Thiorphan-Induced Survival and Proliferation of Rat Thymocytes by Activation of Akt/Survivin Pathway and Inhibition of Caspase-3 Activity

Abstract

The activity of substance P (SP) in the rat thymus seems to be tightly controlled by its bioavailability. In this study, we provide evidence for the expression of the SP-degrading enzyme, neutral endopeptidase (NEP)/CD10, by rat thymocyte subsets, and we illustrate its involvement in the in vivo SP/neurokinin-1 receptor (NK 1 R)-mediated regulation of thymocyte survival and proliferation. NEP/CD10 was expressed at both mRNA and protein levels on a substantial portion (45.5%) of CD5 ϩ thymocytes, namely on the CD4 ϩ CD8 ϩ (double positive; DP) and CD4 ϩ subsets. Continuous administration of thiorphan, a specific NEP/CD10 inhibitor, by means of miniosmotic pumps, enhanced rat thymocyte preprotachykinin-A (PPT-A) and NK 1 R mRNA expression as well as SP and NK 1 R protein levels in an NK 1 R-dependent manner. Thiorphan increased CD10 ϩ CD4 ϩ and CD10 ϩ DP thymocyte numbers, and an NK 1 R antagonist,, abrogated these stimulatory effects. In addition, the NEP/CD10 inhibitor stimulated interleukin (IL)-2 production, IL-2 receptor ␣ chain expression, and concanavalin A-induced proliferation of CD5 ϩ thymocytes, and it inhibited spontaneous and NK 1 R-dependent thymocyte apoptosis. The thiorphan-protective antiapoptotic and proliferative effects involved the activation of Akt serine-threonine kinase, subsequent up-regulation of survivin mRNA, down-regulation of procaspase-3 mRNA levels, and suppression of caspase-3 activity, which were inhibited by SR140333 and mimicked by exogenous SP administration. Overall, our findings suggest that by controlling SP availability, NEP/CD10 negatively regulates thymocyte homeostasis and development.Development of T lymphocytes is a complex process that depends on both stromal cell interactions and the production of soluble factors such as cytokines, peptide hormones, and neuropeptides locally synthesized or released in the thymic microenvironment (Blalock and Smith, 1985). In the thymus, the concentration of biologically active peptides is maintained by a specialized family of enzymes, the endopeptidases (Naquet and Pierres, 1991).The neutral endopeptidase (NEP)-24.11 (EC 3.4.24.11), also known as enkephalinase, neprilysin, common acute lymphoblastic leukemia antigen, or CD10 (Turner and Tanzawa, 1997), is the prototype of a family of membrane-bound zincdependent endopeptidases that regulate the physiological action of a variety of peptides by lowering their extracellular concentration available for receptor binding. Molecular cloning of NEP/CD10 revealed that it is a type II integral membrane protein consisting of a short NH 2 -terminal cytoplasmic domain and a large extracellular domain that contains the active site of the enzyme. It was first isolated as a major 90 to 100-kDa glycoprotein from the renal brush border membrane of rabbits (Kerr and Kenny, 1974).NEP/CD10 is expressed on early normal T-and B-cell progenitors (Shipp and Look, 1993) and on the majority of acute lymphoblastic leukemias and lymphoid malignancies with immature phenotypes (Ritz et al., 1981). Unfracti...

Akt‐mediated anti‐apoptotic effects of substance P in Anti‐Fas‐induced apoptosis of human tenocytes

“…We show that in human tenocytes SP time-dependently activates Akt, a protein kinase that is well known to facilitate cell survival and to prevent apoptotic cell death 13 . This is in accordance with previous results in other cell types 3 , 28 in which SP also activates Akt. Furthermore, the reduction in Anti-Fas-induced apoptosis of human tenocytes seen after SP treatment in this study (as evident by decreased cleavage of PARP), is effectively blocked with an Akt inhibitor, showing that SP's effect is mediated, at least in part, via Akt.…”

CD5-mediated inhibition of TCR signaling proceeds normally in the absence of SHP-1

“…To the best of our knowledge, this is the first study to examine the relevance of SHP-1 in CD5 signaling, by utilizing primary thymocytes from SHP-1-deficient me v mice. Furthermore, given that CD5 and SHP-1 negatively regulate thymocyte development ( 26 , 47 ), we have utilized an H-Y TCR/CD5-overexpressing mouse model to elucidate the physiological and functional importance of SHP-1 for mediating CD5-induced downregulation of both positive and negative selection.…”

Paradoxical expressions of natriuretic peptide receptor-C and neutral endopeptidase account for C-type natriuretic peptide decline during the progression of experimental obstructive nephropathy

“…Specific primers were synthesized according to the published sequences. The sequences of oligonucleotides and sizes of PCR products were, for CNP, sense: 5'-AACATCCCAGACCGCTCATG-3', antisense: 5'-CAA GAA GGG CTT GTC CAA AGG-3'(73 bp) 9 for NPR-B, sense: 5'-AAC GGG CGC ATT GTG TAT ATC TGC GGC-3', antisense: 5'-TTA TCA CAG GAT GGG TCG TCC AAG TCA-3' (692 bp) 10 for NPR-C, sense: 5'-ATA GTG CGC TAC ATC CAG GGC AGT-3', antisense: 5'-TCC AAA GTA ATC ACC AAT GAC CTC CTG GGT ACC TGC-3' (573 bp) 10 for NEP, sense: 5'-CCC CGC CGG CAT TT-3', antisense: 5'-GCC CCC ATA GTT CAA TGA GTT G-3' (66 bp) 11 for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), sense: 5'-TGT GAG GGA GAT GCT CAG TG-3', antisense: 5'-GGC ATT GCT CTC AAT GAC AA-3' (229 bp). 12 One microgram total RNA from the renal tissue of each rat was reverse transcribed into cDNA with an ExScript RT reagent kit (Takara Biotechnology Co. Ltd, Dalian, China).…”