Steroid regulation of autophagic programmed cell death during development

Cheng-Yu Lee 0 1 Eric H. Baehrecke 1 0 Department of Biology, University of Maryland , College Park, Maryland 20742 , USA 1 Center for Agricultural Biotechnology, University of Maryland Biotechnology Institute , College Park, Maryland 20742 , USA SUMMARY Apoptosis and autophagy are morphologically distinct forms of programmed cell death. While autophagy occurs during the development of diverse organisms and has been implicated in tumorigenesis, little is known about the molecular mechanisms that regulate this type of cell death. Here we show that steroid-activated programmed cell death of Drosophila salivary glands occurs by autophagy. Expression of p35 prevents DNA fragmentation and partially inhibits changes in the cytosol and plasma membranes of dying salivary glands, suggesting that caspases are involved in autophagy. The steroid-regulated BR-C, E74A and E93 genes are required for salivary gland cell death. BR-C and E74A mutant salivary glands exhibit vacuole and plasma membrane breakdown, but E93 Programmed cell death plays a critical role during animal development by functioning in the destruction of unneeded cells and tissues (Jacobson et al., 1997; Vaux and Korsmeyer, 1999). The term programmed cell death was established to distinguish physiological or genetic-regulated cell death from necrotic cell destruction (Lockshin and Zakeri, 1991). Genetically regulated cell death is an integral component of normal development, and is used in processes such as limb formation and nervous system remodeling (Robinow et al., 1993; Saunders, 1966). Cell death is also involved in removal of abnormal cells during development, including those that form during tumorigenesis (Thompson, 1995). Morphological studies of developing vertebrate embryos resulted in the definition of three types of physiological cell death (Schweichel and Merker, 1973). The first type, widely known as apoptosis, is found in isolated dying cells that exhibit condensation of the nucleus and cytoplasm, followed by fragmentation and phagocytosis by cells that degrade their contents (Kerr et al., 1972). The second type, known as autophagy, is observed when groups of associated cells or entire tissues are destroyed. These dying cells contain autophagic vacuoles in the cytoplasm that function in the degeneration of cell components. The third type, known as non-lysosomal cell death, is least common, and is characterized by swelling of cavities with membrane borders followed by degeneration without lysosomal activity. While autophagy mutant salivary glands fail to exhibit these changes, indicating that E93 regulates early autophagic events. Expression of E93 in embryos is sufficient to induce cell death with many characteristics of apoptosis, but requires the H99 genetic interval that contains the rpr, hid and grim proapoptotic genes to induce nuclear changes diagnostic of apoptosis. In contrast, E93 expression is sufficient to induce the removal of cells by phagocytes in the absence of the H99 genes. These studies indicate that apoptosis and autophagy utilize some common regulatory mechanisms. fulfills the definition of programmed cell death (Lockshin and Zakeri, 1991), occurs during development of diverse organisms (Clarke, 1990), and has been implicated in tumorigenesis (Bursch et al., 1996; Liang et al., 1999; Schulte-Hermann et al., 1997), little is known about the molecular genetic mechanisms underlying this type of programmed cell death. The morphological characteristics that distinguish apoptosis and autophagy suggest that these cell deaths are regulated by independent mechanisms (Clarke, 1990). Comparison of biochemical changes during lymphocyte apoptosis and insect intersegmental muscle autophagy also indicate that these physiological cell deaths occur by distinct mechanisms (Schwartz et al., 1993). However, recent studies of steroidtriggered cell death of Drosophila larval salivary glands suggest that these cells utilize genes that are part of the conserved apoptosis pathway (Jiang et al., 1997; Lee et al., 2000), even though these cells exhibit characteristics of autophagy (von Gaudecker and Schmale, 1974). Specifically, the caspase dronc and homolog of ced4/Apaf-1 ark, two components of the core apoptotic machinery, increase in transcription immediately prior to salivary gland cell death (Lee et al., 2000). Thus, characterization of the mechanisms governing the regulation of autophagy will identify how these cell deaths differ from those that occur by apoptosis. Steroid hormones activate programmed cell death during animal development (Evans-Storm and Cidlowski, 1995). During insect metamorphosis, the steroid 20-hydroxyecdysone (ecdysone) activates programmed cell death to eliminate unneeded larval cells (Robinow et al., 1993; Truman et al., 1994). Drosophila larval salivary glands are an excellent system for studying the genetic hierarchy that is activated by steroids during programmed cell death. A pulse of ecdysone 10-12 hours after puparium formation (APF) triggers caspase-mediated programmed cell death of Drosophila larval salivary glands (Jiang et al., 1997). Within 4 hours of this rise in hormone titer, salivary glands exhibit several features of programmed cell death including nuclear staining by Acridine Orange, DNA fragmentation, and exposure of phosphatidylserine on the outer leaflet of the plasma membrane (Jiang et al., 1997; S. van den Einde and E.H.B., unpublished). The mechanisms of steroid signaling have been extensively studied in Drosophila larval salivary glands because of the advantages of the giant polytene chromosomes, which form steroid-induced puffs reflecting a transcriptional regulatory hierarchy (Andres and Thummel, 1992; Ashburner et al., 1974; Becker, 1959; Clever, 1964). Previous studies have implicated the ecdysone-regulated genes EcR, usp (ultraspiracle), b FTZF1, BR-C, E74 and E93 in larval salivary gland programmed cell death (Broadus et al., 1999; Hall and Thummel, 1998; Jiang et al., 2000; Lee et al., 2000; Restifo and White, 1992). EcR, usp, b FTZ-F1, BR-C and E74 function in processes other than cell death including the differentiation of adult cells (Bender et al., 1997; Broadus et al., 1999; Fletcher et al., 1995; Hall and Thummel, 1998; Restifo and White, 1992). In contrast, E93 appears to function more specifically in destruction of larval tissues (Lee et al., 2000). EcR, usp and b FTZ-F1 act at the top of this signaling pathway and regulate BR-C, E74 and E93 (Broadus et al., 1999; Woodard et al., 1994). BR-C, E74 and E93 impact on the transcription of programmed cell death genes including rpr (reaper), hid (head involution defective/w; wrinkled), crq (croquemort), Ark and dronc (Nc; Nedd2 like caspase) during larval tissue destruction (Jiang et al., 2000; Lee et al., 2000), suggesting a potential mechanism for steroid-triggered cell death. However, the relationship between the primary steroid response genes BRC, E74 and E93 remains unclear. Although s (...truncated)