Elsevier

Experimental Cell Research

Volume 312, Issue 17, 15 October 2006, Pages 3425-3431
Experimental Cell Research

Research Article
Zyxin is upregulated in the nucleus by thymosin β4 in SiHa cells

https://doi.org/10.1016/j.yexcr.2006.07.021Get rights and content

Abstract

Thymosin β4 is a 43-amino acid actin-binding protein that promotes cell migration and is important in angiogenesis, wound healing, and tumor metastasis. We searched for genes upregulated by thymosin β4 and identified zyxin as increased in SiHa cells in the presence of exogenously added thymosin β4 and when thymosin β4 is overexpressed using adenoviral vectors. Both zyxin and thymosin β4 show increased localization in the nucleus. We conclude that thymosin β4 may exert some of its migration promoting activity via increased zyxin expression.

Introduction

Thymosin β4 is a 43-amino acid peptide that is present in nearly all mammalian cells. Inside the cells, it binds to actin and functions in actin sequestering. It also promotes cell migration and is important in various biological processes, including angiogenesis [1], accelerated dermal and ocular wound healing [2], [3], [4], [5], [6], and the metastatic potential of tumor cells [7], [8], [9], [10], [11]. The central actin-binding site on thymosin β4 is required for its angiogenic and wound healing activity, suggesting that the interaction with actin is functionally important beyond actin sequestration [12]. Another region at the amino end is important for its anti-inflammatory activity [13], [14]. Extracellular thymosin β4 stimulates the migration of endothelial cells, keratinoctyes, and tumor cells, but demonstrates anti-inflammatory activity because it blocks monoctye and neutrophil migration to the peptide fmet-leu-phe. The mechanism for its ability to increase the migration of some cells and inhibit that of others is not known but may be related to its cell surface receptor(s), ability to bind actin, and regulation of proteins/pathways important in cell migration.

Thymosin β4 plays a role in cell-actin cytoskeleton reorganization [15], [16]. Increasing the concentration of thymosin β4 causes a loss of F-actin. The resultant, larger available pool of sequestered actin leads to increased cell motility [17], [18]. A breakdown of organization in the cell cytoskeleton by actin depolymerization is a first step in cell motility, and reorganization/reformation of the cytoskeleton at the leading edge by actin polymerization is important for induction of cell migration [19]. Moreover, cellular responses to extracellular signals often involve extensive rearrangement of the actin cytoskeleton, including localized increases in actin polymerization [20], [21]. Because so many important cell functions depend on the actin cytoskeleton, it is important to understand how it is assembled and the role of thymosin β4.

One protein that has been postulated to play a role in the regulation of the actin cytoskeleton is zyxin [22]. Zyxin is a low-abundance phosphoprotein that is a crucial molecule in cellular focal adhesions where it regulates integrin-regulated cell adhesion [23]. Zyxin is also present in the nucleus, where it modulates gene expression, and may function as a mechanical stimulus [24], [25], [26]. It should also be noted that there is considerable actin in the nucleus [27], [28]. Zyxin has structural features that are compatible with its proposed roles in actin organization and in signal transduction. The proline-rich segment of zyxin contains a number of sequences similar to docking sites for Src homology 3 (SH3). The proline-rich region of zyxin also mediates its activity for the assembly and integrity of the actin cytoskeleton [29], [30]. A second structural feature in zyxin that is consistent with a function in signal transduction is the presence of three copies of a double zinc-finger motif, called the LIM motif [31]. LIM domains are found in a number of proteins that play roles in regulating cell proliferation and differentiation [31], [32], [33]. Zyxin itself contains a functional nuclear export signal (NES) that provides a molecular mechanism for regulating its in vivo distribution [24]. It is not known how zyxin enters into the nucleus or what regulates its synthesis.

Here, we have found that thymosin β4 upregulates zyxin expression and activates its translocation into the nucleus. Thymosin β4 may promote cell migration via zyxin by regulating actin depolymerization and polymerization.

Section snippets

Materials and cells

Thymosin β4 was synthesized by the CBER Facility for Biotechnology Resources (Food and Drug Administration, Bethesda, MD) as previously described [12]. The adenoviral vector and empty shuttle vector were prepared as previously described [34]. The human cervical cancer SiHa cell line was purchased from the American Type Culture Collection (ATCC, Rockville, MD). Rabbit polyclonal thymosin β4 (used at a 1:1000 dilution) was obtained from ALPCO Diagnostics, Windham, NH. Monoclonal GAPDH antibody

Identification of genes upregulated by thymosin β4 and confirmation of zyxin protein expression

Thymosin β4 expression is increased in a dose- (Fig. 1) and time-dependent manner in SiHa cells after viral transfection for 18 h. Using Affymetrix array analysis, we identified upregulated genes in thymosin β4 virus-infected SiHa cells at 18 h (Table 1). Control adenoviral vector transfected cells served as the controls. Although thymosin β4 has a potent effect on cell migration, no genes were upregulated by more than four-fold. Several of the upregulated genes contained LIM domains, including

Discussion

Disruption of focal adhesions and changes in actin organization are key events in cell migration and a hallmark of the transformed phenotype [20], [23], [39]. Actin organizes the cell cytoskeleton to drive cell shape changes that are involved in cytokinesis, cell motility, and cell adhesion. These highly coordinated processes involve many actin-binding proteins: some regulate actin dynamics, others organize actin filaments into distinct networks, and still more use the actin filaments to

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    1

    Present address: Department of Obstetrics and Gynecology, College of Medicine, Ewha Womans University, Seoul, Korea, 158-710.

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