Erika Fröhli

Alpha B-crystallin expression in mouse NIH 3T3 fibroblasts: glucocorticoid responsiveness and involvement in thermal protection.

alpha B-crystallin, a major soluble protein of vertebrate eye lenses, is a small heat shock protein which transiently accumulates in response to heat shock and other kinds of stress in mouse NIH 3T3 fibroblasts. Ectopic expression of an alpha B-crystallin cDNA clone renders NIH 3T3 cells thermoresistant. alpha B-crystallin accumulates in response to the synthetic glucocorticoid hormone dexamethasone. Dexamethasone-treated NIH 3T3 cells become thermoresistant to the same extent as they accumulate alpha B-crystallin. A cell clone in which alpha B-crystallin is superinduced upon heat shock acquires augmented thermotolerance. Expression of the ras oncogene causes a rapid but transient accumulation of alpha B-crystallin within 1 day. Later, sustained ras oncogene expression suppresses the dexamethasone-mediated alpha B-crystallin accumulation. Thus, oncogenic transformation triggered by the ras oncogene interferes with hormone-mediated accumulation of alpha B-crystallin and concomitant acquisition of thermoresistance. Other known heat shock proteins do not accumulate in response to ectopic alpha B-crystallin expression or to dexamethasone treatment. These results indicate that alpha B-crystallin can protect NIH 3T3 fibroblasts from thermal shock.

Syndecan regulates cell migration and axon guidance in C.elegans

During nervous system development, axons that grow out simultaneously in the same extracellular environment are often sorted to different target destinations. As there is only a restricted set of guidance cues known, regulatory mechanisms are likely to play a crucial role in controlling cell migration and axonal pathfinding. Heparan sulfate proteoglycans (HSPGs) carry long chains of differentially modified sugar residues that have been proposed to encode specific information for nervous system development. Here, we show that the cell surface proteoglycan syndecan SDN-1 functions autonomously in neurons to control the neural migration and guidance choices of outgrowing axons. Epistasis analysis suggests that heparan sulfate (HS) attached to SDN-1 can regulate guidance signaling by the Slit/Robo pathway. Furthermore, SDN-1 acts in parallel with other HSPG core proteins whose HS side chains are modified by the C5-epimerase HSE-5, and/or the 2O-sulfotransferase HST-2, depending on the cellular context. Taken together, our experiments show that distinct HS modification patterns on SDN-1 are involved in regulating axon guidance and cell migration in C. elegans.

Alpha B crystallin accumulation is a specific response to Ha-ras and v-mos oncogene expression in mouse NIH 3T3 fibroblasts.

The conditional expression of the v-mos and Ha-ras(EJ) oncogenes in NIH 3T3 cells leads to the accumulation of a 23-kDa protein (p23) (R. Klemenz, S. Hoffmann, R. Jaggi, and A.-K. Werenskiold, Oncogene 4:799-803, 1989). We purified p23 to homogeneity and determined part of the amino acid sequence. The obtained sequence is identical with that of the eye lens protein alpha B crystallin. Northern (RNA) blot and Western immunoblot experiments were performed to demonstrate that alpha B crystallin mRNA and protein do indeed accumulate as a consequence of v-mos and Ha-ras oncogene expression. Comparison of cDNA clones obtained from the mRNA of eye lenses and of oncogene-expressing fibroblasts revealed identity between them. The major transcription initiation site of the alpha B crystallin gene in our experimental system was shown by primer extension experiments to be identical with the one used in eye epithelial cells. In addition, we identified a second minor initiation site 49 nucleotides further upstream. Serum growth factors did not stimulate alpha B crystallin expression in growth-arrested cells.

T1-deficient and T1-Fc-transgenic mice develop a normal protective Th2-type immune response following infection with Nippostrongylus brasiliensis.

The IL‐1 receptor‐related protein T1 is expressed on the surface of Th2, but not Th1 cells. Studies with anti‐T1 monoclonal antibodies have suggested that T1 is critical for development of normal Th2‐type responses. To elucidate the role of T1 in vivo, we generated T1‐deficient mice and a T1‐transgenic strain which secretes soluble T1‐Fc fusion protein into the serum. These were analyzed for the Th2 immune response induced by infection with the parasitic nematode Nippostrongylus brasiliensis. Although Th2 cytokine production by lymph node cells was similar in all groups of N. brasiliensis‐infected mice, a decrease in IL‐5 production by lung lymphocytes was detected in both T1‐deficient and T1‐Fc‐transgenic mice compared to control littermates. This difference in IL‐5 production did not influence blood eosinophilia, but recruitment of eosinophils into lung tissue, especially in T1‐Fc‐transgenic mice was slightly decreased. However, induction of all other immune parameters was normal and both T1‐deficient and T1‐Fc‐transgenic mice were able to clear the parasite infection within 12 days with kinetics similar to those in control mice. Therefore, in contrast to previous suggestions, we conclude that the T1 protein is not obligatory for normal development of Th2 immune responses.

The Caenorhabditis elegans homologue of the proto‐oncogene ect‐2 positively regulates RAS signalling during vulval development

Guanine nucleotide exchange factors (GEFs) regulate the activity of small GTP‐binding proteins in a variety of biological processes. We have identified a gain‐of‐function mutation in the Caenorhabditis elegans GEF ect‐2, the homologue of the mammalian ect2 proto‐oncogene that has an essential role during cytokinesis. Here, we report that, in addition to its known function during mitosis, ECT‐2 promotes the specification of the primary vulval cell fate by activating RAS/mitogen‐activated protein kinase (MAPK) signalling before the end of the S‐phase. Epistasis analysis indicates that ECT‐2 crosstalks to the canonical RAS/MAPK cascade upstream of the RAS GEF SOS‐1 by means of a RHO‐1 signalling pathway. Our results raise the possibility that the transforming activity of the mammalian ect‐2 oncogene could be due to hyperactivation of the RAS/MAPK pathway.

PTEN Negatively Regulates MAPK Signaling during Caenorhabditis elegans Vulval Development

Vulval development in Caenorhabditis elegans serves as an excellent model to examine the crosstalk between different conserved signaling pathways that are deregulated in human cancer. The concerted action of the RAS/MAPK, NOTCH, and WNT pathways determines an invariant pattern of cell fates in three vulval precursor cells. We have discovered a novel form of crosstalk between components of the Insulin and the RAS/MAPK pathways. The insulin receptor DAF-2 stimulates, while DAF-18 PTEN inhibits, RAS/MAPK signaling in the vulval precursor cells. Surprisingly, the inhibitory activity of DAF-18 PTEN on the RAS/MAPK pathway is partially independent of its PIP3 lipid phosphatase activity and does not involve further downstream components of the insulin pathway, such as AKT and DAF-16 FOXO. Genetic and biochemical analyses indicate that DAF-18 negatively regulates vulval induction by inhibiting MAPK activation. Thus, mutations in the PTEN tumor suppressor gene may result in the simultaneous hyper-activation of two oncogenic signaling pathways.

An in vivo EGF receptor localization screen in C. elegans Identifies the Ezrin homolog ERM-1 as a temporal regulator of signaling.

The subcellular localization of the epidermal growth factor receptor (EGFR) in polarized epithelial cells profoundly affects the activity of the intracellular signaling pathways activated after EGF ligand binding. Therefore, changes in EGFR localization and signaling are implicated in various human diseases, including different types of cancer. We have performed the first in vivo EGFR localization screen in an animal model by observing the expression of the EGFR ortholog LET-23 in the vulval epithelium of live C. elegans larvae. After systematically testing all genes known to produce an aberrant vulval phenotype, we have identified 81 genes regulating various aspects of EGFR localization and expression. In particular, we have found that ERM-1, the sole C. elegans Ezrin/Radixin/Moesin homolog, regulates EGFR localization and signaling in the vulval cells. ERM-1 interacts with the EGFR at the basolateral plasma membrane in a complex distinct from the previously identified LIN-2/LIN-7/LIN-10 receptor localization complex. We propose that ERM-1 binds to and sequesters basolateral LET-23 EGFR in an actin-rich inactive membrane compartment to restrict receptor mobility and signaling. In this manner, ERM-1 prevents the immediate activation of the entire pool of LET-23 EGFR and permits the generation of a long-lasting inductive signal. The regulation of receptor localization thus serves to fine-tune the temporal activation of intracellular signaling pathways.

Coordinated Lumen Contraction and Expansion during Vulval Tube Morphogenesis in Caenorhabditis elegans

Morphogenesis is a developmental phase during which cell fates are executed. Mechanical forces shaping individual cells play a key role during tissue morphogenesis. By investigating morphogenesis of the Caenorhabditis elegans hermaphrodite vulva, we show that the force-generating actomyosin network is differentially regulated by NOTCH and EGFR/RAS/MAPK signaling to shape the vulval tube. NOTCH signaling activates expression of the RHO kinase LET-502 in the secondary cell lineage through the ETS-family transcription factor LIN-1. LET-502 induces actomyosin-mediated contraction of the apical lumen in the secondary toroids, thereby generating a dorsal pushing force. In contrast, MAPK signaling in the primary lineage downregulates LET-502 RHO kinase expression to prevent toroid contraction and allow the gonadal anchor cell to expand the dorsal lumen of the primary toroids. The antagonistic action of the MAPK and NOTCH pathways thus controls vulval tube morphogenesis linking cell fate specification to morphogenesis.

LIN-39 and the EGFR/RAS/MAPK pathway regulate C. elegans vulval morphogenesis via the VAB-23 zinc finger protein

Morphogenesis represents a phase of development during which cell fates are executed. The conserved hox genes are key cell fate determinants during metazoan development, but their role in controlling organ morphogenesis is less understood. Here, we show that the C. elegans hox gene lin-39 regulates epidermal morphogenesis via its novel target, the essential zinc finger protein VAB-23. During the development of the vulva, the egg-laying organ of the hermaphrodite, the EGFR/RAS/MAPK signaling pathway activates, together with LIN-39 HOX, the expression of VAB-23 in the primary cell lineage to control the formation of the seven vulval toroids. VAB-23 regulates the formation of homotypic contacts between contralateral pairs of cells with the same sub-fates at the vulval midline by inducing smp-1 (semaphorin) transcription. In addition, VAB-23 prevents ectopic vulval cell fusions by negatively regulating expression of the fusogen eff-1. Thus, LIN-39 and the EGFR/RAS/MAPK signaling pathway, which specify cell fates earlier during vulval induction, continue to act during the subsequent phase of cell fate execution by regulating various aspects of epidermal morphogenesis. Vulval cell fate specification and execution are, therefore, tightly coupled processes.

Systemic Regulation of RAS/MAPK Signaling by the Serotonin Metabolite 5-HIAA

Human cancer is caused by the interplay of mutations in oncogenes and tumor suppressor genes and inherited variations in cancer susceptibility genes. While many of the tumor initiating mutations are well characterized, the effect of genetic background variation on disease onset and progression is less understood. We have used C. elegans genetics to identify genetic modifiers of the oncogenic RAS/MAPK signaling pathway. Quantitative trait locus analysis of two highly diverged C. elegans isolates combined with allele swapping experiments identified the polymorphic monoamine oxidase A (MAOA) gene amx-2 as a negative regulator of RAS/MAPK signaling. We further show that the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA), which is a product of MAOA catalysis, systemically inhibits RAS/MAPK signaling in different organs of C. elegans. Thus, MAOA activity sets a global threshold for MAPK activation by controlling 5-HIAA levels. To our knowledge, 5-HIAA is the first endogenous small molecule that acts as a systemic inhibitor of RAS/MAPK signaling.