Syndecan-4 (SDC4) is a ubiquitously expressed, transmembrane proteoglycan bearing heparan sulfate chains. SDC4 is involved in numerous inside-out and outside-in signaling processes, such as binding and sequestration of growth factors and extracellular matrix components, regulation of the activity of the small GTPase Rac1, protein kinase C-alpha, the level of intracellular calcium, or the phosphorylation of focal adhesion kinase. The ability of this proteoglycan to link the extracellular matrix and actin cytoskeleton enables SDC4 to contribute to biological functions like cell adhesion and migration, cell proliferation, cytokinesis, cellular polarity, or mechanotransduction. The multiple roles of SDC4 in tumor pathogenesis and progression has already been demonstrated; therefore, the expression and signaling of SDC4 was investigated in several tumor types. SDC4 influences tumor progression by regulating cell proliferation as well as cell migration by affecting cell-matrix adhesion and several signaling pathways. Here, we summarize the general role of SDC4 in cell migration and tumor cell motility.

Skeletal muscle is constantly renewed in response to injury, exercise, or muscle diseases. Muscle stem cells, also known as satellite cells, are stimulated by local damage to proliferate extensively and form myoblasts that then migrate, differentiate, and fuse to form muscle fibers. The transmembrane heparan sulfate proteoglycan syndecan-4 plays multiple roles in signal transduction processes, such as regulating the activity of the small GTPase Rac1 (Ras-related C3 botulinum toxin substrate 1) by binding and inhibiting the activity of Tiam1 (T-lymphoma invasion and metastasis-1), a guanine nucleotide exchange factor for Rac1. The Rac1-mediated actin remodeling is required for cell migration. Syndecan-4 knockout mice cannot regenerate injured muscle; however, the detailed underlying mechanism is unknown. Here, we demonstrate that shRNA-mediated knockdown of syndecan-4 decreases the random migration of mouse myoblasts during live-cell microscopy. Treatment with the Rac1 inhibitor NSC23766 did not restore the migration capacity of syndecan-4 silenced cells; in fact, it was further reduced. Syndecan-4 knockdown decreased the directional persistence of migration, abrogated the polarized, asymmetric distribution of Tiam1, and reduced the total Tiam1 level of the cells. Syndecan-4 affects myoblast migration via its role in expression and localization of Tiam1; this finding may facilitate greater understanding of the essential role of syndecan-4 in the development and regeneration of skeletal muscle.

Background and purpose: Cell migration is one of the cornerstones of regeneration processes, as it is necessary for wound healing, and also required for embryonic development, immune system activation, or tumor metastasis formation. Skeletal muscle has a special, advanced dynamism that allows it to adapt to various impacts and recover successfully after an injury, exercise, or muscle disease. Satellite stem cells are activated by local damage during muscle regeneration, and after asymmetric division, myoblasts (i.e., activated satellite cells) migrate to the site of injury, differentiate, and fuse to form muscle fibers. Migration of the cells requires cellular polarization, the creation of leading and trailing edges, as well as the proper orientation and positioning of organelles inside the cell. Efficient migration also requires the presence of an asymmetrical front-to-rear calcium (Ca2+) gradient to regulate focal adhesion assembly and actomyosin contractility. The transmembrane proteoglycan syndecan-4 (SDC4), which is one of the cell surface markers of resting and activated satellite stem cells, is involved in the formation of focal adhesions. Furthermore, SDC4 plays a variety of roles in signal transduction processes, including controlling the function of the small GTPase Rac1 by binding to and inhibiting the activity of T-lymphoma invasion and metastasis-1 (Tiam1), a guanine nucleotide exchange factor for Rac1 (Ras-related C3 botulinum toxin substrate 1) GTPase. Cell migration also requires Rac1-mediated actin remodeling. SDC4 knockout mice are unable to regenerate damaged muscle; however, its underlying precise mechanism is unclear; therefore, our aim was to analyze the role of SDC4 in myoblast migration. Experimental approaches: To achieve SDC4 knockdown, C2C12 murine myoblast cells were transfected stably with plasmids expressing short hairpin RNAs (shRNAs) specific for mouse SDC4 (shSDC4#1 and shSDC4#2) or a scrambled target sequence. To study cell migration, time-lapse images were captured at 37 °C and 5% CO2 using a high-content imaging system for single-cell tracking or wound scratch assay was performed. To evaluate the movement of the single cells, the cell nuclei were tracked with ImageJ and CellTracker software. Super-resolution direct stochastic optical reconstruction microscopy (dSTORM) measurements were performed for the nanoscale analysis of the lamellipodial actin network of the migrating cells. To study the intracellular Ca2+ level, Fluo-4 and Fura Red indicators were applied. Immunofluorescence cytochemistry was performed to analyze the distribution of SDC4, Tiam1, centrosomes, FAK (focal adhesion kinase) or GM130 (anti- Golgi matrix protein of 130 kDa) followed by wide-field fluorescence or confocal microscopy. Image analysis was performed with ImageJ. Rac1 was inhibited by NSC23766 treatment during the measurements (50 µM). Key results: Silencing of SDC4 disrupts the correct polarization of migrating mammalian myoblasts. SDC4 knockdown completely abolished the intracellular Ca2+ gradient, abrogated centrosome reorientation, and thus decreased cell motility, demonstrating the role of SDC4 in cell polarity. Additionally, SDC4 exhibited a polarized distribution during migration. SDC4 knockdown cells exhibited decreases in the total movement distance during migration, maximum and vectorial distances from the starting point, as well as average and maximum cell speeds. Analysis of the dSTORM images of SDC4 knockdown cells revealed nanoscale changes in the actin cytoskeletal architecture, such as decreases in the numbers of branches and individual branch lengths in the lamellipodia of the migrating cells. The Rac1 inhibitor NSC23766 did not restore the migration capacity of SDC4 silenced cells; in fact, it reduced it further. SDC4 knockdown decreased the directional persistence of migration, abrogated the polarized, asymmetric distribution of Tiam1, and reduced the total Tiam1 level of the cells. Conclusion: According to our results, SDC4 affects the migration of C2C12 myoblasts and modulates cell polarity by influencing centrosome positioning, intracellular Ca2+ and Tiam1 distribution. These findings may promote greater understanding the essential role of SDC4 in the embryonic development and postnatal regeneration of skeletal muscle. Given the ubiquitous expression and crucial role of SDC4 in cell migration, we conclude that our findings can facilitate understanding the general role of SDC4 during cell migration.