Proteolytic activation of fatty acid synthase signals pan-stress resolution.

Chronic stress and inflammation are both outcomes and major drivers of many human diseases. Sustained responsiveness despite mitigation suggests a failure to sense resolution of the stressor. Here we show that a proteolytic cleavage event of fatty acid synthase (FASN) activates a global cue for stress resolution in Caenorhabditis elegans. FASN is well established for biosynthesis of the fatty acid palmitate. Our results demonstrate FASN promoting an anti-inflammatory profile apart from palmitate synthesis. Redox-dependent proteolysis of limited amounts of FASN by caspase activates a C-terminal fragment sufficient to downregulate multiple aspects of stress responsiveness, including gene expression, metabolic programs and lipid droplets. The FASN C-terminal fragment signals stress resolution in a cell non-autonomous manner. Consistent with these findings, FASN processing is also seen in well-fed but not fasted male mouse liver. As downregulation of stress responses is critical to health, our findings provide a potential pathway to control diverse aspects of stress responses.

Modulating p38 MAPK signaling by proteostasis mechanisms supports tissue integrity during growth and aging.

The conserved p38 MAPK family is activated by phosphorylation during stress responses and inactivated by phosphatases. C. elegans PMK-1 p38 MAPK initiates innate immune responses and blocks development when hyperactivated. Here we show that PMK-1 signaling is enhanced during early aging by modulating the stoichiometry of non-phospho-PMK-1 to promote tissue integrity and longevity. Loss of pmk-1 function accelerates progressive declines in neuronal integrity and lysosome function compromising longevity which has both cell autonomous and cell non-autonomous contributions. CED-3 caspase cleavage limits phosphorylated PMK-1. Enhancing p38 signaling with caspase cleavage-resistant PMK-1 protects lysosomal and neuronal integrity extending a youthful phase. PMK-1 works through a complex transcriptional program to regulate lysosome formation. During early aging, the absolute phospho-p38 amount is maintained but the reservoir of non-phospho-p38 diminishes to enhance signaling without hyperactivation. Our findings show that modulating the stoichiometry of non-phospho-p38 dynamically supports tissue-homeostasis during aging without hyper-activation of stress response.

Non-Canonical Caspase Activity Antagonizes p38 MAPK Stress-Priming Function to Support Development.

Recent studies have revealed non-canonical activities of apoptotic caspases involving specific modulation of gene expression, such as limiting asymmetric divisions of stem-like cell types. Here we report that CED-3 caspase negatively regulates an epidermal p38 stress-responsive MAPK pathway to promote larval development in C. elegans. We show that PMK-1 (p38 MAPK) primes animals for encounters with hostile environments at the expense of retarding post-embryonic development. CED-3 counters this function by directly cleaving PMK-1 to promote development. Moreover, we found that CED-3 and PMK-1 oppose each other to balance developmental and stress-responsive gene expression programs. Specifically, expression of more than 300 genes is inversely regulated by CED-3 and PMK-1. Analyses of these genes showed enrichment for epidermal stress-responsive factors, including the fatty acid synthase FASN-1, anti-microbial peptides, and genes involved in lethargus states. Our findings demonstrate a non-canonical role for a caspase in promoting development by limiting epidermal stress response programs.

Tag team: Roles of miRNAs and Proteolytic Regulators in Ensuring Robust Gene Expression Dynamics.

Lack of prominent developmental defects arising from loss of many individual miRNAs is consistent with the observations of collaborative networks between miRNAs and roles for miRNAs in regulating stress responses. However, these characteristics may only partially explain the seemingly nonessential nature of many miRNAs. Non-miRNA gene expression regulatory mechanisms also collaborate with miRNA-induced silencing complex (miRISC) to support robust gene expression dynamics. Genetic enhancer screens have revealed roles of miRNAs and other gene repressive mechanisms in development or other cellular processes that were masked by genetic redundancy. Besides discussing the breadth of the non-miRNA genes, we use LIN-28 as an example to illustrate how distinct regulatory systems, including miRNAs and multiple protein stability mechanisms, work at different levels to target expression of a given gene and provide tissue-specific and stage-specific regulation of gene expression.

Coupled Caspase and N-End Rule Ligase Activities Allow Recognition and Degradation of Pluripotency Factor LIN-28 during Non-Apoptotic Development.

Recent findings suggest that components of the classical cell death machinery also have important non-cell-death (non-apoptotic) functions in flies, nematodes, and mammals. However, the mechanisms for non-canonical caspase substrate recognition and proteolysis, and the direct roles for caspases in gene expression regulation, remain largely unclear. Here we report that CED-3 caspase and the Arg/N-end rule pathway cooperate to inactivate the LIN-28 pluripotency factor in seam cells, a stem-like cell type in Caenorhabditis elegans, thereby ensuring proper temporal cell fate patterning. Importantly, the caspase and the E3 ligase execute this function in a non-additive manner. We show that CED-3 caspase and the E3 ubiquitin ligase UBR-1 form a complex that couples their in vivo activities, allowing for recognition and rapid degradation of LIN-28 and thus facilitating a switch in developmental programs. The interdependence of these proteolytic activities provides a paradigm for non-apoptotic caspase-mediated protein inactivation.

Time to move the fat.

In this issue of Genes & Development, Dowen and colleagues (pp. 1515-1528) elegantly unify two previously unconnected aspects of physiology. The investigators provide significant genetic evidence to support a critical link between developmental timing decisions and the regulation of lipid mobilization at the transition to adulthood in Caenorhabditis elegans This novel connection involves cross-tissue signaling from the hypodermis (epidermis) to the intestine to promote reproductive success in the germline.

CED-3 caspase acts with miRNAs to regulate non-apoptotic gene expression dynamics for robust development in C. elegans.

Genetic redundancy and pleiotropism have limited the discovery of functions associated with miRNAs and other regulatory mechanisms. To overcome this, we performed an enhancer screen for developmental defects caused by compromising both global miRISC function and individual genes in Caenorhabditis elegans. Among 126 interactors with miRNAs, we surprisingly found the CED-3 caspase that has only been well studied for its role in promoting apoptosis, mostly through protein activation. We provide evidence for a non-apoptotic function of CED-3 caspase that regulates multiple developmental events through proteolytic inactivation. Specifically, LIN-14, LIN-28, and DISL-2 proteins are known miRNA targets, key regulators of developmental timing, and/or stem cell pluripotency factors involved in miRNA processing. We show CED-3 cleaves these proteins in vitro. We also show CED-3 down-regulates LIN-28 in vivo, possibly rendering it more susceptible to proteasomal degradation. This mechanism may critically contribute to the robustness of gene expression dynamics governing proper developmental control.

Regulation of zinc-responsive Slc39a5 (Zip5) translation is mediated by conserved elements in the 3'-untranslated region.

Translation of the basolateral zinc transporter ZIP5 is repressed during zinc deficiency but Zip5 mRNA remains associated with polysomes and can be rapidly translated when zinc is repleted. Herein, we examined the mechanisms regulating translation of Zip5. The 3'-untranslated region (UTR) of Zip5 mRNA is well conserved among mammals and is predicted by mFOLD to form a very stable stem-loop structure. Three algorithms predict this structure to be flanked by repeated seed sites for miR-328 and miR-193a. RNAse footprinting supports the notion that a stable stem-loop structure exists in this 3'-UTR and electrophoretic mobility shift assays detect polysomal protein(s) binding specifically to the stem-loop structure in the Zip5 3'-UTR. miR-328 and miR-193a are expressed in tissues known to regulate Zip5 mRNA translation in response to zinc availability and both are polysome-associated consistent with Zip5 mRNA localization. Transient transfection assays using native and mutant Zip5 3'-UTRs cloned 3' to luciferase cDNA revealed that the miRNA seed sites and the stem-loop function together to augment translation of Zip5 mRNA when zinc is replete.

Zip4 (Slc39a4) expression is activated in hepatocellular carcinomas and functions to repress apoptosis, enhance cell cycle and increase migration.

BACKGROUND: The zinc transporter ZIP4 (Slc39a4) is important for proper mammalian development and is an essential gene in mice. Recent studies suggest that this gene may also play a role in pancreatic cancer. METHODS/PRINCIPAL FINDINGS: Herein, we present evidence that this essential zinc transporter is expressed in hepatocellular carcinomas. Zip4 mRNA and protein were dramatically elevated in hepatocytes in the majority of human hepatocellular carcinomas relative to noncancerous surrounding tissues, as well as in hepatocytes in hepatocellular carcinomas occurring in farnesoid X receptor-knockout mice. Interestingly, meta-analysis of microarray data in the Geo and Oncomine databases suggests that Zip4 mRNA may also be elevated in many types of cancer. Potential mechanisms of action of ZIP4 were examined in cultured cell lines. RNAi knockdown of Zip4 in mouse Hepa cells significantly increased apoptosis and modestly slowed progression from G(0)/G(1) to S phase when cells were released from hydroxyurea block into zinc-deficient medium. Cell migration assays revealed that RNAi knockdown of Zip4 in Hepa cells depressed in vitro migration whereas forced over-expression in Hepa cells and MCF-7 cells enhanced in vitro migration. CONCLUSIONS: ZIP4 may play a role in the acquisition of zinc by hepatocellular carcinomas, and potentially many different cancerous cell-types, leading to repressed apoptosis, enhanced growth rate and enhanced invasive behavior.

The genetics of essential metal homeostasis during development.

The essential metals copper, zinc, and iron play key roles in embryonic, fetal, and postnatal development in higher eukaryotes. Recent advances in our understanding of the molecules involved in the intricate control of the homeostasis of these metals and the availability of natural mutations and targeted mutations in many of the genes involved have allowed for elucidation of the diverse roles of these metals during development. Evidence suggests that the ability of the embryo to control the homeostasis of these metals becomes essential at the blastocyst stage and during early morphogenesis. However, these metals play unique roles throughout development and exert pleiotropic, metal-specific, and often cell-specific effects on morphogenesis, growth, and differentiation. Herein, we briefly review the major players known to be involved in the homeostasis of each of these essential metals and their known roles in development.

Novel zinc-responsive post-transcriptional mechanisms reciprocally regulate expression of the mouse Slc39a4 and Slc39a5 zinc transporters (Zip4 and Zip5).

Dietary zinc deficiency in mice is accompanied by enhanced expression of the zinc uptake transporter Slc39a4 (Zip4) and repressed expression of Slc39a5 (Zip5) in tissues which regulate zinc homeostasis (intestine, pancreas and visceral yolk sac). Herein, mechanisms controlling this differential expression were investigated. The induction of Zip4 mRNA during zinc deficiency, and its repression in response to zinc repletion were found to reflect changes in Zip4 mRNA stability and not changes in the relative rate of transcription of this gene. During zinc deficiency, ZIP4 protein levels are increased and this protein is localized on the apical membranes. Administration of an oral gavage of zinc caused ZIP4 internalization and degradation in enterocytes and visceral endoderm cells. Similarly, ZIP4 is induced by zinc deficiency in cultured mouse Hepa cells and is rapidly degraded in response to added zinc. Zip5 mRNA abundance does not change in response to zinc, but the translation of this mRNA was found to be zinc-responsive. During zinc deficiency, Zip5 mRNA remains associated with polysomes, while the protein is internalized and degraded in enterocytes, acinar cells and endoderm cells. After zinc-gavage, ZIP5 is rapidly resynthesized and targeted to the basolateral membranes of these cell types.

The mouse acrodermatitis enteropathica gene Slc39a4 (Zip4) is essential for early development and heterozygosity causes hypersensitivity to zinc deficiency.

The human Zip4 gene (Slc39a4) is mutated in the rare recessive genetic disorder of zinc metabolism acrodermatitis enteropathica, but the physiological functions of Zip4 are not well understood. Herein we demonstrate that homozygous Zip4-knockout mouse embryos die during early morphogenesis and heterozygous offspring are significantly underrepresented. At mid-gestation, an array of developmental defects including exencephalia, anophthalmia and severe growth retardation were noted in heterozygous embryos, and at weaning, many (63/280) heterozygous offspring were hydrocephalic, growth retarded and missing one or both eyes. Maternal dietary zinc deficiency during pregnancy exacerbated these effects, whereas zinc excess ameliorated these effects and protected embryonic development of heterozygotes but failed to rescue homozygous embryos. Heterozygous Zip4 embryos were not underrepresented in litters from wild-type mothers, but were approximately 10 times more likely to develop abnormally than were their wild-type littermates during zinc deficiency. Thus, both embryonic and maternal Zip4 gene expressions are critical for proper zinc homeostasis. These studies suggest that heterozygous mutations in the acrodermatitis gene Zip4 may be associated with a wider range of developmental defects than was previously appreciated, particularly when dietary zinc is limiting.

Differential progression of neonatal diethylstilbestrol-induced disruption of the hamster testis and seminal vesicle.

The synthetic estrogen diethylstilbestrol (DES) is now recognized as the prototypical endocrine disruptor. Using a hamster experimental system, we performed a detailed temporal assessment of how neonatal DES-induced disruption progresses in the testis compared to the seminal vesicle. Both morphological and Western blot analyses confirmed that neonatal DES exposure alters androgen responsiveness in the male hamster reproductive tract. We also determined that the disruption phenomenon in the male hamster is manifest much earlier in the seminal vesicle than in the testis and that testis disruption often occurs differently between the pair of organs in a given animal. In the neonatally DES-exposed seminal vesicle, histopathological effects included: (1) general atrophy, (2) lack of exocrine products, (3) epithelial dysplasia, (4) altered organization of stromal cells and extracellular matrix, and (5) striking infiltration with polymorphonuclear leukocytes. Also, the morphological disruption phenomenon in the seminal vesicle was accompanied by a range of up-regulation and down-regulation responses in the whole organ levels of various proteins.