Cellular trafficking of thymosin beta-4 in HEPG2 cells following serum starvation

Abstract

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.

Figure 1. Thymosin β 4 immunoreactivity after 48 h of starvation.

Tβ 4 immunoreactivity in HepG2 cells cultured for 48 h with serum (A and B, magnification 1000×) and without serum (C and D, magnification 1000×). Inserts in A and in C represent one particular of the intranuclear reactivity of the respective pictures (magnification 1000×).

Figure 2. Thymosin β 4 immunoreactivity after 72 h of starvation.

Tβ 4 immunoreactivity in HepG2 cells cultured for 72 h with serum (A and B, magnification 1000×) and without serum (C and D, magnification respectively 400 and 1000×). Inserts in A and in C represent one particular of the intranuclear reactivity of the respective pictures (magnification 1000×).

Figure 3. Thymosin β 4 immunoreactivity after 84 h of starvation.

Tβ 4 immunoreactivity in HepG2 cells cultured for 84 h with serum (A and B, magnification 1000×) and without serum (C and D, magnification 1000×). Inserts in A and in C represent one particular of the intranuclear reactivity of the respective pictures (magnification 1000×).

Figure 4. Thymosin β 4 immunoreactivity 24 and 48 h with complete medium after starvation.

Tβ 4 immunoreactivity in HepG2 cells cultured for 84 h without serum and shifted in complete medium for 24 h (A and B, magnification 400×) and for 48 h (C and D, magnification 400×). Inserts in A and in C represent one particular of the intranuclear reactivity of the respective pictures (magnification 1000×).

Figure 5. XIC peak of Tβ4 evidenced by HPLC-ESI-MS analysis of the cytosolic (CF) and nuclear fractions (NF) of HepG2 cells grown for 48 h.

Enlargements of the chromatographic profiles in the range 17.4–21.1 min are reported. Tβ4 search in CF of the cells grown under starvation and normal conditions, panel A and panel B; Tβ4 search in NF of the cells grown under starvation and normal conditions, panel C and panel D; arrows indicate the absence of the Tβ4 in its chromatographic elution range.