40 ng/ml tumor necrosis factor-alpha (TNF-alpha) or 2 ng/ml interleukin-1alpha (IL-1alpha). 1,25(OH)(2)D(3) did not affect HUVEC viability and proliferation, while TNF-alpha, alone or in combination with the hormone, significantly inhibited HUVEC viability. [(3)H]thymidine incorporation in HUVEC treated with TNF-alpha or IL-1alpha significantly decreased, in the absence or in the presence of the hormone, while the levels of vitamin D receptor markedly increased in the presence of 1,25(OH)(2)D(3) alone or associated with TNF-alpha or IL-1alpha, in comparison to the control. The noteworthy increase in protein levels of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) induced by TNF-alpha was significantly decreased after incubation of the cells with 1,25(OH)(2)D(3), this effect not being seen on E-selectin expression. Neither apoptosis nor nuclear translocation of NF-kappaB, induced in HUVEC by TNF-alpha was influenced by 1,25(OH)(2)D(3) treatment.

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1,25(OH)2D3 and EB 1089 inhibit PBMC proliferation, induce apoptosis in PBMC of healthy subjects and IBD patients, and affect ICAM-1 expression on PBMC and on HUVEC cocultured with PBMC, suggesting that the ICAM-1 downregulation could provide a new target for controlling the recruitment of leukocytes at the sites of inflammation in IBD.

Abstract:The effects of AISI 316L austenitic stainless steel, tested in untreated state or subjected to glow-discharge nitriding (at 10 or 20 hPa) and nitriding ϩ post-oxidizing treatments, on human umbilical vein endothelial cells (HUVEC) and on peripheral blood mononuclear cells (PBMC) were evaluated. All the treated samples showed a better corrosion resistance in PBS and higher surface hardness in comparison with the untreated alloy. In HUVEC put in contact for 72 h with the sample types, proliferation and apoptosis decreased and increased, respectively, in the presence of the nitrided ϩ post-oxidized samples, while only slight differences in cytokine (TNF-␣, IL-6, and TGF-␤1) release were registered. Intercellular adhesion molecule-1 (ICAM-1) increased in HUVEC incubated with all the treated samples, while vascular cell adhesion molecule-1 (VCAM-1) and E-selectin increased in the presence of all the sample types. PBMC incubated for 48 h with the samples showed a decrease in proliferation and an increase in apoptosis in the presence of the untreated samples and the nitrided ϩ post-oxidized ones. All the sample types induced a remarkable increase in TNF-␣ and IL-6 release in PBMC culture medium, while only the untreated sample and the nitrided at 10 hPa induced an increase in ICAM-1 expression. In HUVEC cocultured with PBMC, previously put in contact with the treated AISI 316L samples, increased levels of ICAM-1 were detected. In HUVEC coincubated with the culture medium of PBMC, previously put in contact with the samples under study, a noteworthy increase in ICAM-1, VCAM-1, and E-selectin levels was always registered, with the exception of VCAM-1, which was not affected by the untreated sample. In conclusion, even if the treated samples do not show a marked increase in biocompatibility in comparison with the untreated alloy, their higher corrosion resistance may suggest a better performance as the contact with physiological environment becomes longer.

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