Thymosin beta4 is not always the main beta-thymosin in mammalian platelets.

beta-thymosins constitute a family of highly conserved 5-kDa polypeptides. Thymosin beta(4), the most abundant member of this family, is expressed in most mammalian cell types and is regarded as the main intracellular G-actin sequestering peptide. In addition to this important intracellular function several other activities have been attributed to this peptide. Thymosin beta(4) is released from human platelets and cross-linked to fibrin after activation of platelets with thrombin. While in most mammalian tissues thymosin beta(4) is accompanied by a second member of this peptide family, in human platelets only thymosin beta(4) is present. To elucidate if it is common to mammalian platelets that only one beta-thymosin is present, we analyzed platelets from several mammals for their beta-thymosin content. In human platelets only thymosin beta(4) could be detected, whereas in bovine platelets thymosin beta(9), which is normally the minor beta-thymosin in bovine tissues, was identified as the main beta-thymosin. In rabbit platelets, thymosin beta(4) is not simply replaced by the most homologous thymosin beta(4)(Ala), as might be expected from sequence homology. Thymosin beta(4)(Ala) and thymosin beta(10) were found, but thymosin beta(10) is present in about 2.5-fold higher amounts. Because thymosin beta(4)(Ala) possesses about threefold higher affinity to G-actin, compared to thymosin beta(4), beta(10), and beta(9), we suggest that expression of beta-thymosins is triggered by functional requirements and not sequence homology.

Nuclear localisation of the G-actin sequestering peptide thymosin beta4.

Thymosin beta4 is regarded as the main G-actin sequestering peptide in the cytoplasm of mammalian cells. It is also thought to be involved in cellular events like cancerogenesis, apoptosis, angiogenesis, blood coagulation and wound healing. Thymosin beta4 has been previously reported to localise intracellularly to the cytoplasm as detected by immunofluorescence. It can be selectively labelled at two of its glutamine-residues with fluorescent Oregon Green cadaverine using transglutaminase; however, this labelling does not interfere with its interaction with G-actin. Here we show that after microinjection into intact cells, fluorescently labelled thymosin beta4 has a diffuse cytoplasmic and a pronounced nuclear staining. Enzymatic cleavage of fluorescently labelled thymosin beta4 with AsnC-endoproteinase yielded two mono-labelled fragments of the peptide. After microinjection of these fragments, only the larger N-terminal fragment, containing the proposed actin-binding sequence exhibited nuclear localisation, whereas the smaller C-terminal fragment remained confined to the cytoplasm. We further showed that in digitonin permeabilised and extracted cells, fluorescent thymosin beta4 was solely localised within the cytoplasm, whereas it was found concentrated within the cell nuclei after an additional Triton X100 extraction. Therefore, we conclude that thymosin beta4 is specifically translocated into the cell nucleus by an active transport mechanism, requiring an unidentified soluble cytoplasmic factor. Our data furthermore suggest that this peptide may also serve as a G-actin sequestering peptide in the nucleus, although additional nuclear functions cannot be excluded.

Reduction of thymosin beta4 and actin in HL60 cells during apoptosis is preceded by a decrease of their mRNAs.

Thymosin beta4 (Tbeta4) is the most prominent representative of the beta-thymosins, a family of highly conserved polar 5 kDa peptides. This peptide is now regarded to be the main G-actin sequestering peptide in mammals and therefore plays an important role in organization of the microfilamental system. During apoptosis of cells this microfilamental system is disrupted. Therefore we studied changes in thymosin beta4 and actin content of HL60 cells after induction of apoptosis using cytosine arabinoside (araC). Thymosin beta4 content decreased to about 30% of the control value after incubation for 48 h in the presence of araC. Also the amount of total actin decreased to about half of the control, while total cellular protein remained constant. To further elucidate if the changes of thymosin beta4 and actin content correlate with the gene expression the relative mRNA content of thymosin beta4 and beta-actin was determined using the ribonuclease protection assay (RPA). Already after 24 h the relative amount of mRNA of thymosin beta4 and beta-actin was greatly reduced to 71 and 58%, respectively. Upon a 48 h araC treatment, the mRNA of these two proteins decreased to 15 and 10% compared to the control, whereas the content of total RNA and protein per cell was nearly unchanged. According to our data araC has a significant influence on the transcriptional level of thymosin beta4 and actin.

Thymosin beta4 is released from human blood platelets and attached by factor XIIIa (transglutaminase) to fibrin and collagen.

The beta-thymosins constitute a family of highly conserved and extremely water-soluble 5 kDa polypeptides. Thymosin beta4 is the most abundant member; it is expressed in most cell types and is regarded as the main intracellular G-actin sequestering peptide. There is increasing evidence for extracellular functions of thymosin beta4. For example, thymosin beta4 increases the rate of attachment and spreading of endothelial cells on matrix components and stimulates the migration of human umbilical vein endothelial cells. Here we show that thymosin beta4 can be cross-linked to proteins such as fibrin and collagen by tissue transglutaminase. Thymosin beta4 is not cross-linked to many other proteins and its cross-linking to fibrin is competed by another family member, thymosin beta10. After activation of human platelets with thrombin, thymosin beta4 is released and cross-linked to fibrin in a time- and calcium-dependent manner. We suggest that thymosin beta4 cross-linking is mediated by factor XIIIa, a transglutaminase that is coreleased from stimulated platelets. This provides a mechanism to increase the local concentration of thymosin beta4 near sites of clots and tissue damage, where it may contribute to wound healing, angiogenesis and inflammatory responses.