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Migration of turkey muscle satellite cells is enhanced by the syndecan-4 cytoplasmic domain through the activation of RhoA

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Abstract

Syndecan-4 (S4) is a cell membrane-associated heparan sulfate proteoglycan that forms oligomers in muscle satellite cells. The S4 oligomers activate protein kinase Cα (PKCα) through the S4 cytoplasmic domain and may regulate the activation of ras homolog gene family member A (RhoA), a signal transduction molecule down-stream of PKCα which is thought to influence cell migration. However, little is known about the function of the S4 cytoplasmic domain in satellite cell migration and RhoA activation. The objective of the current study was to determine the function of S4 and its cytoplasmic domain in cell migration and RhoA activation. To study the objective, clones of S4 and S4 without the cytoplasmic domain (S4C) were used in overexpression studies, and small interference RNAs targeting S4 or RhoA were used in knockdown studies. Satellite cell migration was increased by S4 overexpression, but decreased by the knockdown or deletion of the S4 cytoplasmic domain. The RhoA protein was activated by the overexpression of S4, but not with the deletion of the S4 cytoplasmic domain. The treatment of Rho activator II or the knockdown of RhoA also modulated satellite cell migration. Finally, co-transfection (S4 overexpression and RhoA knockdown) and rescue (the knockdown of S4 and the treatment with Rho activator II) studies demonstrated that S4-mediated satellite cell migration was regulated through the activation of RhoA. The cytoplasmic domain of S4 is required for cell migration and RhoA activation which will affect muscle fiber formation.

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Abbreviations

CHO:

Chinese hamster ovary

CON:

Control

DMEM:

Dulbecco’s modified Eagle’s medium

EGTA:

Ethylene glycol tetraacetic acid

EDTA:

Ethylenediaminetetraacetic acid

FGF2:

Fibroblast growth factor 2

GTP:

Guanosine 5′-triphosphate

GDP:

Guanosine 5′-diphosphate

GTPase:

Guanosine 5′-triphosphatase

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

PBS:

Phosphate-buffered saline

PIP2 :

Phosphatidylinositol-4,5-bisphosphate

PKCα:

Protein kinase C α

PT:

Post-transfection

PVDF:

Polyvinylidene fluoride membrane

RBC2:

Randombred control line 2

RBD:

Rho-binding domain

RhoA:

Ras homolog gene family member A

ROCK:

Rho-associated kinase

RT qPCR:

Real-time quantitative polymerase chain reaction

S4:

Syndecan-4

S4C:

Syndecan-4 without the cytoplasmic domain

SDS:

Sodium dodecyl sulfate

siRNA:

Small interfering RNA

SEM:

Standard error of the mean

TBS:

Tris-buffered saline

TBS-T:

Tris-buffered saline containing Tween 20

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Acknowledgments

This research was supported by state and federal funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University, the National Research Initiative Competitive Grant (no. 2009-35503-05176) to S. G. V. from the USDA National Institute of Food and Agriculture, and the National Institute of Food and Agriculture Multistate Project NC-1184 to S.G.V. and D.C.M.

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Authors and Affiliations

  1. Department of Animal Sciences, Ohio Agricultural Research and Development Center, The Ohio State University, 213 Gerlaugh Hall, 1680 Madison Avenue, Wooster, OH, 44691, USA

    Jonghyun Shin & Sandra G. Velleman

  2. Department of Animal Science, South Dakota State University, Brookings, SD, 57007, USA

    Douglas C. McFarland

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  1. Jonghyun Shin
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  2. Douglas C. McFarland
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  3. Sandra G. Velleman
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Correspondence to Sandra G. Velleman.

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Shin, J., McFarland, D.C. & Velleman, S.G. Migration of turkey muscle satellite cells is enhanced by the syndecan-4 cytoplasmic domain through the activation of RhoA. Mol Cell Biochem 375, 115–130 (2013). https://doi.org/10.1007/s11010-012-1534-1

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