Something new under the sun: caspases promote cell survival under moderate heat stress

Plain language summary

Since the discovery of caspases in 1993 (Yuan et al, 1993), they have largely been regarded as essential Cys proteases in apoptosis. However, more recently, it has become evident that caspases also have non‐apoptotic functions in the so‐called caspase‐dependent non‐lethal processes (CDPs) including cell proliferation and cell differentiation (Aram et al, 2017). While most of the non‐apoptotic functions were characterized under normal developmental conditions, a new study by Galasso et al took this examination a step further—to the analysis of moderate stress conditions. The authors now report that caspases have a non‐apoptotic prosurvival function in Drosophila ovarian somatic cells exposed to moderate heat stress by controlling Hedgehog signaling and autophagy (Galasso et al, 2023).

Caspases have a non‐apoptotic prosurvival function in Drosophila ovarian somatic cells exposed to moderate heat stress by controlling Hedgehog signaling and autophagy.

Apoptotic caspases are divided into initiator caspases such as mammalian Caspase‐9 and its Drosophila ortholog Dronc and effector caspases such as Caspase‐3 or Drosophila DrICE. After effector caspases are activated by initiator caspases during apoptosis, they can cleave a large number of intracellular proteins, triggering the death of the cell. However, the development of very specific reporters that can track current, recent, and past caspase activity in Drosophila has revealed that killing of cells is not the only function of caspases and that many cells are exposed to non‐lethal (non‐apoptotic) caspase activity in the course of their lifetime (Tang et al, ²⁰¹⁵; Ding et al, ²⁰¹⁶; Baena‐Lopez et al, ²⁰¹⁸).

In Drosophila ovaries, the germarium is the anterior‐most tissue where both germline and somatic stem cells and their progenies are located. The somatic cells (follicular stem cells, escort, and follicular cells) support the germ cells including the developing oocyte when they develop into egg chambers. After keeping adult female flies for 2 weeks at moderate heat stress (29°C rather than at the standard incubation temperature of 25°C), the authors found that a significant fraction of the somatic cells in the germarium, including follicular stem cells, was exposed to transient activity of the initiator caspase Dronc, but did not die from it, suggesting that this represents a non‐apoptotic activity of Dronc (Galasso et al, ²⁰²³). They also found that the gene expression of Dronc is induced in most somatic cells under these conditions. These observations raised the question about the function of Dronc under moderate heat stress. Conditional inactivation of Dronc in somatic cells resulted in a strong reduction of somatic cell number and loss of somatic cell differentiation markers such as Castor or Fasciclin III. The entire area in the germarium where these cells usually reside was reduced in size in Dronc mutants. The reduction in somatic cell number is likely the result of a delayed cell cycle as Dronc deficiency slowed down the transition through S‐phase (Galasso et al, ²⁰²³). These results suggest that Dronc is required to maintain cell proliferation and differentiation of somatic cells in the germarium under moderate heat stress (Fig 1).

Figure 1. Dronc controls autophagy and Hedgehog (Hh) signaling in somatic ovarian cells under moderate heat stress.

Continued exposure of adult female flies to 29°C triggers a non‐apoptotic response of the initiator caspase Dronc in Drosophila ovarian somatic cells. (Left) Dronc restricts the accumulation of Patched (Ptc) at autophagosomes and inhibits autophagy. (Right) Dronc stimulates the activity of the canonical Hh pathway. Both activities combined ensure the appropriate proliferation and differentiation of ovarian somatic cells under moderate heat stress. The dotted inhibitory lines from Ptc to Hh signaling also indicate the possibility that Ptc may negatively affect the Hh pathway, but given that Ptc accumulates in autophagosomes and not at the plasma membrane, this possibility is an uncertainty.

Interestingly, these phenotypes under moderate heat stress were also observed by inactivation of the entire apoptotic pathway including the caspase activators Reaper, Hid and Grim, the apoptosome component Dark and all effector caspases although there are differences in the degree to which their inactivation affects somatic cell proliferation and differentiation. These results suggest that Dronc does not have an isolated role in ovarian somatic cells under moderate stress, but that the entire apoptotic pathway is engaged in this stress response (Galasso et al, ²⁰²³).

The Hedgehog (Hh) pathway plays an essential role for proliferation of follicular stem cells and differentiation of their progeny (Briscoe & Therond, 2013). Loss of Hh pathway activity causes a very similar proliferation and differentiation phenotype as the loss of Dronc in ovarian somatic cells. Therefore, the authors examined if the non‐apoptotic caspase activation intersects with the Hh pathway under moderate stress. Consistently, loss of Dronc caused downregulation of Hh pathway activity suggesting that Hh signaling requires Dronc. Furthermore, ectopic activation of the Hh pathway rescued the proliferation and differentiation defects observed in Dronc mutants under moderate heat stress suggesting that the Hh pathway acts downstream of Dronc under these conditions (Fig 1). A similar phenomenon was observed in mammalian cells that showed reduced Hh signaling in response to Caspase‐9 knockdown, indicating that Caspase‐induced facilitation of Hh signaling is evolutionary conserved (Galasso et al, ²⁰²³).

Dronc‐deficient somatic cells showed elevated expression levels of Patched (Ptc) protein, a negative regulator of Hh signaling. This upregulation was, however, not observed at the plasma membrane, where Hh signaling takes place, but rather in enlarged vesicles in the cytosol which the authors identified as autophagosomes. Ptc has been shown to induce autophagy independently of canonical Hh signaling in ovarian somatic cells (Singh et al, ²⁰¹⁸). While stressed cells can use autophagy as a resilience mechanism, excessive autophagy can induce autophagic cell death. In the context of heat stress, downregulation of Ptc by RNAi rescued the proliferation and differentiation defects observed in Dronc‐deficient cells. A similar effect was observed when the authors blocked autophagy by inhibiting the autophagy components Atg1 and Atg8 (Galasso et al, ²⁰²³), suggesting that a key function of Dronc is to prevent the accumulation of Ptc protein in autophagosomes and the induction of excessive autophagy under moderate heat stress (Fig 1).

These data highlight a mechanism by which moderately stressed cells maintain autophagy at a level, which is adequate to maintain homeostasis of the stressed cells and the entire tissue, in this case ovarian somatic cells and the germarium. It is possible that this mechanism prevents excessive autophagy, which may otherwise trigger autophagic cell death. It is interesting, though, that these cells chose caspases to keep autophagy non‐apoptotically in check and to maintain their survival. They may have adopted the mutual antagonism between apoptosis and autophagy to their specific needs.

The control of Ptc levels at the autophagosome is not the only function of caspases under moderate heat stress, but also maintain an adequate level of canonical Hh signaling, a pathway critical for proliferation of somatic stem cells and differentiation of their progeny (Briscoe & Therond, 2013). The simultaneous control of Ptc levels at the autophagosome and canonical Hh signaling by caspases likely occurs independently of each other (Fig 1).

There are a number of open questions still to be resolved. How is caspase activation induced under moderate heat stress? While the authors provided evidence that the upstream caspase regulators Reaper, Hid and Grim are involved, this does not answer how these are activated. Furthermore, given that caspases are proteases, what are the proteolytic substrates that are cleaved under these conditions? Are these substrates also cleaved during apoptosis? The answers to the last two questions would address how caspases limit the accumulation of Ptc in autophagosomes and maintain proper Hh function in somatic cells under moderate heat stress. Finally, how is the caspase activity kept at a non‐lethal level under these conditions? It is generally assumed that they are activated at low levels (Florentin & Arama, 2012) which raises the question how? An alternative mechanism to restrain caspase activity includes subcellular sequestration of caspases into a non‐apoptotic compartment (Bergmann, 2018). Future work will address these important and interesting questions.

EMBO reports (2023) 24: e57276