Oxidative Stress Causes Enhanced Secretion of YB-1 Protein that Restrains Proliferation of Receiving Cells

Abstract

The prototype cold-shock Y-box binding protein 1 (YB-1) is a multifunctional protein that regulates a variety of fundamental biological processes including cell proliferation and migration, DNA damage, matrix protein synthesis and chemotaxis. The plethora of functions assigned to YB-1 is strictly dependent on its subcellular localization. In resting cells, YB-1 localizes to cytoplasm where it is a component of messenger ribonucleoprotein particles. Under stress conditions, YB-1 contributes to the formation of stress granules (SGs), cytoplasmic foci where untranslated messenger RNAs (mRNAs) are sorted or processed for reinitiation, degradation, or packaging into ribonucleoprotein particles (mRNPs). Following DNA damage, YB-1 translocates to the nucleus and participates in DNA repair thereby enhancing cell survival. Recent data show that YB-1 can also be secreted and YB-1-derived polypeptides are found in plasma of patients with sepsis and malignancies. Here we show that in response to oxidative insults, YB-1 assembly in SGs is associated with an enhancement of YB-1 protein secretion. An enriched fraction of extracellular YB-1 (exYB-1) significantly inhibited proliferation of receiving cells and such inhibition was associated to a G2/M cell cycle arrest, induction of p21WAF and reduction of Np63 protein level. All together, these data show that acute oxidative stress causes sustained release of YB-1 as a paracrine/autocrine signal that stimulate cell cycle arrest.

Keywords: cold shock proteins; oxidative stress; protein secretion; stress granules.

Figure 1

Y-box-binding protein 1 (YB-1) and Poly(A)-binding protein 1 (PABP1) co-localize and interact in stress granules (SGs) under stress conditions. (a) Confocal immunofluorescence of un-treated (control) HEK293T, and treated with 250 µM sodium arsenite (Na Ars) for 30’, 500 µM H₂O₂ for 1 h or subjected to heat shock at 45 °C for 1 h, stained with α-YB-1 (green) and α-PABP1 (red); yellow and white arrows indicate P-bodies and stress granules respectively; (b) (Upper panel) size and number (lower panel) of SGs after treatments compared to control; statistical analysis was performed using 1-way ANOVA followed by Dunnett’s multiple comparisons test. Levels of significance are indicated (*** p < 0.001, * p = 0.001, see also Supplementary Materials Table S2); (c) Co-immunoprecipitation of HEK293T total protein extracts treated (+) or not (−) with 250 µM Na Ars for 30’; extracts were immunoprecipitated for YB-1 and immunorevealed for PABP1. Input samples immunorevealed with α-PABP1 and α-YB-1 are shown. Each panel is assembled from cropped Western blotting images (see original Western blot file for the original images).

Figure 2

Silencing of YB-1 affects PABP1 positive SGs formation. (a) Western blot of total extract from control (siNC) or 100 nM YB-1 silenced (siYB-1) HEK293T; the degree of reduction of YB-1 protein levels in YB-1 siRNA-treated cells compared with control is indicated beneath each band in the Western blot (where the relative unit 1.0 represents YB-1 levels in cells transfected with the control siRNA). β-tubulin was used as loading control. Each panel is assembled from cropped Western blotting images (see original Western blot file for the original images); (b) (Upper panel) size and number (lower panel) of stress granules (SGs) in YB-1 silenced cells treated with Na Ars compared to siNC (control) cells; statistical analysis was performed using unpaired t-test with Welch’s correction (** p = 0.010 and * p = 0.02 see Supplementary Materials Table S2); (c) Confocal immunofluorescence of control (siNC) and silenced (siYB-1) HEK293T cells, treated or not with 250 µM Na Ars for 30’, stained with α-YB-1 (green) and α-PABP1 (red), nuclei are stained with DAPI (blue).

Figure 3

Na Ars induced oxidative stress promotes YB-1 secretion. Western blot of total protein extracts from HEK293T treated with 250 µM Na Ars (a) or 500 µM H₂O₂ (b) for 30’ and 60’; glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and actinin were used as loading control. The degree of reduction of YB-1 protein levels in treated cells compared with controls is indicated beneath each band in the Western blot (where the relative unit 1.0 represents YB-1 levels in control cells); (c) Western blot of total extract and nuclear/cytoplasmic fractionation of Na Ars treated and untreated HEK293T. GAPDH and PARP were used as loading control for cytoplasm and nucleus, respectively; (d) Western blot of TCA precipitated supernatants of HEK293T cells. Ribonuclease/Angiogenin Inhibitor 1 (RNH1) was used as negative control of cell lysis. Each panel is assembled from cropped Western blotting images (see original Western blot file for the original images).

Figure 4

Extracellular YB-1 (exYB-1) affects HaCaT cell proliferation. Cell proliferation profile of HaCaT cells incubated with indicated concentrations of CCM-YB-1 (a), rYB-1 (b) or Bovine Serum Albumin (BSA) (c); statistical analysis was performed using 2-way ANOVA followed by Dunnett’s multiple comparisons test. Levels of significance are indicated (*** p < 0.001, see also Supplementary Materials Table S2); (d) Crystal violet assay of HaCaT cells treated with 7.5 μg/mL of CCM-YB-1, rYB-1, BSA or left untreated (Control; (bars) optical absorbance at 595 nm is reported on the y-axis; (image) representative colorimetric evaluation. Statistical analysis was performed using 1-way ANOVA followed by Dunnett’s multiple comparisons test. Levels of significance are indicated (*** p < 0.001, see also Supplementary Materials Table S2).

Figure 5

Extracellular YB-1 induced G2/M phase arrest. (a) Cell cycle profile of HaCaT cells treated with 7.5 μg/mL of CCM-YB-1, rYB-1, BSA or left untreated for 48 h. Statistical analysis was performed using 2-way ANOVA followed by Dunnett’s multiple comparisons test. Levels of significance are indicated (*** p < 0.001, * p = 0.01, see also Supplementary Materials Table S2); (b) RT-qPCR analysis of p21waf and ∆np63α in HaCaT cells treated with 7.5 μg/mL of CCM-YB-1, rYB-1, BSA or left untreated (Control for 48 h. Statistical analysis was performed using 1-way ANOVA followed by Dunnett’s multiple comparisons test. Levels of significance are indicated (*** p < 0.001, * p = 0.01, see also Supplementary Materials Table S2); (c,d) Western blot analysis of total extracts of HaCaT cell treated with rYB-1 and CCM-YB1 and revealed with the indicated antibodies. GAPDH and β-tubulin were used as loading control. Each panel is assembled from cropped Western blotting images (see original Western blot file for the original images).