Zyxin: a Mechanotransductor to Regulate Gene Expression

European Review for Medical and Pharmacological Sciences 2019; 23: 413-425 Zyxin: a mechanotransductor to regulate gene expression

Y.-X. WANG1,2, D.-Y. WANG3, Y.-C. GUO1, J. GUO1

1Academy of Medicine and Life Sciences, Nanjing University of Chinese Medicine, Nanjing, China 2The First School of Clinical Medicine, Nanjing University of Chinese Medicine, Nanjing, China 3Model Animal Research Center, Nanjing University, Nanjing, China YuXuan Wang and Dingyu Wang contributed equally to this work

Abstract. – OBJECTIVE: Cells answer to bio- Key Words: chemical, electrical and mechanical signals in Zyxin, Focal adhesions, Nuclear-cytoplasmic com- the environment, which regulate their behavior. munication, Gene expression, Mechanotransductor, Mechanical signals can propagate through me- Cytoskeleton, Clinical diagnosis and treatment. chanically stiff structures like focal adhesions (FAs). Zyxin, a LIM domain protein, is localized primarily at focal adhesion plaques. Growing evidence suggests that zyxin is a vital mechanotransductor to regulate the gene expression. In Introduction this review, we summarize the features of zyxin and the molecular mechanism of how zyxin par- During their lifetimes, cells encounter a vari- ticipate in the cellular activity. ety of stimuli that can dramatically affect their MATERIALS AND METHODS: An English-lan- behavior, such as mechanical stimulation, which guage literature search is based on a key- can raise different kinds of signal pathways and word-based query of multiple databases (MED- regulate physiologic activity1. Dysregulation of LINE, Embase) and bibliographies from identified publications. The references in the select- mechanical force is responsible for a variety of ed paper are also considered as an additional diseases, including neuronal and muscular de- source of data. The search was last updated in generation2, potential immune system disorders3, April 2018; no limitations are applied. hypertension4, and polycystic kidney disease5. RESULTS: Zyxin enhances actin polymer- Cells respond to mechanical properties such ization with the aid of Enabled (Ena)/vasodila- as stiffness, contractility and tensile strength tor-stimulated phosphoprotein (VASP) proteins through punctually, appropriately graded adjust- in response to mechanical tension, to perform 6 its role in stress fibers (SFs) remodeling and re- ments to maintain tissue homeostasis . Also, the pair. Zyxin can translocate from focal adhesions gene expression in living cells can be regulated 7 8-10 (FAs) to the nucleus responds to stretch, and by mechanical stimuli . Multiple studies have regulate gene transcription by interaction with highlighted the mechanosensitive features of the transcription factors like nuclear matrix protein protein zyxin. Zyxin may enter the nucleus as- 4 (NMP4). Misregulation of nuclear functions of sociated with other proteins in response to me- zyxin appears to be associated with pathogenic chanical force stimulation and is exported from effects and diseases, such as prostate cancer the nucleus through intrinsic leucine-rich nuclear and non-small-cell lung cancer. CONCLUSIONS: Zyxin is a crucial ingredi- export sequences (NES). Due to the ability to ent of the cellular mechanotransducing system shuttle between cytoplasm and nucleus, zyxin and can modulate the gene expression. Given may mediate cell function in a force-dependent its clinical relevance, zyxin is also a promising manner, and its ability to detect mechanical force target for the diagnosis and treatment of cer- could be an integral part of the regulation of the tain diseases. Understanding the role of zyxin gene expression11,12. Zyxin has also displayed in force sensing and gene expression regulat- force sensitive translocation to focal adhesions ing provides a compelling challenge for future (FAs) and stress fibers (SFs) in fibroblasts and en- biomechanics studies, and offers attractive evidence for zyxin as a potential diagnostic mark- dothelial cells, helping the remodeling and repair 10 er and therapeutic target for clinical diseases. of SFs . Given the great importance of zyxin in

Corresponding Author: Jun Guo, MD; e-mail: [email protected] 413 Y.-X. Wang, D.-Y. Wang, Y.-C- Guo, J. Guo regulating cellular mechanical activities, under- (82KDa molecular weight), that possesses two standing its role in force sensing and transduc- distinct motifs: N-terminal proline-rich domain tion is meaningful to biomechanics study. Focal and C-terminal LIM domain25,26. The N-termi- adhesions and actin stress fibers are well-known nus of zyxin has been reported to bind some structures that form and develop in a mechanical partners, including the actin filament cross- force-dependent manner13,14. Focal adhesions, al- linker α-actinin27,28, the actin assembly modula- so referred to as focal plaques or focal contacts, tor Ena / VASP 29, the cytoskeletal proteins LIM are an integral component of the transmission of and SH3 domain protein 1 (LASP-1) and the mechanical stimuli15-17. FAs are complex multi- LIM-nebulette (LASP-2)30. Among which, the protein structures that form upon integrin en- VASP family proteins form complexes with gagement with the extracellular matrix (ECM) four proline-rich ActA repeats to facilitate ac- and link the ECM to the intracellular cytoskel- tin-polymerization at FAs and SFs31. The over- eton18. Moreover, FAs serve as critical signaling expressed zyxin LIM domain substitutes the hubs that transmit chemical (extracellular protein endogenous zyxin from FAs would induce ligands) and physical (rigidity, composition) cues the mislocalization of VASP and mammalian about the extracellular environment13. Exoge- Ena8,32. The C-terminus LIM domains of zyxin nous mechanical forces in ECM are transduced that consist of three motifs (termed by the ini- through FAs to the actin cytoskeleton, which tials of LIN-11, Isl-1 and MEC-3) are essential indicates that the FAs and SFs are mechanical for its force sensing function by accumulating mediums for maintaining the force balance in at FAs or force-bearing sites33. LIM domains are the changing mechanical environment and signal dual zinc-finger protein-protein or protein-DNA transmission8,19. It is intriguing to mention that, binding interfaces. Some LIM proteins which only partial focal complexes mature into large localize to the nucleus have been proved to per- and stable focal adhesions, and then could recruit form a transcriptional role9,33. Two leucine-rich many more proteins like zyxin20. Zyxin disso- NES lie in the central region of zyxin are be- ciates from FAs by reducing mechanical loads lieved to mediate nuclear export (Figure 1)34. on the FAs and regains accumulation at FAs by Additionally, the zyxin-nectin interaction proves stretching the substratum8. In other words, the that amino acids 230-280 of zyxin are required application of external forces can change the lo- for localization to cell-cell adhesions35. calization of the FAs protein zyxin. For instance, The functional diversity of LIM proteins sug- zyxin proteins would be recruited to actin stress gests that the LIM domain plays a unique role in fibers when adherent cells are stretched by pull- various cellular processes. ing on the underlying flexible substrate. Mean- while, actin assembly at FAs is enhanced8,10. By contrast, zyxin-deficient cells fail to respond to external strain21. Delocalization of zyxin from FAs or genetic ablation of zyxin leads to unusu- al and integrin-independent migration of cells8. Zyxin is also recruited to stress fiber strain sites for SFs repair and stabilization22. Consequently, zyxin plays as a crucial ingredient of the mechanotransducing system. The focus of this review is the protein zyxin, an adhesion plaque component that has been implicated in signaling events and mechanotransducing system at the adhesive membrane. Figure 1. Molecule structure of zyxin and its domains. The N-terminus of zyxin has four proline-rich ActA repeats Molecular Structure of Zyxin (amino acids 50-120) for the interaction with actin regula- Zyxin is primarily localized at focal adhesion tors VASP and Mena, which also has α-actintin binding sites plaques, actin stress fibers, and cell-ECM and (amino acids 1-50)36. C-terminal LIM domains (after ami- cell-cell junction areas, transiently exist in some no acids 392) contain cysteine/histidine zinc-coordinating 11,23 LIN-11, Isl-1 and MEC-3, which are essential for its location nuclei . FAs are structures located at the ends to FAs, force-induced targeting and protein interactions33,37. of actin fibers and serve as force transmission Two leucine-rich nuclear export sequences and relevant ser- sites24. Zyxin is one of the FAs constituents ine phosphorylation sites lie in the central region of zyxin36.

414 Zyxin: a mechanotransductor to regulate gene expression

Zyxin As a Mechanotransductor to SFs in response to atomic force microscopy Stress fibers, whose formation and develop- (AFM) stylus-driven tension induction49. Zyxin ment are mechanical force-dependent, are formed enhances actin polymerization with the aid of through a combination of actin de novo polym- Ena/VASP proteins in response to mechanical erization that occurs at FAs and the merging tension in FAs, to perform its role in SFs remod- of previously formed fragments38. It has been eling and repair. In other words, mechanical sig- illustrated that actin-regulatory proteins, Arp2/3 nals are transduced into the actin polymerization complex, mammalian Diaphanous (mDia)-related response via zyxin accumulation accompanied formins and Ena/VASP proteins are involved in by Ena/VASP recruitment. the process of actin polymerization39-42. Com- pared to mDia and Arp2/3, Ena/VASP proteins are noticeably localized at FAs, which demon- Externally Applied Force: strates a role of Ena/VASP in the local actin Zyxin Mobilizes to FAs polymerization at FAs. The recruitment of Ena/ VASP to FAs depends on the accumulation of the Zyxin is recruited to Force-Bearing Sites LIM protein zyxin at FAs8. Researechers49,50 showed that zyxin shuttles Hirata et al8 concluded previous experimental from FAs to tension zones within SFs and trig- results from Rottner K, Lele TP and some other gers local recruitment of α-actinin and VASP researchers, then put forward the role of zyxin that thickens and reinforces SFs. In other words, as a crucial element of the mechanotransduc- zyxin accumulates in the sites of externally ing system at FAs. Proteomic studies43,44 have applied forces. However, Uemura et al12 put indicated that LIM domain proteins such as forward their experimental results: zyxin only Hic-5, paxillin, CRP2 and zyxin are sensitive accumulates at the leading edge, it does not to mechanical stress in the actin cytoskeleton. indiscriminately localize to force-bearing sites; Among them, three LIM domain proteins have instead, it is capable of distinguishing between been shown to be recruited to SFs in response these distinct adhesion sites. These results indi- to stretch: Hic-5, CRP2 and zyxin45,46. Zyxin cate that force-dependent zyxin accumulation exhibits an expeditious and intensive mobili- occurs at the leading edge but not at the trailing zation from FAs to actin filaments in response edge of migrating cells. to a unidirectional cyclic stretch of cells, while Scholars51,52 have reported the crucial role of other FAs proteins remain concentrating at the the C-terminus LIM domain of zyxin in medi- substratum attachment sites10. Therefore, zyxin ating zyxin binding to force-bearing sites. The is the only LIM domain protein proved mecha- LIM domain consists of three motifs. Studies noresponsive. have found that multiple zyxin LIM domains fail As mentioned above, zyxin is rapidly mobi- to increase force sensitivity while single zyxin lized from FAs to the remodeling actin filaments LIM domain has the best force-sensitivity. These responds to stretch. Zyxin flows away from FAs results demonstrate the essentiality of all three in synchrony with newly assembled actin bundles LIM motifs for linking binding partners with during SFs assembly45. In answer to a uniaxial zyxin at force-bearing sites, while the impossible cyclic stretch, zyxin-null cells fail to strengthen interaction occurs to multiple LIM domains29. the actin SFs commonly, elucidating zyxin-inde- The yet known partners have been already im- pendent and zyxin-dependent facets of the stretch plied dropping out of the process that zyxin lo- response47. Live-cell imaging technology is ap- calized to force-bearing places, such as p130Cas, plied to investigate zyxin dynamics in response Cysteine-Rich Protein (CRP), and synemin53-56. It to actin SFs thinning. Actin incorporation is high ought to be other zyxin binding partners partici- at zyxin-rich FAs but decreases when over-ex- pating in this process. However, Hoffman et al47 pressed LIM domains replace zyxin36. Moreover, prove that force-induced zyxin phosphorylation after knocking down zyxin’s expression with relies on the activation of the MAPK signaling RNAi, a perinuclear actin cap structure induced pathways, rather than p130Cas, indicating that by mild shear stress would be failed to form, there is the possibility that zyxin would be re- and a reduction happened in the pulling force cruited to force-bearing sites without binding at cell-fibronectin bead contact sites48. Lastly, any partners. The underlying mechanism of how zyxin dissociates from SFs with relief of tension zyxin is recruited to force-bearing sites remains through laser severing and is reversibly recruited to be clarified.

415 Y.-X. Wang, D.-Y. Wang, Y.-C- Guo, J. Guo

Zyxin Recruits VASP to the where zyxin-VASP complexes occur in complex Force-Bearing Sites organizations with suppressed actin regulatory The Ena/VASP family is a growing collection activity65. of related proteins that have been implicated in the Abl signaling pathway and the assembly The LIM domain of Zyxin is Sufficient for of the actin cytoskeleton57. VASP, the vasodi- Force-Dependent Recruitment lator-stimulated phosphoprotein, is a member Many studies tried to find out which molecule of the Ena/VASP family. VASP is identified as recruits zyxin to FAs in a force-dependent man- a prominent 46 kDa substrate for cAMP- and ner and which structure of zyxin play the core role cGMP-dependent kinases in platelets58. Immu- in this process8,12,29. Yi et al56 reported that protein nolocalization works34 have revealed that VASP p130Cas, which is the FAs-associated adapter, in- is localized at integrin-rich adhesion plaque. teracts with the LIM region of zyxin. Crawford69 Molecular cloning of VASP cDNAs established found the direct interaction between zyxin and that human VASP is a protein of 380 aa with a α-actinin and soon after an α-actinin-binding site central proline-rich core that is quite distinct was identified in the N-terminal region of zyx- from the proline sequence found in zyxin59. in26,28. That leads to a long period in which zyxin The proline region of VASP interacts directly N-terminal region was believed to be necessary with profilins, the small actin monomer binding for force-dependent localization of zyxin to FAs. proteins that have been demonstrated in the However, Hirata et al8 observed the accumulation actin filament assembly regulation and signal of the separate LIM region of zyxin at FAs in a transduction60,61. The direct interaction between force-dependent manner. Also, they found that VASP and ActA proteins of bacterium Listeria the force-induced accumulation of endogenous further confirms the role of VASP in actin fil- zyxin at FAs was inhibited by the expression of ament dynamics39. As described above, ActA the LIM region8. These consequences suggest is required for the ability of the bacterium to that the LIM region of zyxin is crucial for the assemble actin filaments on its surface. Inter- force-dependent recruitment of zyxin to FAs, and estingly, VASP has been shown to associate raise the possibility that the LIM domain of zyx- directly with zyxin31. VASP interacts with a in is sufficient for force-dependent recruitment. proline repeat region of ActA that resembles the Uemura et al12 conducted the experiments and sequences found in zyxin26,62. The zyxin-defi- published their results in 2011. By analyzing the cient cells would not determine the position of zyxin mutants with the truncated LIM-domain VASP to FAs anymore63, so does the mislocal- (ΔLIM-GFP) and with only the LIM domains ization of zyxin26. Moreover, zyxin-dependent (LIM-GFP), they concluded that the LIM domain recruitment of VASP to sites of tension-induced of zyxin is sufficient for responding to the trac- cytoskeletal damage is found to regulate actin tion force generated by migrating cells. Further- filament repair64. more, individual or truncated LIM motifs are not We can see the prevailing thought is that zyxin sufficient for force-dependent accumulation, and acts merely as a scaffold protein for VASP bind- zyxin recruitment requires all three LIM motifs. ing26. However, Grange et al65 refute this view To date, this finding has been acknowledged by by identifying the LIM domain-VASP interac- most researchers in the field17,29,70,71. tion. The series of four proline-rich (FPPPPP) motifs can bind zyxin with VASP31,66,67, as well Internally Generated Force: Zyxin Flows as through the LIM domain region68. It is in- Away from FAs to Actin SFs triguing that defects in SFs reinforcement fol- Forces applied to cell-ECM adhesions are lowing stretch stimuli are also observed in cells transmitted across the transmembrane integ- lacking zyxin10, which highlights a critical role rin receptors to the cytoskeleton via molecular for VASP in organizing actin at FAs. They dis- linkages with the FAs30,72. Within the FAs an- cover that zyxin-VASP binding through both choring complex, integrin physically associates the proline-rich motifs and the LIM domains with multiple adaptor proteins involved in sig- alters specific VASP functions; neither individ- nal transduction, such as focal adhesion kinase ual interaction alters VASP’s actin regulatory (FAK), vinculin, talin, p130Cas, and paxillin73-76. activities. Of interest, full-length zyxin dramati- Moreover, the internal movement of actin fila- cally reduces VASP-mediated actin bundling and ments can also be sensed by FAs through a slip- actin assembly. These results suggest a model page-clutch mechanism inside cells77. By means

416 Zyxin: a mechanotransductor to regulate gene expression of conformational changes, some molecules of Force/Stretch Induced Nuclear these complexes, such as talin and paxillin, Translocation and Changes of Zyxin can ‘‘perceive’’ mechanical stimuli and trans- Nuclear Activities duce them into electrical or biochemical signals, FAs proteins combine the actin filaments with subsequently triggering downstream signaling integrins and regulate transmembrane mechan- pathways to modulate cellular physiologic ac- ical force transmission. Zyxin, which acts as tivities78-81. Thus, we can conclude that not only the mechanotransducer, is partly mediated by external forces transmitted across ECM adhe- cytoskeletal tension83,84. Part of this response is sions focus on these FAs sites as already shown, mediated by regulating the physical strength of but also internal forces generated within the actin the FAs that resists cell traction forces to sustain cytoskeleton. To give an example, the recruitment cytoskeletal prestress85. These FAs proteins could of zyxin at force-bearing sites relies on myosin II facilitate to transfer mechanical to chemical sig- and Rho-kinase activation, suggesting that zyxin nals by the Rho pathway, which induces myosin not only responds to the externally applied force II phosphorylation by feedback and produces according to previous data, but also responds cytoskeletal forces86,87. Zyxin, known as FAs pro- to the internally generated actin-myosin force12. tein, alter its binding kinetics in a force-depen- Although this viewpoint has been accepted by dent manner which enables it to shuttle between some researchers70, it still needs further research the cytoplasm and nucleus, and in this manner, to explain how zyxin plays its role in sensing in- zyxin can serve as a transcription factor to reg- ternally generated force. ulate gene expression9,88. Three lines of evidence have demonstrated that zyxin shuttles between Zyxin Mediates SFs Repair nuclear compartments and cytoplasm. First, in Cells will recognize and respond to changes cells that are treated with leptomycin B, an inhib- in cytoskeletal integrity to maintain mechanical itor of Crm1-dependent nuclear export, zyxin ac- homeostasis64. Actin stress fibers come through cumulates in the nucleus85. Second, a leucine-rich bounded, intense, force-mediated elongation and nuclear export signal (NES) has been well-char- decreasing events that settle their function of acterized within the central region of most zyxin stress transmission, followed by SFs repair that proteins, and deletion or mutation of this NES revives this capability64. SF strain sites recruit at also results in nuclear accumulation of zyxin89. least four different proteins found at FAs: zyxin, Third, nuclear accumulation of endogenous zyx- paxillin, α-actinin and VASP19,71. Paxillin, a 68 in is also observed after infection of cells with a kDa FAs protein, has been confirmed the recruit- vaccinia virus90. ment to SF strain sites, so does the zyxin19,82. Growing studies9,91,92 have reported the role Zyxin swiftly accumulates at the damage sites of zyxin in transcriptional responses. Zyxin is of strain-induced SFs, and paxillin recruitment transported into the nucleus respond to applied even precedes zyxin recruitment. The recruit- forces and antisense oligonucleotides against ment and repair process of paxillin is parallel zyxin altered stretch-induced changes in gene to, but independent of, the zyxin repair system19. expression in smooth muscle cells93. However, The repair functions of zyxin are executed by the less is known about the underlying mechanism. actin crosslinker α-actinin and the actin regulator Apart from the proline-rich region, LIM domains VASP, which are recruited to SF strain sites in a of zyxin may also contribute to the process of zyxin dependent manner. Zyxin is recruited first nuclear import94. However, neither is basic. Ac- dependently, in synchrony with VASP, and then cumulation within nuclei is likely to occur via a recruits α-actinin bind the N-terminal region of particular mechanism87. There are some hypoth- zyxin36. Zyxin binding to VASP is required for eses so far. The hydrodynamic characteristics VASP recruitment to either cyclically stretched of chicken zyxin inform that zyxin performs as SFs or to SF strains sites. Mutation of the pro- an elongate monomer of 69 kDa, which is too line-rich ActA repeats in the N-terminal region large to diffuse passively through the nuclear of zyxin eliminates VASP binding to zyxin, pore complex87. Furthermore, zyxin has no tra- which may indicate the binding region on zyxin36. ditional (primary) nuclear localization sequence The mechanism of strain recognition and repair (NLS). Thus, it is possible that zyxin enters the demonstrates the cellular machinery for quick nucleus in association with other NLS-containing modification of cytoskeletal tension respond to proteins or alternative mechanisms9. Zyxin may changes in cell contractility or external forces. use a unique nuclear import mechanism similar

417 Y.-X. Wang, D.-Y. Wang, Y.-C- Guo, J. Guo to that described for the cell adhesion and cell (ETB-R). In practice, zyxin interacts with the signaling protein β-catenin that interacts direct- promoter region of these genes93. Furthermore, ly with the nuclear pore complex87. The zyxin in human cultured endothelial cells that exposed proteins can enter the nucleus by reinforced-as- to cyclic stretch, a nuclear protein-DNA com- sociation with a mutant form of cell adhesion plex forms that, according to supershift analysis, kinase β/proline-rich tyrosine kinase 2 (CAKβ/ contains zyxin, indicating the significant role of PYK2), which abnormally localizes to the nucle- zyxin in stretch-induced endothelial gene expres- us95. Suresh Babu et al96 exhibit a multiple-stage sion93. signaling pathway through the stretch-induced LIM domains have structures related to cer- release of endothelial vasoconstrictor peptide en- tain zinc fingers, which are known to mediate dothelin-1 (ET-1), mediated by the transient re- DNA binding in several transcription factors. ceptor potential channel. Protein kinase G would Zyxin interacts with a variety of nuclear proteins mediate the phosphorylation of zyxin at serine including transcription factors or induces regula- 142, sequentially triggering the translocation of tion of cytoplasmic proteins in the nucleus88,99,100. zyxin to the nucleus. Furthermore, zyxin acts Zyxin acts as coactivators of transcription to reg- as a transducer of transducing the mechanical ulate gene expression85. Proteins regulated by the signal into the nucleus in endothelial cells, where stretch-induced accumulation of zyxin in nuclei it orchestrates the expression of a prominent are as followed: subset of stretch-sensitive genes through a novel DNA-response element97. In vascular smooth 1. 6E6: A yeast two-hybrid library screening muscle cells (VSMCs), within minutes, zyxin determines that zyxin acts as a protein partner translocates from FAs to the nucleus of VSMCs for E6, from Human Papillomavirus (HPV) when exposes to a cyclic strain, and changes the Type 6 and results in E6’s nuclear transloca- expression of the mechanical-sensitive gene98. tion. Cotransfection of E6 from HPV (6E6) ChIP assays revealed that zyxin actually inter- and zyxin leads to the aggregation of zyxin in acts with the promoter region of zyxin-dependent the nucleus, where it can work as an activating genes, such as interleukin-8, VCAM-1, HMNC1, transcription factor. 6E6 can also mobilize Hey-1, HMGCR, and ICAM-193. endogenous zyxin to the nucleus. Moreover, As mentioned above, zyxin not only contrib- when zyxin binds to Gal4-BD for exogenously utes to organizing the actin cytoskeleton but also expressing, the results show that it has inscrip- to the changes in the gene expression occurring tional activation potential and this activity is as an adaptive response to enduring mechan- synergistically enhanced by 6E6 only when the ical strain. The LIM-domains and the similar interacting C-terminal LIM domain is present LIM-domain present in the zyxin homolog, li- in the zyxin construct101. poma-preferred protein, can directly induce gene 2. Akt: Kato et al102 found that atrial natriuretic expression in an artificial assay system. The peptide (ANP) promotes cardiomyocyte sur- phenomenon suggests that zyxin may act as vival by cGMP-dependent nuclear accumula- a transcription factor87. Otherwise, zyxin may tion of zyxin and Akt. Nuclear translocation affect the gene expression exclusively through of zyxin also induces nuclear accumulation of protein-protein interactions, as described for re- activated Akt kinase. Zyxin and activated Akt lated zinc finger proteins like the GATA family participate in a cGMP-dependent signaling of transcription87. cascade leading from ANP receptors to nuclear Here is another supporting proof: long-drawn accumulation of both molecules. Collectively, exposure to enhanced stretch, such as hyper- nuclear accumulation of zyxin and activated tension, can trigger endothelial dysfunction, a Akt may represent a fundamental mechanism hallmark of pathological vascular remodeling that facilitates nuclear-signal transduction and processes93. DNA microarray pathway analyses potentiates cell survival33. Additionally, anoth- of stretch-induced changes in endothelial cell er research shows that zyxin binds to acinus-S, gene expression revealed that zyxin mainly reg- a nuclear speckle protein inducing apoptot- ulates proinflammatory pathways, suggesting a ic, chromatin condensation after cleavage by role for zyxin in vascular remodeling processes. caspases, and restraints its apoptotic action, Testing results of three stretch-sensitive genes which is regulated by Akt103. revealed that zyxin controls the interleukin-8 and 3. HNF-1β: Hepatocyte nuclear factor-1β (HNF- CXCL1 instead of the B-type endothelin receptor 1β), an epithelial tissue-specific transcription

418 Zyxin: a mechanotransductor to regulate gene expression

factor, could regulate the gene expression in is confirmed by the immunoprecipitation of the kidney, liver, intestine, and other organs104. an endogenous-zyxin-complex with the hybrid The LIM-domain protein zyxin is identified myc-Xanf1 protein. By using a set of deletion as a new binding partner of HNF-1β in renal mutants of both proteins, it has been demon- epithelial cells. Zyxin shuttles to the nucle- strated that the combination of the LIM2 do- us with the co-localization of HNF-1β91. The main of zyxin and the Engrailed Homology 1 interaction of the two proteins requires the repressor domain of Xanf1 contributes to the participation of the second LIM domain of interaction of these proteins108. zyxin and the two particular domains of HNF- 8. ZNF384: Zyxin interacts in vitro with ZNF384 1β. The overexpression of zyxin motivates the (zinc finger protein 384, also called the Cas in- transcriptional activity of HNF-1β, while small teracting Zn-finger protein, CIZ, and NMP4), interfering RNA silencing of zyxin inhibits a transcription factor which shuttles between HNF-1β-dependent transcription101. the nucleus and adhesion sites and is involved 4. CBP: Retinoids including all-trans retinoic ac- in osteoblast differentiation. Zyxin interacts id (RA) have been widely used for cancer ther- directly with p130Cas and is postulated to apy. However, the acquired resistance remains link p130Cas to ZNF384. It is intriguing that the main obstacle to RA treatment. Former zyxin or its binding partners have been impli- studies informed that zyxin mediates retinoic cated in the control of the gene expression in acid receptors (RARs) repression by forming two tissues, bone, and smooth muscle, both of a ternary complex with PTOV1 and the RAR which are exquisitely responsive to mechanical coactivator CBP through translocating to the stress109. nucleus in response to RA. Accordingly, it promotes the dissociation of CBP from RAR at the RA-responsive promoter. Consistently, Conclusions RA-induced cancer cell cytotoxicity is signifi- cantly impaired by Zyxin or PTOV1105. In this review, we discuss the FAs protein 5. CARP-1: Zyxin contributes to UV-induced zyxin, also a member of LIM domain pro- apoptosis. Cell cycle and apoptosis regulator teins, which shows mechanosensitive features protein-1 (CARP-1), a 130-kDa nuclear protein that indicate its role as a crucial ingredient of which is co-isolated with zyxin, are identified the mechanotransducing system. In response to by microsequencer analysis. Zyxin connects mechanical force stimulation, zyxin flows away with CARP-1 through its LIM region. Zyxin from FAs to actin stress fibers for SFs remold- lacking the CARP-1 binding region presents ing and repair, enters the nucleus by association lessened proapoptotic activity in response to with other proteins to regulate gene expression. UV-C irradiation106. Mechanical signals are transduced into the actin 6. SIRT1: After treatment with leptomycin B, polymerizing response via zyxin accumulation zyxin accumulates in the nucleus co-localized accompanied by Ena/VASP recruitment. More- with SIRT1 in COS-7 cells. Moreover, the over, zyxin is recruited with VASP and α-acti- SIRT1 deacetylates zyxin suggests that SIRT1 nin, which bind the N-terminal region of zyxin, could interact with nuclear-accumulated zyxin to SF strain sites and mediate SFs repair. How- and regulate its function through deacetyla- ever, there are still many unsolved questions. tion. These consequences raise the possibility Although previous results suggested a model that SIRT1 regulates signal transmission from where zyxin-VASP complexes occur in complex ECM to the nucleus by modulating the func- organizations with suppressed actin regulatory tions of zyxin via deacetylation107. activity, it remains to be clarified65. Further- 7. Xanf1: By using a yeast two-hybrid system, more, apart from zyxin, there are multiple force experiments are designed to seek candidature sensitive modules present at the FAs that are ac- partner protein of the homeodomain transcrip- tivated at distinct locations and regulate specific tion repressor Xanf1, a crucial transcriptional aspects of junction dynamics110. A large number regulator of the early stage of the forebrain of studies have been conducted in this aspects, growth. The LIM domain protein zyxin is but the mechanism they corporate with each identified from the African clawed frog Xen- other in regulating cytoskeletal tension and me- opuslaevis primarily. In the lysate of X. laevis diating nuclear activities has not been revealed embryos, the interaction of zyxin with Xanf1 thoroughly65,106.

419 Y.-X. Wang, D.-Y. Wang, Y.-C- Guo, J. Guo

It is worth noticing that though zyxin is mech- stretch caused by deep inspiration in people with anoresponsive and a potential candidate of mech- asthma. Based on its ability to repair SFs frag- anosensor, there is no direct evidence so far that mentation, zyxin maintains the ASM structure, zyxin directly receives and senses force in cells111. promotes the recovery of contractile force and Although localization and phosphorylation of finally slows airway dilation121. Moreover, several zyxin are sharply altered in response to mechani- studies have revealed the relevance between zyx- cal stimuli to cells10, it is possible that some other in and different kinds of cancer. Prostate cancer molecule may sense force and transduce it into a is a malignant tumor which used to appear mostly signal that modulates zyxin behaviors. Detailed in the male urogenital system122. Zyxin siRNA data mining and bioinformatic analysis have re- treatment inhibited the migration and invasion of vealed that FAs are composed of 180 different DU145 cells. Zyxin expression in tumor tissues kinds of molecules, connected to each other is higher than in normal tissues, suggesting that within a network containing at least 742 interac- zyxin may participate in the growth and invasive- tions112. Zyxin’s recruitment to cytoskeletal struc- ness process of human prostate cancer123. Lung tures under tension might be sensed and driven cancer also has high metastatic potential, which by newly revealed conformational changes, actin is the leading cause of the significant mortali- barbed ends, or post-translational modifications ty124. Zyxin has been determined as a potential in actin or zyxin-binding partners36,113. early diagnostic marker for non-small-cell lung As mentioned above, accumulating evidence cancer125. Therapies ought to be formulated based suggests that zyxin has nuclear functions that on the further understanding of the role of zyxin affect transcription, in addition to their functions aiming at these diseases. Future studies must at focal adhesion plaques. Zyxin interacts with a reveal the mechanism of how zyxin serves as a variety of nuclear proteins, acting as coactivators mechanotransductor in force sensing and regu- of transcription to regulate gene expression85. lation of the gene expression in several diseases, Zyxin may constitute from FAs to the nucleus and that is meaningful for constituting therapeu- through some signaling pathways such as the tic strategies to combat the diseases. Wnt/β-catenin pathway114 and the guanylate kinase CASK115, which have already been clarified their role in mechanotransduction116-118. We think further studies ought to reveal which pathways Conflict of Interest are involved in this process. Moreover, LIM do- The Authors declare that they have no conflict of interests. mains have structures related to certain zinc fingers, which are known to mediate DNA binding in several transcription factors. However, zyxin Acknowledgements is probably not direct transcription factor, as only The work was supported by grants from the National Natu- ral Science Foundation of China (No. 81573409), the Natural the LIM domains of Hic-5 have been shown to Science Foundation of Jiangsu Province (No. BK20161574) have DNA-binding activity, and this has only and the Brand Specialty Building Program of Jiangsu High- been demonstrated in vitro119. Whether zyxin is er Education Institutions (Traditional Chinese Medicine). an actual transcription factor, still needs further exploration. References In certain circumstance, the nuclear misregulation function of zyxin is relevant to pathogenic shitiz ark im elen ngler evchenko 9 1) K , P J, K P, H W, E AJ, L effects and diseases . Zyxin, via its LIM domain A, Kim DH. Control of stem cell fate and function region, interacts with the E6 oncoprotein of HPV by engineering physical microenvironments. Inte- type 6, which is commonly associated with geni- gr Biol (Camb) 2012; 4: 1008-1018. tal warts. The excessive cyclic stretch of vascular 2) Suchyna T, Sachs F. Mechanical and electrical prop- smooth muscle cells leads to the shift in their phe- erties of membranes from dystrophic and normal notype like hypertension. Zyxin modulates the mouse muscle. J Physiol Lond 2007; 581(Pt 1): mechanotransduction of vascular smooth muscle 369 -387. 3) Matheson LA, Maksym GN, Santerre JP, Labow cells by influencing the cytoskeletal structure RS 55,94 . Cyclic biaxial strain affects U937 macro- and signaling pathways . Bronchial hyperre- phage-like morphology and enzymatic activities. sponsiveness of airway smooth muscle (ASM) J Biomed Mater Res A 2006; 76: 52-62. 120 is a characteristic feature of asthma . Zyxin is 4) Huang C. Hypertension, mechanical force, and re- also found to assist the ASM cells to respond to nal disease. Clin Exp Hypertens 2014; 2: 1009.

420 Zyxin: a mechanotransductor to regulate gene expression

5) Delmas P. Polycystins: from mechanosensation to 21) Ngu H, Feng Y, Lu L, Oswald SJ, Longmore GD, Yin gene regulation. Cell 2004; 118: 145-148. FC. Effect of focal adhesion proteins on endotheli- 6) DuFort CC, Paszek MJ, Weaver VM. Balancing forc- al cell adhesion, motility and orientation response es: architectural control of mechanotransduction. to cyclic strain. Ann Biomed Eng 2010; 38: 208- Nat Rev Mol Cell Biol 2011; 12: 308-319. 222. Burridge K, Wittchen ES 7) Wang N. Review of cellular mechanotransduction. 22) . The tension mounts: J Phys D Appl Phys 2017; 50: 233002. stress fibers as force-generating mechanotrans- ducers. J Cell Biol 2013; 200: 9-19. 8) Hirata H, Tatsumi H, Sokabe M. Mechanical forces Mori M, Nakagami H, Koibuchi N, Miura K, Takami Y, facilitate actin polymerization at focal adhesions 23) Koriyama H, Hayashi H, Sabe H, Mochizuki N, Morishi- in a zyxin-dependent manner. J Cell Sci 2008; ta R, Kaneda Y 121: 2795-2804. . Zyxin mediates actin fiber reorga- nization in epithelial-mesenchymal transition and Wang Y, Gilmore TD 9) . Zyxin and paxillin proteins: contributes to endocardial morphogenesis. Mol focal adhesion plaque LIM domain proteins go Biol Cell 2009; 20: 3115-3124. nuclear. Biochim Biophys Acta 2003; 1593: 115- Huttenlocher A, Horwitz AR 120. 24) . Integrins in cell migration. Cold Spring Harb Perspect Biol 2011; 3: Yoshigi M, Hoffman LM, Jensen CC, Yost HJ, Beckerle 10) a005074. MC. Mechanical force mobilizes zyxin from focal Zamir E, Geiger B adhesions to actin filaments and regulates cyto- 25) . Components of cell-matrix ad- skeletal reinforcement. J Cell Biol 2005; 171: 209- hesions. J Cell Sci 2001; 114: 3577-3579. 215. 26) Drees BE, Andrews KM, Beckerle MC. Molecular dis- 11) Nix DA, Beckerle MC. Nuclear-cytoplasmic shut- section of zyxin function reveals its involvement tling of the focal contact protein, zyxin: a poten- in cell motility. J Cell Biol 1999; 147: 1549-1560. tial mechanism for communication between sites 27) Li B, Trueb B. Analysis of the alpha-actinin/zyxin in- of cell adhesion and the nucleus. J Cell Biol 1997; teraction. J. Biol. Chem 2001; 276: 33328-33335. 138: 1139-1147. 28) Reinhard M, Zumbrunn J, Jaquemar D, Kuhn M, Wal- 12) Uemura A, Nguyen TN, Steele AN, Yamada S. The ter U, Trueb B. An alpha-actinin binding site of zyx- LIM domain of Zyxin is sufficient for force-in- in is essential for subcellular zyxin localization duced accumulation of Zyxin during cell migra- and alpha-actinin recruitment. J Biol Chem 1999; tion. Biophys J 2011; 101: 1069-1075. 274: 13410-13418. 13) Burridge K, Fath K, Kelly T, Nuckolls G, Turner C. 29) Steele AN, Sumida GM, Yamada S. Tandem zyxin LIM Focal adhesions – transmembrane junctions be- sequences do not enhance force sensitive accu- tween the extracellular-matrix and the cytoskele- mulation. Biochem Biophys Res Commun 2012; ton. Annu Rev Cell Biol 1988; 4: 487-525. 422: 653-657. 14) Guo J, Wang Y, Sachs F, Meng F. Actin stress in cell 30) Li B, Zhuang L, Trueb B. Zyxin interacts with the reprogramming. Proc Natl Acad Sci U S A 2014; SH3 domains of the cytoskeletal proteins LIM- 111: E5252-5261. nebulette and Lasp-1. J Biol Chem 2004; 279: 15) Fogh BS, Multhaupt AH, Couchman JR. Protein ki- 20401-20410. nase C, focal adhesions and the regulation of cell 31) Drees B, Friederich E, Fradelizi J, Louvard D, Beckerle migration. J Histochem Cytochem 2014; 62: 172- MC, Golsteyn RM. Characterization of the interac- 184. tion between zyxin and members of the Ena/va- 16) Shiokawa S, Yoshimura Y, Nagamatsu S, Sawa H, sodilator-stimulated phosphoprotein family of pro- Hanashi H, Sakai K, Noguchi K, Nakamura Y. Func- teins. J Biol Chem 2000; 275: 22503-22511. tional role of focal adhesion kinase in the process 32) Nix DA, Fradelizi J, Bockholt S, Menichi B, Louvard of inplantation. Mol Hum Reprod 1998; 4: 907- D, Friederich E, Beckerle MC. Targeting of zyxin to 914. sites of actin membrane interaction and to the nu- 17) Oakes PW, Gardel ML. Stressing the limits of focal cleus. J Biol Chem 2001; 276: 34759-34767. adhesion mechanosensitivity. Curr Opin Cell Biol 33) Kadrmas JL, Beckerle MC. The LIM domain: from 2014; 30: 68-73. the cytoskeleton to the nucleus. Nat Rev Mol Cell 18) Bo LJ, Wei B, Li ZH, Wang ZF, Gao Z, Miao Z. Bio- Biol 2004; 5: 920-931. informatics analysis of miRNA expression profile 34) Beckerle MC. Zyxin: zinc fingers at sites of cell ad- between primary and recurrent glioblastoma. Eur hesion. Bioessays 1997; 19: 949-957. Rev Med Pharmacol Sci 2015; 19: 3579-3586. 35) Gregory Call S, Brereton D, Bullard JT, Chung JY, 19) Smith MA, Blankman E, Deakin NO, Hoffman LM, Meacham KL, Morrell DJ, Reeder DJ, Schuler JT, Jensen CC, Turner CE, Beckerle MC. LIM domains Slade AD, Hansen MD. A zyxin-nectin interaction target actin regulators paxillin and zyxin to facilitates zyxin localization to cell-cell adhesions. sites of stress fiber strain. PLoS One 2013; 8: Biochem Biophys Res Commun 2011; 415: 485- e69378. 489. 20) Wu C. Focal adhesion: a focal point in current cell 36) Smith MA, Hoffman LM, Beckerle MC. LIM proteins biology and molecular medicine. Cell Adh Migr in actin cytoskeleton mechanoresponse. Trends 2007; 1: 13-18. Cell Biol 2014; 24: 575-583.

421 Y.-X. Wang, D.-Y. Wang, Y.-C- Guo, J. Guo

37) Velyvis A, Qin J. LIM domain and its binding to tar- 52) Nguyen TN, Uemura A, Shih W, Yamada S. Zyxin-me- get proteins. Madame Curie Bioscience Data- diated actin assembly is required for efficient base Internet. Austin (TX): Landes Bioscience; wound closure. J Biol Chem 2010; 285: 35439- 2000-2013. 35445. 38) Hotulainen P, Lappalainen P. Stress fibers are gen- 53) Schmeichel KL, Beckerle MC. The LIM domain is a erated by two distinct actin assembly mecha- modular protein-binding interface. Cell 1994; 79: nisms in motile cells. J Cell Biol 2006; 173: 383- 211-219. 394. 54) Sun N, Huiatt TW, Paulin D, Li Z, Robson RM. Syn- 39) Beckerle MC. Spatial control of actin filament as- emin interacts with the LIM domain protein zyxin sembly: lessons from Listeria. Cell 1998; 95: 741- and is essential for cell adhesion and migration. 748. Exp Cell Res 2010; 316: 491-505. 40) Blystone SD. Integrating an integrin: a direct route 55) Sun Z, Huang S, Li Z, Meininger GA. Zyxin is in- to actin. Biochim Biophys Acta 2004; 1692: 47-54. volved in regulation of mechanotransduction in 41) Calderwood DA, Shattil SJ, Ginsberg MH. Integrins arteriole smooth muscle cells. Front Physiol 2012; and actin filaments: reciprocal regulation of cell 3: 472. adhesion and signaling. J Biol Chem 2000; 275: 56) Yi J, Kloeker S, Jensen CC, Bockholt S, Honda H, Hi- 22607-22610. rai H, Beckerle MC. Members of the zyxin family 42) Shang Y, Guo XX, Li WW, Rao W, Chen ML, Mu LN, of LIM proteins interact with members of the p130 Li SJ. Cucurbitacin-B inhibits neuroblastoma cell Cas family of signal transducers. J Biol Chem proliferation through up-regulation of PTEN. Eur 2002; 277: 9580-9589. Rev Med Pharmacol Sci 2014; 18: 3297-3303. 57) Krause M, Dent EW, Bear JE, Loureiro JJ, Gertler 43) Kuo JC, Han X, Hsiao CT, Yates JR 3rd, Waterman FB. Ena/VASP proteins: regulators of the actin cy- CM. Analysis of the myosin-II-responsive focal toskeleton and cell migration. Annu Rev Cell Dev adhesion proteome reveals a role for beta-Pix in Biol 2003; 19: 541-564. negative regulation of focal adhesion maturation. 58) Reinhard M, Halbrügge M, Scheer U, Wiegand C, Jock- Nat Cell Biol 2011; 13: 383-393. usch BM, Walter U. The 46/50 kDa phosphopro- 44) Schiller HB, Friedel CC, Boulegue C, Fässler R. tein VASP purified from human platelets is a nov- Quantitative proteomics of the integrin adhesome el protein associated with actin filaments and fo- show a myosin II-dependent recruitment of LIM cal contacts. EMBO J 1992; 11: 2063-2070. domain proteins. EMBO Rep 2011; 12: 259-266. 59) Bachmann C, Fischer L, Walter U, Reinhard M. 45) Guo WH, Wang YL. Retrograde fluxes of focal ad- The EVH2 domain of the vasodilator-stimulated hesion proteins in response to cell migration and phosphoprotein mediates tetramerization, F-actin mechanical signals. Mol Biol Cell 2007; 18: 4519- binding, and actin bundle formation. J Biol Chem 4527. 1999; 274: 23549-23557. 46) Kim-Kaneyama JR, Suzuki W, Ichikawa K, Ohki T, 60) Hüttelmaier S, Mayboroda O, Harbeck B, Jarchau Kohno Y, Sata M, Nose K, Shibanuma M. Uni-axi- T, Jockusch BM, Rüdiger M. The interaction of the al stretching regulates intracellular localization of cell-contact proteins VASP and vinculin is regu- Hic-5 expressed in smooth-muscle cells in vivo. J lated by phosphatidylinositol-4, 5-bisphosphate. Cell Sci 2005; 118: 937-949. Curr Biol 1998; 8: 479-488. 47) Hoffman LM, Jensen CC, Chaturvedi A, Yoshigi M, 61) Hansen SD, Mullins RD. VASP is a processive ac- Beckerle MC. Stretch-induced actin remodeling re- tin polymerase that requires monomeric actin for quires targeting of zyxin to stress fibers and re- barbed end association. J Cell Biol 2010; 191: cruitment of actin regulators. Mol Biol Cell 2012; 571-584. 23: 1846-1859. 62) Machner MP, Urbanke C, Barzik M, Otten S, Se- 48) Mise N, Savai R, Yu H, Schwarz J, Kaminski N, Eick- chi AS, Wehland J, Heinz DW. ActA from Listeria elberg O. Zyxin is a transforming growth factor-β monocytogenes can interact with up to four Ena/ (TGF-β)/Smad3 target gene that regulates lung VASP homology 1 domains simultaneously. J Bi- cancer cell motility via integrin α5β1. J Biol Chem ol Chem 2001; 276: 40096-40103. 2012; 287: 31393-31405. 63) Hoffman LM, Jensen CC, Kloeker S, Wang CL, Yoshigi 49) Colombelli J, Besser A, Kress H, Reynaud EG, Girard P, M, Beckerle MC. Genetic ablation of zyxin causes Caussinus E, Haselmann U, Small JV, Schwarz US, Stelzer Mena/VASP mislocalization, increased motility, EH. Mechanosensing in actin stress fibers revealed and deficits in actin remodeling. J Cell Biol 2006; by a close correlation between force and protein lo- 172: 771-782. calization. J Cell Sci 2009; 122: 1665-1679. 64) Smith MA, Blankman E, Gardel ML, Luettjohann 50) Burridge K, Guilluy C. Focal adhesions, stress fi- L, Waterman CM, Beckerle MC. A zyxin-mediat- bers and mechanical tension. Exp Cell Res 2016; ed mechanism for actin stress fiber maintenance 343: 14-20. and repair. Dev Cell 2010; 19: 365-376. 51) Hansen MD, Beckerle MC. Opposing roles of zyxin/ 65) Grange J, Moody JD, Ascione MP, Hansen MD. Zyx- LPP ACTA repeats and the LIM domain region in in-VASP interactions alter actin regulatory activity cell-cell adhesion. J Biol Chem 2006; 281: 16178- in zyxin-VASP complexes. Cell Mol Biol Lett 2013; 16188. 18: 1-10.

422 Zyxin: a mechanotransductor to regulate gene expression

66) Niebuhr K, Ebel F, Frank R, Reinhard M, Domann 81) Nuckolls GH, Turner CE, Burridge K. Functional E, Carl UD, Walter U, Gertler FB, Wehland J, studies of the domains of talin. J Cell Biol 1990; Chakraborty T. A novel prolinerich motif present in 110: 1635-1644. ActA of Listeria monocytogenes and cytoskeletal 82) Lin ZF, Shen XY, Lu FZ, Ruan Z, Huang HL, Zhen J. proteins is the ligand for the EVH1 domain, a pro- Reveals new lung adenocarcinoma cancer genes tein module present in the Ena/VASP family. EM- based on gene expression. Eur Rev Med Phar- BO J 1997; 16: 5433-5444. macol Sci 2012; 16: 1249-1256. 67) Reinhard M, Rudiger M, Jockusch BM, Walter U. 83) Chan MW, Arora PD, Bozavikov P, McCulloch VASP interaction with vinculin: a recurring theme CA. FAK, PIP5KIgamma and gelsolin coopera- of interactions with proline-rich motifs. FEBS Lett tively mediate force-induced expression of al- 1996; 399: 103-107. pha-smooth muscle actin. J Cell Sci 2009; 122: 68) Moody JD, Grange J, Ascione MP, Boothe D, Bush- 2769-2781. nell E, Hansen MD. A zyxin head-tail interaction 84) Riveline D, Zamir E, Balaban NQ, Schwarz US, Ishi- regulates zyxin-VASP complex formation. Bio- zaki T, Narumiya S, Kam Z, Geiger B, Bershadsky AD. chem Biophys Res Commun 2009; 378: 625-628. Focal contacts as mechanosensors: external- 69) Crawford AW, Michelsen JW, Beckerle MC. An inter- ly applied local mechanical force induces growth action between zyxin and alpha-actinin. J Cell Bi- of focal contacts by an mDia1-dependent and ol 1992; 116: 1381-1393. ROCK-independent mechanism. J Cell Biol 2001; 153: 1175-1186. 70) Shih W, Yamada S. N-cadherin as a key regulator of collective cell migration in a 3D environment. Cell 85) Ezzell RM, Goldmann WH, Wang N, Parashurama Adh Migr 2012; 6: 513-517. N, Ingber DE. Vinculin promotes cell spreading by mechanically coupling integrins to the cytoskele- Watanabe-Nakayama T, Saito M, Machida S, Kishimoto 71) ton. Exp Cell Res 1997; 231: 14-26. K, Afrin R, Ikai A. Requirement of LIM domains for Mammoto A, Huang S, Ingber DE the transient accumulation of paxillin at damaged 86) . Filamin links cell stress fibres. Biol Open 2013; 2: 667-674. shape and cytoskeletal structure to Rho regulation by controlling accumulation of p190RhoGAP Wang N, Butler JP, Ingber DE 72) . Mechanotransduc- in lipid rafts. J Cell Sci 2007; 120: 456-467. tion across the cell surface and through the cyto- Zhao XH, Laschinger C, Arora P, Szászi K, Kapus A, skeleton. Science 1993; 260: 1124-1127. 87) McCulloch CA. Force activates smooth muscle al- Cukierman E, Pankov R, Stevens DR, Yamada KM 73) . Tak- pha-actin promoter activity through the Rho sig- ing cell-matrix adhesions to the third dimension. naling pathway. J Cell Sci 2007; 120: 1801-1809. Science 2001; 294: 1708-1712. 88) Lele TP, Pendse J, Kumar S, Salanga M, Karavitis J, Friedland JC, Lee MH, Boettiger D. 74) Mechanically Ingber DE. Mechanical forces alter zyxin unbind- activated integrin switch controls α5β1 function. ing kinetics within focal adhesions of living cells. Science 2009; 323: 642-644. J Cell Physiol 2006; 207: 187-194. 75) del Rio A, Perez-Jimenez R, Liu R, Roca-Cusachs P, 89) Huang TT, Kudo N, Yoshida M, Miyamoto S. A nucle- Fernandez JM, Sheetz MP. Stretching single talin ar export signal in the N-terminal regulatory do- rod molecules activates vinculin binding. Science main of IκBα controls cytoplasmic localization of 2009; 323: 638-641. inactive NF-κB/IκBα complexes. Proc Natl Acad 76) Sawada Y, Tamada M, Dubin-Thaler BJ, Cherniavska- Sci U S A 2000; 97: 1014-1019. ya O, Sakai R, Tanaka S, Sheetz MP. Force sensing 90) Zeile WL, Condit RC, Lewis JI, Purich DL, Southwick by mechanical extension of the Src family ki- FS. Vaccinia locomotion in host cells: evidence for nase substrate p130Cas. Cell 2006; 127: 1015- the universal involvement of actin-based motility 1026. sequences ABM-1 and ABM-2. Proc Natl Acad 77) Hu K, Ji L, Applegate KT, Danuser G, Waterman-Stor- Sci U S A 1998; 95:13917-13922. er CM. Differential transmission of actin motion 91) Choi YH, McNally BT, Igarashi P. Zyxin regulates within focal adhesions. Science 2007; 315: 111- migration of renal epithelial cells through activa- 115. tion of hepatocyte nuclear factor-1β. Am J Physi- 78) Dingyu W, Fanjie M, Zhengzheng D, Baosheng H, ol Renal Physiol 2013; 305: F100-110. Chao Y, Yi P, Huiwen W, Jun G, Gang H. Regulation 92) Schwartz MA. Integrins and extracellular matrix of intracellular structural tension by talin in the ax- in mechanotransduction. Cold Spring Harb Per- on growth and regeneration. Mol Neurobiol 2016; spect Biol 2010; 2: a005066. 53: 4582-4595. 93) Cattaruzza M, Lattrich C, Hecker M. Focal adhesion 79) Hirata H, Tatsumi H, Lim CT, Sokabe M. Force-de- protein zyxin is a mechanosensitive modulator of pendent vinculin binding to talin in live cells: a gene expression in vascular smooth muscle cells. crucial step in anchoring the actin cytoskeleton to Hypertension 2004; 43: 726-730. focal adhesions. Am J Physiol Cell Physiol 2014; 94) Petit MM, Fradelizi J, Golsteyn RM, Ayoubi TA, Men- 306: C607-620. ichi B, Louvard D, Van de Ven WJ, Friederich E. LPP, 80) Hytonen VP, Vogel V. How force might activate an actin cytoskeleton protein related to zyxin, har- talin’s vinculin binding sites: SMD reveals a struc- bors a nuclear export signal and transcriptional ac- tural mechanism. PLoS Comput Biol 2008; 4: e24. tivation capacity. Mol Biol Cell 2000; 11: 117-129.

423 Y.-X. Wang, D.-Y. Wang, Y.-C- Guo, J. Guo

95) Aoto H, Sasaki H, Ishino M, Sasaki T. Nucle- 107) Fujita Y, Yamaguchi A, Hata K, Endo M, Yamaguchi ar translocation of cell adhesion kinase h/pro- N, Yamashita T. Zyxin is a novel interacting part- line-rich tyrosine kinase 2. Cell Struct Funct ner for SIRT1. BMC Cell Biol 2009; 10: 6. 2002; 27: 47-61. 108) Martynova NIu, Ermolina LV, Eroshkin FM, Gio- 96) Suresh Babu S, Wojtowicz A, Freichel M, Birnbau- eva FK, Zaraĭskiĭ AG. Transcriptional factor mer L, Hecker M, Cattaruzza M. Mechanism of Xanf1 interacts with the focal adhesion pro- stretch-induced activation of the mechanotrans- tein zyxin in the early development of the ducer zyxin in vascular cells. Sci Signal 2012; 5: Xenopus laevis brain. Bioorg Khim 2008; 34: 91. 573-576. 97) Wójtowicz A, Babu SS, Li L, Gretz N, Hecker M, 109) Hervy M, Hoffman L, Beckerle MC. From the Cattaruzza M. Zyxin mediation of stretch-in- membrane to the nucleus and back again: bi- duced gene expression in human endothelial functional focal adhesion proteins. Curr Opin cells. Circ Res 2010; 107: 898-902. Cell Biol 2006; 18: 524-532. 98) Ghosh S, Kollar B, Nahar T, Suresh Babu S, Wo- 110) Oldenburg J, van der Krogt G, Twiss F, Bongaarts jtowicz A, Sticht C, Gretz N, Wagner AH, Korff T, A, Habani Y, Slotman JA, Houtsmuller A, Huve- Hecker M. Loss of the mechanotransducer zyx- neers S, de Rooij J. VASP, zyxin and TES are ten- in promotes a synthetic phenotype of vascular sion-dependent members of Focal Adherens smooth muscle cells. J Am Heart Assoc 2015; Junctions independent of the α-catenin-vinculin 4: e001712. module. Sci Rep 2015; 5: 17225. 99) Yang L, Guerrero J, Hong H, DeFranco DB, Stall- 111) Zeller Kathrin Stephanie. Integrin signaling in cell cup MR. Interaction of the tau2 transcription- adhesion and mechanotransduction. Regula- al activation domain of glucocorticoid receptor tion of P13K, AKT and ROS. Uppsala Universi- with a novel steroid receptor coactivator, Hic- ty Publications, 2012. 5, which localizes to both focal adhesions and 112) Zaidel-Bar R, Geiger B. The switchable integrin the nuclear matrix. Mol Biol Cell 2000; 11: 2007- adhesome. J Cell Sci 2010; 123: 1385-1388. 2018. 113) Janoštiak R, Pataki AC, Brábek J, Rösel D. Mech- 100) Wang Y, Gilmore TD. LIM domain protein Trip6 anosensors in integrin signaling: the emerging has a conserved nuclear export signal, nucle- role of p130Cas. Eur J Cell Biol 2014; 93: 445- ar targeting sequences, and multiple transac- 454. tivation domains. Biochim Biophys Acta 2001; 1538: 260-272. 114) Moon RT, Bowerman B, Boutros M, Perrimon N. The promise and perils of Wnt signaling through 101) Degenhardt YY, Silverstein S. Interaction of zyx- β-catenin. Science 2002; 296: 1644-1646. in, a focal adhesion protein, with the e6 protein from human papillomavirus type 6 results in its 115) Hsueh YP, Wang TF, Yang FC, Sheng M. Nuclear nuclear translocation. J Virol 2001; 75: 11791- translocation and transcription regulation by the 11802. membrane-associated guanylate kinase CASK/ 102) Kato T, Muraski J, Chen Y, Tsujita Y, Wall J, Glem- LIN-2. Nature 2000; 404: 298-302. botski CC, Schaefer E, Beckerle M, Sussman MA. 116) Yao XL, Li L, He XL, Cui L, Kuang W, Tang M. Atrial natriuretic peptide promotes cardiomyo- Activation of β-catenin stimulated by me- cyte survival by cGMP-dependent nuclear ac- chanical strain and estrogen requires es- cumulation of zyxin and Akt. J Clin Invest 2005; trogen receptor in mesenchymal stem cells 115: 2716-2730. (MSCs). Eur Rev Med Pharmacol Sci 2014; 103) Chan CB, Liu X, Tang X, Fu H, Ye K. Akt phos- 18: 3149-3155. phorylation of zyxin mediates its interaction with 117) Simmons RD, Kumar S, Jo H. The role of endothe- acinus-S and prevents acinus-triggered chro- lial mechanosensitive genes in atherosclerosis matin condensation. Cell Death Differ 2007; 14: and omics approaches. Arch Biochem Biophys 1688-1699. 2016; 591: 111-131. 104) Hiesberger T, Bai Y, Shao X, McNally BT, Sin- 118) Jiang M, Qiu J, Zhang L, Lü D, Long M, Chen L, clair AM, Tian X, Somlo S, Igarashi P. Mutation Luo X. Changes in tension regulates prolifer- of hepatocyte nuclear factor-1beta inhibits ation and migration of fibroblasts by remodel- Pkhd1 gene expression and produces renal ing expression of ECM proteins. Exp Ther Med cysts in mice. J Clin Invest 2004; 113: 814- 2016; 12: 1542-1550. 825. 119) Nishiya N, Sabe H, Nose K, Shibanuma M. The 105) Youn H, Kim EJ, Um SJ. Zyxin cooperates with LIM domains of hic-5 protein recognize spe- PTOV1 to confer retinoic acid resistance by re- cific DNA fragments in a zinc-dependent man- pressing RAR activity. Cancer Lett 2013; 331: ner in vitro. Nucleic Acids Res 1998; 26: 4267- 192-199. 4273. 106) Hervy M, Hoffman LM, Jensen CC, Smith M, Beck- 120) Terzano C, Laurendi G, Capoccetta G, Rapisarda erle MC. The LIM protein zyxin binds CARP-1 M, Pacilio R, Petroianni A. The smooth muscle and promotes apoptosis. Genes Cancer 2010; and airway hyperresponsiveness. Eur Rev Med 1: 506-515. Pharmacol Sci 2003; 7: 9-26.

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121) Rosner SR, Pascoe CD, Blankman E, Jensen CC, toshi Ishiguro, Hiroji Uemura, Masatoshi Watanabe. Krishnan R, James AL, Elliot JG, Green FH, Liu JC, Abstract 522: Involvement of Zyxin in aggres- Seow CY, Park JA, Beckerle MC, Paré PD, Fredberg siveness of human prostate cancer. Cancer Res JJ, Smith MA. The actin regulator zyxin reinforc- 2015; 75(15 Supplement): 522. es airway smooth muscle and accumulates in 124) Zhuge BZ, Du BR, Meng XL, Zhang YQ. MASTL airways of fatal asthmatics. PLoS One 2017; 12: is a potential poor prognostic indicator in ER+ e0171728. breast cancer. Eur Rev Med Pharmacol Sci 122) Song XF, Chang H, Liang Q, Guo ZF, Wu JW. 2017; 21: 2413-2420. ZEB1 promotes prostate cancer proliferation 125) Kim YJ, Sertamo K, Pierrard MA, Mesmin C, Kim and invasion through ERK1/2 signaling path- SY, Schlesser M, Berchem G, Domon B. Verifi- way. Eur Rev Med Pharmacol Sci 2017; 21: cation of the biomarker candidates for non- 4032-4038. small-cell lung cancer using a targeted pro- 123) Kei Yamamoto, Yasuki Hori, Jieun Seo, Saho Hashimo- teomics approach. J Proteome Res 2015; 14: to, Yoshihiro Endo, Tadashi Nittami, Yusuke Ito, Hi- 1412-1419.

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