TMSB10, a potential prognosis prediction biomarker, promotes the invasion and angiogenesis of gastric cancer.

BACKGROUND AND AIM: The thymosin beta 10 (TMSB10) was originally identified from the thymus, which plays a key role in the development of many cancers. However, the underlying molecular mechanisms of TMSB10 involved in GC have not been understood. METHODS: We sought to determine the expression of TMSB10 in human GC tissues and illustrate whether it is correlated with the clinical pathologic characteristics and prognosis in GC patients. Its roles and potential mechanisms in regulating tumor growth, invasion, and angiogenesis were evaluated by TMSB10 knockdown/overexpression of GC cells in vitro and ex vivo. RESULTS: Marked overexpression of TMSB10 protein expression was observed in GC cells and tissues, which was associated with the advanced tumor stage and lymph nodes (LN) metastasis of GC patients. Furthermore, prognostic analysis showed that GC patients with high TMSB10 expression had a remarkably shorter survival and acted as an important factor for predicting poor overall survival in GC patients. Moreover, TMSB10 overexpression promoted, while TMSB10 knockdown the proliferation, EMT process, and angiogenesis of GC cells. CONCLUSION: The study highlights that TMSB10 may hold promise as potential prognosis prediction biomarker for the diagnosis of GC and a potential therapeutic target, which will facilitate the development of a novel therapeutic strategy against GC.

Thymosin ß10 promotes tumor-associated macrophages M2 conversion and proliferation via the PI3K/Akt pathway in lung adenocarcinoma.

BACKGROUND: Thymosin ß10 (TMSB10) has been reported to play a protumorigenic role in a majority of solid cancers. However, the existence of TMSB10 in immune microenvironment may contribute to the pathogenesis of lung adenocarcinoma has not been previously explored. METHOD: TAMs-associated TMSB10 expression was evaluated by immunohistochemistry (IHC) in 184 lung adenocarcinomas. Xenograft mice model was established to investigate the effect of TMSB10 shRNA on TAMs phenotypes. The macrophages phenotype associated cytokines IL-6, IL-8, IL-12 and TNF-α were detected by ELISA after treated with TMSB10 shRNA or scramble. Furthermore, the target proteins were detected by immunoblotting. RESULTS: We found that high TAMs-associated TMSB10 expression was significantly correlated with the advanced TNM stage and T3/T4 tumor size. And high TAMs-associated TMSB10 expression was significantly correlated with poor overall and progression-free survival of lung adenocarcinoma, acting as an independent prognostic factor for lung adenocarcinoma. Furthermore, we investigated the biological functions of TMSB10 in macrophages in vivo and in vitro. TMSB10 knockdown dramatically reduced TAMs, THP-1 and RAW264.7 cell proliferation, and promoted macrophages phenotype conversion of M2 to M1, and TMSB10 knockdown reduced the levels of p-Akt (Sec473), p-mTOR (Sec2448) and p-p70S6K (Thr389) without effect on Akt, mTOR and p70S6K expression. CONCLUSIONS: These results demonstrate that TAMs-associated TMSB10 promotes tumor growth through increasing TAMs M2 conversion and proliferation via PI3K/Akt signaling pathway, providing a promising tumor biomarker for predicting prognosis and a potential therapeutic target for lung adenocarcinoma.

Highly effective biosynthesis of N-acetylated human thymosin ß4 (Tß4) in Escherichia coli.

Thymosin ß4 (Tß4) is a multifunctional N-acetylated peptide with distinct activities important at various stages. Due to its potential multiple therapeutic uses in many fields, there is an increasing need of Tß4 at lower costs than with the use of chemical synthesis. In this research, we developed a method to produce rhTß4 with N-acetylation in E. coli. Firstly, the E. coli strain whose chromosome being integrated by the specific N-terminal acetyltransferase ssArd1 was constructed. Secondly, the rhTß4-Intein was constructed, in which rhTß4 was fused to the N-terminus of the smallest mini-intein Spl DnaX. The rhTß4 could be fully acetylated when the rhTß4-Intein was expressed in the engineering strain. After purification, the rhTß4-Intein fusion protein was induced with dithiothreitol (DTT) to release rhTß4 through intein-mediated N-terminal cleavage. Under the optimal conditions, the N-terminal serine residue was shown to be 100% acetylated and the yield of N-acetylated rhTß4 can reach 200 mg per litre. The N-acetylated rhTß4 could be stable at 2-8 °C for 24 months in PBS buffer without protein degradation and concentration change. The N-acetylated rhTß4 also showed the activity of binding with actins from different sources and excellent therapeutic effect on the rats with moderate to severe dry eye disease.

Thymosin beta-4 overexpression correlates with high-risk groups in gastric gastrointestinal stromal tumors: A retrospective analysis by immunohistochemistry.

BACKGROUND: Thymosin beta-4 (Tß4) is a protein that is linked to a number of important biological actions and recently tumor progression and poor prognosis of some tumors. The aim of this study was to evaluate Tß4 expression in gastric GISTs and correlate with some clinicopathological characteristics related with prognosis and clinical outcome in order to add further data to the current literature. METHODS: Tß4 antibody was applied to the 4µm-thick paraffin sections of 57 gastric GISTs by immunohistochemistry. RESULTS: Tß4 expression was found to be directly corrrelated with higher risk groups, tumor size, mitotic count, cellularity, and necrosis while it was inversely correlated with overall survival (OS) by univariate analysis (p=0.000, p=0.001, p=0.000, p=0.025, p=0.023, and p=0.042, respectively). The direct association between Tß4 expression and risk groups were also supported by multivariate analysis (p=0.000, ß=0.497, t=4.374). CONCLUSION: Overexpression of Tß4 was found to be related with predictive characteristics for tumor progression and adverse prognosis. Thus, we suggest that overexpression of Tß4 might play a role in the progression of gastric GISTs and might be used as a potential prognostic tool as well as a target for novel therapies.

Thymosin ß4 overexpression regulates neuron production and spatial distribution in the developing avian optic tectum.

Thymosin ß4 (Tß4), the principal G-actin regulating entity in eukaryotic cells, has also multiple intra- and extracellular functions related to tissue regeneration and healing. While its effect in adult organs is being widely investigated, currently, little is known about its influence on embryonic tissues, i.e., in the developing nervous system. The importance of Tß4 for neural stem cell proliferation in the embryonic chicken optic tectum (OT) has previously been shown by us for the first time. In the present study, using in ovo electroporation, we carried out a quantification of the effects of the Tß4-overexpression on the developing chicken OT between E4 and E6 at the hemisphere as well as cellular level. We precisely examined tissue growth and characterized cells arising from the elevated mitotic activity of progenitor cells. By using spinning-disk confocal laser scanning microscopy, we were able to visualize these effects across whole OT sections. Our experiments now demonstrate more clearly that the overexpression of Tß4 leads to a tangential expansion of the treated OT-hemisphere and that, under these circumstances, overall density of tectal and in particular of postmitotic neuronal cells is increased. Thanks to this new quantitative approach, the present results extend our previous findings that Tß4 is important for the proliferation of progenitor cells, neurogenesis, tangential expansion, and tissue growth in the young embryonic chicken optic tectum. Taken together, our results further illustrate and support the current idea that Tß4 is widely implicated in shaping and maintenance of the nervous system.

Thymosin ß4 and ß10 in Sjögren's syndrome: saliva proteomics and minor salivary glands expression.

BACKGROUND: In the present study, we investigated whether thymosin ß (Tß) in saliva and in minor salivary glands is differentially expressed in patients with primary Sjögren's syndrome (pSS) and patients with autoimmune diseases (systemic sclerosis [SSc], systemic lupus erythematosus [SLE], and rheumatoid arthritis [RA], with and without sicca syndrome [ss]). METHODS: Saliva specimens of nine patients with pSS, seven with ss/SSc, seven with ss/SLE, seven with ss/RA, seven with SSc, seven with SLE, and seven with RA, as well as ten healthy subjects, were analyzed using a high-performance liquid chromatograph coupled with a mass spectrometer equipped with an electrospray ionization source to investigate the presence and levels of Tß4, Tß4 sulfoxide, and Tß10. Immunostaining for Tß4 and Tß10 was performed on minor salivary glands of patients with pSS and ss. RESULTS: Tß4 levels were statistically higher in patients with pSS with respect to the other subgroups. Tß10 was detectable in 66.7 % of patients with pSS and in 42.8 % of those with ss/SSc, while Tß4 sulfoxide was detectable in 44.4 % of patients with pSS and in 42.9 % of those with ss/SSc. Tß10 and Tß4 sulfoxide were not detectable in patients without associated ss and in healthy control subjects. Regarding thymosin immunostaining, all patients had immunoreactivity for Tß10, and a comparable distribution pattern in the four different subgroups of patients was observed. Tß4 immunoreactivity was present in patients with ss/SSc and those with ss/SLE, while it was completely absent in patients with pSS and those with ss/RA. CONCLUSIONS: Our data show that higher salivary Tß expression characterizes patients with pSS, while Tß4 sulfoxide and Tß10 salivary expression was selectively present in patients with sicca symptoms. Moreover, at the immunohistochemical level in patients with pSS, minor salivary glands showed a peculiar pattern characterized by immunostaining for Tß10 in acinar cells in the absence of any reactivity for Tß4. These findings, taken together, suggest a different role for Tß4 and Tß10 in patients with pSS who have ss and other autoimmune disease.

A Tobacco-Derived Thymosin ß4 Concatemer Promotes Cell Proliferation and Wound Healing in Mice.

Thymosin ß4 (Tß4) is a peptide that is known to play important roles in protection, regeneration, and remodeling of injured tissues in humans, and that shows great promise in a range of clinical applications. However, current strategies to Tß4 are insufficient to meet growing demand and have a number of limitations. In this current study we investigated whether expression of recombinant Tß4 in plants, specifically in tobacco (Nicotiana tabacum) leaves, represents an effective approach. To address this question, a 168 bp Tß4 gene optimized for tobacco codon usage bias was constitutively expressed in tobacco as a 4-unit repeat concatemer, fused to a polyhistidine tag. Quantitative polymerase chain reaction and Western blot analyses were used to verify 4×Tß4 expression in 14 transgenic tobacco lines and enzyme-linked immunosorbent assay analysis indicated 4×Tß4 protein concentrations as high as 3 µg/g of fresh weight in the leaves. We observed that direct administration of tobacco-derived Tß4 was more effective than Tß4 either obtained commercially or derived from expression in Escherichia coli at promoting splenocyte proliferation in vitro and wound healing in mice through an endothelial migration assay. This study provides new insights into the development of plant-derived therapeutic proteins and their application by direct administration.

Effect of Thymosin beta4 on the Differentiation and Mineralization of MC3T3-E1 Cell on a Titanium Surface.

Osteoblasts are responsible for the synthesis of bone matrix through the secretion of collagenous and non-collagenous proteins with mineralization. Thymosin beta4 (Tbeta4) is an actin-sequestering peptide that is involved in the regulation of cell proliferation, differentiation and motility. A recent study reported that the inhibition of Tbeta4 mRNA synthesis strongly decreases the level of gene expression of bone sialoprotein (BSP), dentin sialophosphoprotein (DSPP), osteocalcin (OCN), osteonectin (ON) and collagen type I (Col I) with mineralization during differentiation in odontoblasts. Titanium (Ti) is used commonly as an implant material for dental implants, which have strong mechanical potential and good biocompatibility with bone. This study examined whether Tbeta4 can be a potential molecule for promoting the differentiation and mineralization of MC3T3-E1 cells on a Ti surface. Tbeta4 increased the viability of MC3T3-E1 cells during differentiation on Ti discs compared to that of the control. The expression of Tbeta4 mRNA and protein in the Tbeta4-treated MC3T3-E1 cells was higher than the control during differentiation on the Ti discs. In addition, Tbeta4 increased the formation of mineralization nodules and the mRNA expression of alkaline phosphatase (ALP), DSPP, dentin matrix protein1 (DMP1), BSP and Col I compared to that of the control in MC3T3-E1 cells during differentiation on Ti discs. From the results, Tbeta4 increased the viability and promoted the differentiation and mineralization of MC3T3-E1 cells on Ti discs. This highlights the potential use of Tbeta4 for increasing osseointegration through osteoblast differentiation and mineralization on Ti discs.

Expression of thymosin beta-4 in human periodontal ligament cells and mouse periodontal tissue and its role in osteoblastic/cementoblastic differentiation.

A recent report showed that thymosin beta-4 (Tß4) is expressed during the development of tooth germ, but its effect on osteoblastic/cementoblastic differentiation is a controversial topic. Furthermore, the precise expression and function of Tß4 in periodontal tissue remains unclear. Therefore, the purpose of this study was to investigate the immunolocalization of Tß4 in the developing periodontium of mouse, the function of Tß4 in osteoblastic/cementoblastic differentiation, and the underlying mechanism regulating periodontal regeneration in human periodontal ligament cells (hPDLCs), cementoblasts, and osteoblasts. Tß4 expression was observed in differentiating hPDLCs, osteoblasts of the periodontium during development, as well as in mature tissue. Higher Tß4 expression was observed in hPDLCs than in cementoblasts and osteoblasts in the developing periodontium. The expression of Tß4 mRNA and protein gradually increased during PDL cell differentiation. The downregulation of Tß4 expression by Tß4 siRNA transfection inhibited osteoblastic differentiation by decreasing calcium nodule formation, alkaline phosphatase (ALP) activity, and mRNA expression of differentiation markers in hPDLCs, cementoblasts, and osteoblasts. In contrast, Tß4 activation using a Tß4 peptide, promoted these processes by activation of Akt, p38, ERK MAPKs, and the NF-κB pathway. The expression of nuclear NFATc1 was upregulated by Tß4 peptide in hPDLCs. Inhibition of the calcineurin/NFATc1 pathway by cyclosporin A and FK506, attenuated Tß4-induced osteoblastic differentiation and activation of Wnt-related genes, as well as nuclear ß-catenin in hPDLCs. In conclusion, this study demonstrates, for the first time, that Tß4 is expressed in developing periodontal tissue and that its expression is associated with osteoblastic/cementoblastic differentiation. These results suggests that Tß4 is a potential therapeutic target for periodontal regeneration or bone disease.

Prothymosin-α Overexpression Contributes to the Development of Insulin Resistance.

CONTEXT: Prothymosin-α (ProT) is involved in oxidative stress, inflammation, cell proliferation, and apoptosis. Increased oxidative stress and chronic inflammation participate in the pathogenesis of diabetes. A recent study found that ProT is a ligand of toll-like receptor 4, which plays an important role in the development of insulin resistance. However, its physiological role remains poorly understood. OBJECTIVE: The objective was to investigate whether ProT contributes to the development of insulin resistance. DESIGN, SETTINGS, AND PATIENTS: A total of 185 subjects were recruited and classified into nondiabetes (n = 95) and newly diagnosed diabetes (n = 90) groups. Transgenic mice overexpressing ProT were used to investigate the role of ProT in the development of insulin resistance. Lentiviral vectors carrying short hairpin RNA specific for ProT were delivered via the portal vein to silence hepatic ProT expression in mice with high-fat diet-induced insulin resistance. Glucose uptake was determined in L6 myotubes. RESULTS: We show that the serum ProT levels of patients with type 2 diabetes were significantly higher than those of normal individuals (mean ± SEM, 419.8 ± 46.47 vs 246.4 ± 27.89 pg/mL; P < .001). Furthermore, ProT transgenic mice exhibited an insulin-resistant phenotype, whereas the silencing of hepatic ProT expression ameliorated high-fat diet-induced insulin resistance in C57BL/6 mice. In vitro studies reveal that ProT induced insulin resistance through a toll-like receptor 4-nuclear factor-κB-dependent pathway. CONCLUSIONS: Our results support the role for ProT in the development of insulin resistance. Therefore, ProT is a potential novel therapeutic target for type 2 diabetes.

Thymosin α1: burying secrets in the thymus.

Thymosin α1 (Tα1), an epithelial cell (EC)-derived cytokine, has the strong ability to modulate signals delivered through innate immune receptors on dendritic cells (DCs), thus instructing the initiation of appropriate immune responses to T cells. In its ability to activate indoleamine 2,3-dioxygenase 1-dependent tolerogenic programs in DCs, Tα1 pivotally contributes to the maintenance of self-tolerance by regulating the function of regulatory T (Treg) cells. How Tα1 may contribute to the Treg cell ontogeny is not known. The transcriptional regulator autoimmune regulator (AIRE) is known to control central and peripheral tolerance. AIRE is highly expressed in thymic medullary ECs where it controls the ectopic expression of tissue restricted antigens for negative selection. The absence of AIRE-induced tissue-specific antigens in the thymus can lead to autoimmunity in the antigen-expressing target organ. Recently, AIRE protein has been detected in peripheral lymphoid organs, suggesting that peripheral AIRE may play a complementary role. We have addressed the possible relationship between AIRE and Tα1 and discovered an intricate crosstalk, whereby AIRE may promote prothymosin cleavage to Tα1, and Tα1 in turn transcriptionally regulates AIRE expression. Thus, similar to other members of thymic stromal poietins, Tα1 expressed within the thymus and peripheral tissues regulates the EC/DC crosstalk required for salutary immune homeostasis.

Thymosin ß4 promotes hepatoblastoma metastasis via the induction of epithelial-mesenchymal transition.

Hepatoblastoma (HB) is the most common malignant hepatic tumor in children and complete surgical resection offers the highest possibility for cure in this disease. Tumor metastasis is the principle obstacle to the development of efficient treatments for patients with HB. The present study aimed to measure the expression levels of thymosin ß4 (Tß4) in liver samples from patients with HB and to investigate the involvement of Tß4 in HB metastasis. The expression of Tß4 was significantly higher in liver samples from patients with metastatic HB and in the HepG2 metastatic HB cell line, compared with that in adjacent healthy liver samples and in the L02 healthy hepatic cell line. By contrast, the expression levels of epithelial-cadherin (E-cadherin) and cytosolic accumulation of ß-catenin, the two most prominent markers involved in epithelial-mesenchymal transition (EMT), were reduced in liver specimens from patients with metastatic HB compared with that of healthy adjacent control tissue. HepG2 cells were transfected with small interfering-RNA in order to downregulate Tß4 gene expression. This resulted in a reduced cell migratory capacity compared with control cells. Tß4 gene expression knockdown significantly inhibited transforming growth factor ß1-mediated-EMT in vitro by upregulating the expression of E-cadherin. The results of the present study suggested that Tß4 may promote HB metastasis via the induction of EMT, and that Tß4 may therefore be a target for the development of novel treatments for patients with HB.

Increased expression of prothymosin-α, independently or combined with TP53, correlates with poor prognosis in colorectal cancer.

Human prothymosin-α (PTMA) plays an important role in tumorigenesis, and its overexpression triggers a TP53 response. In this study, we identified that PTMA expression was up-regulated at both the transcriptional and translational level in tumor tissue compared to that in adjacent normal tissue. PTMA overexpression was significantly associated with the depth of tumor invasion, lymph node metastasis (LNM), distant metastasis, advanced AJCC stage, and tumor differentiation. There was also a significant association between PTMA over-expression and mutant TP53 expression (r=0.515, P < 0.001). Survival analysis revealed that the disease-free survival (DFS) and overall survival (OS) rates were significantly lower among patients with PTMA- and TP53-positive tumors. Hence, PTMA might play an important role in the progression of CRC, and the assessment of both PTMA and mutant TP53 expression can help predict colon cancer prognosis.

Thymosin ß4 up-regulation of microRNA-146a promotes oligodendrocyte differentiation and suppression of the Toll-like proinflammatory pathway.

Thymosin ß4 (Tß4), a G-actin-sequestering peptide, improves neurological outcome in rat models of neurological injury. Tissue inflammation results from neurological injury, and regulation of the inflammatory response is vital for neurological recovery. The innate immune response system, which includes the Toll-like receptor (TLR) proinflammatory signaling pathway, regulates tissue injury. We hypothesized that Tß4 regulates the TLR proinflammatory signaling pathway. Because oligodendrogenesis plays an important role in neurological recovery, we employed an in vitro primary rat embryonic cell model of oligodendrocyte progenitor cells (OPCs) and a mouse N20.1 OPC cell line to measure the effects of Tß4 on the TLR pathway. Cells were grown in the presence of Tß4, ranging from 25 to 100 ng/ml (RegeneRx Biopharmaceuticals Inc., Rockville, MD), for 4 days. Quantitative real-time PCR data demonstrated that Tß4 treatment increased expression of microRNA-146a (miR-146a), a negative regulator the TLR signaling pathway, in these two cell models. Western blot analysis showed that Tß4 treatment suppressed expression of IL-1 receptor-associated kinase 1 (IRAK1) and tumor necrosis factor receptor-associated factor 6 (TRAF6), two proinflammatory cytokines of the TLR signaling pathway. Transfection of miR-146a into both primary rat embryonic OPCs and mouse N20.1 OPCs treated with Tß4 demonstrated an amplification of myelin basic protein (MBP) expression and differentiation of OPC into mature MBP-expressing oligodendrocytes. Transfection of anti-miR-146a nucleotides reversed the inhibitory effect of Tß4 on IRAK1 and TRAF6 and decreased expression of MBP. These data suggest that Tß4 suppresses the TLR proinflammatory pathway by up-regulating miR-146a.

Loss of nuclear prothymosin-α expression is associated with disease progression in human superficial bladder cancer.

In this paper, we report a study on the clinical relevance of prothymosin-α expression and its correlation with intratumoral Foxp3(+) and CD8(+) lymphocytes (Foxp3(+)TIL and CD8(+)TIL) in bladder cancer patients. We used immunohistochemical staining for prothymosin-α, Foxp3, and CD8 on 101 tumor specimens harvested by endoscopic resection. The results were correlated with clinicopathological variables and clinical outcome in bladder cancer patients, particularly in 73 patients with superficial disease, using the log-rank test and Cox proportional hazard model. Overall, of the tumors, 30 % were negative, 34 % showed nuclear, and 37 % showed cytoplasmic prothymosin-α expression. Foxp3(+)TILs were detected in 11 % of patients (nonnuclear vs. nuclear, p = 0.096). Patients with a history of urothelial carcinoma have a higher frequency of nonnuclear prothymosin-α expression than those without (p = 0.016, chi-square test). By univariate and multivariate analyses of cases with superficial disease, grade and stage were identified as independent predictors for recurrence-free survival (p = 0.016 and 0.016, respectively). Higher stage and nonnuclear prothymosin-α expression independently predict shorter progression-free survival (p = 0.006 and 0.043, respectively). The presence of Foxp3(+)TILs was significantly associated with disease progression by univariate analysis (p = 0.022), but not by multivariate analysis (p = 0.147). In vitro assays showed that J82 cells which express ectopically nuclear prothymosin-α exhibit higher growth rate and secrete less TGF-ß1 than those with cytoplasmic expression or control cells. Altogether, prothymosin-α expression is a determinant of disease progression in superficial bladder cancer. Foxp3(+)TILs tend to be found more often in bladder cancer with nonnuclear prothymosin-α expression. Future study is required to unravel their interaction.

Thymosin beta 4 and thymosin beta 10 expression in hepatocellular carcinoma.

Thymosin beta 4 (Tß4) and thymosin beta 10 (Tß10) are two members of the beta-thymosin family involved in many cellular processes such as cellular motility, angiogenesis, inflammation, cell survival and wound healing. Recently, a role for beta-thymosins has been proposed in the process of carcinogenesis as both peptides were detected in several types of cancer. The aim of the present study was to investigate the expression pattern of Tß4 and Tß10 in hepatocellular carcinoma (HCC). To this end, the expression pattern of both peptides was analyzed in liver samples obtained from 23 subjects diagnosed with HCC. Routinely formalin-fixed and paraffin-embedded liver samples were immunostained by indirect immunohistochemistry with polyclonal antibodies to Tß4 and Tß10. Immunoreactivity for Tß4 and Tß10 was detected in the liver parenchyma of the surrounding tumor area. Both peptides showed an increase in granular reactivity from the periportal to the periterminal hepatocytes. Regarding HCC, Tß4 reactivity was detected in 7/23 cases (30%) and Tß10 reactivity in 22/23 (97%) cases analyzed, adding HCC to human cancers that express these beta-thymosins. Intriguing finding was seen looking at the reactivity of both peptides in tumor cells infiltrating the surrounding liver. Where Tß10 showed a strong homogeneous expression, was Tß4 completely absent in cells undergoing stromal invasion. The current study shows expression of both beta-thymosins in HCC with marked differences in their degree of expression and frequency of immunoreactivity. The higher incidence of Tß10 expression and its higher reactivity in tumor cells involved in stromal invasion indicate a possible major role for Tß10 in HCC progression.

Thymosin ß 4 gene silencing decreases stemness and invasiveness in glioblastoma.

Thymosin beta 4 is a pleiotropic actin-sequestering polypeptide that is involved in wound healing and developmental processes. Thymosin beta 4 gene silencing promotes differentiation of neural stem cells whereas thymosin beta 4 overexpression initiates cortical folding of developing brain hemispheres. A role of thymosin beta 4 in malignant gliomas has not yet been investigated. We analysed thymosin beta 4 staining on tissue microarrays and performed interrogations of the REMBRANDT and the Cancer Genome Atlas databases. We investigated thymosin beta 4 expression in seven established glioma cell lines and seven glioma-initiating cell lines and induced or silenced thymosin beta 4 expression by lentiviral transduction in LNT-229, U87MG and GS-2 cells to study the effects of altered thymosin beta 4 expression on gene expression, growth, clonogenicity, migration, invasion, self-renewal and differentiation capacity in vitro, and tumorigenicity in vivo. Thymosin beta 4 expression increased with grade of malignancy in gliomas. Thymosin beta 4 gene silencing in LNT-229 and U87MG glioma cells inhibited migration and invasion, promoted starvation-induced cell death in vitro and enhanced survival of glioma-bearing mice. Thymosin beta 4 gene silencing in GS-2 cells inhibited self-renewal and promoted differentiation in vitro and decreased tumorigenicity in vivo. Gene expression analysis suggested a thymosin beta 4-dependent regulation of mesenchymal signature genes and modulation of TGFß and p53 signalling networks. We conclude that thymosin beta 4 should be explored as a novel molecular target for anti-glioma therapy.

A thymosin beta15-like peptide promotes intersegmental myotome extension in the chicken embryo.

Beta-thymosins constitute a group of small actin-sequestering peptides. These highly conserved peptides are involved in cytoskeleton dynamics and can influence different cell properties such as motility, substrate adhesion, shape and chemotaxis. As a marker for tumour metastasis, the mammalian thymosin beta15 is believed to have an important diagnostic relevance in cancer prognosis, although little is known about its physiological function. In order to study the role of thymosin beta15(avian) in embryogenesis, we cloned the chicken and quail orthologues of thymosin beta15 and used the chicken as a model for vertebrate development. Avian thymosin beta15, the first known non-mammalian thymosin beta15-like gene, encodes a peptide that possesses a cysteine at position one after the methionine which is a significant difference compared to its mammalian counterparts. Thymosin beta15(avian) expression starts at an early stage of development. The expression pattern changes rapidly with development and differs from that of the related thymosin beta4 gene. The most prominent expression domain is seen in developing muscles of limbs and trunk. Gain-of-function experiments revealed that thymosin beta15(avian) has a function in normal myotome development. Ectopic over-expression of thymosin beta15(avian) leads to premature elongation of myotome cells trespassing segment borders. We conclude that thymosin beta15(avian) has a still undescribed function in promoting myocyte elongation.

Comparative expression and regulation of TMSB4X in male reproductive tissues of rats and chickens.

Thymosin ß4 (TMSB4X) belongs to a class of highly conserved small proteins that are present in a high abundance in immune tissues, where it participates in various biological activities, including anti-inflammation, wound healing, apoptosis, and cell survival. However, little is known about the expression and regulation of TMSB4X in reproductive tissues. The aim of this study was to examine the expression of rat Tmsb4x and chicken TMSB4X genes during testicular and epididymal development. Rat Tmsb4x was strongly detected in the spermatogonia and spermatocytes of testis at early postnatal development and transmitted to Leydig cells at sexual maturation. Also, rat Tmsb4x was detected at an increased level in the epididymis during postnatal development. When compared to the rat, expression of the chicken TMSB4X gene was low in the testis and epididymal region, and the mRNA localization was also unexpected. Three experiments were performed to examine the regulation of rat Tmsb4x in the epididymis: after elimination of Leydig cells using ethylene dimethane sulfonate (EDS); after destruction of the testis by cryptorchidism; and after castration. EDS-treated and castrated rats were injected with testosterone propionate. The expression of Tmsb4x was significantly reduced in the epididymis of EDS-treated, and castrated rats. In contrast, Tmsb4x was significantly enhanced in the epididymis after testosterone treatment. The expression of rat Tmsb4x was also regulated in the epididymis after cryptorchidism. Collectively, the expression of Tmsb4x was strongly detected in the testis and epididymis of rats, and was highly regulated in the epididymis by testosterone.