Myoblast Migration and Directional Persistence Affected by Syndecan-4-Mediated Tiam-1 Expression and Distribution

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

Syndecan-4 affects myogenesis via Rac1-mediated actin remodeling and exhibits copy-number amplification and increased expression in human rhabdomyosarcoma tumors

Skeletal muscle demonstrates a high degree of regenerative capacity repeating the embryonic myogenic program under strict control. Rhabdomyosarcoma is the most common sarcoma in childhood and is characterized by impaired muscle differentiation. In this study, we observed that silencing the expression of syndecan-4, the ubiquitously expressed transmembrane heparan sulfate proteoglycan, significantly enhanced myoblast differentiation, and fusion. During muscle differentiation, the gradually decreasing expression of syndecan-4 allows the activation of Rac1, thereby mediating myoblast fusion. Single-molecule localized superresolution direct stochastic optical reconstruction microscopy (dSTORM) imaging revealed nanoscale changes in actin cytoskeletal architecture, and atomic force microscopy showed reduced elasticity of syndecan-4-knockdown cells during fusion. Syndecan-4 copy-number amplification was observed in 28% of human fusion-negative rhabdomyosarcoma tumors and was accompanied by increased syndecan-4 expression based on RNA sequencing data. Our study suggests that syndecan-4 can serve as a tumor driver gene in promoting rabdomyosarcoma tumor development. Our results contribute to the understanding of the role of syndecan-4 in skeletal muscle development, regeneration, and tumorigenesis

The phosphomimetic mutation of syndecan-4 binds and inhibits Tiam1 modulating Rac1 activity in PDZ interaction-dependent manner

The small GTPases of the Rho family comprising RhoA, Rac1 and Cdc42 function as molecular switches controlling several essential biochemical pathways in eukaryotic cells. Their activity is cycling between an active GTP-bound and an inactive GDP-bound conformation. The exchange of GDP to GTP is catalyzed by guanine nucleotide exchange factors (GEFs). Here we report a novel regulatory mechanism of Rac1 activity, which is controlled by a phosphomimetic (Ser179Glu) mutant of syndecan-4 (SDC4). SDC4 is a ubiquitously expressed transmembrane, heparan sulfate proteoglycan. In this study we show that the Ser179Glu mutant binds strongly Tiam1, a Rac1-GEF reducing Rac1-GTP by 3-fold in MCF-7 breast adenocarcinoma cells. Mutational analysis unravels the PDZ interaction between SDC4 and Tiam1 is indispensable for the suppression of the Rac1 activity. Neither of the SDC4 interactions is effective alone to block the Rac1 activity, on the contrary, lack of either of interactions can increase the activity of Rac1, therefore the Rac1 activity is the resultant of the inhibitory and stimulatory effects. In addition, SDC4 can bind and tether RhoGDI1 (GDP-dissociation inhibitor 1) to the membrane. Expression of the phosphomimetic SDC4 results in the accumulation of the Rac1-RhoGDI1 complex. Co-immunoprecipitation assays (co-IP-s) reveal that SDC4 can form complexes with RhoGDI1. Together, the regulation of the basal activity of Rac1 is fine tuned and SDC4 is implicated in multiple ways

The activity of Rac1 is modulated by SDC4.

<p>(A) The level of active Rac1-GTP was monitored in MCF-7 cells expressing different SDC4 constructs by pull-down assay employing CRIB domain of Pak1. The Ser179Glu cells contained the least Rac1-GTP among the cell lines. Administration of non-hydrolysable GTP-γS equalized the amount of pulled-down Rac1, while the high access of GDP eliminated it. Rac1 and GFP were used as loading control. Bar diagrams represent the quantification of the ratio of Rac1-GTP and the endogenous Rac1 normalized to MCF-7 cells. According to this the Rac1 activity of the control cells was reduced 3-fold in the Ser179Glu cells and was increased by 15% in the ΔPDZ cells. In the 4th slab SDC4 was probed on the CRIB-based Rac1 assay. In the 5th panel Rac1 was tested in GFP co-IPs. The pattern is similar to that of Rac1 received in the Rac-assay. MCF-7 line served as a negative control. The results of panel A are representatives of at least 5 independent experiments; data are reported as mean ± SEM (n = 5). (B) Pak1 kinase was probed in the SDC4 co-IPs. The most abundant Pak1 was detected in the Ser179Glu mutant cells. Pak1 was used as loading control.</p

Schematic representation of the assumed mechanism of the regulation of Rac1 activity by SDC4.

<p>The activation cycle of Rac1 is regulated by several factors. GEFs catalyze the exchange of GDP for GTP. The intrinsic GTPase activity can be accelerated by GAPs. The GDIs can extract Rac1 from the membrane keeping it mostly in GDP-bound form in the cytosol. The red arrows show the potential regulatory sites of the phosphomimetic SDC4, 1) inhibiting Tiam1 a Rac1-GEF and 2) inducing the accumulation of the cytosolic Rac1-RhoGDI1 complex.</p

Schematic representation of the assumed mechanism of the inhibition of Tiam1 by phosphomimetic SDC4.

<p>(A) The only connection of SDC4 to Tiam1 via PDZ binding site or Glu¹⁷⁹ does not interfere with Tiam1 activity; however the simultaneous interactions block the enzyme activity. (B) Structure of Tiam1 contains two pleckstrin homology domains (PHn and PHc). Rac1 binding site is located between the PHn and PHc domains. In pull down experiment PH domain was identified as interaction site, and PHn was shown to regulate binding of GTPases [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187094#pone.0187094.ref010" target="_blank">10</a>] thus we suppose that phosphomimetic SDC4 can interact with the PDZ and PHn domains simultaneously to exclude Rac1.</p

SDC4 controls the activity of Tiam1.

<p>(A) Schematic representation of SDC4 clones. The Ser179 of the cytoplasmic domain of SDC4 was mutated to non-phosphorylatable Ala (Ser179Ala) or, phosphomimetic Glu (Ser179Glu), respectively. The type II PDZ binding site was deleted in the Ser179 mutants generating ΔPDZ constructs. (B) Co-IPs were carried out with anti-Tiam1 antibody from the lysates of the different SDC4 lines and the subsequent immunoblot was probed with anti-GFP-HRP (GFP::SDC4) or anti-Rac1 antisera, respectively. Tiam1, Rac1 and GFP::SDC4 were used as loading controls. The strongest interactions were detected in the Ser179Glu and ΔPDZ-Ser179Glu cell lines. The Rac1 was greatly reduced in Tiam1 co-IP in the lysates of Ser179Glu cells only. The bar diagrams show the relative amount of the ectopic SDC4 normalized to wt-SDC4 cells, and pulled-down Rac1 normalized to MCF-7 cells. The results of panel B are representatives at least 3 independent experiments; data are reported as mean ± SEM (n = 3). (C) The protein level of Tiam1 and the Rac1 activity was monitored in non-treated MCF7 cells and upon transfection with scrambled sequence of Tiam1 shRNA construct, administration of NSC23766, transfection with Tiam1 shRNA, and in Ser179Glu SDC4 cells, respectively. Rac1 was used as loading control. Bar diagrams represent the quantification of the level of Tiam1 protein normalized to the nt MCF-7 cells and the ratio of Rac1-GTP and the endogenous Rac1 normalized to MCF-7 cells. The Tiam1 shRNA reduced the expression of Tiam1, and the Rac1 activity, too. The administration of NSC23766 and the expression of Ser179Glu SDC4 decreased the activity of Rac1 solely, the level of Tiam1 was unchanged. The use of the scrambled sequence of Tiam1 shRNA did not influence the expression level of Tiam1 and the Rac1-GTP either.</p

Pituitary Adenylate Cyclase Activating Polypeptide Has Inhibitory Effects on Melanoma Cell Proliferation and Migration In Vitro

Pituitary adenylate cyclase activating polypeptide (PACAP) is an endogenous neuropeptide which is distributed throughout the body. PACAP influences development of various tissues and exerts protective function during cellular stress and in some tumour formation. No evidence is available on its role in neural crest derived melanocytes and its malignant transformation into melanoma. Expression of PACAP receptors was examined in human skin samples, melanoma lesions and in a primary melanocyte cell culture. A2058 and WM35 melanoma cell lines, representing two different stages of melanoma progression, were used to investigate the effects of PACAP. PAC1 receptor was identified in melanocytes in vivo and in vitro and in melanoma cell lines as well as in melanoma lesions. PACAP administration did not alter viability but decreased proliferation of melanoma cells. With live imaging random motility, average speed, vectorial distance and maximum distance of migration of cells were reduced upon PACAP treatment. PACAP administration did not alter viability but decreased proliferation capacity of melanoma cells. On the other hand, PACAP administration decreased the migration of melanoma cell lines towards fibronectin chemoattractant in the Boyden chamber. Furthermore, the presence of the neuropeptide inhibited the invasion capability of melanoma cell lines in Matrigel chambers. In summary, we provide evidence that PACAP receptors are expressed in melanocytes and in melanoma cells. Our results also prove that various aspects of the cellular motility were inhibited by this neuropeptide. On the basis of these results, we propose PACAP signalling as a possible target in melanoma progression

Transcriptome-based screening of ion channels and transporters in a migratory chondroprogenitor cell line isolated from late-stage osteoarthritic cartilage

Abstract Chondrogenic progenitor cells (CPCs) may be used as an alternative source of cells with potentially superior chondrogenic potential compared to mesenchymal stem cells (MSCs), and could be exploited for future regenerative therapies targeting articular cartilage in degenerative diseases such as osteoarthritis (OA). In this study, we hypothesised that CPCs derived from OA cartilage may be characterised by a distinct channelome. First, a global transcriptomic analysis using Affymetrix microarrays was performed. We studied the profiles of those ion channels and transporter families that may be relevant to chondroprogenitor cell physiology. Following validation of the microarray data with quantitative reverse transcription-polymerase chain reaction, we examined the role of calcium-dependent potassium channels in CPCs and observed functional large-conductance calcium-activated potassium (BK) channels involved in the maintenance of the chondroprogenitor phenotype. In line with our very recent results, we found that the KCNMA1 gene was upregulated in CPCs and observed currents that could be attributed to the BK channel. The BK channel inhibitor paxilline significantly inhibited proliferation, increased the expression of the osteogenic transcription factor RUNX2, enhanced the migration parameters, and completely abolished spontaneous Ca2+ events in CPCs. Through characterisation of their channelome we demonstrate that CPCs are a distinct cell population but are highly similar to MSCs in many respects. This study adds key mechanistic data to the in-depth characterisation of CPCs and their phenotype in the context of cartilage regeneration