Faculty Bibliography

RATIONALE: Shear stress (SS) has an established role in atherosclerotic plaque localization, but how it exerts its protective effect is not fully understood. OBJECTIVE: To test the hypothesis that SS may downregulate angiotensin type 1 receptors (AT(1)Rs). Angiotensin II has been shown to be proinflammatory and to promote atherosclerosis. METHODS AND RESULTS: Using immunohistochemistry, we found a pronounced expression of AT(1)R in the inner, atheroprone regions of the aortic arch of C57BL/6 and endothelial NO synthase-deficient (eNOS(-/-)) mice but not eNOS-overexpressing mice. In human umbilical vein endothelial cells (HUVECs), laminar SS (15 dyn/cm(2)) induced a biphasic decrease in AT(1)R protein expression characterized by a first reduction at 1 hour (31+/-4% of static control, P<0.01), partial recovery at 3 hours (65+/-9%), and a second more prolonged decline at 6, 12, and 24 hours (48+/-9%, 36+/-9%, 33+/-5%, respectively, P<0.05). One and 24 hours of SS significantly reduced fluorescent angiotensin binding compared to static HUVECs. Shear-induced downregulation of AT(1)R was abolished by treatment with protein kinase A and G inhibitors or N(G)-nitro-l-arginine methyl ester (L-NAME). Fittingly, stimulating static HUVECs with an NO donor decreased AT(1)R protein levels. RT-PCR revealed a significant (P<0.05) decrease of AT(1)R mRNA in HUVECs exposed to SS during 3 (6+/-2% of static control), 6 (4+/-1%), 12 (4+/-1%), and 24 hours (15+/-4%), suggesting a transcriptional downregulation of AT(1)R at length. Finally, angiotensin-induced vascular cell adhesion molecule was abated in HUVECs exposed to SS and in the outer aortic arch of mice. CONCLUSIONS: Our results demonstrate that SS may convey some of its atheroprotective effects through downregulation of AT(1)R in endothelial cells.

The Rnd proteins (Rnd1, Rnd2 and Rnd3/RhoE) form a distinct branch of the Rho family of small GTPases. Altered Rnd3 expression causes changes in cytoskeletal organization and cell cycle progression. Rnd3 functions to decrease RhoA activity, but how Rnd3 itself is regulated to cause these changes is still under investigation. Unlike other Rho family proteins, Rnd3 is regulated not by GTP/GDP cycling, but at the level of expression and by post-translational modifications such as prenylation and phosphorylation. We show in the present study that, upon PKC (protein kinase C) agonist stimulation, Rnd3 undergoes an electrophoretic mobility shift and its subcellular localization becomes enriched at internal membranes. These changes are blocked by inhibition of conventional PKC isoforms and do not occur in PKCalpha-null cells or to a non-phosphorylatable mutant of Rnd3. We further show that PKCalpha directly phosphorylates Rnd3 in an in vitro kinase assay. Additionally, we provide evidence that the phosphorylation status of Rnd3 has a direct effect on its ability to block signalling from the Rho-ROCK (Rho-kinase) pathway. These results identify an additional mechanism of regulation and provide clarification of how Rnd3 modulates Rho signalling to alter cytoskeletal organization

ABSTRACT: OBJECTIVE: Alzheimer's disease (AD) is a progressive neurodegenerative disease of the central nervous system (CNS). Recently, an increased interest in the role diet plays in the pathology of AD has resulted in a focus on the detrimental effects of diets high in cholesterol and fat and the beneficial effects of caloric restriction. The current study examines how dietary composition modulates cerebral amyloidosis and neuronal integrity in the TgCRND8 mouse model of AD. METHODS: From 4 wks until 18 wks of age, male and female TgCRND8 mice were maintained on one of four diets: (1) reference (regular) commercial chow; (2) high fat/low carbohydrate custom chow (60 kcal% fat/30 kcal% protein/10 kcal% carbohydrate); (3) high protein/low carbohydrate custom chow (60 kcal% protein/30 kcal% fat/10 kcal% carbohydrate); or (4) high carbohydrate/low fat custom chow (60 kcal% carbohydrate/30 kcal% protein/10 kcal% fat). At age 18 wks, mice were sacrificed, and brains studied for (a) wet weight; (b) solubilizable Abeta content by ELISA; (c) amyloid plaque burden; (d) stereologic analysis of selected hippocampal subregions. RESULTS: Animals receiving a high fat diet showed increased brain levels of solubilizable Abeta, although we detected no effect on plaque burden. Unexpectedly, brains of mice fed a high protein/low carbohydrate diet were 5% lower in weight than brains from all other mice. In an effort to identify regions that might link loss of brain mass to cognitive function, we studied neuronal density and volume in hippocampal subregions. Neuronal density and volume in the hippocampal CA3 region of TgCRND8 mice tended to be lower in TgCRND8 mice receiving the high protein/low carbohydrate diet than in those receiving the regular chow. Neuronal density and volume were preserved in CA1 and in the dentate gyrus. INTERPRETATION: Dissociation of Abeta changes from brain mass changes raises the possibility that diet plays a role not only in modulating amyloidosis but also in modulating neuronal vulnerability. However, in the absence of a study of the effects of a high protein/low carbohydrate diet on nontransgenic mice, one cannot be certain how much, if any, of the loss of brain mass exhibited by high protein/low carbohydrate diet-fed TgCRND8 mice was due to an interaction between cerebral amyloidosis and diet. Given the recent evidence that certain factors favor the maintenance of cognitive function in the face of substantial structural neuropathology, we propose that there might also exist factors that sensitize brain neurons to some forms of neurotoxicity, including, perhaps, amyloid neurotoxicity. Identification of these factors could help reconcile the poor clinicopathological correlation between cognitive status and structural neuropathology, including amyloid pathology

The c-abl proto-oncogene encodes a nonreceptor tyrosine kinase involved in many cellular processes, including signaling from growth factor and antigen receptors, remodeling the cytoskeleton, and responding to DNA damage and oxidative stress. Many downstream pathways are affected by c-Abl. Elevated c-Abl kinase activity can inhibit NF-kappaB activity by stabilizing the inhibitory protein IkappaB alpha, raising the possibility that c-Abl-deficient cells might have increased NF-kappaB activity. We examined the levels of NF-kappaB activity in primary mouse embryonic fibroblasts (MEFs) derived from wild-type and c-Abl knockout mice and found that the knockout MEFs indeed exhibited elevated NF-kappaB activity in response to stimulation as well as constitutively elevated NF-kappaB activity. Thus, endogenous c-Abl is a negative regulator of basal and inducible NF-kappaB activity. Examination of various points of NF-kappaB regulation revealed that unstimulated c-Abl knockout MEFs do not exhibit an increase in IkappaB alpha degradation, p65/RelA nuclear translocation, or DNA binding of NF-kappaB subunits. They do, however, show reduced levels of the histone deacetylase HDAC1, a negative regulator of basal NF-kappaB activity. Unstimulated c-Abl knockout MEFs are less responsive to induction of NF-kappaB activity by trichostatin A, an HDAC inhibitor, suggesting that c-Abl might play a role in the HDAC-mediated repression of basal NF-kappaB activity.

The role of clathrin adaptor proteins in sorting cargo in the biosynthetic and recycling routes is an area of intense research. In this issue, Delevoye et al. (2009. J. Cell Biol. doi:10.1083/jcb.200907122) show that a close interaction between the clathrin adaptor AP-1 and a kinesin motor KIF13A is essential for delivering melanogenic enzymes from recycling endosomes to nascent melanosomes and for organelle biogenesis.

The tafazzin gene encodes a phospholipid-lysophospholipid transacylase involved in cardiolipin metabolism, but it is not known why it forms multiple transcripts as a result of alternative splicing. Here we studied the intracellular localization, enzymatic activity, and metabolic function of four isoforms of human tafazzin and three isoforms of Drosophila tafazzin upon expression in different mammalian and insect systems. When expressed in HeLa cells, all isoforms were localized in mitochondria except for the B-form of Drosophila tafazzin, which was associated with multiple intracellular membranes. Among the human isoforms, only full-length tafazzin (FL) and tafazzin lacking exon 5 (Delta5) had transacylase activity, and only these two isoforms were able to restore a normal cardiolipin pattern, normal respiratory activity of mitochondria, and male fertility in tafazzin-deficient flies. Both FL and Delta5 were associated with large protein complexes in 293T cell mitochondria, but treatment with alkali and proteinase K suggested that the Delta5 isoform was more integrated into the hydrophobic core of the membrane than the FL isoform. Although all Drosophila isoforms showed transacylase activity in vitro, only the A-form supported cardiolipin remodeling in flies. The data suggest that humans express two mitochondrial isoenzymes of tafazzin that have similar transacylase activities but different membrane topologies. Furthermore, the data show that the expression of human tafazzin in flies creates cardiolipin with a Drosophila pattern, suggesting that the characteristic fatty acid profile of cardiolipin is not determined by the substrate specificity of tafazzin

Muscle-specific kinase (MuSK) is an essential receptor tyrosine kinase for the establishment and maintenance of the neuromuscular junction (NMJ). Activation of MuSK by agrin, a neuronally derived heparan-sulfate proteoglycan, and LRP4 (low-density lipoprotein receptor-related protein-4), the agrin receptor, leads to clustering of acetylcholine receptors on the postsynaptic side of the NMJ. The ectodomain of MuSK comprises three immunoglobulin-like domains and a cysteine-rich domain (Fz-CRD) related to those in Frizzled proteins, the receptors for Wnts. Here, we report the crystal structure of the MuSK Fz-CRD at 2.1 A resolution. The structure reveals a five-disulfide-bridged domain similar to CRDs of Frizzled proteins but with a divergent C-terminal region. An asymmetric dimer present in the crystal structure implicates surface hydrophobic residues that may function in homotypic or heterotypic interactions to mediate co-clustering of MuSK, rapsyn, and acetylcholine receptors at the NMJ

We describe a role for diacylglycerol in the activation of Ras and Rap1 at the phagosomal membrane. During phagocytosis, Ras density was similar on the surface and invaginating areas of the membrane, but activation was detectable only in the latter and in sealed phagosomes. Ras activation was associated with the recruitment of RasGRP3, a diacylglycerol-dependent Ras/Rap1 exchange factor. Recruitment to phagosomes of RasGRP3, which contains a C1 domain, parallels and appears to be due to the formation of diacylglycerol. Accordingly, Ras and Rap1 activation was precluded by antagonists of phospholipase C and of diacylglycerol binding. Ras is dispensable for phagocytosis but controls activation of extracellular signal-regulated kinase, which is partially impeded by diacylglycerol inhibitors. By contrast, cross-activation of complement receptors by stimulation of Fcgamma receptors requires Rap1 and involves diacylglycerol. We suggest a role for diacylglycerol-dependent exchange factors in the activation of Ras and Rap1, which govern distinct processes induced by Fcgamma receptor-mediated phagocytosis to enhance the innate immune response

The transcription factors that bind to EpRE's play a key role in the regulation of phase II genes. In this study, we examined whether c-Jun, a partner of Nrf2 in binding to EpRE's, requires phosphorylation by JNK for binding and transcriptional activation. We used chromatin immunoprecipitation assays to measure the recruitment of transcription factors to EpRE sequences in NQO2, GCLC, and GCLM; Western analysis for phosphorylation of JNK; and EpRE-driven reporters along with a JNK-specific inhibitor peptide to determine the potential importance of c-Jun phosphorylation. Human bronchial epithelial (HBE1) and human hepatoma (HepG2) cells were exposed to 4-hydroxy-2-nonenal (HNE), and differences in the regulation of the same EpRE sequences were examined. We found that binding of c-Jun to EpRE sequences increased subsequent to HNE exposure in HepG2 cells; however, in HNE-exposed HBE1 cells, the binding of only phosphorylated c-Jun to the three EpRE sequences increased. Despite the increase in binding of phosphorylated c-Jun, reporter assays for EpRE's showed that inhibition of c-Jun phosphorylation had variable effects on basal and HNE-induced transcription of GCLC and GCLM in HBE1 cells. Thus, in terms of its role in mediating HNE induction of EpRE-mediated transcription, c-Jun seems to be a partner of Nrf2 and, whereas its phosphorylated form may predominate in one cell type versus another, the effects of phosphorylation of c-Jun on transcription can vary with the gene. This contrasts markedly with the well-established requirement for phosphorylation of c-Jun in the activation of AP-1/TRE-mediated transcription.

We have recently identified two promoters, distal and proximal for rat mitochondrial glycerophosphate acyltransferase (mtGPAT). Here we are reporting further characterization of the promoters. Insulin and epidermal growth factor (EGF) stimulated while leptin and glucagon inhibited the luciferase activity of the distal promoter and the amounts of the distal transcript. Conversely, luciferase activity of the proximal promoter and proximal transcript remained unchanged due to these treatments. Only the distal promoter has binding sites for carbohydrate response element binding protein (ChREBP) and sterol regulatory element binding protein-1 (SREBP-1). Electromobility shift assays and chromatin immunoprecipitation assays demonstrated that ChREBP and SREBP-1 bind to the mtGPAT distal promoter. Insulin and EGF increased while glucagon and leptin decreased the binding of SREBP-1 and ChREBP to the distal promoter. Thus, the distal promoter is the regulatory promoter while the proximal promoter acts constitutively for rat mtGPAT gene under the influence of hormones and growth factor.