Intestinal organoid deleted for the key pro-apoptotic ARTS protein, which regulates cell death in the stem cell niche. As a consequence of apoptotic resistance, the intestinal stem cell niche undergoes expansion and displays enhanced activity of the Wnt/β-catenin pathway. Organoid is stained for β-catenin and 4′,6 -diamidino-2-phenylindole (DAPI). Image rendered using color-coded projections for each focal plane. PHOTO: YARON FUCHS

Stem cells are classically defined by their unlimited proliferative potential and capacity to differentiate into diverse cell types. For many years, investigations in the stem cell field have focused specifically on the self-renewal and differentiation aspects, leaving the mechanisms of stem cell elimination relatively unexplored (1). What may at first appear to be a trivial question—how can an “immortal” self-renewing stem cell commit cellular suicide?—struck me as biologically important. Are there distinct mechanisms enabling such elimination, I wondered, and, if so, to what extent does this process affect tissue regeneration?

Apoptosis as an Aid to Healing With these questions in mind, I set out to examine the importance of various apoptotic machinery proteins in a model system known to be dependent on stem cells: the hair follicle. I found that loss of the pro-apoptotic factor Septin4 (Sept4)/ARTS protected hair follicle stem cells against cell death, leading to their expansion (2). Normally, hair follicle stem cells do not contribute to epidermal homeostasis. However, in response to wounding, they exit their niche and participate in repopulating the epidermis. Upon wound infliction, I found that mice deficient for the Sept4 locus that encodes ARTS (denoted Sept4/ARTS-/-) could rapidly generate neo-epidermis, resulting in considerably smaller scars. This accelerated tissue repair was accompanied by a pronounced regeneration of de novo hair follicles from the wound bed, which normally only form in large numbers during embryonic development (2). By performing genetic lineage-tracing experiments, I showed that apoptotic-resistant hair follicle stem cells were responsible for the enhanced healing and de novo hair follicle regeneration. Moreover, I was able to determine that X-linked inhibitor of apoptosis (XIAP) served as a direct biochemical target of ARTS in hair follicle stem cells (2). My lab has discovered that ARTS and XIAP have similar effects in the intestinal epithelium, a system rapidly replenished by actively dividing stem cells. Loss of ARTS function, mediated through elevated XIAP, protected Lgr5+ intestinal stem cells against apoptosis, increased their numbers, and led to substantial improvement in intestinal regeneration (3). Notably, we also found that the Sept4/ARTS-/- stem cell niche displayed enhanced Wnt/β-catenin activation and augmented cell proliferation, and that these cells could give rise to massive cystic-like intestinal organoids (3). These studies demonstrate the importance of apoptosis in restricting stem cell expansion and enabling proper repair, suggesting that it serves as a defense mechanism against irreparably damaged stem cells and the emergence of cancer. Furthermore, these findings suggest that transiently targeting apoptotic pathways in hair follicle or intestinal stem cells may offer therapeutic benefits to promote wound healing and regeneration. We are presently in the process of developing specific inhibitors and activators of the apoptotic machinery proteins to improve stem cell–dependent skin and intestinal regeneration.

Death Begets Death Traditionally, apoptosis has been regarded as an isolated process that does not affect surrounding tissues. However, it is becoming increasingly clear that cells undergoing apoptosis in response to stress and injury can secrete mitogenic and morphogenic factors to stimulate growth and repair in their surroundings. Large groups of cells often undergo coordinated death during development and under conditions of severe tissue injury (4). One example of communal cell death is the regressive phase of the hair follicle, which undergoes cycles of growth (anagen), death (catagen), and rest (telogen). During catagen, all cells in the lower portion of the hair follicle are eliminated by apoptosis in a very rapid and highly synchronized manner (5). However, it was unclear how this cohort behavior could be achieved. I showed that, during the catagenic phase, hair follicle cells undergoing apoptosis induced nonautonomous apoptosis in neighboring cells. Through this previously unknown mechanism, which we termed “apoptosis-induced-apoptosis” (AiA), dying cells released the long-range death factor TNF-α (tumor necrosis factor–α) to induce additional cell killing and ensure progression of the hair cycle. Thus, AiA provided a mechanism to explain the cohort behavior of dying cells seen both during normal development and in pathological conditions (6).

An Unexpected Role for the Key Executioner of Apoptosis In my quest to understand how epidermal stem cells use apoptotic machinery proteins, I noted that a very large number of proliferating sebaceous gland cells expressed cleaved (active) caspase-3—a key executioner of the apoptotic cascade. Intriguingly, these cells appeared to be vital and did not display any of the known morphological characteristics of an apoptotic cell. Captivated by this initial observation, my lab set out to characterize the nonapoptotic functions of caspase-3. Contrary to our expectations, deletion or inhibition of caspase-3 resulted in diminished proliferation and cell number, decreased organ size, and marked impairment in sebaceous gland regeneration (7). Exploring the underlying mechanism, we discovered that caspase-3 controls the activity of YAP, a fundamental regulator of tissue regeneration and organ size. Our data indicated that caspase-3 could liberate YAP by cleaving α-catenin, which sequesters YAP in the cytoplasm. Accordingly, activation of caspase-3 facilitated YAP-dependent organ size augmentation (7). Because XIAP serves as the main endogenous inhibitor of caspase-3, we examined its role in the caspase-3–YAP module and found that it generates a negative feedback loop that prevents organ overgrowth. Thus, our results demonstrated that the apoptotic machinery could be refocused to orchestrate precise organ size (7). Ongoing work in my lab now indicates that the caspase-3–YAP module heavily contributes to wound healing and tumorigenesis of the skin. We are currently developing new strategies for targeting caspase-3 activation in different tumor settings and in diverse regenerative models.

Looking Forward We have taken the first steps toward understanding the specific mechanisms that underlie stem cell elimination, determining the effect dying cells have on the tissue environment, and identifying the molecular switches that dictate the “life or death” functions of apoptotic proteins. The next question is, how should we go about harnessing and refocusing the devastating power of programmed cell death to combat tumor formation and instruct tissue regeneration? It is my hope that answers gained from these studies may soon be translated into new approaches for regenerative medicine and tumor therapy.

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