Fibroblast growth factor 2 and protein kinase C alpha are involved in syndecan-4 cytoplasmic domain modulation of turkey myogenic satellite cell proliferation Yan Song a , Douglas C. McFarland b , Sandra G. Velleman a, ⁎ a Department of Animal Sciences, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, OH 44691, USA b Department of Animal and Range Sciences, South Dakota State University, Brookings, SD 57007, USA abstract article info Article history: Received 8 June 2011 Received in revised form 2 September 2011 Accepted 4 September 2011 Available online 10 September 2011 Keywords: Glycosaminoglycan Muscle N-linked glycosylated chain Satellite cell Syndecan-4 cytoplasmic domain Turkey Syndecan-4 core protein is composed of extracellular, transmembrane, and cytoplasmic domains. The cytoplas- mic domain functions in transmitting signals into the cell through the protein kinase C alpha (PKCα) pathway. The glycosaminoglycan (GAG) and N-linked glycosylated (N-glycosylated) chains attached to the extracellular domain influence cell proliferation. The current study investigated the function of syndecan-4 cytoplasmic domain in combination with GAG and N-glycosylated chains in turkey muscle cell proliferation, differentiation, fibroblast growth factor 2 (FGF2) responsiveness, and PKCα membrane localization. Syndecan-4 or syndecan-4 without the cytoplasmic domain and with or without the GAG and N-glycosylated chains were transfected or co-transfected with a small interfering RNA targeting syndecan-4 cytoplasmic domain into turkey muscle satellite cells. The over- expression of syndecan-4 mutants increased cell proliferation but did not change differentiation. Syndecan-4 mu- tants had increased cellular responsiveness to FGF2 during proliferation. Syndecan-4 increased PKCα cell membrane localization, whereas the syndecan-4 mutants decreased PKCα cell membrane localization compared to syndecan-4. However, compared to the cells without transfection, syndecan-4 mutants increased cell membrane localization of PKCα. These data indicated that the syndecan‐4 cytoplasmic domain and the GAG and N-glycosylated chains are critical in syndecan-4 regulating satellite cell proliferation, responsiveness to FGF2, and PKCα cell mem- brane localization. © 2011 Elsevier Inc. All rights reserved. 1. Introduction Muscle growth and development can be divided into 2 periods, hyper- plasia and hypertrophy. Hyperplasia occurs during the embryonic period and is characterized by an increase in muscle fiber number. Hypertrophy happens after birth or hatch and is characterized by the enlargement of existing muscle fibers. Satellite cells are responsible for hypertrophy by proliferating, differentiating, and donating their nuclei to adjacent muscle fibers (Moss and Leblond, 1970). The increased number of nuclei in muscle fibers leads to increased protein synthesis and muscle fiber size enlargement. Satellite cells are myogenic stem cells that are located between the sarcolemma and basement membrane (Mauro, 1961). The activity of satellite cells is critical for muscle growth and development. Fibroblast growth factor 2 (FGF2), a strong stimulator of cell proliferation and in- hibitor of cell differentiation (Dollenmeier et al., 1981) and the extra- cellular environment are two important factors that regulate the activity of satellite cells (Velleman, 1999). The extracellular matrix (ECM) is composed of proteins and polysaccharides which is secreted by cells and can influence cell behavior by transducing signals into the cell. Proteoglycans are a major group of molecules in the ECM. According to the types of glycosaminoglycans (GAG) attached to the core protein, proteoglycans can be divided into four groups, heparan sulfate, derma- tan sulfate, keratan sulfate, and chondroitin sulfate proteoglycans. The heparan sulfate proteoglycans are involved in cell to substratum adhe- sion (LeBaron et al., 1988; Haugen et al., 1992; Sanderson et al., 1992; Morgan et al., 2007; Farach-Carson et al., 2008), cell to cell adhesion (Cole et al., 1986; Reyes et al., 1990; Stanley et al., 1995), and signaling mediated by growth factors and cell adhesion molecules. Syndecans are a group of membrane-associated proteoglycans. There are four syndecans expressed in mammals, syndecan-1 through -4. Syndecan-1 and syndecan-3 belong to one subfamily whereas syn- decan-2 and syndecan-4 belong to another, based on sequence Comparative Biochemistry and Physiology, Part A 161 (2012) 44–52 Abbreviations: BSA, bovine serum albumin; DMEM, Dulbecco's modified eagle medium; ECM, extracellular matrix; FGF2, fibroblast growth factor 2; GAG, glycos- aminoglycan; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; N-glycosylated, N-linked glycosylated; PBS, phosphate buffered saline; pCMS, pCMS-EGFP; PIP 2 , phosphati- dylinositol 4,5-bisphosphate; PKCα, protein kinase C alpha; S4, wild-type syndecan-4; S4C, syndecan-4 without cytoplasmic domain; S4C-N0, syndecan-4 without cytoplasmic domain and N-linked glycosylated chains; S4C-S0, syndecan-4 without cytoplasmic domain and glycosaminoglycan chains; S4C-S0N0, syndecan-4 without cytoplasmic domain, N- linked glycosylated chains, and glycosaminoglycan chains; siRNA, small interfering RNA; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; SEM, standard error of the mean; TBS-T, Tris-buffered saline. ⁎ Corresponding author at: Department of Animal Science, Ohio Agricultural Re- search and Development Center, The Ohio State University, 1680 Madison Avenue, Wooster, OH 44691, USA. Tel.: +1 330 263 3905; fax: +1 330 263 3949. E-mail addresses: song.249@osu.edu (Y. Song), douglas.mcfarland@sdstate.edu (D.C. McFarland), Velleman.1@osu.edu (S.G. Velleman). 1095-6433/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2011.09.001 Contents lists available at SciVerse ScienceDirect Comparative Biochemistry and Physiology, Part A journal homepage: www.elsevier.com/locate/cbpa