dSTK10 inhibits ectopic JNK-induced cell death in development
Given that JNK signaling is of vital importance in regulating cell death, to identify additional factors that regulate JNK-mediated cell death, we have previously performed a genetic screen with ectopic JNK-induced cell death in the Drosophila eye [5, 35]. Compared with the GMR-Gal4 controls (Fig. 1A, F), ectopic expression of a constitutive active form of Hep (HepCA) triggered strong cell death posterior to the morphogenetic furrow (MF) in the eye imaginal discs (Fig. 1G), indicated by Acridine Orange (AO) staining that detects dying cells [36], and produced a small eye phenotype in the adults (Fig. 1B). dSTK10 was identified as a potential inhibitor of JNK-mediated cell death, as GMR>HepCA-induced cell death and small eye phenotype were enhanced in heterozygous dSTK10KG04837 mutants, but were significantly suppressed by overexpression of dSTK10 (Fig. 1C-E, H-J).
To investigate whether dSTK10 inhibits JNK-mediated cell death in other tissues, we turned to the developing wing. Compared with the controls (Fig. 1K, P), expression of Hep along the anterior/posterior (A/P) compartment border driven by patched (ptc)-Gal4 [37] resulted in increased cell death in the wing imaginal discs (Fig. 1Q), and generated a loss of anterior cross vein (ACV) phenotype in the adult wings (Fig. 1L). Both phenotypes were considerably impeded by ectopic expression of dSTK10, but remained unaffected by that of LacZ (Fig.1 M-O, R-T). Collectively, these results indicate that dSTK10 negatively regulates ectopic Hep-induced JNK-mediated cell death during eye and wing development.
Depletion of dSTK10 triggers apoptotic cell death in development
Given that dSTK10 is an inhibitor of ectopic JNK-mediated cell death, we wonder whether dSTK10 is physiologically required for preventing cell death in development. To test this, we depleted dSTK10 with three independent RNA interference (RNAi), whose knockdown efficiencies were confirmed by the quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay (Fig. 2P). Compared with the GMR-Gal4 or ptc-Gal4 controls, cell death was significantly increased upon dSTK10 knockdown in the corresponding areas of 3rd instar larval eye (Fig. 2A-E) or wing (Fig. 2F-J) discs, suggesting that endogenous dSTK10 inhibits cell death in normal development.
Apoptosis is the major form of cell death in Drosophila, which is mediated by the cleavage and activation of caspases [38, 39]. To check whether dSTK10 regulates apoptotic cell death, we utilized an anti-cleaved Dcp-1 antibody that specifically recognizes the activated form of the effector caspase Dcp-1 [40]. We found that knockdown of dSTK10 induced strong apoptosis in the wing discs (Fig. 2K-O). Consistently, dSTK10 depletion-induced cell death was efficiently blocked by expressing P35, a viral caspase inhibitor [41], but not a lacZ RNAi (Fig. S1). Taken together, these data suggest that dSTK10 is physiologically required for preventing apoptotic cell death in normal development.
Depletion of dSTK10 promotes JNK pathway activation
As JNK signaling plays significant roles in regulating apoptotic cell death [42-45], dSTK10 depletion-triggered developmental apoptosis may depend on JNK pathway activation. In agreement with this assumption, knockdown of dSTK10, but not lacZ, by ptc-Gal4 resulted in upregulated expression of TRE-RFP (Fig. 3A-D, A’-D’), a general reporter of JNK signaling [46, 47]. Moreover, JNK phosphorylation, detected by an antibody specific to the phosphorylated JNK (p-JNK) [5, 48], was considerably elevated upon dSTK10 depletion (Fig. 3E-H, E’-H’). Together, these data suggest that loss of dSTK10 potentiates JNK signaling in development.
JNK signaling is required for loss of dSTK10-induced apoptotic cell death
Given that dSTK10 depletion activates JNK and apoptosis, we hypothesized that loss of dSTK10 might promotes cell death through activating JNK signaling. In agreement with this speculation, we found that ptc-Gal4 (Fig. 4A) driven dSTK10 depletion-caused cell death (Fig. 4B) was soundly suppressed by knocking down bsk, which encodes the Drosophila JNK, or expressing a dominant negative form of Bsk (BskDN) or Puc, an inhibitor of JNK [18, 19] (Fig. 4H-K). Consistently, loss of dSTK10-induced apoptosis, visualized by anti-cleaved Dcp-1 (cDcp-1) antibody staining, was remarkably impeded by BskDN (Fig. 4L, M, P, Q). In conclusion, these results suggest that dSTK10 depletion triggers JNK-dependent apoptotic cell death.
dSTK10 functions upstream of or in parallel to Hep
To understand how dSTK10 regulates JNK signaling-mediated cell death, we performed genetic epistasis analysis between dSTK10 and core components of the Egr-JNK pathway. We found that dSTK10 knockdown-induced cell death was significantly impeded by depletion of hep, but not by that of egr, dTRAF2, dTAK1 or lacZ serving as a negative control (Fig. 4A-G, K). Consistently, loss of dSTK10-induced Dcp-1 activation was abolished by expression of hep-IR or BskDN, but remained unaffected upon depletion of egr (Fig.4L-Q). Taken together, these results suggest that dSTK10 may act upstream of or in parallel to Hep to regulate JNK-mediated apoptotic cell death.
dSTK10 modulates JNK-dependent tissue homeostasis in normal development
Strict regulation of cell death, which contributes to the adjustment of cell number and elimination of unwanted cells, is vital for tissue homeostasis and organism fitness [49]. To characterize the physiological function of dSTK10 in tissue homeostasis, we generated RNAi-mediated knockdown clones in the wing discs by the Flippase (FLP)-Flp recognition target (FRT)-mediated genetic mosaic technique [50]. Compared with the control clones, dSTK10 depletion resulted in decreased clone (GFP+) sizes, which were appreciably restored by expressing BskDN (Fig. 5A-D). Furthermore, dSTK10 knockdown either in the posterior compartment by en-Gal4 or in the wing pouch by nub-Gal4 resulted in diminished sizes of corresponding areas, which were largely recovered by blocking Bsk activity (Fig. 5E-L). Together, these data suggest that dSTK10 is required to maintain JNK-dependent tissue homeostasis in normal development.
Human STK10 rescues dSTK10 depletion-induced developmental defects in Drosophila
dSTK10 encodes a member of the Sterile-20 kinase family, sharing 46% similarity and 32% identity with its human ortholog STK10. To check whether STK10 has retained the developmental functions of dSTK10 in Drosophila, we produced UAS-STK10 transgenic flies and checked whether expression of STK10 could rescue dSTK10 loss-caused developmental defects. We noted that expression of dSTK10 or STK10 could largely rescue ptc>dSTK10-IR-induced L3-L4 area reduction in adult wings (Fig. 6A-E) and robust cell death along A/P compartment boundary in larval wing discs (Fig. 6F-J). In addition, area reduction in the posterior compartment of en>dSTK10-IR wing discs was efficiently rescued by expressing dSTK10 or STK10 (Fig. 6K-O). Thus, these data demonstrate that the developmental functions of dSTK10 are evolutionarily conserved by STK10.
dSTK10 function in the JNK pathway is retained by STK10
As the JNK pathway is evolutionarily conserved from fly to human, and STK10 is able to rescue dSTK10 loss-induced developmental defects, it is plausible that STK10 has retained the regulatory role of dSTK10 in the JNK pathway. Consistently, GMR>Egr-triggered cell death in the larval eye discs and the small eye phenotype in the adults were effectively restored by expressing STK10, while hep depletion was included as a positive control (Fig. S2). Since Egr triggers both JNK-dependent and JNK-independent cell death in development [12, 35, 51], to determine whether STK10 modulates JNK-mediated cell death, we activated JNK signaling by overexpressing the JNK kinase Hep. We found that expression of STK10 markedly inhibited GMR>HepCA-caused small eye in adults (Fig. 7A-D) and increased AO staining in larval eye discs (Fig. 7E-H), and ptc>Hep-induced loss-of-ACV phenotype in adult wings (Fig. 7I-L) and cell death in larval wing discs (Fig. 7M-P). Hence, we conclude that STK10 could functionally substitute for dSTK10 to regulate JNK-dependent cell death in Drosophila.
dSTK10 and STK10 modulates physiological JNK‐mediated cell death
The above results suggest that dSTK10 negatively regulates ectopic JNK-mediated cell death, triggered by overexpression of Egr or Hep, yet it remains unknown whether dSTK10 modulates the physiological JNK signaling. It has been reported that disruption of cell polarity promotes JNK-mediated cell death in development [52, 53]. Consistently, knockdown of the cell polarity gene scrib along the A/P compartment boundary by ptc-Gal4 induced severe cell death in wing discs (Fig. 8F, G) and ACV loss in adult wings (Fig. 8A, B). These phenotypes, caused by the activation of endogenous JNK signaling [5], were effectively inhibited by expressing dSTK10 or STK10 (Fig. 8C-E, H-J). Importantly, ptc>scrib-IR-increased cell death was potently aggravated in heterozygous dSTK10 mutants (Fig. 8K-M). Together, these results indicated that dSTK10 modulates physiological JNK signaling-induced cell death in development.
Finally, to examine whether STK10 regulates JNK activity in human cells, we knockdown STK10 in SW480 human colon cancer cells by two independent siRNA (Fig. S3), and found that p-JNK level was up-regulated whereas total JNK protein level remained unaffected (Fig. 8N). Cumulatively, our findings support that dSTK10/STK10 regulate JNK pathway in a conserved manner from Drosophila to human cells.