Phenotypic characterization of epiphycan-deficient and epiphycan/biglycan double-deficient mice.

OBJECTIVE: To characterize the in vivo role epiphycan (Epn) has in cartilage development and/or maintenance. METHODS: Epn-deficient mice were generated by disrupting the Epn gene in mouse embryonic stem cells. Epn/biglycan (Bgn) double-deficient mice were produced by crossing Epn-deficient mice with Bgn-deficient mice. Whole knee joint histological sections were stained using van Gieson or Fast green/Safranin-O to analyze collagen or proteoglycan content, respectively. Microarray analysis was performed to detect gene expression changes within knee joints. RESULTS: Epn-deficient and Epn/Bgn double-deficient mice appeared normal at birth. No significant difference in body weight or femur length was detected in any animal at 1 month of age. However, 9-month Epn/Bgn double-deficient mice were significantly lighter and had shorter femurs than wild type mice, regardless of gender. Male Epn-deficient mice also had significantly shorter femurs than wild type mice at 9 months. Most of the deficient animals developed osteoarthritis (OA) with age; the onset of OA was observed earliest in Epn/Bgn double-deficient mice. Message RNA isolated from Epn/Bgn double-deficient knee joints displayed increased matrix protein expression compared with wild type mice, including other small leucine-rich proteoglycan (SLRP) members such as asporin, fibromodulin and lumican. CONCLUSION: Similar to other previously studied SLRPs, EPN plays an important role in maintaining joint integrity. However, the severity of the OA phenotype in the Epn/Bgn double-deficient mouse suggests a synergy between these two proteins. These data are the first to show a genetic interaction involving class I and class III SLRPs in vivo.

Regulation of longevity and stress resistance by Sch9 in yeast.

The protein kinase Akt/protein kinase B (PKB) is implicated in insulin signaling in mammals and functions in a pathway that regulates longevity and stress resistance in Caenorhabditis elegans. We screened for long-lived mutants in nondividing yeast Saccharomyces cerevisiae and identified mutations in adenylate cyclase and SCH9, which is homologous to Akt/PKB, that increase resistance to oxidants and extend life-span by up to threefold. Stress-resistance transcription factors Msn2/Msn4 and protein kinase Rim15 were required for this life-span extension. These results indicate that longevity is associated with increased investment in maintenance and show that highly conserved genes play similar roles in life-span regulation in S. cerevisiae and higher eukaryotes.

A selective genetic analysis of the Syracuse high- and low-avoidance (SHA/Bru and SLA/Bru) strains of rats (Rattus norvegicus).

Selective breeding of Long-Evans rats for good and poor avoidance learning in a two-way shuttle box resulted in the Syracuse strains that differ markedly in the selected phenotypes. These phenotypes have many associated traits, five of which are studied here: emotionality (open-field defecation), Pavlovian fear conditioning (CER suppression), passive avoidance training (punishment), size (weight) of the adrenal glands and adrenal concentration of corticosterone. Specifically, animals of the low-avoidance strain are more emotional, show greater fear conditioning, exhibit faster passive avoidance learning, and have larger adrenal glands in which adrenal corticosterone levels are lower than those of the high-avoidance strain. A reciprocal dihybrid cross of the two strains produced F1 hybrids, which were used to produce the segregating second filial and high and low backcross generations from which animals displaying the extreme high- and low-avoidance phenotypes were selected for study of the associated traits. Measurement of the five traits in these high and low phenotypic animals indicated that all five remain significantly associated with the avoidance phenotypes, in the expected direction, and comparably in all three segregating generations. The results indicate that the hypothesis of a major gene controlling avoidance learning must be rejected and that the few (2-3) genetic units thought to be involved may be closely linked to those that mediate these five associated characters, or express all five pleiotropically.

The effects of extended training and acute administration of an anxiolytic on avoidance learning and intertrial responding in the Syracuse strains of rats.

Male and female animals of the SHA/Bru and SLA/Bru strains of rats were given extended two-way avoidance training in the shuttle box at the rate of 30 trials per day for 11 days. SLA/Bru animals increased their avoidance responses (AVRs) from approximately 10 to roughly 25%, whereas animals of the SHA/Bru strain remained unchanged at approximately 100% AVRs. SHA/Bru animals made a number of intertrial responses (ITRs) early in the experiment; these declined after about 3 days to the low level made by SLA/Bru animals. Chlordiazepoxide (CDP) had no effect on AVRs in animals of either strain, and had no effect on ITRs made by animals of the SHA/Bru strain, but increased ITRs, in a dose-dependent way, in animals of the SLA/Bru strain. These results are interpreted in terms of the well-established genetic difference in emotional reactivity between animals of the two strains and in terms of genetically determined differences in sensitivity to anxiolytic drugs such as CDP.

Effect of ligand conformation on melanoma cell alpha3beta1 integrin-mediated signal transduction events: implications for a collagen structural modulation mechanism of tumor cell invasion.

The importance of three-dimensional interactions between receptors with their respective ligands has been extensively explored during the binding process, but considerably less so for postbinding events such as induction of signaling pathways. Tumor cell receptor association with basement membrane proteins is believed to facilitate the metastatic process. Melanoma and ovarian carcinoma cells have been shown to utilize the alpha3beta1 integrin to bind to models of the alpha1(IV)531-543 sequence from basement membrane (type IV) collagen [Miles, A. J., et al. (1994) J. Biol. Chem. 269, 30939-30945; Miles, A. J., et al. (1995) J. Biol. Chem. 270, 29047-29050]. In the present study, the effects of ligand three-dimensional structure on possible signal transduction pathways induced by alpha3beta1 integrin binding have been evaluated. Human melanoma cell binding to type IV collagen resulted in Tyr phosphorylation of p125(FAK), consistent with prior studies correlating beta1 integrin subunit binding to collagen and p125(FAK) Tyr phosphorylation. Cross-linking of an anti-alpha3 integrin subunit monoclonal antibody also induced p125(FAK) Tyr phosphorylation. Incubation of melanoma cells with single-stranded or triple-helical peptide models of alpha1(IV)531-543 induced Tyr phosphorylation of intracellular proteins. Immunoprecipitation analysis identified one of these proteins as pp125(FAK). Induction of p125(FAK) Tyr phosphorylation was enhanced and the time of induction was shortened when the ligand was used in triple-helical conformation. Subsequent clustering of either the single-stranded or the triple-helical ligand also increased the level of p125(FAK) phosphorylation compared to unclustered ligand. The clustered triple-helical peptide ligand induced more rapid paxillin Tyr phosphorylation than the single-stranded ligand. In addition, the induction of activated proteases was found to be more rapid due to ligand triple helicity. Overall, these studies have shown that (i) a model of an isolated sequence from type IV collagen, alpha1(IV)531-543, can induce alpha3beta1 integrin-mediated signal transduction in melanoma cells and (ii) ligand conformation (secondary, tertiary, and/or quaternary structure) can directly influence several alpha3beta1 integrin-mediated signal transduction events. The effects of ligand conformation suggest that a "collagen structural modulation" mechanism may exist for tumor cell invasion, whereby triple-helical collagen promotes cell binding and induction of signal transduction, subsequently leading to collagen dissolution by proteases, decreased signal transduction, and enhanced tumor cell motility.