Biology Contribution
Bax and Bak Do Not Exhibit Functional Redundancy in Mediating Radiation-Induced Endothelial Apoptosis in the Intestinal Mucosa

https://doi.org/10.1016/j.ijrobp.2007.11.043Get rights and content

Purpose

To address in vivo the issue of whether Bax and Bak are functionally redundant in signaling apoptosis, capable of substituting for each other.

Methods and Materials

Mice were exposed to whole-body radiation, and endothelial cell apoptosis was quantified using double immunostaining with TUNEL and anti-CD31 antibody. Crypt survival was determined at 3.5 days after whole-body radiation by the microcolony survival assay. Actuarial animal survival was calculated by the product-limit Kaplan-Meier method, and autopsies were performed to establish cause of death.

Results

Radiation exposure of Bax- and Bak-deficient mice, both expressing a wild-type acid sphingomyelinase (ASMase) phenotype, indicated that Bax and Bak are both mandatory, though mutually independent, for the intestinal endothelial apoptotic response. However, neither affected epithelial apoptosis at crypt positions 4–5, indicating specificity toward endothelium. Furthermore, Bax deficiency and Bak deficiency each individually mimicked ASMase deficiency in inhibiting crypt lethality in the microcolony assay and in rescuing mice from the lethal gastrointestinal syndrome.

Conclusions

The data indicate that Bax and Bak have nonredundant functional roles in the apoptotic response of the irradiated intestinal endothelium. The observation that Bax deficiency and Bak deficiency also protect crypts in the microcolony assay provides strong evidence that the microvascular apoptotic component is germane to the mechanism of radiation-induced damage to mouse intestines, regulating reproductive cell death of crypt stem cell clonogens.

Introduction

Although Bax and Bak are regarded as prototypical proapoptotic Bcl-2 multidomain proteins, early studies showed that murine embryonic fibroblasts (MEFs) deficient in either Bax or Bak alone displayed intact apoptosis. Rather, double deficiency of both Bax and Bak was required to endow resistance to apoptotic stimuli, indicating functional redundancy for Bax and Bak in this system (1). Similar functional overlap has been described in stress-induced apoptosis of thymocytes (2) and B lymphocytes (3), in developmental apoptosis of the retina during embryogenesis (4), and in the photoreceptor death response of retinal cells in the adult mouse (5). These studies led to a widely held perception that Bak and Bax are redundant regulators of intrinsic mitochondrial apoptosis and that simultaneous inactivation of both is required to negate this mode of cell death. However, studies of mouse neuronal or intestinal epithelial cells 6, 7 and human colon cancer cell lines (8) suggested that Bax and Bak do not necessarily overlap in apoptotic function, because Bax deficiency alone imparted maximal resistance to stress-induced apoptosis in these systems. Furthermore, Przemeck et al.(9) reported that either Bak or Bax deficiency alone conferred apoptotic resistance upon epithelial cells in the stem cell/proliferative zones of the gastric corpus and antral mucosa of Bax−/− or Bak−/− mice treated with whole-body radiation (WBR).

To further address this issue in vivo, we used a model of radiation-induced damage to murine small intestines, recently reported to be critically dependent on induction of early-phase apoptosis in the intestinal microvascular network 10, 11. It has long been accepted that induction of radiation damage to intestinal mucosa requires depletion of the clonogenic stem cell compartment, residing at the bottom of the intestinal crypt of Lieberkühn (12). The intestinal stem cell clonogenic system consists of pluripotent stem cells and uncommitted progenitor clonogens, and its complete depletion eradicates the crypt–villus unit, whereas surviving stem cell clonogens, even one per crypt, are fully capable of regenerating a functional crypt (12).

Immunohistochemical and labeling studies with TdR and BrdUrd suggested that the earliest detectable response of the crypt stem cell clonogen compartment is a temporary dose-dependent delay in progression through a late S-phase checkpoint and mitotic arrest (13), apparently signaled by radiation-induced DNA double-strand breaks. Resumption of mitotic activity at 36–48 h leads to a rapid depletion of crypt stem cell clonogens succumbing to the reproductive (also termed clonogenic or postmitotic) cell death mechanism 12, 14. This model implies that the primary direct interaction of radiation with gastrointestinal (GI) stem cell clonogens is required and sufficient for induction of crypt lethality.

The latter notion has been recently challenged by studies demonstrating that crypt stem cell clonogen lethality after exposure to single doses within the clinically relevant range of 8–15 Gy is conditionally linked to an early wave of acid sphingomyelinase (ASMase)-mediated apoptosis in endothelium of the intestinal microvascular system 10, 11. Acid sphingomyelinase is a phospholipase C that hydrolyses sphingomyelin to generate the second messenger ceramide, which initiates a generic pathway of stress-induced apoptosis (15). Whereas endothelial cells are specifically enriched in a secretory variant of ASMase, this cell type is particularly sensitive to radiation-induced apoptosis in vitro and in vivo via ASMase activity (15). Genetic inactivation of ASMase in SV129/C57BL/6 mice attenuated intestinal microvascular apoptosis, preserved crypt stem cell clonogens, and protected mice against lethality from the GI syndrome 10, 11, indicating that microvascular endothelium and the crypt stem cell clonogens constitute a linked primary target system for radiation in inducing GI damage. A similar endothelial–stem cell linked system was described in experimental mouse tumor systems 16, 17. Although the mouse intestinal experiments did not definitively exclude a direct impact of ASMase deficiency on stem cell radiation responses, the tumor xenograft data precluded this possibility, because the endothelial–stem cell linkage was still observed with tumors transplanted into asmase−/− hosts despite the tumor stem cells being wild type for ASMase (17). The mechanism of this linkage requires substantive further investigation, but our recent preliminary data have suggested that microvascular dysfunction regulates reproductive cell death by attenuating repair of radiation-induced DNA dsb (Kolesnick and Fuks, unpublished data). Thus, our model of engagement of dysfunctional microvasculature in radiation-induced tissue damage is consistent with a basic tenet of classical radiobiology, that the determinant mode of tissue stem cell and clonogen lethality is reproductive cell death (18).

The discovery of the linked crypt stem cell clonogen/endothelial response was enabled by use of a combination of tissue readout systems, including immunohistochemical double staining of the GI mucosa with specific endothelial cell surface markers and TUNEL, clonogenic crypt survival assays at 3.5 days after irradiation, and mouse survival patterns after irradiation coupled with autopsy examinations to assess cause of death 11, 12, 14. The crypt survival assay is based on the ability to histologically identify typical hyperchromatic regenerative crypts at 3.5 days after irradiation, each assumed to represent progeny of at least one stem cell clonogen per crypt that survives exposure to radiation 12, 19. The number of surviving crypts after exposure to low radiation doses (<14 Gy) is usually sufficient to support a complete recovery of the mucosa, whereas at higher doses massive stem cell clonogen loss leads to irreversible collapse of the crypt–villus system, mucosal denudation, and animal death at 5–7 days from the GI syndrome, which can be definitively confirmed only by autopsy 11, 12, 14.

Although genetic (asmase−/−) attenuation of crypt stem cell clonogen lethality strongly suggested linkage to a microvascular component, a hypothetical possibility remains that another of the pleiotropic events signaled by ASMase activation might be a critical effector of crypt stem cell clonogen lethality. To address this issue, we used here apoptosis-resistant Bax−/− or Bak−/− mice, both displaying a normal ASMase phenotype, to test the hypothesis that endothelial apoptosis per se regulates crypt stem cell lethality after high-dose radiation. The present experiments show that either Bax deficiency or Bak deficiency alone significantly attenuated the GI endothelial apoptotic response of irradiated mice and consequently protected against crypt stem cell clonogen lethality, rescuing mice from the GI syndrome. In contrast, there were no detectable effects of Bax deficiency or Bak deficiency on the p53-mediated epithelial apoptosis at positions 4–5 of irradiated crypts, indicating specificity of the radioprotective Bax/Bak deficiency effects on the endothelial component of the irradiated GI phenotype. These studies provide strong evidence that microvascular dysfunction, rather than another event regulated by ASMase signaling, impacts the fate of irradiated crypt stem cell clonogens.

Section snippets

Mice

C3H/HeJ and C3HeB/FeJ male and female mice, 8–12 weeks old, were purchased from Jackson Laboratories (Bar Harbor, ME). SV129/C57BL/6asmase−/− and C57BL/6Bax−/− mice were bred in our colony and genotyped as described previously 20, 21. SV129/C57BL/6Bak−/− mice were inbred as described elsewhere (1). Backcrossing the asmase−/− genotype onto the C3HeB/FeJ background was performed by mating wild-type C3HeB/FeJ females with male SV129/C57BL/6asmase−/− mice. Male F1 mice with asmase+/− genotype were

Bax and Bak deficiencies block radiation-induced GI endothelial cell apoptosis

Although in our previous studies we defined an endothelial apoptotic component in the pathogenesis of radiation damage to the intestines by use of a genetic model of asmase deficiency (11), a question still remained whether the asmase null mutation conferred radiation protection by elimination of another event regulated by ASMase signaling. To address this issue we initially explored whether apoptosis-refractory C57BL/6Bax−/− mice (21), shown to express wild-type ASMase (27), mimic the asmase−/−

Discussion

The present studies involving Bax- and Bak-deficient mice, both expressing a wild-type asmase phenotype, indicate that both Bax and Bak are mandatory for the apoptotic pathway induced by radiation in intestinal microvascular endothelium, and are mutually independent without evidence of functional overlap. Thus, the Bax- and Bak-deficient phenotypes mimic the effect of ASMase deficiency on microvascular dysfunction and organ fate. Lack of detectable impact of Bax deficiency or Bak deficiency on

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