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Thymosin beta-4 (Tβ4) is an ubiquitous multi-functional regenerative peptide, related to many critical biological processes, with a dynamic and flexible conformation which may influence its functions and its subcellular distribution. For these reasons, the intracellular localization and trafficking of Tβ4 is still not completely defined and is still under investigation in in vivo as well as in vitro studies. In the current study we used HepG2 cells, a human hepatoma cell line; cells growing in normal conditions with fetal bovine serum expressed high levels of Tβ4, restricted to the cytoplasm until 72 h. At 84 h, a diffuse Tβ4 cytoplasmic immunostaining shifted to a focal perinuclear and nuclear reactivity. In the absence of serum, nuclear reactivity was localized in small granules, evenly dispersed throughout the entire nuclear envelop, and was observed as earlier as at 48 h. Cytoplasmic immunostaining for Tβ4 in HepG2 cells under starvation appeared significantly lower at 48 h and decreased progressively at 72 and at 84 h. At these time points, the decrease in cytoplasmic staining was associated with a progressive increase in nuclear reactivity, suggesting a possible translocation of the peptide from the cytoplasm to the nuclear membrane. The normal immunocytochemical pattern was restored when culture cells submitted to starvation for 84 h received a new complete medium for 48 h. Mass spectrometry analysis, performed on the nuclear and cytosolic fractions of HepG2 growing with and without serum, showed that Tβ4 was detectable only in the cytosolic and not in the intranuclear fraction. These data suggest that Tβ4 is able to translocate from different cytoplasmic domains to the nuclear membrane and back, based on different stress conditions within the cell. The punctuate pattern of nuclear Tβ4 immunostaining associated with Tβ4 absence in the nucleoplasm suggest that this peptide might be localized in the nuclear pores, where it could regulate the pore permeability.

Background: Thymosin beta(4), its sulfoxide, and thymosin beta(10) were detected in whole saliva of human pre-term newborns by reversed-phase high performance chromatography coupled to electrospray ion-trap mass spectrometry., Methodology/principal Findings: Despite high inter-individual variability, concentration of beta-thymosins increases with an inversely proportional trend to postmenstrual age (PMA: gestational age plus chronological age after birth) reaching a value more than twenty times higher than in adult whole saliva at 190 days (27 weeks) of PMA (thymosin beta(4) concentration: more than 2.0 micromol/L versus 0.1 micromol/L). On the other hand, the ratio between thymosin beta(4) and thymosin beta(10) exhibits a constant value of about 4 along all the range of PMA (190-550 days of PMA) examined. In order to investigate thymosin beta(4) origin and to better establish the trend of its production as a function of gestational age (GA), immunohistochemical analysis of major and minor salivary glands of different pre-term fetuses were carried out, starting from 84 days (12 weeks) of gestational age. Reactive granules were seen in all glands with a maximum of expression around 140-150 days of GA, even though with high inter- and intra-individual variability. In infants and adults reactive granules in acinar cells were not observed, but just a diffuse cytoplasmatic staining in ductal cells., Significance: This study outlines for the first time that salivary glands during foetal life express and secrete peptides such as beta-thymosins probably involved in the development of the oral cavity and its annexes. The secretion increases from about 12 weeks till to about 21 weeks of GA, subsequently it decreases, almost disappearing in the period of expected date of delivery, when the gland switches towards the secretion of adult specific salivary peptides. The switch observed may be an example of further secretion switches involving other exocrine and endocrine glands during foetal development.