Molecular analysis of fiber type-specific expression of murine myostatin promoter

2004 ◽  
Vol 287 (4) ◽  
pp. C1031-C1040 ◽  
Author(s):  
Mônica Senna Salerno ◽  
Mark Thomas ◽  
Davanea Forbes ◽  
Trevor Watson ◽  
Ravi Kambadur ◽  
...  
Keyword(s):  
Promoter Activity ◽  
Fiber Type ◽  
Muscle Growth ◽  
Quadriceps Muscle ◽  
Negative Regulator ◽  
Specific Expression ◽  
Myostatin Gene ◽  
E Boxes

Myostatin is a negative regulator of muscle growth, and absence of the functional myostatin protein leads to the heavy muscle phenotype in both mouse and cattle. Although the role of myostatin in controlling muscle mass is established, little is known of the mechanisms regulating the expression of the myostatin gene. In this study, we have characterized the murine myostatin promoter in vivo. Various constructs of the murine myostatin promoter were injected into the quadriceps muscle of mice, and the reporter luciferase activity was analyzed. The results indicate that of the seven E-boxes present in the 2.5-kb fragment of the murine myostatin promoter, the E5 E-box plays an important role in the regulation of promoter activity in vivo. Furthermore, the in vitro studies demonstrated that MyoD preferentially binds and upregulates the murine myostatin promoter activity. We also analyzed the activity of the bovine and murine promoters in murine skeletal muscle and showed that, despite displaying comparable levels of activity in murine myoblast cultures, bovine myostatin promoter activity is much weaker than murine myostatin promoter in mice. Finally, we demonstrate that in vivo, the 2.5-kb region of the murine myostatin promoter is sufficient to drive the activity of the reporter gene in a fiber type-specific manner.

Regulation of myostatin expression and myoblast differentiation by FoxO and SMAD transcription factors

2007 ◽  
Vol 292 (1) ◽  
pp. C188-C199 ◽  
Author(s):  
David L. Allen ◽  
Terry G. Unterman
Keyword(s):  
Transcription Factors ◽  
Promoter Activity ◽  
Transforming Growth Factor ◽  
Active Form ◽  
Muscle Growth ◽  
Myoblast Differentiation ◽  
Binding Assays ◽  
Myostatin Gene

Myostatin, a member of the transforming growth factor (TGF)-β family, plays an important role in regulating skeletal muscle growth and differentiation. Here we examined the role of FoxO1 and SMAD transcription factors in regulating myostatin gene expression and myoblast differentiation in C2C12 myotubes in vitro. Both myostatin and FoxO1 mRNA expression were greater in fast- vs. slow-twitch skeletal muscles in vivo. Moreover, expression of a constitutively active form of FoxO1 increased myostatin mRNA and increased activity of a myostatin promoter reporter construct in differentiated C2C12 myotubes. Mutagenesis of highly conserved FoxO or SMAD binding sites significantly decreased myostatin promoter activity, and binding assays showed that both FoxO1 and SMADs bind to their respective sites in the myostatin promoter. Treatment with TGF-β and/or overexpression of SMAD2, -3, or -4 also resulted in a significant increase in myostatin promoter activity. Treatment with TGF-β along with overexpression of SMAD2 and FoxO1 resulted in the largest increase in myostatin promoter activity. Finally, TGF-β treatment and SMAD2 overexpression greatly potentiated FoxO1-mediated suppression of myoblast differentiation. Together these data demonstrate that FoxO1 and SMAD transcription factors regulate the expression of myostatin and contribute to the control of muscle cell growth and differentiation.

scholarly journals The β-cell specific transcription factor Nkx6.1 inhibits glucagon gene transcription by interfering with Pax6

2007 ◽  
Vol 403 (3) ◽  
pp. 593-601 ◽  
Author(s):  
Benoit R. Gauthier ◽  
Yvan Gosmain ◽  
Aline Mamin ◽  
Jacques Philippe
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Gene Transcription ◽  
Promoter Activity ◽  
Islet Cells ◽  
Gene Activation ◽  
Physical Interaction ◽  
Specific Expression

The transcription factor Nkx6.1 is required for the establishment of functional insulin-producing β-cells in the endocrine pancreas. Overexpression of Nkx6.1 has been shown to inhibit glucagon gene expression while favouring insulin gene activation. Down-regulation resulted in the opposite effect, suggesting that absence of Nkx6.1 favours glucagon gene expression. To understand the mechanism by which Nkx6.1 suppresses glucagon gene expression, we studied its effect on the glucagon gene promoter activity in non-islet cells using transient transfections and gel-shift analyses. In glucagonoma cells transfected with an Nkx6.1-encoding vector, the glucagon promoter activity was reduced by 65%. In BHK21 cells, Nkx6.1 inhibited by 93% Pax6-mediated activation of the glucagon promoter, whereas Cdx2/3 and Maf stimulations were unaltered. Although Nkx6.1 could interact with both the G1 and G3 element, only the former displayed specificity for Nkx6.1. Mutagenesis of the three potential AT-rich motifs within the G1 revealed that only the Pax6-binding site preferentially interacted with Nkx6.1. Chromatin immunoprecipitation confirmed interaction of Nkx6.1 with the glucagon promoter and revealed a direct competition for binding between Pax6 and Nkx6.1. A weak physical interaction between Pax6 and Nkx6.1 was detected in vitro and in vivo suggesting that Nkx6.1 predominantly inhibits glucagon gene transcription through G1-binding competition. We suggest that cell-specific expression of the glucagon gene may only proceed when Nkx6.1, in combination with Pdx1 and Pax4, are silenced in early α-cell precursors.

scholarly journals Androgens induce growth of the limb skeletal muscles in a rapamycin-insensitive manner

2018 ◽  
Vol 315 (4) ◽  
pp. R721-R729 ◽  
Author(s):  
Michael L. Rossetti ◽  
David H. Fukuda ◽  
Bradley S. Gordon
Keyword(s):  
Skeletal Muscle ◽  
Fiber Type ◽  
Muscle Growth ◽  
Total Protein Content ◽  
Cross Sectional ◽  
S6 Kinase ◽  
Mechanistic Target Of Rapamycin ◽  
Ribosomal S6 Kinase

Signaling through the mechanistic target of rapamycin complex 1 (mTORC1) has been well defined as an androgen-sensitive transducer mediating skeletal muscle growth in vitro; however, this has yet to be tested in vivo. As such, male mice were subjected to either sham or castration surgery and allowed to recover for 7 wk to induce atrophy of skeletal muscle. Then, castrated mice were implanted with either a control pellet or a pellet that administered rapamycin (~2.5 mg·kg−1·day−1). Seven days postimplant, a subset of castrated mice with control pellets and all castrated mice with rapamycin pellets were given once weekly injections of nandrolone decanoate (ND) to induce muscle growth over a six-week period. Effective blockade of mTORC1 by rapamycin was noted in the skeletal muscle by the inability of insulin to induce phosphorylation of ribosomal S6 kinase 1 70 kDa (Thr389) and uncoordinated-like kinase 1 (Ser757). While castration reduced tibialis anterior (TA) mass, muscle fiber cross-sectional area, and total protein content, ND administration restored these measures to sham levels in a rapamycin-insensitive manner. Similar findings were also observed in the plantaris and soleus, suggesting this rapamycin-insensitive effect was not specific to the TA or fiber type. Androgen-mediated growth was not due to changes in translational capacity. Despite these findings in the limb skeletal muscle, rapamycin completely prevented the ND-mediated growth of the heart. In all, these data indicate that mTORC1 has a limited role in the androgen-mediated growth of the limb skeletal muscle; however, mTORC1 was necessary for androgen-mediated growth of heart muscle.

scholarly journals A novel mechanism of myostatin regulation by its alternative splicing variant during myogenesis in avian species

2015 ◽  
Vol 309 (10) ◽  
pp. C650-C659 ◽  
Author(s):  
Sangsu Shin ◽  
Yan Song ◽  
Jinsoo Ahn ◽  
Eunsoo Kim ◽  
Paula Chen ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Muscle Development ◽  
Muscle Growth ◽  
Avian Species ◽  
Negative Regulator ◽  
Cell Proliferation And Differentiation ◽  
Exon 1 ◽  
Splicing Isoforms

Myostatin ( MSTN) is a key negative regulator of muscle growth and development, and an increase of muscle mass is achieved by inhibiting MSTN signaling. In the current study, five alternative splicing isoforms of MSTN mRNAs in avian species were identified in various tissues. Among these five, three truncated forms of myostatin, MSTN-B, -C, and -E created premature stop codons and produced partial MSTN prodomains encoded from exon 1. MSTN-B is the second dominant isoform following full-length MSTN-A, and their expression was dynamically regulated during muscle development of chicken, turkey, and quail in vivo and in vitro. To clarify the function of MSTN-B, two stable cell lines of quail myoblasts (QM7) were generated to overexpress MSTN-A or MSTN-B. Interestingly, MSTN-B promoted both cell proliferation and differentiation similar to the function of the MSTN prodomain to counteract the negative role of MSTN on myogenesis. The coimmunoprecipitation assay revealed that MSTN-B binds to MSTN-A and reduces the generation of mature MSTN. Furthermore, the current study demonstrated that the partial prodomain encoded from exon 1 is critical for binding of MSTN-B to MSTN-A. Altogether, these data imply that alternative splicing isoforms of MSTN could negatively regulate pro-myostatin processing in muscle cells and prevent MSTN-mediated inhibition of myogenesis in avian species.

Probing the 14-3-3 Isoform-Specificity Profile of Interactions Stabilized by Fusicoccin A

2020 ◽  
Author(s):  
James Frederich ◽  
Ananya Sengupta ◽  
Josue Liriano ◽  
Ewa A. Bienkiewicz ◽  
Brian G. Miller
Keyword(s):  
Protein Interactions ◽  
Neurological Diseases ◽  
Adapter Proteins ◽  
Protein Protein Interactions ◽  
Specific Expression ◽  
Individual Client ◽  
Isoform Specificity ◽  
Fusicoccin A

Fusicoccin A (FC) is a fungal phytotoxin that stabilizes protein–protein interactions (PPIs) between 14-3-3 adapter proteins and their phosphoprotein interaction partners. In recent years, FC has emerged as an important chemical probe of human 14-3-3 PPIs implicated in cancer and neurological diseases. These previous studies have established the structural requirements for FC-induced stabilization of 14-3-3·client phosphoprotein complexes; however, the effect of different 14-3-3 isoforms on FC activity has not been systematically explored. This is a relevant question for the continued development of FC variants because there are seven distinct isoforms of 14-3-3 in humans. Despite their remarkable sequence and structural similarities, a growing body of experimental evidence supports both tissue-specific expression of 14-3-3 isoforms and isoform-specific functions <i>in vivo</i>. Herein, we report the isoform-specificity profile of FC <i>in vitro</i>using recombinant human 14-3-3 isoforms and a focused library of fluorescein-labeled hexaphosphopeptides mimicking the C-terminal 14-3-3 recognition domains of client phosphoproteins targeted by FC in cell culture. Our results reveal modest isoform preferences for individual client phospholigands and demonstrate that FC differentially stabilizes PPIs involving 14-3-3s. Together, these data provide strong motivation for the development of non-natural FC variants with enhanced selectivity for individual 14-3-3 isoforms.

Non-Viral Vectors for Gene Delivery

2018 ◽  
Vol 9 (1) ◽  
pp. 4-11 ◽  
Author(s):  
Aparna Bansal ◽  
Himanshu
Keyword(s):  
Gene Therapy ◽  
Viral Vectors ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Expression System ◽  
Target Cells ◽  
Specific Expression ◽  
Naked Dna

Introduction: Gene therapy has emerged out as a promising therapeutic pave for the treatment of genetic and acquired diseases. Gene transfection into target cells using naked DNA is a simple and safe approach which has been further improved by combining vectors or gene carriers. Both viral and non-viral approaches have achieved a milestone to establish this technique, but non-viral approaches have attained a significant attention because of their favourable properties like less immunotoxicity and biosafety, easy to produce with versatile surface modifications, etc. Literature is rich in evidences which revealed that undoubtedly, non–viral vectors have acquired a unique place in gene therapy but still there are number of challenges which are to be overcome to increase their effectiveness and prove them ideal gene vectors. Conclusion: To date, tissue specific expression, long lasting gene expression system, enhanced gene transfection efficiency has been achieved with improvement in delivery methods using non-viral vectors. This review mainly summarizes the various physical and chemical methods for gene transfer in vitro and in vivo.

scholarly journals Overexpression of the Oral Mucosa-Specific microRNA-31 Promotes Skin Wound Closure

2019 ◽  
Vol 20 (15) ◽  
pp. 3679 ◽  
Author(s):  
Lin Chen ◽  
Alyne Simões ◽  
Zujian Chen ◽  
Yan Zhao ◽  
Xinming Wu ◽  
...  
Keyword(s):  
Wound Healing ◽  
Oral Mucosa ◽  
Wound Closure ◽  
Expression Patterns ◽  
Skin Wound ◽  
Skin Wound Healing ◽  
Specific Expression ◽  
Keratinocyte Proliferation

Wounds within the oral mucosa are known to heal more rapidly than skin wounds. Recent studies suggest that differences in the microRNAome profiles may underlie the exceptional healing that occurs in oral mucosa. Here, we test whether skin wound-healing can be accelerating by increasing the levels of oral mucosa-specific microRNAs. A panel of 57 differentially expressed high expresser microRNAs were identified based on our previously published miR-seq dataset of paired skin and oral mucosal wound-healing [Sci. Rep. (2019) 9:7160]. These microRNAs were further grouped into 5 clusters based on their expression patterns, and their differential expression was confirmed by TaqMan-based quantification of LCM-captured epithelial cells from the wound edges. Of these 5 clusters, Cluster IV (consisting of 8 microRNAs, including miR-31) is most intriguing due to its tissue-specific expression pattern and temporal changes during wound-healing. The in vitro functional assays show that ectopic transfection of miR-31 consistently enhanced keratinocyte proliferation and migration. In vivo, miR-31 mimic treatment led to a statistically significant acceleration of wound closure. Our results demonstrate that wound-healing can be enhanced in skin through the overexpression of microRNAs that are highly expressed in the privileged healing response of the oral mucosa.

scholarly journals Transcriptome analysis of gravitational effects on mouse skeletal muscles under microgravity and artificial 1 g onboard environment

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Risa Okada ◽  
Shin-ichiro Fujita ◽  
Riku Suzuki ◽  
Takuto Hayashi ◽  
Hirona Tsubouchi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Muscle Mass ◽  
Muscle Atrophy ◽  
Transcriptome Analysis ◽  
Fiber Type ◽  
Expression Profiles ◽  
Space Station ◽  
Gene Expression Profiles

AbstractSpaceflight causes a decrease in skeletal muscle mass and strength. We set two murine experimental groups in orbit for 35 days aboard the International Space Station, under artificial earth-gravity (artificial 1 g; AG) and microgravity (μg; MG), to investigate whether artificial 1 g exposure prevents muscle atrophy at the molecular level. Our main findings indicated that AG onboard environment prevented changes under microgravity in soleus muscle not only in muscle mass and fiber type composition but also in the alteration of gene expression profiles. In particular, transcriptome analysis suggested that AG condition could prevent the alterations of some atrophy-related genes. We further screened novel candidate genes to reveal the muscle atrophy mechanism from these gene expression profiles. We suggest the potential role of Cacng1 in the atrophy of myotubes using in vitro and in vivo gene transductions. This critical project may accelerate the elucidation of muscle atrophy mechanisms.

scholarly journals TGFβ signalling acts as a molecular brake of myoblast fusion

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Julie Melendez ◽  
Daniel Sieiro ◽  
David Salgado ◽  
Valérie Morin ◽  
Marie-Julie Dejardin ◽  
...  
Keyword(s):  
Dynamic Control ◽  
Muscle Growth ◽  
Myoblast Fusion ◽  
In Vivo Studies ◽  
In Vitro Assays ◽  
Receptor Complex ◽  
Tgfβ Signalling ◽  
Molecular Brake

AbstractFusion of nascent myoblasts to pre-existing myofibres is critical for skeletal muscle growth and repair. The vast majority of molecules known to regulate myoblast fusion are necessary in this process. Here, we uncover, through high-throughput in vitro assays and in vivo studies in the chicken embryo, that TGFβ (SMAD2/3-dependent) signalling acts specifically and uniquely as a molecular brake on muscle fusion. While constitutive activation of the pathway arrests fusion, its inhibition leads to a striking over-fusion phenotype. This dynamic control of TGFβ signalling in the embryonic muscle relies on a receptor complementation mechanism, prompted by the merging of myoblasts with myofibres, each carrying one component of the heterodimer receptor complex. The competence of myofibres to fuse is likely restored through endocytic degradation of activated receptors. Altogether, this study shows that muscle fusion relies on TGFβ signalling to regulate its pace.

scholarly journals Estrogen Up-Regulates Hepatic Expression of Suppressors of Cytokine Signaling-2 and -3 in Vivo and in Vitro

Endocrinology ◽  
2004 ◽  
Vol 145 (12) ◽  
pp. 5525-5531 ◽  
Author(s):  
Gary M. Leong ◽  
Sofia Moverare ◽  
Jesena Brce ◽  
Nathan Doyle ◽  
Klara Sjögren ◽  
...  
Keyword(s):  
Promoter Activity ◽  
Hepatoma Cells ◽  
Cytokine Signaling ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Wild Type ◽  
Hepatic Expression ◽  
Suppressors Of Cytokine Signaling

Abstract Suppressors of cytokine signaling (SOCS) are important negative regulators of cytokine action. We recently reported that estrogen stimulates SOCS-2 expression and inhibits GH signaling in kidney cells. The effects of estrogen on SOCS expression in other tissues are unclear. The aim of this study was to investigate in vivo and in vitro whether estrogen affected SOCS expression in the liver, a major target organ of GH. The in vivo hepatic effects of estrogen on ovariectomized mice lacking estrogen receptor (ER)-α, ERβ, or both and their wild-type littermates were examined by DNA microarray analysis. In vitro, the effects of estrogen on SOCS expression in human hepatoma cells were examined by reverse transcription quantitative PCR. Long-term (3 wk) estrogen treatment induced a 2- to 3-fold increase in hepatic expression of SOCS-2 and -3 in wild-type and ERβ knockout mice but not in those lacking ERα or both ER subtypes. Short-term treatment (at 24 h) increased the mRNA level of SOCS-3 but not SOCS-2. In cultured hepatoma cells, estrogen increased SOCS-2 and -3 mRNA levels by 2-fold in a time- and dose-dependent manner (P &lt; 0.05). Estrogen induced murine SOCS-3 promoter activity by 2-fold (P &lt; 0.05) in constructs containing a region between nucleotides −1862 and −855. Moreover, estrogen and GH had additive effects on the SOCS-3 promoter activity. In summary, estrogen, via ERα, up-regulated hepatic expression of SOCS-2 and -3, probably through transcriptional activation. This indicates a novel mechanism of estrogen regulation of cytokine action.

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