ABSTRACT
The thiazolidinedione (TZD), rosiglitazone, has previously been found to tyrosine-phosphorylate Cbl and activate Cbl-dependent phosphatidylinositol (PI) 3-kinase and atypical protein kinase Cs (aPKCs) while stimulating glucose transport in 3T3/L1 adipocytes. Presently, the role of Cbl in rosiglitazone action was further assessed in both 3T3/L1 and human adipocytes by expressing Y371F and/or Y731F mutant forms of Cbl that nullified the functionality of canonical pYXXM motifs in Cbl. These mutants diminished the interaction of Cbl with the p85 subunit of PI 3-kinase and inhibited subsequent increases in Cbl-dependent PI 3-kinase activity, aPKC activity, and glucose transport. These mutants also inhibited the interaction of Cbl with Crk, which has been implicated in the activation of other PI 3-kinase-independent signaling factors that have been found to be required during activation of glucose transport by insulin and other agonists. We conclude that pYXXM motifs in Cbl serve to activate PI 3-kinase-dependent and possibly PI 3-kinase-independent pathways that are required for TZD-dependent glucose transport in adipocytes.
Subject(s)
Adipocytes/enzymology , Glucose/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Protein Kinase C/metabolism , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins/physiology , Thiazolidinediones/pharmacology , Ubiquitin-Protein Ligases , 3T3-L1 Cells , Adipocytes/drug effects , Adipocytes/metabolism , Amino Acid Motifs , Animals , Biological Transport/drug effects , Cells, Cultured , Deoxyglucose/antagonists & inhibitors , Deoxyglucose/metabolism , Enzyme Activation/drug effects , Enzyme Activation/genetics , Humans , Insulin/pharmacology , Isoenzymes , Mice , Mutagenesis, Site-Directed , Phosphoinositide-3 Kinase Inhibitors , Protein Binding , Protein Subunits/metabolism , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-cbl , Proto-Oncogene Proteins c-crk , Thiazolidinediones/antagonists & inhibitorsABSTRACT
Articular cartilage exhibits little intrinsic repair capacity, and new tissue engineering approaches are being developed to promote cartilage regeneration using cellular therapies. The goal of this study was to examine the chondrogenic potential of adipose tissue-derived stromal cells. Stromal cells were isolated from human subcutaneous adipose tissue obtained by liposuction and were expanded and grown in vitro with or without chondrogenic media in alginate culture. Adipose-derived stromal cells abundantly synthesized cartilage matrix molecules including collagen type II, VI, and chondroitin 4-sulfate. Alginate cell constructs grown in chondrogenic media for 2 weeks in vitro were then implanted subcutaneously in nude mice for 4 and 12 weeks. Immunohistochemical analysis of these samples showed significant production of cartilage matrix molecules. These findings document the ability of adipose tissue-derived stromal cells to produce characteristic cartilage matrix molecules in both in vitro and in vivo models, and suggest the potential of these cells in cartilage tissue engineering.
