Space radiation triggers persistent stress response, increases senescent signaling, and decreases cell migration in mouse intestine

Abstract

Proliferative gastrointestinal (GI) tissue is radiation-sensitive, and heavy-ion space radiation with its high-linear energy transfer (high-LET) and higher damaging potential than low-LET γ-rays is predicted to compromise astronauts' GI function. However, much uncertainty remains in our understanding of how heavy ions affect coordinated epithelial cell migration and extrusion, which are essential for GI homeostasis. Here we show using mouse small intestine as a model and BrdU pulse labeling that cell migration along the crypt-villus axis is persistently decreased after a low dose of heavy-ion ⁵⁶Fe radiation relative to control and γ-rays. Wnt/β-catenin and its downstream EphrinB/EphB signaling are key to intestinal epithelial cell (IEC) proliferation and positioning during migration, and both are up-regulated after ⁵⁶Fe radiation. Conversely, factors involved in cell polarity and adhesion and cell-extracellular matrix interactions were persistently down-regulated after ⁵⁶Fe irradiation-potentially altering cytoskeletal remodeling and cell extrusion. ⁵⁶Fe radiation triggered a time-dependent increase in γH2AX foci and senescent cells but without a noticeable increase in apoptosis. Some senescent cells acquired the senescence-associated secretory phenotype, and this was accompanied by increased IEC proliferation, implying a role for progrowth inflammatory factors. Collectively, this study demonstrates a unique phenomenon of heavy-ion radiation-induced persistently delayed IEC migration involving chronic sublethal genotoxic and oncogenic stress-induced altered cytoskeletal dynamics, which were seen even a year later. When considered along with changes in barrier function and nutrient absorption factors as well as increased intestinal tumorigenesis, our in vivo data raise a serious concern for long-duration deep-space manned missions.

Keywords: DNA damage; SASP; cytoskeleton remodeling; intestinal epithelial cell migration; senescence.

Fig. 1.

Long-term decrease of IEC migration after ⁵⁶Fe radiation relative to γ-rays. (A and B) Sections of the jejunal–ilial region of mouse intestine 7 and 60 d after sham, γ-, or ⁵⁶Fe radiation exposure were immunohistochemically stained for BrdU 24 h after an i.p. BrdU injection and nuclei were counterstained with hematoxylin; a representative image from each group is presented showing slower IEC migration after ⁵⁶Fe radiation. (Scale bars, 10 μm.) (C) IEC migration distance was measured by counting the number of cells between the BrdU-stained top-most cell in the villus and the bottom-most cell in the crypt in 7- and 60-d postexposure samples; *, significant relative to control; **, significant relative to γ-rays. Arrows show cell migration height as well as highest labeled cell. Statistical significance is set at P < 0.05 and error bars represent mean ± SEM.

Fig. 2.

Dysregulation of Wnt/β-catenin and EphB/EphrinB signaling after ⁵⁶Fe radiation. (A) Immunoblots show decreased Wnt antagonist Dkk1, increased phospho-GSK3β, increased activated β-catenin, and increased EphB/EphrinB after ⁵⁶Fe. (B) Immunofluorescence image showing increased EphB3 after ⁵⁶Fe radiation. (Scale bars, 5 μm.) (C) Putative TCF/LEF family of transcription factor binding sites on the promoter of the EphrinB1 and EphB3 genes relative to transcription start sites. While four binding sites are on the EphrinB1 promoter, three binding sites are on the EphB3 promoter. Gray boxes, binding sites; numbers, location of the binding-site end positions; colored lines and the numbers above, PCR primer span and the number of primer pairs; red lines, PCR nonamplification; blue lines, successful PCR amplification. (D) Quantitative RT-PCR results from immunoprecipitated DNA show enhanced β-catenin binding to the EphrinB1 promoter after ⁵⁶Fe radiation. (E) qRT-PCR results showing enhanced β-catenin binding to the EphB3 promoter after ⁵⁶Fe radiation; *, significant relative to control; **, significant relative to γ-rays. Statistical significance is set at P < 0.05 and error bars represent mean ± SEM.

Fig. 3.

Decreased cell polarity and adhesion protein expression after ⁵⁶Fe radiation. (A) Representative immunoblot images showing altered expression of factors involved in cytoskeleton (Cdc42, Rock1, Mlck), cell polarity (Par3), and cell adhesion (E-cadherin) dynamics 7 (Left) and 60 (Right) d after ⁵⁶Fe irradiation relative to γ-rays. (B) Intestinal sections were stained using anti-Par3 antibody, and representative images are presented showing decreased Par3 expression after ⁵⁶Fe radiation. (Scale bars, 5 μm.) (C) Intestinal sections stained for the cell adhesion marker E-cadherin, and representative images are presented showing decreased E-cadherin after ⁵⁶Fe radiation. (Scale bars, 10 μm.)

Fig. 4.

Heavy-ion radiation decreases expression of key cellular tight junction proteins and alters microtubule dynamics in mouse intestine. (A) Representative immunoblot images of cellular tight junction proteins showing radiation-induced altered expression of Occludin, Claudin1, Zo-1, and Scrib. (B) Intestinal sections were stained for Zo-1, and representative images are presented showing decreased Zo-1 after ⁵⁶Fe radiation. (Scale bars, 5 μm.) (C) Intestinal sections were stained for Scrib, and a representative image for each group is presented showing decreased Scrib expression after ⁵⁶Fe radiation. (Scale bars, 10 μm.) (D) Immunoblot analysis of microtubule-associated proteins was performed, and representative images are presented showing radiation-induced altered expression of Tau, phospho-Tau, Crmp2, and Map1b.

Fig. 5.

Persistent deregulation of integrins and NH₂-terminal villin after heavy-ion radiation exposure. (A) Schematic representation of integrin (green bars)-mediated interaction between the epithelial cell and stroma in the intestinal villus, and the red box highlights the area presented in the immunofluorescence images. (B) Sections were stained for β1-integrin, and representative images are presented showing increased β1-integrin expression after ⁵⁶Fe radiation. (Scale bars, 5 μm.) (C) Immunoblots showing radiation-induced increased expression of β1- and β7-integrins after ⁵⁶Fe radiation. (D) Sections were immunohistochemically stained for NH₂-terminal villin, an intestinal villus tip cell apoptosis marker. Representative images captured at 10× microscopic magnification are presented showing decreased expression of NH₂-terminal villin in areas near the villus tip after ⁵⁶Fe radiation. (Scale bars, 10 μm.)

Fig. 6.

Long-term up-regulation of proliferative markers and down-regulation of cell migration factors along with increased intestinal metaplasia after ⁵⁶Fe radiation. (A) Representative immunoblot images are presented showing increased β-catenin, EphB3, and β1-integin and decreased expression of E-cadherin, Par3, Zo-1, and Map1b 12 mo after ⁵⁶Fe radiation. (B) E-cadherin expression was assessed by immunofluorescence in intestinal sections, and representative images are presented showing decreased expression after ⁵⁶Fe radiation. (Scale bars, 10 μm.) (C) Representative immunofluorescence images of Par3 from intestinal sections are presented showing decreased expression after ⁵⁶Fe radiation. (Scale bars, 10 μm.) (D) Expression of GC-C, a marker of intestinal metaplasia, is shown in representative images. (Scale bars, 10 μm.)

Fig. 7.

Altered cell migration factors and increased intestinal tumorigenesis in APC^1638N/+ mice. (A) Increased intestinal tumor frequency is presented graphically showing higher tumorigenesis after ⁵⁶Fe relative to sham and γ-radiation. (B) Immunoblot images showing increased EphB3 and β1-integrin expression and decreased Claudin1, Zo-1, and Map1b expression in tumor and adjacent normal tissues from APC^1638N/+ mice. (C) Representative images of Par3 showing decreased expression in normal and tumor tissues after ⁵⁶Fe radiation. (Scale bars, 10 μm.) (D) Immunofluorescence images showing decreased expression of E-cadherin in normal and tumor tissues after ⁵⁶Fe radiation. (Scale bars, 10 μm.) (E) Representative images showing increased expression of Cyclin D1 in both normal and tumor tissues after ⁵⁶Fe radiation. (Scale bars, 10 μm.) (F and G) Intestinal sections were stained using Alcian blue to score for mucin-producing goblet cells, a differentiated cell type in intestine, in normal and tumor tissues, and results are presented graphically showing decreased number after ⁵⁶Fe radiation; *, significant relative to control; **, significant relative to γ-rays. Statistical significance is set at P < 0.05 and error bars represent mean ± SEM.

Fig. 8.

Ongoing DNA damage, induction of cellular senescence, and acquisition of the senescence-associated secretory phenotype 12 mo after ⁵⁶Fe radiation. (A and B) γH2AX foci counts are graphically plotted demonstrating long-term persistence of DNA damage after ⁵⁶Fe relative to γ-rays and control. Representative images from 12-mo samples showing increased DNA double-strand breakage after ⁵⁶Fe radiation. HPF, high-power field. (Scale bars, 5 μm.) (C and D) Numbers of SA-β-gal–positive cells are plotted graphically showing persistently higher senescence induction after ⁵⁶Fe radiation. (Scale bars, 10 μm.) (E) Quantitative RT-PCR results show increased expression of p16 and p21 after ⁵⁶Fe radiation. (F) Representative costaining images showing Glb1+IL8–positive cells (white arrowheads) in ⁵⁶Fe samples. (Scale bars, 5 μm.) (G) Quantification of Glb1+IL8 cells per 20× field is presented graphically showing higher numbers in ⁵⁶Fe relative to control and γ-rays. (H) Quantitative RT-PCR results showing increased expression of IL6, Ptges, Faim2, and Opg after ⁵⁶Fe radiation; *, significant relative to control; **, significant relative to γ-rays. Statistical significance is set at P < 0.05 and error bars represent mean ± SEM.

Fig. 9.

Proposed model for altered IEC migration and pathophysiology after heavy-ion space radiation. Unlike low-LET γ-radiation, ⁵⁶Fe ions trigger long-term perturbation in IECs, including direct damage with the generation of senescent cells and long-term signaling abnormalities consistent with SASP responses. Ongoing oxidative stress can further exacerbate signaling events affecting proliferation and differentiation, cell–cell interactions, cell–ECM interaction, as well as generation of additional senescent cells. The presence of senescent cells in crypts indicates that these events impact stem cell function.