calpain and sphingosine-1-phosphate

In anaplastic thyroid cancer C643 cells, sphingosine 1-phosphate (S1P) attenuates migration by activating the S1P2 receptor and the Rho-ROCK pathway. In the present study, we show that stimulating C643 cells with S1P decreases the expression, secretion and activity of matrix metalloproteinase-2 (MMP2), and to a lesser extent MMP9. Using receptor-specific antagonists, and S1P2 siRNA, we showed that the inhibition of expression of MMP2 is mediated through S1P2. Furthermore, S1P inhibited calpain activity, and inhibiting calpain pharmacologically, inhibited the effect of S1P on MMP2 expression and activity, and on MMP9 activity. S1P treatment increased Rho activity, and by incubating cells with the Rho inhibitor C3 transferase or the ROCK inhibitor Y27632, the S1P-induced inhibition of invasion and MMP2 expression and activity was abolished. We conclude that S1P attenuates the invasion of C643 cells by activating S1P2 and the Rho-ROCK pathway, by decreasing calpain activity, and by decreasing the expression, secretion and activity of MMP2 and, to a lesser extent, MMP9. Our results thus unveil a novel function for the S1P2 receptor in attenuating thyroid cancer cell invasion.

Topics: Amides; Calpain; Cell Line, Tumor; Cell Movement; Gene Expression Regulation, Neoplastic; Humans; Lysophospholipids; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Pyridines; Receptors, Lysosphingolipid; rho-Associated Kinases; Sphingosine; Thyroid Carcinoma, Anaplastic

The bioactive lipid sphingosine-1-phosphate (S1P) has emerged as a potent inducer of cancer cell migration and invasion. Previously, we have shown that S1P induces invasion of ML-1 follicular thyroid cancer cells via S1P receptors 1 and 3 (S1P1,3). Matrix metalloproteinases (MMPs) are zinc-dependent proteolytic enzymes used by cells for degradation of the extracellular matrix during invasion and migration. In the present study, we examined the role of MMP2 and MMP9 for S1P-induced invasion of ML-1 cells, and found that S1P regulates the secretion and activity of MMP2 and MMP9 via S1P1,3. Both pharmacological inhibitors and siRNA knockdown of MMP2 and MMP9 could attenuate S1P-induced invasion. Additionally, we show that calpains and Rac1 mediate S1P-induced secretion of MMP2 and MMP9. In conclusion, MMP2 and MMP9 participate in S1P-evoked follicular ML-1 thyroid cancer cell invasion.

Topics: Adenocarcinoma, Follicular; Calpain; Cell Line, Tumor; Cell Movement; Humans; Lysophospholipids; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; rac1 GTP-Binding Protein; Sphingosine; Thyroid Neoplasms

Endothelial cells normally line the vasculature and remain quiescent. However, these cells can be rapidly stimulated to undergo morphogenesis and initiate new blood vessel formation given the proper cues. This study reports a new mechanism for initiating angiogenic sprout formation that involves vimentin, the major intermediate filament protein in endothelial cells. Initial studies confirmed vimentin was required for sphingosine 1-phosphate (S1P)- and growth factor (GF)-induced endothelial cell invasion, and vimentin was cleaved by calpains during invasion. Calpains were predominantly activated by GF and were required for sprout initiation. Because others have reported membrane type 1-matrix metalloproteinase (MT1-MMP) is required for endothelial sprouting responses, we tested whether vimentin and calpain acted upstream of MT1-MMP. Both calpain and vimentin were required for successful MT1-MMP membrane translocation, which was stimulated by S1P. In addition, vimentin complexed with MT1-MMP in a manner that required both the cytoplasmic domain of MT1-MMP and calpain activation, which increased the soluble pool of vimentin in endothelial cells. Altogether, these data indicate that pro-angiogenic signals converge to activate calpain-dependent vimentin cleavage and increase vimentin solubility, which act upstream to facilitate MT1-MMP membrane translocation, resulting in successful endothelial sprout formation in three-dimensional collagen matrices. These findings help explain why S1P and GF synergize to stimulate robust sprouting in 3D collagen matrices.

Topics: Calpain; Cell Membrane; Cells, Cultured; Endothelial Cells; Humans; Lysophospholipids; Matrix Metalloproteinase 14; Protein Transport; Proteolysis; Solubility; Sphingosine; Vimentin

We have recently reported that the bioactive lipid sphingosine-1-phosphate (S1P), usually signaling proliferation and anti-apoptosis induces neuronal death when generated by sphingosine-kinase2 and when accumulation due to S1P-lyase deficiency occurs. In the present study, we identify the signaling cascade involved in the neurotoxic effect of sphingoid-base phosphates. We demonstrate that the calcium-dependent cysteine protease calpain mediates neurotoxicity by induction of the endoplasmic reticulum stress-specific caspase cascade and activation of cyclin-dependent kinase5 (CDK5). The latter is involved in an abortive reactivation of the cell cycle and also enhances tau phosphorylation. Neuroanatomical studies in the cerebellum document for the first time that indeed neurons with abundant S1P-lyase expression are those, which degenerate first in S1P-lyase-deficient mice. We therefore propose that an impaired metabolism of glycosphingolipids, which are prevalent in the central nervous system, might be linked via S1P, their common catabolic intermediate, to neuronal death.

Topics: Aldehyde-Lyases; Animals; Apoptosis; Calcium; Calpain; Caspase 12; Caspase 9; Cell Cycle; Cerebellum; Cyclin-Dependent Kinase 5; Glycosphingolipids; Lysophospholipids; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitochondria; Nerve Degeneration; Neurons; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine; tau Proteins

The vascular endothelium continually senses and responds to biochemical and mechanical stimuli to appropriately initiate angiogenesis. We have shown previously that fluid wall shear stress (WSS) and sphingosine 1-phosphate (S1P) cooperatively initiate the invasion of human umbilical vein endothelial cells into collagen matrices (Kang, H., Bayless, K. J., and Kaunas, R. (2008) Am. J. Physiol. Heart Circ. Physiol. 295, H2087-2097). Here, we investigated the role of calpains in the regulation of endothelial cell invasion in response to WSS and S1P. Calpain inhibition significantly decreased S1P- and WSS-induced invasion. Short hairpin RNA-mediated gene silencing demonstrated that calpain 1 and 2 were required for WSS and S1P-induced invasion. Also, S1P synergized with WSS to induce invasion and to activate calpains and promote calpain membrane localization. Calpain inhibition results in a cell morphology consistent with reduced matrix proteolysis. Membrane type 1-matrix metalloproteinase (MT1-MMP) has been shown by others to regulate endothelial cell invasion, prompting us to test whether calpain acted upstream of MT1-MMP. S1P and WSS synergistically activated MT1-MMP and induced cell membrane localization of MT1-MMP in a calpain-dependent manner. Calpain activation, MT1-MMP activation and MT1-MMP membrane localization were all maximal with 5.3 dynes/cm(2) WSS and S1P treatment, which correlated with maximal invasion responses. Our data show for the first time that 5.3 dynes/cm(2) WSS in the presence of S1P combine to activate calpains, which direct MT1-MMP membrane localization to initiate endothelial sprouting into three-dimensional collagen matrices.

Topics: Calpain; Cell Membrane; Endothelial Cells; Endothelium, Vascular; Extracellular Matrix; Gene Silencing; Humans; Lysophospholipids; Matrix Metalloproteinase 14; Microscopy, Fluorescence; Neovascularization, Pathologic; Shear Strength; Sphingosine; Stress, Mechanical; Subcellular Fractions; Umbilical Veins

Sphingosine 1-phosphate (S1P) and ceramide have been implicated in both autophagy and apoptosis. However, the roles of these sphingolipid metabolites in the links between these two processes are not completely understood. Depletion of S1P phosphohydrolase-1 (SPP1), which degrades intracellular S1P, induces the unfolded protein response and endoplasmic reticulum stress-induced autophagy (Lépine, S., Allegood, J. C., Park, M., Dent, P., Milstien, S., and Spiegel, S. (2011) Cell Death Differ. 18, 350-361). Surprisingly, however, treatment with doxorubicin, which by itself also induced autophagy, markedly reduced the extent of autophagy mediated by depletion of SPP1. Concomitantly, doxorubicin-induced apoptosis was greatly enhanced by down-regulation of SPP1. Autophagy and apoptosis seemed to be sequentially linked because inhibiting autophagy with 3-methyladenine also markedly attenuated apoptosis. Moreover, silencing Atg5 or the three sensors of the unfolded protein response, IRE1α, ATF6, and PKR-like eIF2α kinase (PERK), significantly decreased both autophagy and apoptosis. Doxorubicin stimulated calpain activity and Atg5 cleavage, which were significantly enhanced in SPP1-depleted cells. Inhibition or depletion of calpain not only suppressed Atg5 cleavage, it also markedly decreased the robust apoptosis induced by doxorubicin in SPP1-deficient cells. Importantly, doxorubicin also increased de novo synthesis of the pro-apoptotic sphingolipid metabolite ceramide. Elevation of ceramide in turn stimulated calpain; conversely, inhibiting ceramide formation suppressed Atg5 cleavage and apoptosis. Hence, doxorubicin switches protective autophagy in SPP1-depleted cells to apoptosis by calpain-mediated Atg5 cleavage.

Topics: Activating Transcription Factor 6; Adenine; Antibiotics, Antineoplastic; Apoptosis; Autophagy; Autophagy-Related Protein 5; Calpain; Cell Line, Tumor; Ceramides; Down-Regulation; Doxorubicin; eIF-2 Kinase; Endoribonucleases; Female; Gene Silencing; Humans; Lysophospholipids; Membrane Proteins; Microtubule-Associated Proteins; Phosphoric Monoester Hydrolases; Protein Serine-Threonine Kinases; Proteolysis; Sphingosine

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.

Topics: Animals; Calpain; Cell Differentiation; Cell Line; Connexin 43; Lysophospholipids; Mice; Muscle Development; Muscle, Skeletal; Myoblasts, Skeletal; Patch-Clamp Techniques; Protein Kinase C-alpha; RNA, Small Interfering; Signal Transduction; Sphingosine; TRPC Cation Channels