Spexin Promotes the Proliferation and Differentiation of C2C12 Cells In Vitro—The Effect of Exercise on SPX and SPX Receptor Expression in Skeletal Muscle In Vivo

SPX (spexin) and its receptors GalR2 and GalR3 (galanin receptor subtype 2 and galanin receptor subtype 3) play an important role in the regulation of lipid and carbohydrate metabolism in human and animal fat tissue. However, little is still known about the role of this peptide in the metabolism of muscle. The aim of this study was to determine the impact of SPX on the metabolism, proliferation and differentiation of the skeletal muscle cell line C2C12. Moreover, we determined the effect of exercise on the SPX transduction pathway in mice skeletal muscle. We found that increased SPX, acting via GalR2 and GalR3 receptors, and ERK1/2 phosphorylation stimulated the proliferation of C2C12 cells (p < 0.01). We also noted that SPX stimulated the differentiation of C2C12 by increasing mRNA and protein levels of differentiation markers Myh, myogenin and MyoD (p < 0.01). SPX consequently promoted myoblast fusion into the myotubule (p < 0.01). Moreover, we found that, in the first stage (after 2 days) of myocyte differentiation, GalR2 and GalR3 were involved, whereas in the last stage (day six), the effect of SPX was mediated by the GalR3 isoform. We also noted that exercise stimulated SPX and GalR2 expression in mice skeletal muscle as well as an increase in SPX concentration in blood serum. These new insights may contribute to a better understanding of the role of SPX in the metabolism of skeletal muscle.

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Impact of vitamin D and vitamin D receptor TaqI polymorphism in primary human myoblasts

Endocrine Connections ◽

10.1530/ec-19-0194 ◽

2019 ◽

Vol 8 (7) ◽

pp. 1070-1081

Author(s):

Amarjit Saini ◽

Linda Björkhem-Bergman ◽

Johan Boström ◽

Mats Lilja ◽

Michael Melin ◽

...

Keyword(s):

Vitamin D ◽

Vitamin D Receptor ◽

Cellular Response ◽

Myoblast Differentiation ◽

Differentiation Markers ◽

Vdr Polymorphism ◽

Proliferation And Differentiation ◽

Myoblast Proliferation

The CC genotype of the vitamin D receptor (VDR) polymorphism TaqI rs731236 has previously been associated with a higher risk of developing myopathy compared to TT carriers. However, the mechanistic role of this polymorphism in skeletal muscle is not well defined. The effects of vitamin D on patients genotyped for the VDR polymorphism TaqI rs731236, comparing CC and TT carriers were evaluated. Primary human myoblasts isolated from 4 CC carriers were compared with myoblasts isolated from four TT carriers and treated with vitamin D in vitro. A dose-dependent inhibitory effect on myoblast proliferation and differentiation was observed concurrent with modifications of key myogenic regulatory factors. RNA sequencing revealed a vitamin D dose–response gene signature enriched with a higher number of VDR-responsive elements (VDREs) per gene. Interestingly, the greater the expression of muscle differentiation markers in myoblasts, the more pronounced was the vitamin D-mediated response to suppress genes associated with myogenic fusion and myotube formation. This novel finding provides a mechanistic explanation to the inconsistency regarding previous reports of the role of vitamin D in myoblast differentiation. No effects in myoblast proliferation, differentiation or gene expression were related to CC vs TT carriers. Our findings suggest that the VDR polymorphism TaqI rs731236 comparing CC vs TT carriers did not influence the effects of vitamin D on primary human myoblasts and that vitamin D inhibits myoblast proliferation and differentiation through key regulators of cell cycle progression. Future studies need to employ strategies to identify the primary responses of vitamin D that drive the cellular response towards quiescence.

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004.Control of skeletal muscle cell proliferation and differentiation

Reproduction Fertility and Development ◽

10.1071/srb05abs004 ◽

2005 ◽

Vol 17 (9) ◽

pp. 63

Author(s):

M. Grounds

Keyword(s):

Skeletal Muscle ◽

Cell Proliferation ◽

Multinucleated Cells ◽

Cell Proliferation And Differentiation ◽

Proliferation And Differentiation ◽

The Matrix ◽

Key Issues

Skeletal muscle is formed by mononucleated precursor cells (myoblasts) that cease cell proliferation to start differentiation; this results in fusion between the myoblasts to form multinucleated cells (myotubes) that continue to differentiate (and fuse with more muscle cells) and mature into myofibres. Myogenesis has been widely used as a model to study in vitro factors controlling cell proliferation and differentiation. Condition in vitro may not reflect what happens in the more complex in vivo environment. Some of the key issues are what activates quiescent myoblasts in mature skeletal muscle in vivo, and what controls the switch between proliferation and differentiation? The role of the matrix, and molecules such as MyoD, p53, NFAT and IGF-1 will be considered.

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Ang-(1-7) Protects Skeletal Muscle Function in Aged Mice

10.21203/rs.3.rs-294231/v1 ◽

2021 ◽

Author(s):

Xiaoli Huang ◽

Jiao Song ◽

Yangyang Jiang ◽

Xue Yang ◽

Li Cao ◽

...

Keyword(s):

Skeletal Muscle ◽

Muscle Function ◽

Glucose Transporter ◽

C2c12 Cells ◽

Protective Effects ◽

Skeletal Muscle Function ◽

Aged Mice ◽

Age Related ◽

The Impact

Abstract Background: The angiotensin-converting enzyme 2 (ACE2)/angiotensin 1-7 (Ang-(1-7)) axis has been shown to perform a protective task in the decline of the function of skeletal muscle correlated with the process of aging. In the present investigation, the protective effects of ACE2 in mitigating the age-associated decline of skeletal function and identified the potential underlying molecular mechanism mediating the process have been extensively evaluated.Methods: We measured the expression levels of Ang-(1-7) in C57BL/6J mice of different ages and correlated these levels with measures of skeletal muscle function. Also, we determine the expression of myocyte enhancer factor 2A (MEF2A) were detected in ACE2 knockout (ACE2KO) and correlated with muscle function. We then treated ACE2KO aged mice for 4 weeks with Ang-(1-7) and characterized the levels of MEF2A and skeletal muscle function before and after treatment. We assessed the impact of Ang-(1-7) on the growth and differentiation of C2C12 cells in vitro and assessed changes in the glucose transporter type 4 (Glut4) expression.Results: Aged mice showed reduced skeletal muscle function and levels of Ang-(1-7) expression in comparison to young and middle-aged mice. In ACE2KO mice, skeletal muscle function and MEF2A protein expression were significantly lower than in age-matched WT mice. After 1 month of the treatment of Ang-(1-7), the function of skeletal muscle related to the aged ACE2KO mice improved, however, the expression of MEF2A protein was similar to that in the untreated group. In C2C12 cells, Ang-(1-7) was shown to increased cell growth and differentiation characteristics along with the upregulated expression of Glut4.Conclusions: The axis of ACE2/ Ang-(1-7) has a protective task in skeletal muscle and the administration of exogenous Ang-(1-7) can delay the age-related decline in the functions of skeletal muscle.

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Isoform-specific role of Na/K-ATPase α1 in skeletal muscle

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00275.2017 ◽

2018 ◽

Vol 314 (6) ◽

pp. E620-E629 ◽

Cited By ~ 6

Author(s):

Laura C. Kutz ◽

Shreya T. Mukherji ◽

Xiaoliang Wang ◽

Amber Bryant ◽

Isabel Larre ◽

...

Keyword(s):

Skeletal Muscle ◽

Soleus Muscle ◽

Genetic Manipulation ◽

Muscle Growth ◽

Specific Role ◽

Muscle Performance ◽

Tissue Mass ◽

The Impact

The distribution of Na/K-ATPase α-isoforms in skeletal muscle is unique, with α1 as the minor (15%) isoform and α2 comprising the bulk of the Na/K-ATPase pool. The acute and isoform-specific role of α2 in muscle performance and resistance to fatigue is well known, but the isoform-specific role of α1 has not been as thoroughly investigated. In vitro, we reported that α1 has a role in promoting cell growth that is not supported by α2. To assess whether α1 serves this isoform-specific trophic role in the skeletal muscle, we used Na/K-ATPase α1-haploinsufficient (α1+/−) mice. A 30% decrease of Na/K-ATPase α1 protein expression without change in α2 induced a modest yet significant decrease of 10% weight in the oxidative soleus muscle. In contrast, the mixed plantaris and glycolytic extensor digitorum longus weights were not significantly affected, likely because of their very low expression level of α1 compared with the soleus. The soleus mass reduction occurred without change in total Na/K-ATPase activity or glycogen metabolism. Serum analytes including K+, fat tissue mass, and exercise capacity were not altered in α1+/− mice. The impact of α1 content on soleus muscle mass is consistent with a Na/K-ATPase α1-specific role in skeletal muscle growth that cannot be fulfilled by α2. The preserved running capacity in α1+/− is in sharp contrast with previously reported consequences of genetic manipulation of α2. Taken together, these results lend further support to the concept of distinct isoform-specific functions of Na/K-ATPase α1 and α2 in skeletal muscle.

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Myoblast Adhesion, Proliferation and Differentiation on Human Elastin-Like Polypeptide (HELP) Hydrogels

Journal of Applied Biomaterials & Functional Materials ◽

10.5301/jabfm.5000331 ◽

2017 ◽

Vol 15 (1) ◽

pp. 43-53 ◽

Cited By ~ 2

Author(s):

Paola D'Andrea ◽

Deborah Civita ◽

Michela Cok ◽

Luisa Ulloa Severino ◽

Francesca Vita ◽

...

Keyword(s):

Skeletal Muscle ◽

Extracellular Matrix ◽

Matrix Protein ◽

Myogenic Differentiation ◽

Adhesion Formation ◽

Genetically Engineered ◽

C2c12 Cells ◽

Type Iv ◽

Proliferation And Differentiation

Background The biochemical, mechanical and topographic properties of extracellular matrix are crucially involved in determining skeletal muscle cell morphogenesis, proliferation and differentiation. Human elastin-like polypeptides (HELPs) are recombinant biomimetic proteins designed to mimic some properties of the native matrix protein; when employed as myoblast adhesion substrates, they stimulate in vitro myogenesis. Given the influence that the biophysical properties of extracellular matrix have on skeletal muscle cells, the aim of this work was to investigate the effects of HELP hydrogels on myoblasts’ viability and functions. Methods We recently synthesized a novel polypeptide, HELPc, by fusing the elastin-like backbone to a 41aa sequence present in the α2 chain of type IV collagen, containing two arginyl-glycyl-aspartic acid (RGD) motifs. To obtain hydrogels, the enzymatic cross-linking of the HELPc was accomplished by transglutaminase. Here, we employed both non-cross-linked HELPc glass coatings and cross-linked HELPc hydrogels at different monomer densities, as adhesion substrates for C2C12 cells, used as a myoblast model. Results By comparing cell adhesion, proliferation and differentiation, we revealed several striking differences. Depending on support rigidity, adhesion to HELPc substrates dictated cell morphology, spreading, focal adhesion formation and cytoskeletal organization. Hydrogels greatly stimulated cell proliferation, particularly in low-serum medium, and partially inhibited myogenic differentiation. Conclusions On the whole, the results underline the potential of these genetically engineered polypeptides as a tool for dissecting crucial steps in myogenesis.

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Loss of splicing factor IK impairs normal skeletal muscle development

BMC Biology ◽

10.1186/s12915-021-00980-y ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Hye In Ka ◽

Hyemin Seo ◽

Youngsook Choi ◽

Joohee Kim ◽

Mina Cho ◽

...

Keyword(s):

Skeletal Muscle ◽

Muscle Development ◽

Myogenic Differentiation ◽

Mrna Splicing ◽

C2c12 Cells ◽

Splicing Factor ◽

Skeletal Muscle Development

Abstract Background IK is a splicing factor that promotes spliceosome activation and contributes to pre-mRNA splicing. Although the molecular mechanism of IK has been previously reported in vitro, the physiological role of IK has not been fully understood in any animal model. Here, we generate an ik knock-out (KO) zebrafish using the CRISPR/Cas9 system to investigate the physiological roles of IK in vivo. Results The ik KO embryos display severe pleiotropic phenotypes, implying an essential role of IK in embryonic development in vertebrates. RNA-seq analysis reveals downregulation of genes involved in skeletal muscle differentiation in ik KO embryos, and there exist genes having improper pre-mRNA splicing among downregulated genes. The ik KO embryos display impaired neuromuscular junction (NMJ) and fast-twitch muscle development. Depletion of ik reduces myod1 expression and upregulates pax7a, preventing normal fast muscle development in a non-cell-autonomous manner. Moreover, when differentiation is induced in IK-depleted C2C12 myoblasts, myoblasts show a reduced ability to form myotubes. However, inhibition of IK does not influence either muscle cell proliferation or apoptosis in zebrafish and C2C12 cells. Conclusion This study provides that the splicing factor IK contributes to normal skeletal muscle development in vivo and myogenic differentiation in vitro.

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Role of the 807 C/T Polymorphism of the α2 Gene in Platelet GP Ia Collagen Receptor Expression and Function

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614605 ◽

1999 ◽

Vol 81 (06) ◽

pp. 951-956 ◽

Cited By ~ 59

Author(s):

J. Corral ◽

R. González-Conejero ◽

J. Rivera ◽

F. Ortuño ◽

P. Aparicio ◽

...

Keyword(s):

Venous Thrombosis ◽

Cell Types ◽

Receptor Expression ◽

Case Control Studies ◽

Platelet Surface ◽

Vitro Analysis ◽

And Function ◽

In Vitro Analysis

SummaryThe variability of the platelet GP Ia/IIa density has been associated with the 807 C/T polymorphism (Phe 224) of the GP Ia gene in American Caucasian population. We have investigated the genotype and allelic frequencies of this polymorphism in Spanish Caucasians. The T allele was found in 35% of the 284 blood donors analyzed. We confirmed in 159 healthy subjects a significant association between the 807 C/T polymorphism and the platelet GP Ia density. The T allele correlated with high number of GP Ia molecules on platelet surface. In addition, we observed a similar association of this polymorphism with the expression of this protein in other blood cell types. The platelet responsiveness to collagen was determined by “in vitro” analysis of the platelet activation and aggregation response. We found no significant differences in these functional platelet parameters according to the 807 C/T genotype. Finally, results from 3 case/control studies involving 302 consecutive patients (101 with coronary heart disease, 104 with cerebrovascular disease and 97 with deep venous thrombosis) determined that the 807 C/T polymorphism of the GP Ia gene does not represent a risk factor for arterial or venous thrombosis.

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NLRP7 Promotes Choriocarcinoma Growth and Progression through the Establishment of an Immunosuppressive Microenvironment

Cancers ◽

10.3390/cancers13122999 ◽

2021 ◽

Vol 13 (12) ◽

pp. 2999

Author(s):

Deborah Reynaud ◽

Roland Abi Nahed ◽

Nicolas Lemaitre ◽

Pierre-Adrien Bolze ◽

Wael Traboulsi ◽

...

Keyword(s):

Immune Response ◽

Tumor Cells ◽

Major Gene ◽

Critical Role ◽

Orthotopic Model ◽

Independent Manner ◽

Maternal Immune Response ◽

The Impact

The inflammatory gene NLRP7 is the major gene responsible for recurrent complete hydatidiform moles (CHM), an abnormal pregnancy that can develop into gestational choriocarcinoma (CC). However, the role of NLRP7 in the development and immune tolerance of CC has not been investigated. Three approaches were employed to define the role of NLRP7 in CC development: (i) a clinical study that analyzed human placenta and sera collected from women with normal pregnancies, CHM or CC; (ii) an in vitro study that investigated the impact of NLRP7 knockdown on tumor growth and organization; and (iii) an in vivo study that used two CC mouse models, including an orthotopic model. NLRP7 and circulating inflammatory cytokines were upregulated in tumor cells and in CHM and CC. In tumor cells, NLRP7 functions in an inflammasome-independent manner and promoted their proliferation and 3D organization. Gravid mice placentas injected with CC cells invalidated for NLRP7, exhibited higher maternal immune response, developed smaller tumors, and displayed less metastases. Our data characterized the critical role of NLRP7 in CC and provided evidence of its contribution to the development of an immunosuppressive maternal microenvironment that not only downregulates the maternal immune response but also fosters the growth and progression of CC.

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Increased Expression of Retinol-Binding Protein 4 in Ovarian Endometrioma and Its Possible Role in the Pathogenesis of Endometriosis

International Journal of Molecular Sciences ◽

10.3390/ijms22115827 ◽

2021 ◽

Vol 22 (11) ◽

pp. 5827

Author(s):

Jae Chul Lee ◽

Sung Hoon Kim ◽

Young Sang Oh ◽

Ju Hee Kim ◽

Sa Ra Lee ◽

...

Keyword(s):

Retinol Binding Protein ◽

Endometrial Stromal Cells ◽

Endometrial Cells ◽

Retinol Binding Protein 4 ◽

Ovarian Endometrioma ◽

Proliferation And Differentiation ◽

In Vitro Effects ◽

Ovarian Endometriomas

Although endometriosis is a benign disease characterized by the presence of endometrial tissues outside the uterus, ectopic endometrial cells can exhibit malignant biological behaviors. Retinol-binding protein4 (RBP4) is a novel adipocyte-derived cytokine, which has important roles in regulating insulin sensitivity and energy metabolism. RBP4 is a potent modulator of gene transcription, and acts by directly controlling cell growth, invasiveness, proliferation and differentiation. Here, we evaluated the possible role of RBP4 in the pathogenesis of endometriosis. We compared the levels of RBP4 in the tissues and peritoneal fluid (PF) of women with and without endometriosis and evaluated the in vitro effects of RBP4 on the viability, invasiveness, and proliferation of endometrial stromal cells (ESCs). RBP4 levels were significantly higher in the PF of the women in the endometriosis group than in the controls. RBP4 immunoreactivity was significantly higher in the ovarian endometriomas of women with advanced stage endometriosis than those of controls. In vitro treatment with human recombinant-RBP4 significantly increased the viability, bromodeoxyuridine expression, and invasiveness of ESCs. Transfection with RBP4 siRNA significantly reduced ESC viability and invasiveness. These findings suggest that RBP4 partakes in the pathogenesis of endometriosis by increasing the viability, proliferation and invasion of endometrial cells.

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Phenotypic Susceptibility to Bevirimat in Isolates from HIV-1-Infected Patients without Prior Exposure to Bevirimat

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01784-09 ◽

2010 ◽

Vol 54 (6) ◽

pp. 2345-2353 ◽

Cited By ~ 39

Author(s):

Nicolas A. Margot ◽

Craig S. Gibbs ◽

Michael D. Miller

Keyword(s):

Recombinant Viruses ◽

Gag Polyprotein ◽

New Class ◽

Major Mutation ◽

Hiv Drugs ◽

The Impact ◽

First Time ◽

Hiv 1

ABSTRACT Bevirimat (BVM) is the first of a new class of anti-HIV drugs with a novel mode of action known as maturation inhibitors. BVM inhibits the last cleavage of the Gag polyprotein by HIV-1 protease, leading to the accumulation of the p25 capsid-small peptide 1 (SP1) intermediate and resulting in noninfectious HIV-1 virions. Early clinical studies of BVM showed that over 50% of the patients treated with BVM did not respond to treatment. We investigated the impact of prior antiretroviral (ARV) treatment and/or natural genetic diversity on BVM susceptibility by conducting in vitro phenotypic analyses of viruses made from patient samples. We generated 31 recombinant viruses containing the entire gag and protease genes from 31 plasma samples from HIV-1-infected patients with (n = 21) or without (n = 10) prior ARV experience. We found that 58% of the patient isolates tested had a >10-fold reduced susceptibility to BVM, regardless of the patient's ARV experience or the level of isolate resistance to protease inhibitors. Analysis of mutants with site-directed mutations confirmed the role of the V370A SP1 polymorphism (SP1-V7A) in resistance to BVM. Furthermore, we demonstrated for the first time that a capsid polymorphism, V362I (CA protein-V230I), is also a major mutation conferring resistance to BVM. In contrast, none of the previously defined resistance-conferring mutations in Gag selected in vitro (H358Y, L363M, L363F, A364V, A366V, or A366T) were found to occur among the viruses that we analyzed. Our results should be helpful in the design of diagnostics for prediction of the potential benefit of BVM treatment in HIV-1-infected patients.

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