Beta-catenin is a mediator of the response of fibroblasts to irradiation

Abstract

Radiation causes soft tissue complications that include fibrosis and deficient wound healing. beta-Catenin, a key component in the canonical Wnt-signaling pathway, is activated in fibrotic processes and wound repair and, as such, could play a role in mediating cellular responses to irradiation. beta-Catenin can form a transcriptionally active complex with members of the Tcf family. A reporter mouse model, in addition to human cell cultures, was used to demonstrate that ionizing radiation activates beta-catenin-mediated, Tcf-dependent transcription both in vitro and in vivo. Furthermore, radiation activates beta-catenin via a Wnt-mediated mechanism, as in the presence of dickkopf-1, an inhibitor of Wnt receptor activation, beta-catenin levels did not increase after irradiation. Fibroblast cell cultures were derived from mice expressing either null or stabilized beta-catenin alleles. Cells expressing stabilized beta-catenin alleles had a higher proliferation rate and formed more colony-forming units than wild-type or null cells after irradiation. Wound healing was studied in these same mice after irradiation. There was a positive correlation between the tensile strength of the wound, the expression levels of type 1 collagen in the skin, and beta-catenin levels. Mice treated with lithium showed increased beta-catenin levels and increased wound strength. beta-Catenin mediates the effects of ionizing radiation in fibroblasts, and its modulation has the potential to decrease the severity of radiation-induced soft tissue complications.

Figure 1

β-Catenin-mediated Tcf-dependent transcription is activated after irradiation of fibroblasts. A: A representative Western analysis showing an increase in β-catenin protein level starting 1 hour after irradiation. B: β-Galactosidase assays showing an increase in Tcf-dependent transcriptional activation with irradiation. Data are given as means and 95% confidence intervals for relative Tcf activity. *P < 0.05 compared to the zero time point. C: Subcellular location of β-catenin as examined using immunohistochemistry. There is nuclear localization of β-catenin after irradiation or with lithium treatment as a positive control. Top: Cells stained with an antibody to β-catenin. Bottom: Cells stained with DAPI to identify the nuclei. D: The expression of both p21 and p53 increases after irradiation. p21 data are given as RT-PCR and p53 as a Western blot. Gapdh and actin are shown as loading controls. E: There is a decrease in the number of colony-forming units after irradiation. Data are given as means and 95% confidence intervals. F: Primary human skin fibroblast cell cultures, as well as a primary human fibroblast cell line, show an elevated β-catenin protein level. G: Epithelial cell lines (labeled breast cancer, breast epithelial, and hepatocarcinoma) and a keratinocyte cell line (labeled keratinocyte cell line) show no substantial changes in β-catenin after exposure to irradiation. A primary keratinocyte cell culture (labeled keratinocyte) also showed no substantial change in β-catenin protein level after irradiation.

Figure 2

β-Catenin protein level and Tcf-dependent transcriptional activation during wound repair in mice after irradiation. A: Western analysis from mouse wound tissues. IR is from irradiated tissues and non-IR is from wounds that were not radiated. There is an increase in β-catenin protein level after irradiation. The blots were stripped and reprobed using an antibody for actin as a loading control. B: β-Galactosidase assays show an increase in Tcf-dependent transcriptional activation with irradiation. Data are given as means and 95% confidence intervals for relative activity. There is a significant (P < 0.05) difference between cells after irradiation and control cells.

Figure 3

Irradiation modulates β-catenin through a Wnt-dependent mechanism. A: The level of expression of β-catenin is lower in irradiated cells, as detected using RT-PCR (lanes labeled IR are from irradiated cells). B and C: Treatment with cycloheximide inhibits the elevation of β-catenin after irradiation. B: Representative Western analysis. C: Relative density of the β-catenin compared to control protein as detected using densitometry. *P < 0.05 compared to nonirradiated cells without cycloheximide treatment. D and E: After irradiation there is an increase in phospho-Ser-9-GSK3β levels similar to that seen in cells treated with lithium. Western analysis using a phospho-Ser-9-GSK3β antibody is shown in D. The blot was stripped and reprobed with an antibody to total GSK3β. E: Relative level of Ser-9-GSK3β to total GSK3β as measured using densitometry. *P < 0.05 compared to nonirradiated cells. F and G: Treatment with Dkk-1 abolishes the increase in β-catenin protein levels. F: Representative Western analysis. G: Relative level of β-catenin compared to control protein as determined using densitometry. *P < 0.05 compared to nonirradiated cells without Dkk-1 treatment. H: There is a higher level of expression of Wnt3a and Wnt 4 after irradiation in fibroblasts as detected using RT-PCR.

Figure 4

β-Catenin regulates the proliferation rate and clonogenic survival of irradiated fibroblasts. A: The expected changes in β-catenin levels are confirmed using Western analysis, with the expected smaller size of the protein band from cells expressing the Catnb^lox(ex3) alleles identified. B: Proliferation as measured using BrdU incorporation. The proportion of cells that incorporate BrdU is shown as means and 95% confidence intervals. *P < 0.05 from the same radiation exposure in wild-type cells. There is a significant difference between radiated and nonirradiated wild-type cells, but not between radiated and nonirradiated β-catenin-null or stabilized cells. C: Number of colonies formed after irradiation. Data are shown as means and 95% confidence intervals. Cell cultures from mice expressing Catnb^lox(ex3) alleles have a greater proliferation rate and more colony-forming units than wild-type controls. *P < 0.05 from the same irradiation exposure in wild-type cells. There is a significant difference between irradiated and nonirradiated wild-type cells, but not between irradiated and nonirradiated β-catenin-null or stabilized cells.

Figure 5

β-Catenin regulates the ultimate tensile strength of healing wounds in mice and the ability of irradiation to induce collagen expression. A: The expected changes in β-catenin levels were confirmed using Western analysis, with the expected smaller size of the protein band from cells expressing the Catnb^lox(ex3) alleles identified. B: Ultimate tensile strength in megapascals shown as means and 95% confidence intervals, showing an increase in strength when β-catenin protein is stabilized and a decrease when null alleles are expressed. *P < 0.05 from the nonirradiated wounds. C: Expression of type I collagen relative to Gapdh in normal unwounded skin exposed to irradiation in mice. *P < 0.05 from the nonirradiated skin. There is an increase in expression of type 1 collagen in mice expressing wild-type or stabilized β-catenin alleles, but not in mice expressing null alleles.