Thymosin ß4 Is an Endogenous Iron Chelator and Molecular Switcher of Ferroptosis.

Thymosin ß4 (Tß4) was extracted forty years agofrom calf thymus. Since then, it has been identified as a G-actin binding protein involved in blood clotting, tissue regeneration, angiogenesis, and anti-inflammatory processes. Tß4 has also been implicated in tumor metastasis and neurodegeneration. However, the precise roles and mechanism(s) of action of Tß4 in these processes remain largely unknown, with the binding of the G-actin protein being insufficient to explain these multi-actions. Here we identify for the first time the important role of Tß4 mechanism in ferroptosis, an iron-dependent form of cell death, which leads to neurodegeneration and somehow protects cancer cells against cell death. Specifically, we demonstrate four iron2+ and iron3+ binding regions along the peptide and show that the presence of Tß4 in cell growing medium inhibits erastin and glutamate-induced ferroptosis in the macrophage cell line. Moreover, Tß4 increases the expression of oxidative stress-related genes, namely BAX, hem oxygenase-1, heat shock protein 70 and thioredoxin reductase 1, which are downregulated during ferroptosis. We state the hypothesis that Tß4 is an endogenous iron chelator and take part in iron homeostasis in the ferroptosis process. We discuss the literature data of parallel involvement of Tß4 and ferroptosis in different human pathologies, mainly cancer and neurodegeneration. Our findings confronted with literature data show that controlled Tß4 release could command on/off switching of ferroptosis and may provide novel therapeutic opportunities in cancer and tissue degeneration pathologies.

Thymosin fraction 5 re-evaluated after 35 years by high-resolution mass spectrometry.

OBJECTIVES: We reevaluated a lyophilized sample of thymosin fraction 5, stored for 37 years at room temperature, by high-resolution mass spectrometry in terms of stability and yet uncharacterized polypeptides that could be biological important substances. METHODS: A top-down proteomic platform based on high-performance liquid chromatography (HPLC) coupled to high-resolution LTQ-Orbitrap mass spectrometry (MS) was applied to molecular characterization of polypeptides present in thymosin fraction 5. RESULTS: We detected more than 100 monoisotopic masses corresponding to thymosin ß4 and truncated forms of ubiquitin, prothymosin α, thymosin ß4, and thymosin ß9. Additionally, we discovered a new polypeptide present in thymosin fraction 5 and identified it as intact SH3 domain-binding glutamic acid-rich-like protein 3. CONCLUSION: In spite of the well-known proteolytic processes inherent to the preparation of thymosin fraction 5, still uncharacterized polypeptides as well as truncated forms of already well-known thymosins are present in fraction 5 after long-term storage. Therefore, continuing characterization of thymosin fraction 5 is even nowadays highly promising.

Loss of Glycine Transporter 1 Causes a Subtype of Glycine Encephalopathy with Arthrogryposis and Mildly Elevated Cerebrospinal Fluid Glycine.

Glycine is a major neurotransmitter that activates inhibitory glycine receptors and is a co-agonist for excitatory glutamatergic N-methyl-D-aspartate (NMDA) receptors. Two transporters, GLYT1 and GLYT2, regulate extracellular glycine concentrations within the CNS. Dysregulation of the extracellular glycine has been associated with hyperekplexia and nonketotic hyperglycinemia. Here, we report four individuals from two families who presented at birth with facial dysmorphism, encephalopathy, arthrogryposis, hypotonia progressing to hypertonicity with startle-like clonus, and respiratory failure. Only one individual survived the respiratory failure and was weaned off ventilation but has significant global developmental delay. Mildly elevated cerebrospinal fluid (CSF) glycine and normal serum glycine were observed in two individuals. In both families, we identified truncating mutations in SLC6A9, encoding GLYT1. We demonstrate that pharmacologic or genetic abolishment of GlyT1 activity in mice leads to mildly elevated glycine in the CSF but not in blood. Additionally, previously reported slc6a9-null mice and zebrafish mutants also display phenotypes consistent with the affected individuals we examined. Our data suggest that truncating SLC6A9 mutations lead to a distinct human neurological syndrome hallmarked by mildly elevated CSF glycine and normal serum glycine.

Functional consequence of fibulin-4 missense mutations associated with vascular and skeletal abnormalities and cutis laxa.

Fibulin-4 is a 60kDa calcium binding glycoprotein that has an important role in development and integrity of extracellular matrices. It interacts with elastin, fibrillin-1 and collagen IV as well as with lysyl oxidases and is involved in elastogenesis and cross-link formation. To date, several mutations in the fibulin-4 gene (FBLN4/EFEMP2) are known in patients whose major symptoms are vascular deformities, aneurysm, cutis laxa, joint laxity, or arachnodactyly. The pathogenetic mechanisms how these mutations translate into the clinical phenotype are, however, poorly understood. In order to elucidate these mechanisms, we expressed fibulin-4 mutants recombinantly in HEK293 cells, purified the proteins in native forms and analyzed alterations in protein synthesis, secretion, matrix assembly, and interaction with other proteins in relation to wild type fibulin-4. Our studies show that different mutations affect these properties in multiple ways, resulting in fibulin-4 deficiency and/or impaired ability to form elastic fibers. The substitutions E126K and C267Y impaired secretion of the protein, but not mRNA synthesis. Furthermore, the E126K mutant showed less resistance to proteases, reduced binding to collagen IV and fibrillin-1, as well as to LTBP1s and LTBP4s. The A397T mutation introduced an extra O-glycosylation site and deleted binding to LTBP1s. We show that fibulin-4 binds stronger than fibulin-3 and -5 to LTBP1s, 3, and 4s, and to the lysyl oxidases LOX and LOXL1; the binding of fibulin-4 to the LOX propeptide was strongly reduced by the mutation E57K. These findings show that different mutations in the fibulin-4 gene result in different molecular defects affecting secretion rates, protein stability, LOX-induced cross-linking, or binding to other ECM components and molecules of the TGF-ß pathway, and thus illustrate the complex role of fibulin-4 in connective tissue assembly.

High-resolution mass spectrometry for thymosins detection and characterization.

OBJECTIVES: The aim of this study was to characterize ß and α thymosins and their proteoforms in various tissues and bodily fluids by mass spectrometry and to look at their association with a wide variety of pathologies. METHODS: A top-down proteomic platform based on high-performance liquid chromatography (HPLC) coupled to high-resolution LTQ-Orbitrap mass spectrometry (MS) was applied to the characterization of naturally occurring peptides. RESULTS: In addition to thymosin ß4 (Tß4) and ß10 (Tß10), several post-translational modifications of both these peptides were identified not only in bodily fluids but also in normal and pathological tissues of different origins. The analysis of tissue specimens allowed the characterization of different C-terminal truncated forms of Tß4 and Tß10 together with other proteolytic fragments. The sulfoxide derivative of both Tß4 and Tß10 and the acetylated derivatives at lysine residues of Tß4 were also characterized. Different proteoforms of prothymosin α, parathymosin α, thymosin α1 and thymosin α11 together with diverse proteolytic fragments were identified too. CONCLUSION: The clinical and prognostic significance and the origin of these proteoforms have to be deeply investigated.

Identification of PDGF-BB binding to thymosin ß4 by chemical cross-linking.

INTRODUCTION: The purpose of our work was to identify unknown interaction partners of thymosin ß4 (Tß4). It was suggested that Tß4 could be an antifibrotic drug for treatment of liver fibrogenesis, because Tß4 prevents the platelet-derived growth factor-BB (PDGF-BB)-induced activation of hepatic stellate cells (HSCs). Very little information is available how Tß4 counteracts the PDGF-BB-induced activation of HSCs. We propose the hypothesis that Tß4 could bind directly to PDGF-BB and thereby reduce the concentration of free PDGF-BB available for binding to the PDGF-ß receptor. METHODS: To prove our suggestion of a direct interaction between Tß4 and PDGF-BB, we carried out chemical as well as photochemical cross-linking experiments between the two pure proteins in vitro. RESULTS: We identified an interaction between Tß4 and PDGF-BB by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) cross-linking as well as through biotin label transfer using a bifunctional photoactivatable derivative of Tß4. In an in vitro system, PDGF-BB was identified as the first extracellular partner interacting with Tß4. This interaction could influence PDGF-BB binding to its receptor and abolish PDGF-BB-related effects. CONCLUSION: Direct interaction of Tß4 with extracellular factors should be considered as a potential mechanism to explain the pleiotropic effects of ß-thymosins.

MRTF-A controls vessel growth and maturation by increasing the expression of CCN1 and CCN2.

Gradual occlusion of coronary arteries may result in reversible loss of cardiomyocyte function (hibernating myocardium), which is amenable to therapeutic neovascularization. The role of myocardin-related transcription factors (MRTFs) co-activating serum response factor (SRF) in this process is largely unknown. Here we show that forced MRTF-A expression induces CCN1 and CCN2 to promote capillary proliferation and pericyte recruitment, respectively. We demonstrate that, upon G-actin binding, thymosin ß4 (Tß4), induces MRTF translocation to the nucleus, SRF-activation and CCN1/2 transcription. In a murine ischaemic hindlimb model, MRTF-A or Tß4 promotes neovascularization, whereas loss of MRTF-A/B or CCN1-function abrogates the Tß4 effect. We further show that, in ischaemic rabbit hindlimbs, MRTF-A as well as Tß4 induce functional neovascularization, and that this process is inhibited by angiopoietin-2, which antagonizes pericyte recruitment. Moreover, MRTF-A improves contractile function of chronic hibernating myocardium of pigs to a level comparable to that of transgenic pigs overexpressing Tß4 (Tß4tg). We conclude that MRTF-A promotes microvessel growth (via CCN1) and maturation (via CCN2), thereby enabling functional improvement of ischaemic muscle tissue.

Peptide labeling with photoactivatable trifunctional cadaverine derivative and identification of interacting partners by biotin transfer.

A new photoactivatable trifunctional cross-linker, cBED (cadaverine-2-[6-(biotinamido)-2-(p-azidobenzamido) hexanoamido]ethyl-1,3'-dithiopropionate), was synthesized by chemical conversion of sulfo-SBED (sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azidobenzamido) hexanoamido]ethyl-1,3'-dithiopropionate) with cadaverine. This cross-linker was purified by reversed-phase high-performance liquid chromatography (RP-HPLC) and characterized using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis. cBED is based on sulfo-SBED that has a photoactivatable azido group, a cleavable disulfide bond for label transfer methods, and a biotin moiety for highly sensitive biotin/avidin detection. By ultraviolet (UV) light, the azido group is converted to a reactive nitrene, transforming transient bindings of interacting structures to covalent bonds. In contrast to the sulfo-N-hydroxysuccinimide (sulfo-NHS) moiety of sulfo-SBED, which attaches quite unspecifically to amino groups, cBED includes a cadaverine moiety that can be attached by transglutaminase more specifically to certain glutamine residues. For instance, thymosin ß4 can be labeled with cBED using tissue transglutaminase. By high-resolution HPLC/ESI-MS (electrospray ionization-mass spectrometry) and tandem MS (MS/MS) of the trypsin digest, it was established that glutamine residues at positions 23 and 36 were labeled, whereas Q39 showed no reactivity. The covalent binding of cBED to thymosin ß4 did not influence its G-actin sequestering activity, and the complex could be used to identify new interaction partners. Therefore, cBED can be used to better understand the multifunctional role of thymosin ß4 as well as of other proteins and peptides.

In vivo imaging using fluorescent antibodies to tumor necrosis factor predicts therapeutic response in Crohn's disease.

As antibodies to tumor necrosis factor (TNF) suppress immune responses in Crohn's disease by binding to membrane-bound TNF (mTNF), we created a fluorescent antibody for molecular mTNF imaging in this disease. Topical antibody administration in 25 patients with Crohn's disease led to detection of intestinal mTNF(+) immune cells during confocal laser endomicroscopy. Patients with high numbers of mTNF(+) cells showed significantly higher short-term response rates (92%) at week 12 upon subsequent anti-TNF therapy as compared to patients with low amounts of mTNF(+) cells (15%). This clinical response in the former patients was sustained over a follow-up period of 1 year and was associated with mucosal healing observed in follow-up endoscopy. These data indicate that molecular imaging with fluorescent antibodies has the potential to predict therapeutic responses to biological treatment and can be used for personalized medicine in Crohn's disease and autoimmune or inflammatory disorders.

Thymosin beta4 inhibits ADF/cofilin stimulated F-actin cycling and hela cell migration: reversal by active Arp2/3 complex.

F-actin treadmilling plays a key part in cell locomotion. Because immunofluorescence showed colocalisation of thymosin beta4 (Tß4) with cofilin-1 and Arp2/3 complex in lamellipodia, we analyzed combinations of these proteins on F-actin-adenosine triphosphate (ATP)-hydrolysis, which provides a measure of actin treadmilling. Actin depolymerising factor (ADF)/cofilin stimulated treadmilling, while Tß4 decreased treadmilling, presumably by sequestering monomers. Tß4 added together with ADF/cofilin also inhibited the treadmilling, relative to cofilin alone, but both the rate and extent of depolymerization were markedly enhanced in the presence of both these proteins. Arp2/3 complex reversed the sequestering activity of Tß4 when equimolar to actin, but not in the additional presence of cofilin-1 or ADF. Transfection experiments to explore the effects of changing the intracellular concentration of Tß4 in HeLa cells showed that an increase in Tß4 resulted in reduced actin filaments bundles and narrower lamellipodia, and a conspicuous decrease of cell migration as seen by two different assays. In contrast, cells transfected with a vector leading to Tß4 knockdown by small interfering RNA (siRNA) displayed prominent actin filament networks within the lamellipodia and the leading lamella and enhanced migration. The experiments reported here demonstrate the importance of the interplay of these different classes of actin-binding proteins on cell behaviour.

Thymosin ß4 and tissue transglutaminase. Molecular characterization of cyclic thymosin ß4.

Thymosin ß4 is the prototype of ß-thymosins and is present in almost every mammalian cell. It is regarded to be the main intracellular G-actin sequestering peptide. Thymosin ß4 serves as a specific glutaminyl substrate for guinea pig transglutaminase. In the absence of an appropriate additional aminyl donor an ε-amino group of thymosin ß4 serves also as an aminyl substrate and an intramolecular bond is formed concomitantly NH3 (17 Da) is lost. The molecular mass of the product is 4,949.6 Da. This is 16.3 Da less than the molecular mass of thymosin ß4 (4,965.9 Da). Digestion with endopeptidases and Edman degradation of the fragments identified the exact position of the ring forming isopeptide bond. In spite of 3 glutaminyl and 9 lysyl residues of thymosin ß4 only one isopeptide bond between Lys16 and Gln36 was formed (cyclic thymosin ß4). These two amino acid residues are conserved in all ß-thymosins. Cyclic thymosin ß4 still forms a complex with G-actin albeit the stability of the complex is about one fiftieth of the stability of the thymosin ß4 × G-actin complex.

High-resolution HPLC-ESI-MS characterization of the contact sites of the actin-thymosin ß(4) complex by chemical and enzymatic cross-linking.

Thymosin ß4 sequesters actin by formation of a 1:1 complex. This transient binding in the complex was stabilized by formation of covalent bonds using the cross-linking agents 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and a microbial transglutaminase. The localization of cross-linking sites was determined after separating the products using SDS-PAGE by tryptic in-gel digestion and high-resolution HPLC-ESI-MS. Three cross-linked fragments were identified after chemical cross-linking, indicating three contact sites. Because the cross-linked fragments were detected simultaneously with the corresponding non-cross-linked fragments, the three contact sites were not formed in parallel. K3 of thymosin ß4 was cross-linked to E167 of actin, K18 or K19 of thymosin ß4 to one of the first three amino acids of actin (DDE), and S43 of thymosin ß4 to H40 of actin. The imidazole ring of histidine was proven to be an acyl acceptor for carbodiimide-mediated cross-linking. Molecular modeling proved an extended conformation of thymosin ß4 along the subdomains 1 to 3 of actin. The enzymatic cross-linking using a microbial transglutaminase led to the formation of three cross-linking sites. Q41 of actin was cross-linked to K19 of thymosin ß4, and K61 of actin to Q39 of thymosin ß4. The third cross-linking site was identified between Q41 of actin and Q39 of thymosin ß4, which are simultaneously cross-linked to K16, K18, or K19 of thymosin ß4. When both cross-linking reactions are taken together, the complex formation of actin by thymosin ß4 is more likely to be flexible than rigid and is localized along the subdomains 1 to 3 of actin.

Identification of interaction partners of ß-thymosins: application of thymosin ß4 labeled by transglutaminase.

In this review, we identify potential interaction partners of the ß-thymosin family. The proteins of this family are highly conserved peptides in mammals and yet only one intracellular (G-actin) and one cell-surface protein (ß subunit of F(1) -F(0) ATP synthase) were identified as interaction partners of thymosin ß4. Cross-linking experiments may be a possible approach to discover additional proteins that interact with the ß-thymosin family. It has previously been shown that thymosin ß4 can be labeled at its glutaminyl residues with various cadaverines using tissue transglutaminase. Here, we illuminate recent results and give an outlook on upcoming work in the field.

Antibodies in research of thymosin ß4: investigation of cross-reactivity and influence of fixatives.

Antibodies against thymosin ß4 are available from various sources and have been used in immunohistochemistry, ELISA, and Western blot analyses. None of these antibodies have been fully characterized for specificity and influence of fixation techniques. This presents a difficulty because many tissues express more than one member of the ß-thymosin family; in addition, highly homologous sequences are typical elements of ß-thymosins. It is also important to scrutinize the influence of fixatives on the antibody-binding capability. Fixatives such as formaldehyde are well known as cross-linking reagents. Chemical modifications within the thymosin ß4 molecule might change the putative epitope recognized by the antibody. These considerations suggest that investigations on thymosin ß4 antibodies available to the scientific community are important and necessary before any experiment can be performed to exclude cross-reactivity with other ß-thymosins that are coexistent in the examined tissue and to prove antibody binding after fixation steps.

Thymosin ß4: a multi-functional regenerative peptide. Basic properties and clinical applications.

INTRODUCTION: Thymosin ß(4), a low molecular weight, naturally-occurring peptide plays a vital role in the repair and regeneration of injured cells and tissues. After injury, thymosin ß(4), is released by platelets, macrophages and many other cell types to protect cells and tissues from further damage and reduce apoptosis, inflammation and microbial growth. Thymosin ß(4) binds to actin and promotes cell migration, including the mobilization, migration, and differentiation of stem/progenitor cells, which form new blood vessels and regenerate the tissue. Thymosin ß(4) also decreases the number of myofibroblasts in wounds, resulting in decreased scar formation and fibrosis. AREAS COVERED: This article will cover the many thymosin ß(4) activities that directly affect the repair and regeneration cascade with emphasis on its therapeutic uses and potential. Our approach has been to evaluate the basic biology of the molecule as well as its potential for clinical applications in the skin, eye, heart and brain. EXPERT OPINION: The considerable advances in our understanding of the functional biology and mechanisms of action of thymosin ß(4) have provided the scientific foundation for ongoing and projected clinical trials in the treatment of dermal wounds, corneal injuries and in the regeneration and repair of heart and CNS tissue following ischemic insults and trauma.

The beta-thymosins: intracellular and extracellular activities of a versatile actin binding protein family.

The beta-thymosins are N-terminally acetylated peptides of about 5 kDa molecular mass and composed of about 40-44 amino acid residues. The first member of the family, thymosin beta4, was initially isolated from thymosin fraction 5, prepared in five steps from calf thymus. Thymosin beta4 was supposed to be specifically produced and released by the thymic gland and to possess hormonal activities modulating the immune response. Various paracrine effects have indeed been reported for these peptides such as cardiac protection, angiogenesis, stimulation of wound healing, and hair growth. Besides these paracrine effects, it was noted that beta-thymosins occur in high concentration in the cytoplasm of many eukaryotic cells and bind to the cytoskeletal component actin. Subsequently it became apparent from in vitro experiments that they preferentially bind to monomeric (G-)actin and stabilize it in its monomeric form. Due to this ability the beta-thymosins are the main intracellular actin sequestering factor, i.e., they posses the ability to remove monomeric actin from the dynamic assembly and disassembly processes of the actin cytoskeleton that constantly occur in activated cells. In this review we will concentrate on the intracellular activity and localization of the beta-thymosins, i.e., their modulating effect on the actin cytoskeleton.

Thymosin beta4 is an essential paracrine factor of embryonic endothelial progenitor cell-mediated cardioprotection.

BACKGROUND: Prolonged myocardial ischemia results in cardiomyocyte loss despite successful revascularization. We have reported that retrograde application of embryonic endothelial progenitor cells (eEPCs) provides rapid paracrine protection against ischemia-reperfusion injury. Here, we investigated the role of thymosin beta4 (Tbeta4) as a mediator of eEPC-mediated cardioprotection. METHODS AND RESULTS: In vitro, neonatal rat cardiomyocytes were subjected to hypoxia-reoxygenation in the absence or presence of eEPCs with or without Tbeta4 short hairpin RNA (shRNA) transfection. In vivo, pigs (n=9 per group) underwent percutaneous left anterior descending artery occlusion for 60 minutes on day 1. After 55 minutes of ischemia, control eEPCs (5x10(6) cells) or cells transfected with Tbeta4 shRNA when indicated or 15 mg Tbeta4 alone were retroinfused into the anterior interventricular vein. Segmental endocardial shortening in the infarct zone at 150-bpm atrial pacing, infarct size (triphenyl tetrazolium chloride viability and methylene blue exclusion), and inflammatory cell influx (myeloperoxidase activity) were determined 24 hours later. Survival of neonatal rat cardiomyocytes increased from 32+/-4% to 90+/-2% after eEPC application, an effect sensitive to shRNA transfection compared with Tbeta4 (45+/-7%). In vivo, infarct size decreased with eEPC application (38+/-4% versus 54+/-4% of area at risk; P<0.01), an effect abolished by Tbeta4 shRNA (62+/-3%). Segmental subendocardial shortening improved after eEPC treatment (22+/-3% versus -3+/-4% of control area) unless Tbeta4 shRNA was transfected (-6+/-4%). Retroinfusion of Tbeta4 mimicked eEPC application (infarct size, 37+/-3%; segmental endocardial shortening, 34+/-7%). Myeloperoxidase activity (3323+/-388 U/mg in controls) was decreased by eEPCs (1996+/-546 U/mg) or Tbeta4 alone (1455+/-197 U/mg) but not Tbeta4 shRNA-treated eEPCs (5449+/-829 U/mg). CONCLUSIONS: Our findings show that short-term cardioprotection derived by regional application of eEPCs can be attributed, at least in part, to Tbeta4.

Myelin proteolipid protein (PLP) as a marker antigen of central nervous system contaminations for routine food control.

Spreading transmissible spongiform encephalopathies (TSE) have been widely attributed to transmission by ingestion of mammalian central nervous system (CNS) tissue. Reliable exclusion of this epidemiological important route of transmission relies on an effective surveillance of food contamination. Here, myelin proteolipid protein (PLP) is identified as a specific and largely heat-resistant marker for detection of food contaminations by CNS tissue. PLP is a component of oligodendritic glial sheaths of neuronal processes that is specifically expressed in the CNS. A highly selective polyclonal antibody was developed directed against an epitope present in the full-length PLP protein, but absent from the developmentally regulated splice variant DM-20. In combination with a hydrophobic extraction of PLP from tissue samples, the antibody reliably detected PLP from spinal cord, cerebellum, and cortex of different mammalian species. Consistent with earlier reports on PLP expression, no cross-reactivity was observed with peripheral nerve or extraneural tissue, except for a very faint signal obtained with heart. When applied to an artificial CNS contamination present in sausages, the antibody reliably detected a low concentration (1%) of the contaminant. Application of heat, as used during conventional sausage manufacturing, led to a predominant alteration of arginine residues in the PLP protein and a partial loss of immunoreactivity. In contrast, a stretch of hydrophilic amino acids(112-122) proved to be heat-resistant, preserving the immunogenicity of this PLP epitope during heating. Taken together, the excellent CNS specificity of PLP immunodetection and the presence of a heat-resistant epitope have permitted the development of a highly sensitive immunoassay for CNS contamination in routine food control.

Subcellular distribution of thymosin beta4.

The localization of Oregon Green cadaverine-labeled thymosin beta(4), its fragments, and variants was investigated in cytoplasm-depleted A431 cells and in microinjected cells without and with fixation. The studied thymosin beta(4) variants included substitutions of the lysine residues within the basic cluster (14-KSKLKK-19) and the actin-binding motif (17-LKKTETQ-23). In contrast to Oregon Green cadaverine, none of the variants or fragments of thymosin beta(4) could pass the intact nuclear pore of cytoplasm-depleted cells and were hence excluded from the nucleus. However, an equal distribution of all thymosin beta(4) variants was observed in living cells. The nuclear localization is neither dependent on the actin-binding ability of thymosin beta(4) nor on its basic lysine cluster. The equal distribution of the beta-thymosins, the ability of the fragments thymosin beta(4)(1-26) and beta(4)(27-43) to enter the nucleus in intact cells immediately after injection, and their exclusion from cytoplasm-depleted nuclei make it unlikely that they are transported by a single transport protein. A passive but regulated diffusion could explain the described ability of thymosin beta(4) to shuttle into the nucleus.

Thymosin beta4 upregulates the expression of hepatocyte growth factor and downregulates the expression of PDGF-beta receptor in human hepatic stellate cells.

Hepatic stellate cells (HSCs) are the main producers of type I collagen in the liver, and therefore are responsible, in part, for the fibrous scar observed in cirrhotic livers. Although there is no approved treatment for this deadly disease, drugs inducing HSC apoptosis in animals (gliotoxin) and hepatocyte regeneration in man (hepatocyte growth factor [HGF]), have been used successfully in ameliorating liver fibrosis. In this communication we investigated whether thymosin beta(4) (Tbeta(4)), an actin-sequestering peptide that prevents scarring of the heart after a myocardial infarction and that prevents kidney fibrosis in animals, has the potential to be used to treat liver fibrosis. To this end we studied whether the administration of Tbeta(4) to HSCs could alter the expression of genes encoding for extracellular matrix components, as well as those required for differentiation of HSCs. Our preliminary findings show that Tbeta(4) had no effect on the expression of alpha2 (I) collagen, tissue inhibitor of metalloproteinases-1, and matrix metalloproteinase-2 mRNAs. However, it upregulated the expression of HGF and downregulated the expression of platelet-derived growth factor-beta receptor mRNAs in these cells. Overall, these findings suggest that Tbeta(4) has antifibrogenic potential.