Differentiation and proliferation of respiration-deficient human myoblasts

182

163

To characterize the regulatory pathways involved in the inhibition of cell differentiation induced by the impairment of mitochondrial activity, we investigated the relationships occurring between organelle activity and myogenesis using an avian myoblast cell line (QM7). The inhibition of mitochondrial translation by chloramphenicol led to a potent block of myoblast differentiation. Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone and oligomycin, which affect the organelle at different levels, exerted a similar influence. In addition, we provided evidence that this phenomenon was not the result of an alteration in cell viability. Conversely, overexpression of the mitochondrial T3 receptor (p43) stimulated organelle activity and strongly potentiated myoblast differentiation. The involvement of mitochondrial activity in an actual regulation of myogenesis is further supported by results demonstrating that the muscle regulatory gene myogenin, in contrast to CMD1 (chicken MyoD) and myf5, is a specific transcriptional target of mitochondrial activity. Whereas myogenin mRNA and protein levels were down-regulated by chloramphenicol treatment, they were up-regulated by p43 overexpression, in a positive relationship with the expression level of the transgene. We also found that myogenin or CMD1 overexpression in chloramphenicol-treated myoblasts did not restore differentiation, thus indicating that an alteration in mitochondrial activity interferes with the ability of myogenic factors to induce terminal differentiation.Recent studies emphasize that mitochondria, in addition to their well known involvement in the regulation of energy metabolism, are implicated in the regulation of cell growth and differentiation. In particular, mitochondrial events are involved in the preliminary steps of apoptosis (1), and inhibition of mitochondrial activity, either by deleting mtDNA (rho°cells) or by blocking translation in the organelle, has been shown to stop or decrease the proliferation of different cell lines (2-4). Furthermore, the general activity of the organelle, not restricted to energy production, is implicated in such regulation (5, 6). In addition, mitochondrial protein synthesis inhibition is associated with the impairment of differentiation of different cell lines, such as mouse erythroleukemia (7) and mastocytoma cells (8), neurons (9), and human (10), avian (11) or murine myoblasts (12). In agreement with these data, several pathologies are associated with mitochondrial disorders, even if the links between mitochondrial genome rearrangements or activity and pathological symptoms are not always clearly established. Despite these reports, little is known about the molecular mechanisms involved in these regulations. First, the exclusive use of inhibitors of mitochondrial function in previous reports was not fully adapted to demonstrating the occurrence of an actual regulatory pathway involving mitochondrial activity in the regulation of cell differentiation. Second, the nature of the molecular signals underlying the...

Section: Regulation Of the Expression Of A Specific Set Of Nuclear Gesupporting

confidence: 61%

Mitochondrial Activity Is Involved in the Regulation of Myoblast Differentiation through Myogenin Expression and Activity of Myogenic Factors

Rochard¹,

Rodier²,

Casas³

et al. 2000

182

163

“…The control parental L6 GLUT4myc myocytes were maintained for the same time period in normal culture condition. Since the depletion of mtDNA in myoblast treated with EtBr severely impairs the formation of myotube (33), which makes L6 GLUT4myc myocyte differentiation less quantitative and less reproducible, and since L6 GLUT4myc myocytes without differentiation possess GLUT4 recycling compartments, insulin-sensitive glucose uptake and the insulin signaling intermediates similar to primary muscle cells (34 -39), we used L6 GLUT4myc myocytes to study how insulin stimulates glucose utilization. Myocytes were deprived of serum for 5 h prior to all experimental manipulations.…”

Section: Methodsmentioning

confidence: 99%

Depletion of Mitochondrial DNA Causes Impaired Glucose Utilization and Insulin Resistance in L6 GLUT4myc Myocytes

Park

Choi

et al. 2005

Mitochondrial dysfunction contributes to a number of human diseases, such as hyperlipidemia, obesity, and diabetes. The mutation and reduction of mitochondrial DNA (mtDNA) have been suggested as factors in the pathogenesis of diabetes. To elucidate the association of cellular mtDNA content and insulin resistance, we produced L6 GLUT4myc myocytes depleted of mtDNA by long term treatment with ethidium bromide. L6 GLUT4myc cells cultured with 0.2 g/ml ethidium bromide (termed depleted cells) revealed a marked decrease in cellular mtDNA and ATP content, concomitant with a lack of mRNAs encoded by mtDNA. Interestingly, the mtDNA-depleted cells showed a drastic decrease in basal and insulin-stimulated glucose uptake, indicating that L6 GLUT4myc cells develop impaired glucose utilization and insulin resistance. The repletion of mtDNA normalized basal and insulin-stimulated glucose uptake. The mRNA level and expression of insulin receptor substrate (IRS)-1 associated with insulin signaling were decreased by 76 and 90% in the depleted cells, respectively. The plasma membrane (PM) GLUT4 in the basal state was decreased, and the insulin-stimulated GLUT4 translocation to the PM was drastically reduced by mtDNA depletion. Moreover, insulin-stimulated phosphorylation of IRS-1 and Akt2/protein kinase B were drastically reduced in the depleted cells. Those changes returned to control levels after mtDNA repletion. Taken together, our data suggest that PM GLUT4 content and insulin signal pathway intermediates are modulated by the alteration of cellular mtDNA content, and the reductions in the expression of IRS-1 and insulin-stimulated phosphorylation of IRS-1 and Akt2/protein kinase B are associated with insulin resistance in the mtDNA-depleted L6 GLUT4myc myocytes.

“…Inhibition of mitochondrial activity either by deleting mtDNA (rho°cells) or by blocking translation in the organelle has been shown to arrest or decrease proliferation in various cell lines (5)(6)(7)(8)(9). Mitochondrial protein synthesis inhibition is associated with the impairment of differentiation in different cell types, including mouse erythroleukemia (10) and mastocytoma cells (11), neurons (12), and human (13), avian (14), or murine myoblasts (15). The coordination of mitochondrial and nuclear genetic systems in the cell is necessary for proper mitochondrial biogenesis and cellular functioning.…”

mentioning

confidence: 99%

Down-regulation of the Mitochondrial Translation System during Terminal Differentiation of HL-60 cells by 12-O-Tetradecanoyl-1-phorbol-13-acetate

Takeuchi

Ueda

2003

Mitochondrial (mt) biogenesis depends on both the nuclear and mt genomes, and a coordination of these two genetic systems is necessary for proper cell functioning. Little is known about the regulatory mechanisms of mt translation or about the expression of mt translation factors. Here, we studied the expression of mt translation factors during 12-O-tetradecanoyl-1-phorbol-13-acetate (TPA)-induced terminal differentiation of HL-60 cells. For all mt translation factors investigated, mRNA expression was markedly downregulated in a coordinate and specific manner, whereas mRNA levels for the cytoplasmic translation factors showed only a slight reduction. An actinomycin D chase study and nuclear run-on assay revealed that the TPAinduced decrease in mt elongation factor Tu (EF-Tumt) mRNA mainly results from decreased mRNA stability. Polysome analysis showed that there was no significant translational control of mt translation factor (EF-Tumt, ribosomal proteins L7/L12mt and S12mt) mRNA expression during differentiation. Thus, the decreased protein level of one of these mt translation factors (EF-Tumt) simply reflects its decreased mRNA level. It was also demonstrated by pulse labeling of mt translation products that the down-regulation of mt translational activity is actually associated with down-regulated mt translation factor expression during cellular differentiation. Our results illustrate that the regulatory mechanisms of mt translational activity upon terminal differentiation (in response to the growth arrest) is different to that of the cytoplasmic system, where the control of mRNA translational efficiency of major translation factors is the central mechanism for their down-regulation.Mitochondrial (mt) biogenesis depends on both the nuclear and mitochondrial genomes (1, 2). Mitochondrial DNA (mtDNA) 1 is present in 10 3 -10 4 copies per cell and encodes 13proteins, which are critical subunits of the respiratory chain complexes I, III, IV, and V, as well as 2 ribosomal RNAs and 22 transfer RNAs, which are necessary for mitochondrial protein synthesis (3). Nuclear genes encode the majority of the respiratory chain subunits and all protein components necessary for maintenance and expression of mtDNA. Mitochondria play pivotal roles in eukaryotic cells in producing cellular energy and essential metabolites as well as in controlling apoptosis by integrating numerous death signals (4). Recently, evidence has emerged that mitochondria are also implicated in the regulation of cell growth and differentiation. Inhibition of mitochondrial activity either by deleting mtDNA (rho°cells) or by blocking translation in the organelle has been shown to arrest or decrease proliferation in various cell lines (5-9). Mitochondrial protein synthesis inhibition is associated with the impairment of differentiation in different cell types, including mouse erythroleukemia (10) and mastocytoma cells (11), neurons (12), and human (13), avian (14), or murine myoblasts (15). The coordination of mitochondrial and nuclear genetic systems in t...