ABSTRACT
Desmin is the main intermediate filament (IF) protein of muscle cells. In skeletal muscle, desmin IFs form a scaffold that interconnects the entire contractile apparatus with the subsarcolemmal cytoskeleton and cytoplasmic organelles. The interaction between desmin and the sarcolemma is mediated by a number of membrane proteins, many of which are Ca2+-sensitive. In the present study, we analyzed the effects of the Ca2+ chelator EGTA (1.75 mM) on the expression and distribution of desmin in C2C12 myoblasts grown in culture. We used indirect immunofluorescence microscopy and reverse transcription polymerase chain reaction (RT-PCR) to analyze desmin distribution and expression in C2C12 cells grown in the presence or absence of EGTA. Control C2C12 myoblasts showed a well-spread morphology after a few hours in culture and became bipolar when grown for 24 h in the presence of EGTA. Control C2C12 cells showed a dense network of desmin from the perinuclear region to the cell periphery, whereas EGTA-treated cells showed desmin aggregates in the cytoplasm. RT-PCR analysis revealed a down-regulation of desmin expression in EGTA-treated C2C12 cells compared to untreated cells. The present results suggest that extracellular Ca2+ availability plays a role in the regulation of desmin expression and in the spatial distribution of desmin IFs in myoblasts, and is involved in the generation and maintenance of myoblast cell shape.
Subject(s)
Calcium/metabolism , Cell Shape/physiology , Desmin/metabolism , Intermediate Filaments/metabolism , Muscle, Skeletal/chemistry , Myoblasts/physiology , Animals , Chelating Agents/pharmacology , Desmin/drug effects , Desmin/genetics , Down-Regulation , Egtazic Acid/pharmacology , Extracellular Matrix , Intermediate Filaments/drug effects , Mice , Microscopy, Fluorescence , Rabbits , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Desmin is the main intermediate filament (IF) protein of muscle cells. In skeletal muscle, desmin IFs form a scaffold that interconnects the entire contractile apparatus with the subsarcolemmal cytoskeleton and cytoplasmic organelles. The interaction between desmin and the sarcolemma is mediated by a number of membrane proteins, many of which are Ca2+-sensitive. In the present study, we analyzed the effects of the Ca2+ chelator EGTA (1.75 mM) on the expression and distribution of desmin in C2C12 myoblasts grown in culture. We used indirect immunofluorescence microscopy and reverse transcription polymerase chain reaction (RT-PCR) to analyze desmin distribution and expression in C2C12 cells grown in the presence or absence of EGTA. Control C2C12 myoblasts showed a well-spread morphology after a few hours in culture and became bipolar when grown for 24 h in the presence of EGTA. Control C2C12 cells showed a dense network of desmin from the perinuclear region to the cell periphery, whereas EGTA-treated cells showed desmin aggregates in the cytoplasm. RT-PCR analysis revealed a down-regulation of desmin expression in EGTA-treated C2C12 cells compared to untreated cells. The present results suggest that extracellular Ca2+ availability plays a role in the regulation of desmin expression and in the spatial distribution of desmin IFs in myoblasts, and is involved in the generation and maintenance of myoblast cell shape.
Subject(s)
Animals , Mice , Rabbits , Calcium/metabolism , Cell Shape/physiology , Desmin/metabolism , Intermediate Filaments/metabolism , Muscle, Skeletal/chemistry , Myoblasts/physiology , Chelating Agents/pharmacology , Down-Regulation , Desmin/drug effects , Desmin/genetics , Extracellular Matrix , Egtazic Acid/pharmacology , Intermediate Filaments/drug effects , Microscopy, Fluorescence , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Hepatitis C treatment with interferon alpha-2b (IFN-alpha) and ribavirin has been related to decreased bone mineral density. The aim of this study was to investigate the in vitro effects of different concentrations of ribavirin and IFN-alpha on osteoblast-like cells. Human osteoblast-like cells obtained by the outgrowth of cells from bone chips were exposed to ribavirin (0.1-10 microg/mL) or IFN-alpha (0.1-1000 UI/mL). At regular time-points, cultures were harvested for posterior analysis. Alkaline phosphatase (ALP) activity was determined on days 7 and 14, and cell growth was accessed by C3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and cell count on days 1, 3, 5, and 7. Flow cytometry analysis was used for investigating cell death on days 1, 3, 5, and 7. IFN-alpha affected ALP expression only at the higher concentration (1000 UI/mL) after 7 days (P < 0.05). No effects were detected in cell growth. In ribavirin treated cultures, concentrations higher than 2.5 microg/mL were associated with a decrease in ALP activity within 7 and 14 days (P < 0.01 and P < 0.001, respectively). Furthermore, the reduction in cell growth was dose-dependent and was detected after the fifth day. This decrease can be explained by an increase in the number of dead cells and a decrease in cell proliferation. In conclusion, our experiments demonstrated that ribavirin reduced, in a time- and dose-dependent manner, the number of metabolically active cells through a decrease in proliferation and an increase in cell death, and induced an impairment in osteoblast differentiation. These negative effects of ribavirin on osteblast-like cells might contribute to the bone loss reported in vivo.
