The molecular mechanisms that enable multicellular organisms to sense and modulate their responses to hyperosmotic environments are poorly understood. Here, we employ Caenorhabditis elegans to characterize the response of a multicellular organism to osmotic stress and establish a genetic screen to isolate mutants that are osmotic stress resistant (OSR). In this study, we describe the cloning of a novel gene, osr-1, and demonstrate that it regulates osmosensation, adaptation, and survival in hyperosmotic environments. Whereas wild-type animals exposed to hyperosmotic conditions rapidly lose body volume, motility, and viability, osr-1(rm1) mutant animals maintain normal body volume, motility, and viability even upon chronic exposures to high osmolarity environments. In addition, osr-1(rm1) animals are specifically resistant to osmotic stress and are distinct from previously characterized osmotic avoidance defective (OSM) and general stress resistance age-1(hx546) mutants. OSR-1 is expressed in the hypodermis and intestine, and expression of OSR-1 in hypodermal cells rescues the osr-1(rm1) phenotypes. Genetic epistasis analysis indicates that OSR-1 regulates survival under osmotic stress via CaMKII and a conserved p38 MAP kinase signaling cascade and regulates osmotic avoidance and resistance to acute dehydration likely by distinct mechanisms. We suggest that OSR-1 plays a central role in integrating stress detection and adaptation responses by invoking multiple signaling pathways to promote survival under hyperosmotic environments.
WATER loss, due to conditions of high salinity and drought, affects growth and development of multiple organisms in their natural habitats. Cellular structure and protein function are highly dependent upon aqueous environments, and an imbalance in water homeostasis can cause irreversible damage with detrimental effects on organisms. To protect themselves against dehydration, diverse organisms (e.g., bacteria, fungi, tardigrades, rotifers, nematodes, and desert resurrection plants) have developed survival strategies to detect and withstand cellular dehydration in hyperosmotic environments (Potts 1994; Clegg 2001; Crowe 2002). The synthesis and accumulation of compatible osmolytes (glycerol, trehalose, sucrose, and myo-inositol) is a universal osmoadaptive response of diverse organisms to cellular dehydration (Yanceyet al. 1982) and is suggested to stabilize membranes and native proteins and regulate cell volume (Croweet al. 1984; Welch and Brown 1996).
The eukaryotic response to osmotic stress has been extensively investigated in the budding yeast, Saccharomyces cerevisiae. In yeast, perturbations in the osmotic environment are detected by a two-component system (SLN1-YPD1-SSK1; Maedaet al. 1994; Posaset al. 1996) and other sensors, such as SHO1 (Maedaet al. 1995) and MSB2 (O'Rourke and Herskowitz 2002; O'Rourkeet al. 2002), that regulate activation of the osmoadaptive HOG/p38 pathway (Brewsteret al. 1993) and glycerol biosynthesis (Hohmann 2002).
In contrast to unicellular organisms such as yeast, much less is known about sensors, signal transduction pathways, and mechanisms that constitute the response to high osmolarity in multicellular organisms. In mammals, osmotic stress results in activation of an adaptive response that involves three different mitogen-activated protein (MAP) kinases (SAPKs), p38, JNK, and ERK5 (de Nadalet al. 2002). A novel osmosensing scaffold protein has been recently discovered and shown to activate the mammalian p38 MAPK pathway in osmotic stress conditions, in a manner strikingly similar to that seen in yeast (Uhliket al. 2003). Yet another osmosensor is the VR-OAC receptor (TRPV4), which has been suggested to have a role in sensing systemic osmotic and hydrostatic pressure (Liedtkeet al. 2000).
The nematode Caenorhabditis elegans has emerged as a powerful genetic model for studying osmosensation in multicellular organisms (Culotti and Russell 1978; Bargmannet al. 1990). In addition to adaptive responses (as described above for mammals and yeast), C. elegans worms have also developed mechanisms by which they avoid hyperosmotic environments. Mutants defective in sensing high osmolarity (OSM) have been described (Culotti and Russell 1978; Colbertet al. 1997; Hartet al. 1999). Unlike wild-type animals, OSM mutants fail to avoid environments of high osmolarity, due at least partly to defective function of the polymodal osmosensory ASH neurons (Colbertet al. 1997; Hartet al. 1999). However, the entire complement of molecular mechanisms by which multicellular organisms integrate responses that lead to detection, adaptation, and survival under osmotic stress remain poorly understood.
To address these issues, we have employed C. elegans as a model system and characterized its response to hyperosmotic environments. Here we report on the cloning and characterization of a novel gene, osr-1, and show that in C. elegans, OSR-1 regulates multiple aspects of the osmotic stress physiology via distinct genetic pathways to promote survival under hyperosmotic environments.
MATERIALS AND METHODS
Strains and culture conditions: C. elegans worms were cultured at 22° under standard growth conditions (Brenner 1974). Strains used in this study were Bristol strain N2 (wild-type), osr-1 (rm1), unc-73(e936);dpy-5(e61), age-1(hx546), daf-16(mgdf50), osm-9 (ky10), ocr-2(ak47), osm-10(n1052), unc-43(n1186), sek-1(km4), nsy-1 (ky397), pmk-3(ok169), jnk-1(jk7), jkk-1(km2), and mek-1(ks54).
Genetic screens for osmotic stress resistant mutants: Standard EMS mutagenesis (Brenner 1974) was performed on N2 strain hermaphrodites. The F2 generation (∼20,000 haploid genomes) was screened for mutants that maintained normal swimming behavior on NGM agar plates containing 500 mm NaCl (high-salt plates) for >15 min (acute motility assay). Putative mutants that showed normal motility in this assay were isolated for further analysis.
Mapping and cloning of osr-1: Standard mapping procedures (Brenner 1974) were used to map osr-1 to chromosome I. We used three-factor mapping to identify osr-1 on –0.2 map units (MU) between dpy-5 and unc-73. Individual cosmids from the identified genetic interval were injected into osr-1(rm1) animals at 40 ng/μl with 100 ng/μl myo-2::gfp plasmid as a co-injection marker. Rescue experiments were performed with at least three independent transgenic lines.
Behavioral and stress resistance assays: Worms were placed on high-salt plates containing 500 mm NaCl and scored for their motility over a period of 10 min (acute motility assay) and after 1, 5, and 12 hr (chronic adaptation assay). Osmotic stress survival assays were performed on the high-salt plates seeded with Escherichia coli (OP50). To score for viability, worms were collected from the salt plates using a recovery buffer (300 mm NaCl in M9), transferred to regular NGM plates (50 mm NaCl), and allowed to recover overnight before scoring for viability. Osmotic avoidance behavior (OSM) was quantified as the percentage of worms that crossed a 2-cm ring of 4 m NaCl or 8 m fructose on an NGM agar plate, within 5 min. Nose touch response, 1-octanol repellent assays (Hartet al. 1999), and heat (Lithgowet al. 1995) and oxidative stress (Leeet al. 2003) experiments were performed as described previously. Statistical significance between mutants in the behavioral and stress assays was determined using the two-tailed t-test and by one-way ANOVA test.
Molecular biology: The functional osr-1::gfp operon construct and the tissue-specific expression constructs were generated using the Gateway system (Invitrogen, Carlsbad, CA; Walhoutet al. 2000). The rescuing fragment (pASRM1) or the heterologous promoter::osr-1 cDNA was inserted into a pEntry vector containing the SL2-gfp operon cassette. The promoters used were dpy-7 (hypodermal), vha-6 (intestine; Wanget al. 2002), and F25B3.3 (pan-neuronal; Altun-Gultekinet al. 2001). The full transcription unit of osr-1 was determined using a partial expressed sequence tag (EST; yk563c9) obtained from Yuji Kohara and a SMART RACE cDNA kit (CLONTECH, Palo Alto, CA). Further details of plasmid construction are available on request.
RNAi experiments: RNAi experiments were performed using a previously described feeding method (Timmonset al. 2001). For osr-1 RNAi, pAS1 was constructed by subcloning the osr-1 cDNA (1932 bp) into the L4440 vector. For pmk-1 RNAi, we used the pDK177 plasmid (Kimet al. 2002). Eggs were hatched on E. coli (HT115) carrying the L4440 expression vector (control groups), HT115 carrying pDK177 plasmid (pmk-1), or the plasmid pAS1 (osr-1) and allowed to grow for 3 days (adult stage). For knockdown of both pmk-1 and osr-1, eggs were hatched on plates containing equal amounts of bacteria expressing dsRNA for both pmk-1 and osr-1. Animals that were grown on the RNAi plates were exposed to 500 mm NaCl for 24 hr and scored for viability as mentioned above. Efficiency of the feeding RNAi was confirmed using the onestep RT-PCR kit (Invitrogen).
To establish a metazoan model system for osmotic stress signaling, we characterized the organismal response of C. elegans to hyperosmotic stress. When wild-type (N2 strain) animals were exposed to acute osmotic stress (10 min, 500 mm NaCl), we observed a complete loss of motility and reduction in body volume of the worms (Figures 1, A, B, C, D, and 2A). Remarkably, upon transfer to normal growth medium (50 mm NaCl), these animals recovered their original body size and regained normal motility within minutes (Figure 1, E, F, G). However, prolonged exposure of N2 worms to high salt concentrations severely affects motility and viability. After 5 hr of exposure to 500 mm NaCl, most N2 animals were immotile and those that were motile (43%; Figure 2B), exhibited a sluggish swimming behavior. This prolonged exposure to high osmotic stress was eventually lethal as after 24 hr 87% of the wild-type animals died (Figure 2C).
To identify genes mediating the osmotic stress responses in C. elegans, we designed a genetic screen to isolate mutants that are osmotic stress resistant (OSR) at 500 mm NaCl. Mutants that maintained normal motility on the high-salt plates for 15 min were isolated for further analysis. Among the 104 isolated mutant strains, we have identified four different complementation groups: osr-1, osr-2, osr-3, and osr-4. In this study we report on the cloning and characterization of osr-1.
osr-1(rm1) mutants are specifically resistant to osmotic stress: We found that osr-1(rm1) animals have an enhanced ability to resist hyperosmotic stress (Figures 1 and 2). In contrast to N2 animals, osr-1(rm1) animals exhibit normal swimming behavior (Figure 1, H, I, J, K) when exposed to a transient osmotic stress (10 min, 500 mm NaCl) and remain viable (88%) even after 24 hr (Figure 2C). osr-1(rm1) animals are also resistant to osmotic stress caused by sorbitol, glucose, sucrose, and KCl, demonstrating that osr-1(rm1) animals have a general resistance to hyperosmotic stress (data not shown).
To determine whether the osr-1(rm1) mutation is specific to osmotic stress or confers resistance to multiple stresses, as is observed for long-lived mutants of the insulin-like signaling (ILS) pathway (Kenyonet al. 1993; Larsen 1993; Dormanet al. 1995; Lithgowet al. 1995; Murakami and Johnson 1996; Kimuraet al. 1997; Tissenbaum and Ruvkun 1998), we tested for the ability of osr-1(rm1) animals to survive heat-shock (35°) or oxidative stress (300 mm paraquat; Figure 2, D and E). In contrast to age-1(hx546) animals, osr-1(rm1) animals were sensitive to both stresses (P < 0.05). Moreover, osr-1(rm1) animals did not exhibit other phenotypes associated with general stress resistance as seen in other ILS pathway mutants, such as extended life span or constitutive dauer formation (data not shown). Thus, osr-1(rm1) animals are a new class of mutants that are resistant specifically to osmotic stress.
OSR-1 and AGE-1 have critical roles in resistance to chronic osmotic stress: We explored the possibility that the general stress resistant mutant, age-1(hx546), might be also resistant to osmotic stress. However, as shown in Figure 2A, exposure of age-1(hx546) animals to 500 mm NaCl caused a complete loss of motility and reduction in body volume within 11 min, similar to wild-type animals. This demonstrates that age-1(hx546) animals are not preadapted to acute osmotic stress like osr-1(rm1) animals (Figure 2A). In addition, and in contrast to osr-1 (rm1) animals, age-1(hx546) animals have a wild-type phenotype for osmotic avoidance (Figure 2F). Interestingly, age-1(hx546) animals exhibited motility and viability similar to that of the osr-1(rm1) animals upon prolonged exposures to high osmotic environments (Figure 2, B and C). These results indicate that although AGE-1 does not regulate osmotic avoidance or resistance to acute osmotic stress, both OSR-1 and AGE-1 regulate survival under conditions of prolonged hyperosmotic stress.
osr-1(rm1) animals display an osmotic avoidance abnormality phenotype: The observed osmotolerance of osr-1(rm1) animals led us to investigate whether OSR-1 is involved in detection of high osmolarity environments as was described previously for osmotic avoidance defective (OSM) mutants (Culotti and Russell 1978; Bargmannet al. 1990). osr-1(rm1) animals exhibited an osmotic avoidance abnormality phenotype as they fail to avoid regions of 4 m NaCl or 8 m fructose (Figure 2F and data not shown). The osmotic avoidance defect of osr-1(rm1) animals is quantitatively similar to that seen in the previously characterized osm mutants, such as osm-9(ky10) (Colbertet al. 1997), osm-10(n1052) (Hartet al. 1999) and ocr-2(ak47) (Tobinet al. 2002).
Osmosensation in C. elegans is dependent on the integrity of osmosensory organs (amphids and phasmids) and function of the ASH neurons; the defective osmotic avoidance phenotype in osm-9(ky10), osm-10(n1052), and ocr-2(ak47) has been ascribed to impaired function of the ASH neurons (Colbertet al. 1997; Hartet al. 1999; Tobinet al. 2002). On the basis of lipophilic dye staining, we could not detect any morphological abnormalities in the osmosensory organs (amphids and phasmids) of osr-1(rm1) animals (A. Solomon and R. Morimoto, unpublished results). In addition, osr-1(rm1) animals have normal responses to nose touch and the repellent, 1-octanol (data not shown), which are also mediated by the polymodal ASH neurons via distinct signaling pathways (Kaplan and Horvitz 1993; Hartet al. 1999). Although we cannot rule out that the ASH neurons are not impaired for osmosensation in osr-1(rm1) animals, our results demonstrate that at least the mechanosensory and chemosensory responses mediated by the same neurons are normal in these animals.
We have also found that, unlike osr-1(rm1) animals, other osmotic avoidance defective mutants, osm-9(ky10), ocr-2(ak47), and osm-10(n1052), are not resistant to either acute or chronic osmotic stress. Also, none of these mutations affect osmotic stress tolerance in OSR-1 deficient animals (data not shown). This demonstrates that the osmosensory pathway defined by OSM-9, OCR-2, and OSM-10 does not mediate acute or chronic resistance to osmotic stress, and that the hyperresistance of osr-1(rm1) animals is independent of the pathways regulated by the OSM genes tested by us.
Positional cloning, sequence analysis, and expression patterns of osr-1: osr-1 was cloned using standard two- and three-factor mapping and transformation rescue approaches (Figure 3A). Two cosmids, C32E12 and F56A3, with an overlapping region of 11 kb gave a full rescue of the osr-1(rm1) phenotypes. In this overlapping region, we identified a 4-kb minimal rescuing fragment (pASRM1) containing a single predicted gene, C32E12.3, that completely rescued all the osr-1(rm1) phenotypes. We found in osr-1(rm1) worms a G-to-A mutation at a predicted splice acceptor site in the transcription unit of C32E12.3 (Figure 3B). Introduction of this mutation in pASRM1 completely eliminated its ability to rescue the osr-1(rm1) animals' phenotypes. In addition, RNAi of osr-1 in wild-type animals results in osr-1(rm1)-like phenotypes. We conclude that the osr-1 gene corresponds to C32E12.3.
The osr-1 gene encodes a predicted protein of 643 amino acids (Figure 3C) and lacks any domains that would suggest a specific molecular function. Proteins with significant similarity to OSR-1 were found only in the genomes of C. briggsae (86% overall identity to CBE 12837) and the parasitic nematode Strongyloides ratti (48% identity in partial EST kt82b03.y1).
To identify the tissues in which OSR-1 is expressed and functions, we placed the rescuing fragment, pASRM1, in an artificial operon with green fluorescent protein (GFP). This construct fully rescued the osr-1(rm1) phenotypes and GFP fluorescence was seen in the hypodermis and intestine during all developmental stages (Figure 4, A and B). To test which tissues have functional importance, we performed rescue experiments using heterologous promoter constructs including hypodermal (Pdpy-7::osr-1::gfp), intestinal (Pvha-6::osr-1::gfp; Wanget al. 2002) and pan-neuronal (PF25B3.3::osr-1::gfp) promoters (Altun-Gultekinet al. 2001). We found that only hypodermal expression of osr-1 cDNA rescues the osr-1(rm1) phenotypes (Figure 4C).
OSR-1 genetically interacts with CaMKII and a conserved p38 MAPK cascade: To understand the mechanisms by which OSR-1 regulates osmotic stress responses in C. elegans, we tested the potential role(s) of the C. elegans homologs of HOG/p38 MAPK signaling pathway (Brewsteret al. 1993) in mediating phenotypes seen in osr-1(rm1) animals. In C. elegans, there are multiple MAPK encoding genes implicated in development and resistance to abiotic and biotic stresses (Kurz and Ewbank 2003). The Esp pathway (NSY-1/MAPKKK → SEK-1/MAPKK → PMK-1/p38/MAPK), has been shown to function in pathogen resistance (Kimet al. 2002), and the neuronal symmetry (Nsy) pathway (UNC-43/CaMKII → NSY-1 SEK-1 → unknown MAPK) mediates asymmetric neuronal cell fate in AWC sensory neurons (Sagastiet al. 2001; Tanaka-Hinoet al. 2002).
Downregulation of pmk-1/p38 in osr-1(rm1) worms by feeding RNAi significantly reduced their ability to survive chronic osmotic stress (Figure 5A; P < 0.05). Similarly, null mutations, of NSY-1 and SEK-1, completely suppressed the ability of osr-1 deficient animals to survive osmotic stress (Figure 5A). These results led us to investigate the potential relationship between OSR-1 and the most upstream component of the Nsy pathway, UNC-43. As shown in Figure 5A, UNC-43, the only identified CaMKII in C. elegans (Reineret al. 1999), was also required for viability under chronic osmotic stress.
The interactions between OSR-1 and PMK-1/p38 appear to be specific because OSR-1 does not interact with two other C. elegans p38 homologs (Bermanet al. 2001), PMK-2 and PMK-3 (A. Solomon and R. Morimoto, unpublished results). Mutations in genes that mediate heavy metal resistance, including JNK-1, JKK-1 (Villanuevaet al. 2001), and MEK-1 (Kogaet al. 2000), also do not suppress the OSR-1 phenotypes (Figure 5A and data not shown). Because the forkhead transcription factor, DAF-16, promotes general stress resistance and longevity (Murakami and Johnson 1996; Ogget al. 1997; Henderson and Johnson 2001), we also tested for the effect of osr-1 RNAi in a null mutant of daf-16 (Ogget al. 1997). As shown in Figure 5A, the ability of OSR-1 to regulate survival in hyperosmotic environments is not dependent on DAF-16. Taken together, our results indicate that OSR-1 functions upstream of or in parallel to UNC-43/CaMKII and a conserved p38 pathway (NSY-1 → SEK-1 → PMK-1) in promoting survival under chronic osmotic stress (Figure 5B).
We also investigated the role of the PMK-1/p38 pathway in regulating resistance to acute dehydration and osmotic avoidance defects seen in osr-1(rm1) animals. Interestingly, we find that osr-1(rm1), pmk-1(RNAi) (N = 175); nsy-1(ky379), osr-1(RNAi) (N = 1259); and sek-1 (km4), osr-1(RNAi) (N = 376) animals remained resistant to the acute effects of osmotic stress by maintaining normal swimming behavior (100% motile animals after 10 min). In addition, these animals retain the OSM-like phenotype, as ∼57% of these animals cross the 4 m NaCl ring (data not shown), similar to the phenotypes seen in osm-9(ky10) and osr-1(rm1) animals (P > 0.05; see Figure 2F).
In this study, we have used the osmotic stress response of C. elegans as a model for stress signaling in a multicellular organism. We established a novel genetic screen to identify genes that are critical regulators of the osmotic stress response in a whole animal and report the cloning and characterization of a novel gene, osr-1. On the basis of the phenotypes of osr-1(rm1) mutant animals, we suggest that OSR-1 regulates the ability of C. elegans to (i) detect and avoid high osmolarity, (ii) resist acute exposures to osmotic stress, and (iii) maintain viability upon prolonged exposure to osmotic stress. osr-1(rm1) animals are not resistant to heat and oxidative stress and do not display phenotypes associated with mutants of the ILS pathway, such as extended life span and constitutive dauer formation. Thus, OSR-1 specifically regulates osmotic stress responses in C. elegans.
How might OSR-1 regulate the different osmotic stress-induced responses in C. elegans? Osmotic upshifts in multiple organisms cause perturbations in membrane-dependent transport processes and activation of protein kinase cascades and lead to the accumulation of osmolytes, such as glycerol (Hohmann 2002). Our genetic epistasis analysis demonstrates that OSR-1 regulates a conserved p38 MAPK pathway. This pathway has been shown to induce production of glycerol in yeast cells during osmotic upshifts (Hohmann 2002). We also found that osr-1(rm1) animals accumulate high levels of glycerol (1455 ± 26 nmol of glycerol per milligram of protein; A. Solomon and R. Morimoto, unpublished results) even under normal growth conditions, while wild-type animals have undetectable levels. Since accumulation of glycerol has been previously implicated in osmotic stress tolerance in yeast and different species of nematodes including C. elegans (Hohmann 2002; Qiu and Bedding 2002; Lamitinaet al. 2003), we hypothesize that activation of the p38 MAPK cascade and glycerol accumulation might be sufficient to explain the osmotic stress tolerance of osr-1(rm1) animals.
We demonstrate that pmk-1 downregulation suppresses partially (>50%) the chronic osmotic stress resistance phenotype of osr-1(rm-1) animals, and it does not affect resistance to acute dehydration and osmotic avoidance defects in these animals. These findings raise the intriguing possibility that OSR-1 regulates these responses via other, yet to be identified, mechanisms (Figure 5B). This is also supported by our observations that null mutants of the upstream component of the p38 pathway, SEK-1 and NSY-1, also do not suppress the osmotic avoidance or acute dehydration phenotypes of osr-1 deficient animals.
For the observed acute resistance to osmotic stress seen in osr-1(rm1) animals, we suggest that it might be related to alterations in cuticle structure, as we find that mutations in cuticle collagen genes, DPY-10 and DPY-2 (Levyet al. 1993), result in an osr-1(rm1)-like resistance phenotype in acute and chronic osmotic stress conditions (A. Solomon and R. Morimoto, unpublished results). We are currently investigating potential genetic interactions between cuticle component genes and osr-1 to test this idea further.
We additionally found that the age-1(hx546) mutants are also resistant to the effects of chronic exposure to osmotic stress but, unlike osr-1(rm1) animals, they are not osmotic avoidance defective or resistant to acute osmotic stress. We are currently investigating the possibility that OSR-1 and AGE-1 regulate resistance to chronic osmotic stress through overlapping mechanisms. In our studies, we isolated an additional 104 OSR-like mutants and have so far characterized four different complementation groups. Understanding the interrelationships among these genes, age-1, and osr-1-dependent pathways will reveal in greater detail how OSR-1 functions to regulate osmotic stress responses in C. elegans.
Osmotic avoidance defective phenotypes have been previously described for the OSM class of mutants (Culotti and Russell 1978). Mutations in OSM-9, OCR-2, and OSM-10 influence the function of the osmosensory ASH neurons (Colbertet al. 1997; Hartet al. 1999; Tobinet al. 2002). However, we have found no evidence for impaired ASH neuron function in osr-1(rm1) animals. Also, osm-9(ky10), ocr-2(ak47), and osm-10(n1052) mutant animals are not resistant to either acute or chronic osmotic stress; moreover, none of these mutations suppress the osmotic stress resistance of osr-1 deficient animals (data not shown). These results demonstrate that the ASH neurons and the pathways mediated by the OSM genes (OSM-9, OSM-10, and OCR-2) play a limited role, if any, in the ability of OSR-1 to regulate resistance to osmotic stress.
Consistent with previous suggestions of an important role for the hypodermis in osmoregulation in C. elegans (Petalcorinet al. 1999), we found that only hypodermal expression of osr-1 cDNA rescues the osr-1(rm1) phenotypes. However, we cannot rule out the possibility that the hypodermal promoter (dpy-7) is not completely off in neurons or other tissues. Weak expression in the nervous system, for example, could indeed explain the partial rescue of the osmotic avoidance phenotype of osr-1(rm-1) animals.
As seen in yeast and mammals (Hanet al. 1994; de Nadalet al. 2002), the p38 MAPK signaling pathway has an important role in mediating osmotic stress tolerance in C. elegans. However, as is the case with other signaling pathways such as the RAS pathway, the upstream regulators of this conserved MAPK cascade may differ between metazoans and unicellular organisms like yeast. For C. elegans, we suggest that CaMKII regulates a p38 pathway in an OSR-1-dependent manner (Figure 5B); CaMKII has also been implicated in osmotic responses in mammalian kidney cells (Tokudaet al. 2002). In contrast, the HOG/p38 cascade in S. cerevisiae is regulated by a two-component system (SLN1-YPD1-SSK1; Maedaet al. 1994; Posaset al. 1996) and no orthologs of the two-component system have been identified yet in animals.
Our genetic data indicate that OSR-1 negatively regulates multiple osmotic stress responses in C. elegans probably via distinct mechanisms. We favor the interpretation that under normal conditions, OSR-1 functions to enable worms to avoid hyperosmotic environments. However, in hyperosmotic environments, OSR-1 functions are likely inhibited, which in turn activates distinct signaling pathways to promote adaptation and survival. Such a mechanism bears a striking resemblance to the reported inactivation of the response regulator, Ssk1, in the two-component system (SLN1-YPD1-SSK1) of S. cerevisiae during conditions of high osmolarity (Posaset al. 1996). We anticipate that further characterization of OSR-1 and the identification of its interacting partners and other osr-1-like genes will greatly facilitate the understanding of how metazoans integrate multiple responses and survive in hyperosmotic environments.
We thank the members of the Morimoto lab for their help and discussion and Jim Morley, Michelle Lee, Jason Merchant, Marta Camilot, Peter G. Czarnecki, and Luca Jahreiss for their advice and assistance in this project. Special thanks go to Heather Brignull and Anat Ben-Zvi for extensive comments and discussion on the manuscript and Sue Fox for her technical support. We appreciate the efforts of Alan Coulson for cosmids; Yuji Kohara for cDNA clones; Theresa Stiernagle and the Caenorhabditis Genetics Center for strains; and Mark Edgley, Kunihiro Matsumoto, Ding Xue, Mario deBono, Cathy Savage-Dunn, Kim Dennis, and Frederick M. Ausubel for strains and vectors. We also thank Danielle Thierry-Mieg from the transcriptom project for sharing information on 1F768. A.S. was a recipient of a postdoctoral fellowship from the Rothschild Foundation. G.J.B. is a recipient of a Burroughs Wellcome Fund Career Award in the Biomedical Sciences. R.I.M. is supported by grants from the National Institutes of Health (National Institute of General Medical Sciences) and the Huntington Disease Society Coalition for the Cure.
Communicating editor: B. J. Meyer
Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession no. AY360470.
- Received October 26, 2003.
- Accepted January 29, 2004.
- Copyright © 2004 by the Genetics Society of America