Francesca Bini

Regulation of transient receptor potential canonical channel 1 (TRPC1) by sphingosine 1-phosphate in C2C12 myoblasts and its relevance for a role of mechanotransduction in skeletal muscle differentiation.

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 channels 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.

Effects of S1P on skeletal muscle repair/regeneration during eccentric contraction

Skeletal muscle regeneration is severely compromised in the case of extended damage. The current challenge is to find factors capable of limiting muscle degeneration and/or potentiating the inherent regenerative program mediated by a specific type of myoblastic cells, the satellite cells. Recent studies from our groups and others have shown that the bioactive lipid, sphingosine 1‐phosphate (S1P), promotes myoblast differentiation and exerts a trophic action on denervated skeletal muscle fibres. In the present study, we examined the effects of S1P on eccentric contraction (EC)‐injured extensor digitorum longus muscle fibres and resident satellite cells. After EC, skeletal muscle showed evidence of structural and biochemical damage along with significant electrophysiological changes, i.e. reduced plasma membrane resistance and resting membrane potential and altered Na+ and Ca2+ current amplitude and kinetics. Treatment with exogenous S1P attenuated the EC‐induced tissue damage, protecting skeletal muscle fibre from apoptosis, preserving satellite cell viability and affecting extracellular matrix remodelling, through the up‐regulation of matrix metalloproteinase 9 (MMP‐9) expression. S1P also promoted satellite cell renewal and differentiation in the damaged muscle. Notably, EC was associated with the activation of sphingosine kinase 1 (SphK1) and with increased endogenous S1P synthesis, further stressing the relevance of S1P in skeletal muscle protection and repair/regeneration. In line with this, the treatment with a selective SphK1 inhibitor during EC, caused an exacerbation of the muscle damage and attenuated MMP‐9 expression. Together, these findings are in favour for a role of S1P in skeletal muscle healing and offer new clues for the identification of novel therapeutic approaches to counteract skeletal muscle damage and disease.

Functional interaction between TRPC1 channel and connexin-43 protein: a novel pathway underlying S1P action on skeletal myogenesis

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 Ca2+-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.

New signalling pathway involved in the anti-proliferative action of vitamin D3 and its analogues in human neuroblastoma cells. A role for ceramide kinase

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.

Sphingosine-1-phosphate signaling in skeletal muscle cells.

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid regulator of numerous important physiological and pathological processes in mammalian and nonmammalian cells. There are emerging evidence that many cell types can produce and release S1P; therefore, the quantification of its intracellular and extracellular content as well as the activity of sphingosine kinase (SphK), the enzyme responsible of S1P synthesis, is crucial to attribute to the SphK/S1P axis a functional significance in response to many different stimuli and in physiopathological conditions.This chapter describes experimental procedures to measure intracellular S1P formation in skeletal muscle cells and skeletal muscle fibers by using sphingolipid precursors. It also underlines the relevance of measuring S1P production in specific cellular compartments in order to attribute to S1P signaling a role in the biology of skeletal muscle cells.

Vitamin D3 protects against Aβ peptide cytotoxicity in differentiated human neuroblastoma SH- SY5Y cells: A role for S1P1/p38MAPK/ATF4 axis.

&NA; Besides its classical function of bone metabolism regulation, 1alpha, 25‐dihydroxyvitamin D3 (1,25(OH)2D3), 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)2D3. We have found that 1,25(OH)2D3 prevents amyloid‐&bgr; peptide (A&bgr;(1−42)) cytotoxicity both in differentiated SH‐SY5Y human neuroblastoma cells and in vivo. In differentiated SH‐SY5Y cells, A&bgr;(1−42) strongly reduces the sphingosine‐1‐phosphate (S1P)/ceramide (Cer) ratio while 1,25(OH)2D3 partially reverts this effect. 1,25(OH)2D3 reverts also the A&bgr;(1−42)‐induced reduction of sphingosine kinase activity. We have also studied the crosstalk between 1,25(OH)2D3 and S1P signaling pathways downstream to the activation of S1P receptor subtype S1P1. Notably, we found that 1,25(OH)2D3 prevents the reduction of S1P1 expression promoted by A&bgr;(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)2D3 protects from aggregated A&bgr;(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)2D3 and its structural analogue ZK191784 in counteracting the A&bgr;(1−42) peptide‐induced toxicity through the modulation of S1P/S1P1/p38MAPK/ATF4 pathway in differentiated SH‐SY5Y cells. Highlights1,25(OH)2D3 increases the level of the pro‐survival S1P while decreases that of pro‐apoptotic ceramide in SH‐SY5Y cells.The neuroprotective effect of 1,25(OH)2D3 against the &bgr;‐amyloid‐induced toxicity involves the receptor subtype S1P1 signaling.Downstream signaling of S1P1 in 1,25(OH)2D3‐induced neuroprotection involves p38MAPK/ERK/ATF4 axis.1,25(OH)2D3‐analogue ZK191784 mimics hormone action in neuroprotection against &bgr;‐amyloid‐ induced toxicity.