High level expression of transfected beta- and gamma-actin genes differentially impacts on myoblast cytoarchitecture

The impact of the human beta- and gamma-actin genes on myoblast cytoarchitecture was examined by their stable transfection into mouse C2 myoblasts. Transfectant C2 clones expressing high levels of human beta-actin displayed increases in cell surface area. In contrast, C2 clones with high levels of human gamma-actin expression showed decreases in cell surface area. The changes in cell morphology were accompanied by changes in actin stress-fiber organization. The beta-actin transfectants displayed well-defined filamentous organization of actin; whereas the gamma-actin transfectants displayed a more diffuse organization of the actin cables. The role of the beta-actin protein in generating the enlarged cell phenotype was examined by transfecting a mutant form of the human beta-actin gene. Transfectant cells were shown to incorporate the aberrant actin protein into stress-fiber-like structures. High level expression of the mutant beta-actin produced decreases in cell surface area and disruption of the actin microfilament network similar to that seen with transfection of the gamma-actin gene. In contrast, transfection of another mutant form of the beta-actin gene which encodes an unstable protein had no impact on cell morphology or cytoarchitecture. These results strongly suggest that it is the nature of the encoded protein that determines the morphological response of the cell. We conclude that the relative gene expression of beta- and gamma-actin is of relevance to the control of myoblast cytoarchitecture. In particular, we conclude that the beta- and gamma-actin genes encode functionally distinct cytoarchitectural information.

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Transfection of nonmuscle beta- and gamma-actin genes into myoblasts elicits different feedback regulatory responses from endogenous actin genes

The Journal of Cell Biology ◽

10.1083/jcb.117.4.787 ◽

1992 ◽

Vol 117 (4) ◽

pp. 787-797 ◽

Cited By ~ 51

Author(s):

C Lloyd ◽

G Schevzov ◽

P Gunning

Keyword(s):

Actin Cytoskeleton ◽

Feedback Regulation ◽

Cytochalasin D ◽

Actin Gene ◽

Down Regulation ◽

Actin Genes ◽

Beta Actin ◽

Actin Mrna ◽

Actin Protein

We have examined the role of feedback-regulation in the expression of the nonmuscle actin genes. C2 mouse myoblasts were transfected with the human beta- and gamma-actin genes. In gamma-actin transfectants we found that the total actin mRNA and protein pools remained unchanged. Increasing levels of human gamma-actin expression resulted in a progressive down-regulation of mouse beta- and gamma-actin mRNAs. Transfection of the beta-actin gene resulted in an increase in the total actin mRNA and protein pools and induced an increase in the levels of mouse beta-actin mRNA. In contrast, transfection of a beta-actin gene carrying a single-point mutation (beta sm) produced a feedback-regulatory response similar to that of the gamma-actin gene. Expression of a beta-actin gene encoding an unstable actin protein had no impact on the endogenous mouse actin genes. This suggests that the nature of the encoded actin protein determines the feedback-regulatory response of the mouse genes. The role of the actin cytoskeleton in mediating this feedback-regulation was evaluated by disruption of the actin network with Cytochalasin D. We found that treatment with Cytochalasin D abolished the down-regulation of mouse gamma-actin in both the gamma- and beta sm-actin transfectants. In contrast, a similar level of increase was observed for the mouse beta-actin mRNA in both control and transfected cells. These experiments suggest that the down-regulation of mouse gamma-actin mRNA is dependent on the organization of the actin cytoskeleton. In addition, the mechanism responsible for the down-regulation of beta-actin may be distinct from that governing gamma-actin. We conclude that actin feedback-regulation provides a biochemical assay for differences between the two nonmuscle actin genes.

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Two-level regulation of cardiac actin gene transcription: muscle-specific modulating factors can accumulate before gene activation.

Molecular and Cellular Biology ◽

10.1128/mcb.6.6.2137 ◽

1986 ◽

Vol 6 (6) ◽

pp. 2137-2148 ◽

Cited By ~ 47

Author(s):

A Minty ◽

H Blau ◽

L Kedes

Keyword(s):

Cell Line ◽

Gene Activation ◽

C2c12 Cell ◽

C2c12 Cells ◽

Actin Gene ◽

High Level Expression ◽

Actin Genes ◽

Cardiac Actin ◽

Specific Factors ◽

High Level

We have previously proposed that the upstream regions of the human cardiac actin gene contain sequences that interact with muscle-specific factors with direct high-level transcription of this gene in differentiated muscle cells. In this study we showed that these factors already accumulate in the dividing myoblasts of the mouse C2C12 cell line before differentiation of the cells. The endogenous cardiac actin gene in the C2C12 line is expressed only at a low level in myoblasts but at a high level when these cells differentiate into multinucleate myotubes. In contrast, human cardiac actin genes stably introduced into C2C12 cells show high-level expression in both myoblasts and myotubes, indicating that the endogenous cardiac actin gene is repressed in myoblasts by a mechanism which does not affect transfected genes. In a second muscle cell line (the rat L8 cell line), the level of expression of transfected cardiac actin genes increases when these cells differentiate into myotubes, paralleling the expression of the endogenous sarcomeric actin genes. We suggest that the level of transcriptional modulating factors is low in L8 myoblasts and increases when these cells differentiate into myotubes. Our results demonstrate that at least two steps are necessary for high-level cardiac actin gene expression: activation of the gene and subsequent modulation of its transcriptional activity. Furthermore, the results indicate that the two regulatory steps can be dissociated and that the factors involved in modulation are distinct from those involved in gene activation.

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Differential regulation of tropomyosin isoform organization and gene expression in response to altered actin gene expression.

The Journal of Cell Biology ◽

10.1083/jcb.121.4.811 ◽

1993 ◽

Vol 121 (4) ◽

pp. 811-821 ◽

Cited By ~ 33

Author(s):

G Schevzov ◽

C Lloyd ◽

D Hailstones ◽

P Gunning

Keyword(s):

Gene Expression ◽

Cell Surface ◽

Surface Area ◽

Stress Fiber ◽

Actin Gene ◽

Cell Surface Area ◽

Protein Levels ◽

Differential Pattern ◽

Myoblast Cells ◽

Twofold Decrease

Phenotypically altered C2 myoblast cells, generated by the stable transfection of human nonmuscle actin genes (Schevzov, G., C. Lloyd, and P. Gunning. 1992. J. Cell Biol. 117:775-786), exhibit a differential pattern of tropomyosin cellular organization and isoform gene expression. The beta-actin transfectants displaying a threefold increase in the cell surface area, showed no significant changes in the pattern of organization of the high M(r) tropomyosin isoform, Tm 2, or the low M(r) tropomyosin isoform, Tm 5. In contrast, the gamma- and beta sm-actin gene transfectants, exhibiting a twofold decrease in the cell surface area, had an altered organization of Tm 2 but not Tm 5. In these actin transfectants, Tm 2 did not preferentially segregate into stress fiber-like structures and the intensity of staining was greatly diminished. Conversely, a well-defined stress fiber-like organization of Tm 5 was observed. The pattern of organization of these tropomyosin isoforms correlated with their expression such that a profound decrease in Tm 2 expression was observed both at the transcript and protein levels, whereas Tm 5 remained relatively unchanged. These results suggest that relative changes in nonmuscle actin gene expression can affect the organization and expression of tropomyosin in an isoform specific manner. Furthermore, this apparent direct link observed between actin and tropomyosin expression suggests that nonpharmacological signals originating in the cytoskeleton can regulate cytoarchitectural gene expression.

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Two-level regulation of cardiac actin gene transcription: muscle-specific modulating factors can accumulate before gene activation

Molecular and Cellular Biology ◽

10.1128/mcb.6.6.2137-2148.1986 ◽

1986 ◽

Vol 6 (6) ◽

pp. 2137-2148

Author(s):

A Minty ◽

H Blau ◽

L Kedes

Keyword(s):

Cell Line ◽

Gene Activation ◽

C2c12 Cell ◽

C2c12 Cells ◽

Actin Gene ◽

High Level Expression ◽

Actin Genes ◽

Cardiac Actin ◽

Specific Factors ◽

High Level

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High-Level Expression of Toll-Like Receptors on Dendritic Cells in Adult Patients with Burns on ≥90% of Total Body Surface Area (TBSA)

Medical Science Monitor ◽

10.12659/msm.897433 ◽

2016 ◽

Vol 22 ◽

pp. 3493-3499

Author(s):

Xu Zhang ◽

Na Li ◽

Yan Meng ◽

Renjing Zhang ◽

Jinjun Bian ◽

...

Keyword(s):

Dendritic Cells ◽

Surface Area ◽

Body Surface Area ◽

Body Surface ◽

Total Body Surface Area ◽

Adult Patients ◽

Toll Like Receptors ◽

High Level Expression ◽

Total Body ◽

High Level

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MiR-26a-5p inhibits GSK3β expression and promotes cardiac hypertrophy in vitro

PeerJ ◽

10.7717/peerj.10371 ◽

2020 ◽

Vol 8 ◽

pp. e10371

Author(s):

Liqun Tang ◽

Jianhong Xie ◽

Xiaoqin Yu ◽

Yangyang Zheng

Keyword(s):

Cell Surface ◽

Cardiac Hypertrophy ◽

Surface Area ◽

Myocardial Cell ◽

Immunofluorescence Staining ◽

Cell Surface Area ◽

Direct Target

Background The role of miR-26a-5p expression in cardiac hypertrophy remains unclear. Herein, the effect of miR-26a-5p on cardiac hypertrophy was investigated using phenylephrine (PE)-induced cardiac hypertrophy in vitro and in a rat model of hypertension-induced hypertrophy in vivo. Methods The PE-induced cardiac hypertrophy models in vitro and vivo were established. To investigate the effect of miR-26a-5p activation on autophagy, the protein expression of autophagosome marker (LC3) and p62 was detected by western blot analysis. To explore the effect of miR-26a-5p activation on cardiac hypertrophy, the relative mRNA expression of cardiac hypertrophy related mark GSK3β was detected by qRT-PCR in vitro and vivo. In addition, immunofluorescence staining was used to detect cardiac hypertrophy related mark α-actinin. The cell surface area was measured by immunofluorescence staining. The direct target relationship between miR-26a-5p and GSK3β was confirmed by dual luciferase report. Results MiR-26a-5p was highly expressed in PE-induced cardiac hypertrophy. MiR-26a-5p promoted LC3II and decreased p62 expression in PE-induced cardiac hypertrophy in the presence or absence of lysosomal inhibitor. Furthermore, miR-26a-5p significantly inhibited GSK3β expression in vitro and in vivo. Dual luciferase report results confirmed that miR-26a-5p could directly target GSK3β. GSK3β overexpression significantly reversed the expression of cardiac hypertrophy-related markers including ANP, ACTA1 and MYH7. Immunofluorescence staining results demonstrated that miR-26a-5p promoted cardiac hypertrophy related protein α-actinin expression, and increased cell surface area in vitro and in vivo. Conclusion Our study revealed that miR-26a-5p promotes myocardial cell autophagy activation and cardiac hypertrophy by regulating GSK3β, which needs further research.

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Sequential activation of alpha-actin genes during avian cardiogenesis: vascular smooth muscle alpha-actin gene transcripts mark the onset of cardiomyocyte differentiation.

The Journal of Cell Biology ◽

10.1083/jcb.107.6.2575 ◽

1988 ◽

Vol 107 (6) ◽

pp. 2575-2586 ◽

Cited By ~ 222

Author(s):

D L Ruzicka ◽

R J Schwartz

Keyword(s):

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Cardiac Cells ◽

Actin Gene ◽

Heart Tube ◽

Specific Expression ◽

Actin Genes ◽

Beta Actin ◽

Smooth Muscle Alpha Actin ◽

Alpha Actin

The expression of cytoplasmic beta-actin and cardiac, skeletal, and smooth muscle alpha-actins during early avian cardiogenesis was analyzed by in situ hybridization with mRNA-specific single-stranded DNA probes. The cytoplasmic beta-actin gene was ubiquitously expressed in the early chicken embryo. In contrast, the alpha-actin genes were sequentially activated in avian cardiac tissue during the early stages of heart tube formation. The accumulation of large quantities of smooth muscle alpha-actin transcripts in epimyocardial cells preceded the expression of the sarcomeric alpha-actin genes. The accumulation of skeletal alpha-actin mRNAs in the developing heart lagged behind that of cardiac alpha-actin by several embryonic stages. At Hamburger-Hamilton stage 12, the smooth muscle alpha-actin gene was selectively down-regulated in the heart such that only the conus, which subsequently participates in the formation of the vascular trunks, continued to express this gene. This modulation in smooth muscle alpha-actin gene expression correlated with the beginning of coexpression of sarcomeric alpha-actin transcripts in the epimyocardium and the onset of circulation in the embryo. The specific expression of the vascular smooth muscle alpha-actin gene marks the onset of differentiation of cardiac cells and represents the first demonstration of coexpression of both smooth muscle and striated alpha-actin genes within myogenic cells.

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Molecular cloning and characterization of mutant and wild-type human beta-actin genes

Molecular and Cellular Biology ◽

10.1128/mcb.4.10.1961-1969.1984 ◽

1984 ◽

Vol 4 (10) ◽

pp. 1961-1969

Author(s):

J Leavitt ◽

P Gunning ◽

P Porreca ◽

S Y Ng ◽

C S Lin ◽

...

Keyword(s):

Homologous Recombination ◽

Human Fibroblasts ◽

Actin Gene ◽

Actin Genes ◽

Beta Actin ◽

Gene Libraries ◽

Actin Mrna ◽

Dna Fragment ◽

Specific Sequences

There are more than 20 beta-actin-specific sequences in the human genome, many of which are pseudogenes. To facilitate the isolation of potentially functional beta-actin genes, we used the new method of B. Seed (Nucleic Acids Res. 11:2427-2446, 1983) for selecting genomic clones by homologous recombination. A derivative of the pi VX miniplasmid, pi AN7 beta 1, was constructed by insertion of the 600-base-pair 3' untranslated region of the beta-actin mRNA expressed in human fibroblasts. Five clones containing beta-actin sequences were selected from an amplified human fetal gene library by homologous recombination between library phage and the miniplasmid. One of these clones contained a complete beta-actin gene with a coding sequence identical to that determined for the mRNA of human fibroblasts. A DNA fragment consisting of mostly intervening sequences from this gene was then used to identify 13 independent recombinant copies of the analogous gene from two specially constructed gene libraries, each containing one of the two types of mutant beta-actin genes found in a line of neoplastic human fibroblasts. The amino acid and nucleotide sequences encoded by the unmutated gene predict that a guanine-to-adenine transition is responsible for the glycine-to-aspartic acid mutation at codon 244 and would also result in the loss of a HaeIII site. Detection of this HaeIII polymorphism among the fibroblast-derived clones verified the identity of the beta-actin gene expressed in human fibroblasts.

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