Identification and characterization of protein-tyrosine-phosphatases in human skeletal muscle systems.

摘要

Protein tyrosine phosphatases (PTPases) have been implicated in such cellular processes as differentiation, proliferation, and metabolism. In this study, PTPase expression was investigated in human skeletal muscle and during differentiation in a human myoblast-to-myotube cell culture system. The objectives were to identify candidate PTPases whose pattern of expression would suggest selective involvement in differentiated skeletal muscle and to characterize expression of those PTPases that have been implicated in regulation of insulin receptor signal transduction in other tissues.; Following initial cloning of 10 PTPases from human skeletal muscle, expression of 8 of these PTPases was confirmed at the mRNA level in isolated human myoblasts. Three receptor-like PTPases were identified (LAR, PTPα, and PTPσ) along with 5 non-receptor PTPases (PTP1B, TCPTP, CL-6, PTPD1, and PEST). Focusing on those PTPases implicated in tyrosine kinase receptor signal transduction (LAR, PTPα, and PTPσ, and PTP1B), semi-quantitative RT-PCR was used to examine changes in mRNA expression as a function of cell density and during differentiation of human myoblasts to myotubes. Only PTPα mRNA increased with cell density, increasing by 2–2.5 fold as cell density increased from 50% to 100%. In contrast, PTPσ was the PTPase that changed most dramatically during differentiation, with mRNA levels increasing by 4–5 fold as myoblasts fused to myotubes in 2 to 5 days. PTP1B mRNA expression also increased by 100% by 5 days of differentiation.; Protein expression correlated with mRNA levels at most time points. LAR and PTP1B did not increase as a function of either cell density in myoblasts or differentiation to myotubes. In contrast, PTPσ increased modestly with cell density and by 2–2.5 fold with differentiation, in agreement with the sizeable increases in PTPσ mRNA levels.; Cell surface expression of both LAR and PTPσ was confirmed by immunocytochemistry in human myotubes. Analysis of human skeletal muscle also demonstrated abundant expression of LAR. Expression was predominantly in Type 1 slow twitch muscle fibers and appeared to be localized to the T tubule system. Extrapolating from evidence in liver, our results indicate that LAR is a candidate PTPase for regulation of insulin receptor signaling pathway in skeletal muscle. The expression pattern of PTPσ in skeletal muscle indicates that this PTPase also has an increased role during skeletal muscle differentiation or in the terminally differentiated muscle.