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.
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.
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 PKCa 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/PKCa 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.
Besides its classical function of bone metabolism regulation, 1alpha, 25-dihydroxyvitamin D3 (1,25(OH)D), 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)D We have found that 1,25(OH)D 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)D partially reverts this effect. 1,25(OH)D reverts also the Aβ(1-42)-induced reduction of sphingosine kinase activity. We have also studied the crosstalk between 1,25(OH)D and S1P signaling pathways downstream to the activation of S1P receptor subtype S1P1. Notably, we found that 1,25(OH)D 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)D 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)D 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.
The complete nucleotide sequence of the Bacillus coagulans plasmid pBC1 was determined. The sequence revealed an open reading frame encoding a polypeptide of 259 amino acids. This open reading frame shows sequence similarity to genes coding for replication-associated proteins in a group of gram-positive bacterial plasmids known to replicate via single-stranded intermediates. A region required for replication in cis, when the intact replicon is supplied in trans, was identified as well. (Difco Laboratories) was supplemented with antibiotics as indicated. Erythromycin (5 ,ug/ml for B. subtilis) and chloramphenicol (12.5 and 5 ,ug/ml for E. coli and B. subtilis, respectively) were used. Transformation of B. subtilis or E. coli competent cells with plasmid DNA and all manipulations of DNA were performed as previously described (3,20). pBC1 plasmid was extracted from B. coagulans Zu196I as previously described (5). The DNA sequence was determined by the dideoxy-chain termination method (26) after different DNA restriction fragments were subcloned in the pGEM4Z plasmid (Promega Biotec, Madison, Wis.) by using the strategy shown in Fig. 1. A T7 DNA polymerase sequencing kit (Pharmacia, Uppsala, Sweden) and SP6 and T7 promoters (Promega) were used for sequencing and as primers, respectively.By examination of possible ORFs, we found only one large frame (Fig. 2) Fig. 2) complementary to the 3' end of 16S rRNA (3'-UCUUUCCUCCAGUAG-5') of B. subtilis (23), at position -9 to -13 from the ATG initiation codon. Potential promoter regions following consensus sequences pointed out by Graves and Rabinowitz (10) were found. The most plausible area for transcriptional initiation seems to be the 5'-TATATC-3' sequence (at positions -33 to -38 from the ATG codon) for the -10 region and the 5'-GTGTTT-3' sequence (at positions -64 to -69 from the ATG codon) for the -35 region. Translation of ORF E resulted in a polypeptide of 259 amino acids with-a predicted molecular mass of 30.3 kDa. The amino acid sequence of ORF E was compared with those of the replication proteins of gram-positive bacterial plasmids such as pFTB14 from Bacillus amyloliquefaciens (24), pC194 (16) and pUB110 from Staphylococcus aureus (21), pLP1 from Lactobacillus plantarum (2k, and pBAA1 from B. subtilis (7) and also with the phage PX174 replication protein by using the CLUSTAL homology search system (14). As shown in Fig. 3, significant homologies were observed between the ORF E product and the proteins involved in gram-positive bacterial plasmid replication. This result strongly suggests that the ORF E-encoded protein is implicated in pBC1 replication; thus, this protein was named RepE. As in other published replication protein sequences, a tyrosine residue is present at position 171. This amino acid was described as the linkage site of the pX174 protein A and the DNA when nicking occurs at the plus-strand origin (29). At position 161 began a sequence (5'-TTTCTTATCTTGATA-3') ( Fig. 2) which was identical to the known plus-strand origin of replication sequences o...
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.
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