The cytosolic isoform of glutaredoxin 2 promotes cell migration and invasion

https://doi.org/10.1016/j.bbagen.2020.129599Get rights and content

Highlights

  • Grx2c induces cell motility and invasiveness through redox regulation of proteins.

  • Redox signaling profoundly affects phosphorylation signaling.

  • Grx2c may be relevant in the development and progression of cancer.

Abstract

Backround

Cytosolic glutaredoxin 2 (Grx2c) controls axonal outgrowth and is specifically induced in many cancer cell lines. We thus hypothesized that Grx2c promotes cell motility and invasiveness.

Methods

We characterized the impact of Grx2c expression in cell culture models. We combined stable isotope labeling, phosphopeptide enrichment, and high-accuracy mass spectrometry to characterize the underlying mechanisms.

Results

The most prominent associations were found with actin dynamics, cellular adhesion, and receptor-mediated signal transduction, processes that are crucial for cell motility. For instance, collapsin response mediator protein 2, a protein involved in the regulation of cytoskeletal dynamics, is regulated by Grx2c through a redox switch that controls the phosphorylation state of the protein as well. Cell lines expressing Grx2c showed dramatic alterations in morphology. These cells migrated two-fold faster and gained the ability to infiltrate a collagen matrix.

Conclusions

The expression of Grx2c promotes cell migration, and may negatively correlate with cancer-specific survival.

General significance

Our results imply critical roles of Grx2c in cytoskeletal dynamics, cell adhesion, and cancer cell invasiveness.

Introduction

Cell motility and invasion, hallmark features of disseminating and metastasizing cancer cells, require an orchestrated interplay of signaling and effector molecules that regulate the continuous re-arrangement of the actin cytoskeleton. These dynamics are directly controlled by numerous actin-binding proteins that stabilize filaments, and promote elongation, severing, or nucleation of filaments [[1], [2], [3], [4]]. The spatio-temporal regulation of these effectors, for instance through receptor-associated kinases or small GTPases of the Rho family, facilitates the formation of cellular protrusions, the contraction of filament bundles, and the re-modeling of cell-cell as well as cell-matrix contacts. A key regulator of actin re-modeling is the actin-related protein 2 and 3 (Arp2/3) complex, a nucleation/branching factor essential for the formation and dynamics of lamellopodia and invadopodia [4,5]. The activity of this hetero-heptameric protein complex is controlled by nucleation-promoting factors, mostly members of the Wiskott-Aldrich syndrome protein (WASP) family protein complexes, such as WASP, N-WASP, and WAVE 1–3. These complexes are recruited to the membrane and activated by Rho GTPases, most notably Rac1 and CDC42, phosphoinositide binding, and other protein-protein interactions [6,7]. Further on, cytoskeletal dynamics, cell motility, and invasion are intimately connected to the interactions of a cell with other cells and the extracellular matrix. These interactions are mediated by integral membrane proteins, for instance cadherins and integrins, that assemble intracellular signaling complexes and serve, via mediator proteins, as an anchor for the organization of actin filaments [8,9].

Glutaredoxins (Grxs) are glutathione-dependent oxidoreductases that catalyze the reduction of protein disulfides, for instance in ribonucleotide reductase [10], and the reversible formation and reduction of protein-glutathione mixed disulfides [11], see also [[12], [13], [14]]. Mammalian genomes encode four Grxs and some Grx domain containing proteins [15]. The gene for glutaredoxin 2 (Grx2, GLRX2) gives rise to alternative transcript variants through mechanisms of alternative splicing and transcription initiation [[16], [17], [18]]. Three protein isoforms of human Grx2 were described, mitochondrial Grx2a, and the cytosolic/nuclear Grx2b and Grx2c; while Grx2a is expressed ubiquitously in all tissues, Grx2b and Grx2c could only be detected in spermatogenic and cancer cells in adult humans [17]. In mice, Grx2a and Grx2c are conserved and both transcribed ubiquitously [18] and also the genomes of other vertebrate species, like zebrafish, contain genes that encode homologues to cytosolic Grx2c. Zebrafish with silenced expression of cytosolic Grx2 fail to develop an axonal scaffold and lose essentially all types of neurons by apoptotic cell death [19]. These defects could be rescued by re-introduction of wildtype Grx2c, but not by the introduction of mutants that could not catalyze the reduction of protein disulfides [19]. The process of axonal path-finding is facilitated by growth cones, actin-based structures at the tip of the growing axons or neurites, and guided by external permissive and repulsive signals that lead to the remodeling of the growth cone [20]. These processes resemble in many aspects the migration of entire cells.

The functions of the cytosolic Grx2c in the establishment of axonal scaffolds and spermatogenesis [17,19], a process that includes the transmigration of spermatogenic cells through the close Sertoli cell formation, led us to hypothesize that Grx2c may promote both the motility and invasion behavior of cells in general. To test this hypothesis, we have generated cell lines that express cytosolic Grx2c [21] at low to moderate levels – with dramatic effects on cytoskeletal dynamics, morphology, motility, and invasion. We performed high-accuracy mass spectrometry to characterize the influence of Grx2c expression on the levels and phosphorylation of proteins controlling cytoskeletal dynamics, cell adhesion, and migration. Analysis of a small set of patient samples implies roles of Grx2c during carcinogenesis.

Section snippets

Results

We hypothesized that cytosolic Grx2c contributes to the development of cancer cells by promoting cell migration and invasion. To investigate this assumption, we have analyzed different transiently transfected cancer cell lines and a stably transfected HeLa clone. Wildtype (wt) HeLa and the clone mildly overexpressing cytosolic Grx2 were established in [21]. The latter was originally annotated tGrx2-HeLa before the Grx2c isoform was identified in mammals. Enoksson et al. confirmed an increased

Discussion

Our study provides strong evidence that Grx2c does not only control axonal outgrowth and guidance [19] [25], but may also promote both the motility and invasiveness of non-neuronal cells. The combined proteomic and phosphoproteomic approach and the specific analysis for pathways altered in the Grx2c-expressing cells demonstrated multiple specific alterations in the two major processes controlling cell migration and infiltration: cell adhesion and control of cytoskeletal dynamics. Fig. 5

Materials

Chemicals and enzymes were purchased from Sigma-Aldrich (St. Louis MO, USA), unless otherwise stated, and were of analytical grade or better. Antibodies: CRMP2 (Sigma-Aldrich, C2993); GAPDH (Sigma-Aldrich, G9545); α-Tubulin (Sigma-Aldrich, T9026); additional antibodies were listed in the suppl. materials.

Electrophoresis and western blotting

SDS-PAGE and western blots were run using pre-casted TGX stain-free gels (4–20%, Biorad, Hercules CA, USA), and PVDF membranes (Macherey & Nagel, Düren, Germany), blue native electrophoresis

Author contributions

MG, NK, FH, MS, and CHL conceived and designed this study, MG, ER, JM, LK, KM, EMH, SR, ES, SW, and CHL performed and analyzed the experiments.

Funding

This work was supported by the Robert Bosch Stiftung (Stuttgart, Germany), the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation, to CHL: SFB593-N01, LI 984/3-1 (SPP 1710), LI 984/4-1, GRK1947-A1) and the Bundesministerium für Bildung und Forschung (to FH: 03Z1CN21, to KM: 03Z2DN11).

Declaration of Competing Interest

The authors declare no conflicts of interest.

Acknowledgments

The authors express their gratitude to C. Cott, M. Harms, K. Ventz, S. Oesteritz, and M. Werling for support and assistance. The authors thank M. Schwarzländer for his assistance with the measurement and the provision of the Grx1-roGFP2 sensor.

The results shown here are in part based upon data generated by the TCGA Research Network. We would like to thank ‘The Cancer Genome Atlas’ initiative, all tissue donors, and investigators who contributed to the acquisition and analysis of the samples

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