Overexpression of dnIKK in mesenchymal stem cells leads to increased migration and decreased invasion upon TNFα stimulation
Introduction
Human mesenchymal stem cells (hMSC) represent the continuous cellular source for tissue repair and regeneration. The regenerative potential of hMSC is mainly based on their high self-renewal capacity paired with the ability to differentiate into various tissues including bone, cartilage and muscle [1]. Recruitment of hMSC to the sites of tissue injury requires active migration towards chemokines or cytokines and invasion by proteolytic interaction with the extracellular matrix [1], [2], [3]. In vitro studies on invasion are routinely carried out using three-dimensional (3-D) assays with extracellular matrix (ECM) barriers. Experimental data have shown that tumor necrosis-factor α (TNFα) is a strong stimulus for hMSC invasion [4], [5]. As a ligand of TNF receptor I (p55) and II (p75), TNFα activates the canonical nuclear factor kappa B (NFκB) pathway. In unstimulated cells, the binding of NFκB to IκB in the cytoplasm prevents its translocation into the nucleus [6]. Upon stimulation, specific IκB kinases (IKK-1 and -2) phosphorylate IκB, causing its rapid degradation by proteasomes [7], [8]. The release of NFκB from IκB results in the transfer of NFκB into the nucleus, where it binds to the promoter regions of its target genes. IKK-2 is described as a rate-limiting step of the NFκB pathway [9] and is crucial for TNFα-mediated invasion of hMSC [4]. Invasion through extracellular matrix requires upregulation of ECM binding receptors and proteolytic enzymes. Consequently, CD44 and matrix metalloproteinase (MMP) 9 upregulation is found in hMSC upon TNFα mediated invasion [4], [5]. Reduction of IKK-2 activity inhibits upregulation of these genes, resulting in impaired invasive capacity.
Beside the observed upregulation of proteolytic enzymes and the increase of invasion capacity, TNFα is also known to be a strong chemoattractant in two-dimensional (2-D) migration [10], [11]. Cell migration is broadly scrutinized in in vitro studies as directed cell movement on different surfaces [12]. It results from a three-step cycle of polarized cell extension and substrate binding through leading pseudopods, followed by actin-based contraction of the cell body and release of adhesive bonds at the trailing edge [13]. However, it is unknown whether TNFα induced 2-D migration of hMSC is also mediated via IKK-2. Therefore, the aim of the present study was to evaluate the involvement of IKK-2 on 2-D migration of hMSC upon TNFα stimulation. Elucidating the impact of IKK-2 on this distinct cellular process may contribute to a more profound understanding of cytokine guided hMSC recruitment to the sites of tissue injury.
Section snippets
Cells and cell culture
Experiments were carried out using a single-cell-derived human mesenchymal stem cell line expressing hTERT after lentiviral gene transfer, termed SCP-1. Previous studies proved this cell line to be a reliable, standardized and well-characterized cell model analyzing mechanisms of hMSC signal transduction and biological response [14]. SCP-1 cells were additionally transduced with the dominant-negative mutant (K44A) IKK-2 (SCP-1 dnIKK) according to our previously published protocol [2]. Cells
Lentiviral transduction and characterization of SCP-1 dnIKK cells
Phase contrast observation during cell culturing revealed no morphological differences between SCP-1 and SCP-1 dnIKK cells (Fig. 1A). Semi-quantitative RT-PCR was performed to demonstrate the efficiency of hTERT and dnIKK lentiviral transduction as well as to investigate potential changes in TNFα receptor expression. SCP-1 cells highly expressed hTERT and expression levels remained unchanged after transduction of dnIKK-2. No hTERT expression was detected in untransduced hMSC. A basal expression
Discussion
Regeneration of mesenchymal tissues requires the recruitment of mesenchymal stem cells as the continuous cellular source for tissue repair. This process necessitates directed cell migration and invasion across extracellular matrix barriers [18], [19]. TNFα, among other proinflammatory cytokines, is released at the sites of injury, attracting hMSC and promoting invasion by upregulation of metalloproteinases [20]. TNFα induced invasion is mediated via the NFkB pathway and strongly depends on
Acknowledgments
Florian Haasters was supported by the Friedrich-Baur-Foundation, LMU Munich (project 0018/2008). Wolf Christian Prall and Florian Haasters acknowledge the Faculty of Medicine, LMU Munich (FöFoLe, project 565 and project 660, respectively).
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