Coordinated inhibition of C/EBP by Tribbles in multiple tissues is essential for Caenorhabditis elegans development

C. elegans Tribbles NIPI-3 was identified on the basis of its roles in host defense [20, 21]. Here, through generation and analyses of null alleles, we find nipi-3 to be essential for animal development and viability. Remarkably, the larval arrest and lethality caused by complete loss of nipi-3 is fully suppressed by loss of cebp-1, a C/EBP bZIP transcription factor, or by loss of function in the PMK-1/p38 MAPK cascade including tir-1/SARM, nsy-1/MAPKKK, sek-1/MAPKK and pmk-1/MAPK. Our data show that complete elimination of the function of nipi-3 causes abnormally high expression of CEBP-1, and activation of PMK-1 MAPK. This then disrupts development and leads to death. The level of sek-1 mRNA is increased in nipi-3(0) mutants but not in cebp-1(0) or in nipi-3 cebp-1 animals. The level of phosphorylated (active) PMK-1 follows the same trend. Coupled with our ChIP-seq analyses and genetic epistasis data, this suggests that CEBP-1 acts as a direct positive regulator of sek-1. The PMK-1 pathway is therefore activated when CEBP-1 expression is high in nipi-3(0). On the other hand, cebp-1 expression levels remain high in nipi-3(0); pmk-1(0) animals, confirming that CEBP-1 does not act downstream of the PMK-1 pathway. Together, these results suggest that NIPI-3 negatively regulates the PMK-1 MAPK cascade, via CEBP-1, to promote animal viability and development (Fig. 6?h).

In innate immunity, however, nipi-3 cell-autonomously promotes or enhances the same p38 kinase cascade to activate host defense in the epidermis [20]. It has been shown that overexpression of sek-1 in the epidermis rescues the block of AMP induction in nipi-3 mutants upon fungal infection [20], and an overexpression of nipi-3 provokes an increase in the constitutive expression of AMP which is dependent on the p38 cascade [21]. It is intriguing that NIPI-3 appears to be capable of activating or inhibiting PMK-1/p38 in the epidermis at different times or under different conditions (infection versus development). How might NIPI-3 achieve this dual role under different stresses and in altered cellular contexts? As Tribbles proteins are well known to act as adaptors, NIPI-3 might be regulated via binding with other co-factors only present under specific circumstances. Indeed, we find that other upstream and downstream components of the epidermal immune response cascade are not involved in the developmental regulation described here. Thus, the core PMK-1/p38 MAPK cassette has evolved context-specific functions depending on different upstream regulators or co-factors [40, 41]. Members of the Tribbles family in other species have been mostly studied in the context of cell proliferation, adipocyte tissue differentiation, energy metabolism and immunity, where they function in a cell-autonomous manner. Our discovery of the opposing roles of NIPI-3 in development and in the immune response illustrates how cellular context can alter the function of highly conserved signalling molecules.

Negative regulation of C/EBP by Tribbles has been observed throughout the animal kingdom. Drosophila and mammalian Tribbles bind and degrade C/EBP proteins [1, 7, 13–15]. We find that C. elegans NIPI-3 represses the transcription of cebp-1, which has important functional consequences in vivo. This form of regulation has not been reported in other organisms. Given its nuclear localization, NIPI-3 may inhibit the transcription of cebp-1 by interfering with other transcription factor(s). The promoter of cebp-1 contains putative CEBP-1 binding consensus motifs, raising the possibility that NIPI-3, by binding to CEBP-1, may also alter the transcriptional activity of CEBP-1.

NIPI-3 is required to control CEBP-1 levels in multiple tissues for animal development and viability. Consistent with the inhibition of CEBP-1 expression by NIPI-3 in the epidermis and neurons, simultaneous expression of nipi-3(+) in both tissues makes a noticeable contribution to animal development in nipi-3(0) mutants, compared with nipi-3(+) expression in single tissues. Conversely, simultaneous expression of cebp-1(+) in both epidermis and neurons causes noticeable defects in animal development in nipi-3(0) cebp-1(0) mutants, compared with cebp-1(+) expression in single tissues. Thus, a tightly regulated coordination of these two genes’ interactions in multiple tissues is required to ensure proper development.

A key conclusion from our study is that the precise control of CEBP-1 and PMK-1/p38 MAPK pathways in multiple tissues is critical for organismal development. NIPI-3 acts as a master regulator to prevent improper activation of CEBP-1 and PMK-1, whose hyperactivation during development has deleterious consequences. Interestingly, hyperactivation of PMK-1/p38 was previously shown to block larval development when the endoplasmic reticulum unfolded protein response was altered [42]. Moreover, innate immune activation with a xenobiotic that provides protection from bacterial infection in the adult has been shown to provoke a growth delay during development [43]. Subsequently, an elegant genetic suppressor screen revealed that mutations in the PMK-1/p38 MAPK pathway suppressed this developmental phenotype [44]. Thus, the NIPI-3/CEBP-1 axis is a key mechanism by which immune effector expression is held in check during nematode development.

During normal development, both CEBP-1 and PMK-1 are maintained at a basal level by NIPI-3. The levels of inducible signalling from these pathways are, however, important for animals to protect themselves or to promote repair. For instance, following fungal infection, NIPI-3 promotes the PMK-1/p38 MAPK signalling pathway in the epidermis [20]. Thus, animals can successfully defend themselves from fungal infection with activated PMK-1 locally in the epidermis, while survival is not affected as PMK-1 remains inactive in other tissues. Similarly, CEBP-1 is known to play a key role in neuronal stress responses [22, 23, 45], and we identified potential CEBP-1 target genes that are involved in different stress responses. Moreover, a concomitant study has identified NIPI-3 as a negative regulator of CEBP-1 in intestinal defense against the bacterial toxin ToxA (McEwan et al., personal communication). An important challenge for the future will be to understand how NIPI-3 regulates its downstream pathways and how NIPI-3 itself is regulated depending on developmental and environmental conditions. Understanding the molecular mechanism of this systemic, coordinated regulation should advance our knowledge of how animal development can be maintained in the face of environmental stresses.