ABSTRACT
BTBD1 is a recently cloned BTB-domain-containing protein particularly expressed in skeletal muscle and interacting with DNA topoisomerase 1 (Topo1), a key enzyme of cell survival. We have previously demonstrated that stable overexpression of a N-terminal truncated BTBD1 inhibited ex vivo myogenesis but not adipogenesis of pluripotent C2C12 cells. Here, BTBD1 expression was studied in three models of cellular differentiation: myogenesis (C2C12 cells), adipogenesis (3T3-L1 cells) and osteogenesis (hMADS cells). BTBD1 mRNA was found to be upregulated during myogenesis. At the opposite, we have not observed BTBD1 upregulation in an altered myogenesis cellular model and we observed a downregulation of BTBD1 mRNA expression in adipogenesis. Interestingly, amounts of Topo1 protein, but not Topo1 mRNA, were found to be modulated at the opposite of BTBD1 mRNA. No variation of BTBD1 expression was measured during osteogenesis. Taken together, these results indicate that BTBD1 mRNA is specifically regulated during myogenic and adipogenic differentiation, in relation with Topo1 expression. Moreover, they corroborate observations made previously with truncated BTBD1 and show that BTBD1 is a key protein of balance between adipogenesis and myogenesis. Finally, a transcriptome analysis gave molecular clues to decipher BTBD1 role, with an emphasis on the involvement in ubiquitin/proteasome degradation pathway.
Subject(s)
Adipogenesis/genetics , DNA-Binding Proteins/metabolism , Muscle Development/genetics , Osteogenesis/genetics , Transcription Factors/metabolism , Animals , Cell Differentiation/genetics , DNA Topoisomerases, Type I/genetics , DNA Topoisomerases, Type I/metabolism , DNA-Binding Proteins/genetics , Gene Deletion , Gene Expression Regulation, Developmental , Humans , Mesoderm/cytology , Mesoderm/metabolism , Mice , Proteasome Endopeptidase Complex/metabolism , RNA, Messenger/metabolism , Transcription Factors/genetics , Transcription, Genetic , Ubiquitin/metabolismABSTRACT
Involvement of mitogen-activated protein (MAPK) in inflammatory bowel disease (IBD) remains enigmatic. We sought to evaluate the expression and activity of p38 and JNK MAPK in IBD and 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis; and the effects of a p38 inhibitor, SB203580, in TNBS colitis. P38 and JNK were quantified in colonic mucosa of 28 IBD patients and 19 controls and in 77 TNBS or control mice treated or not with SB203580. Colitis severity was assessed by survival, macroscopic and microscopic scoring, and molecular markers. Expression and activity of p38 and JNK were similar in IBD patients and controls and not modified by inflammation. In mice, p38 and JNK expression or activity did not increase following the induction of colitis. SB203580 decreased the p38 activity but displayed no clinical nor biological therapeutic effect. In conclusion, these results minimize the role of p38 and JNK in inflammatory colitis and the interest of p38 as a therapeutic target in IBD.
Subject(s)
Colitis, Ulcerative/metabolism , Inflammatory Bowel Diseases/metabolism , JNK Mitogen-Activated Protein Kinases/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Adult , Animals , Blotting, Western , Colitis, Ulcerative/chemically induced , Colitis, Ulcerative/drug therapy , Disease Models, Animal , Enzyme Inhibitors/therapeutic use , Female , Gene Expression , Humans , Imidazoles/therapeutic use , Inflammatory Bowel Diseases/genetics , Interleukin-1/genetics , Intestinal Mucosa/metabolism , Male , Mice , Mice, Inbred BALB C , Middle Aged , Polymerase Chain Reaction , Prognosis , Pyridines/therapeutic use , RNA, Messenger/genetics , Trinitrobenzenesulfonic Acid/toxicity , Tumor Necrosis Factor-alpha/genetics , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitorsABSTRACT
The molecular signalling pathways governing skeletal muscle differentiation remain unclear. Recent work has demonstrated that both the phosphatidylinositol 3-kinase (PI3K)/Akt and p38 pathways play important roles in myogenesis. Here, we describe the interactions between these pathways in C2C12 cells. Overall, our results suggest that Akt acts downstream of p38 in myogenic cell differentiation. Activating the p38 pathway results in the concurrent activation of Akt; conversely, activating Akt does not affect p38. We have analysed Akt messenger RNA and protein levels in a C2C12 cell line stably expressing a dominant negative (DN) form of the p38 activator MKK3. Compared to control cells, this cell line exhibits reduced levels of Akt messenger RNA and total protein. In addition, blocking the p38 pathway during differentiation inhibits Akt activation. Our results show for the first time that p38 can directly affect Akt at the transcriptional level as well as at the protein activation level during myogenic differentiation.
Subject(s)
Muscles/cytology , Muscles/enzymology , Transcriptional Activation , p38 Mitogen-Activated Protein Kinases/physiology , Animals , Blotting, Northern , Cell Differentiation , Cell Line , Enzyme Inhibitors/pharmacology , Genes, Dominant , Immunoblotting , Mice , Models, Biological , Myogenin/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Plasmids/metabolism , Protein Binding , Protein Biosynthesis , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Transcription, Genetic , Transfection , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
The signal transduction pathways connecting cell surface receptors to the activation of muscle-specific promoters and leading to myogenesis are still largely unknown. Recently, a contribution of the p38 mitogen-activated protein kinase (MAPK) pathway to this process was evoked through the use of pharmacological inhibitors. We used several mutants of the kinases composing this pathway to modulate the activity of the muscle-specific myosin light chain and myogenin promoters in C2C12 cells by transient transfections. In addition, we show for the first time, using a stable C2C12 cell line expressing a dominant-negative form of the p38 activator MAPK kinase (MKK)3, that a functional p38 MAPK pathway is indeed required for terminal muscle cell differentiation. The most obvious phenotype of this cell line, besides the inhibition of the activation of p38, is its inability to undergo terminal differentiation. This phenotype is accompanied by a drastic inhibition of cell cycle and myogenesis markers such as p21, p27, MyoD, and troponin T, as well as a profound disorganization of the cytoskeleton.
