Faculty Bibliography

Dendritic cells initiate the immune response by presenting antigen in the context of varying levels of costimulation. The maturation state of the dendritic cell determines the quantity and quality (Th1, Th2) of the subsequent T cell response. Members of the NF-kappaB family of transcription factors have previously been implicated in dendritic cell development. Here, we used a mouse with a homozygous c-Rel deletion to investigate the role of c-Rel in the function of bone marrow derived dendritic cells. When direct presentation was evaluated, we found c-Rel(-/-) dendritic cells induce less allogeneic T cell stimulation than c-Rel(+/+) dendritic cells. In addition, T cell encounters with c-Rel(-/-) dendritic cells generate less IFN-gamma and IL-4 when compared to those with c-Rel(+/+) DCs. A similar degree of functional compromise was observed in antigen-specific T cells that were stimulated by c-Rel(-/-) dendritic cells. Functional deficits were not linked to differences in the ability to undergo maturation per se, as LPS exposure induced similar morphologic and cell surface changes in both c-Rel(+/+) and cRel(-/-) DCs. Although LPS induced a compensatory increase in the nuclear activity of fellow NF-kappaB family members, RelB and p65, LPS exposure was unable to negate the deficiencies in autologous T cell proliferation and cytokine production associated with the loss of c-Rel in dendritic cells. Taken together, our study supports a unique and non-redundant role for c-Rel in dendritic cell costimulatory capacity

Cellular activities are primarily initiated, modulated and sustained by multifunctional molecules (cytokines and growth factors) that are secreted into the extracellular space and that signal through membrane-bound, high-affinity receptors. In contrast to the fairly well understood mechanisms that mediate the specificity of signal transduction within the confined and compartmentalized environment of the cell, significantly less is known about the mechanisms that regulate the availability of signaling molecules in the extracellular milieu. Recent findings have implicated the participation of extracellular protein macroaggregates in signaling events controlling patterning and morphogenesis. The results suggest a functional coupling between the tissue-specific organization of collagenous and elastic macroaggregates and their ability to perform instructive as well as structural functions. These observations open the way to a novel understanding in these poorly understood and critically important areas of cell and developmental biology

Reactive oxygen species play an important role in development of lung injury. Neonates exhibit a high risk of developing acute and/or chronic lung disorder, often associated with surfactant deficiency, and in parallel they show low vitamin E concentration. We investigated whether the vitamin E status of adult rats affects the content of phospholipids (PL) in bronchoalveolar lavage and alveolar type II cells. Phosphatidylcholine (PtdCho) is the dominant and functional most important PL in lung surfactant. Therefore, we determined its formation via de novo synthesis and reacylation of lyso-PtdCho in type II cells. Vitamin E depletion caused a decrease of PL content in bronchoalveolar lavage and type II cells and decreased glycerol-3-phosphate O-acyltransferase (G3P-AT) activity, de novo synthesis of PtdCho, and reacylation of lyso-PtdCho in type II cells. Preincubation of type II cell homogenates with dithiothreitol restored the activity of G3P-AT and de novo synthesis but inhibited reacylation. Reacylation was strongly reduced by chelerythrine-mediated inhibition of protein kinase C. We conclude that antioxidant and PKC-modulating properties of vitamin E regulate de novo synthesis of PtdCho and reacylation of lyso-PtdCho in alveolar type II cells. Vitamin E depletion reduced the two pathways of PL synthesis and caused a decrease of PL content in alveolar surfactant of rats

BACKGROUND: LDL receptor-deficient 'apolipoprotein (apo)-B100-only' mice (Ldlr-/-Apob100/100 have elevated LDL cholesterol levels on a chow diet and develop severe aortic atherosclerosis. We hypothesized that both the hypercholesterolemia and the susceptibility to atherosclerosis could be eliminated by switching off hepatic lipoprotein production. METHODS AND RESULTS: We bred Ldlr-/-Apob100/100 mice that were homozygous for a conditional allele for Mttp (the gene for microsomal triglyceride transfer protein) and the inducible Mx1-Cre transgene. In these animals, which we called 'Reversa mice,' the hypercholesterolemia could be reversed, without modifying the diet or initiating a hypolipidemic drug, by the transient induction of Cre expression in the liver. After Cre induction, hepatic Mttp expression was virtually eliminated (as judged by quantitative real-time PCR), hepatic lipoprotein secretion was abolished (as judged by electron microscopy), and LDLs were virtually eliminated from the plasma. Intestinal lipoprotein production was unaffected. In mice fed a chow diet, Cre induction reduced plasma cholesterol levels from 233.9+/-46.0 to 37.2+/-6.5 mg/dL. In mice fed a high-fat diet, cholesterol levels fell from 525.7+/-32.2 to 100.6+/-14.3 mg/dL. The elimination of hepatic lipoprotein production completely prevented both the development of atherosclerosis and the changes in gene expression that accompany atherogenesis. CONCLUSIONS: We developed mice in which hypercholesterolemia can be reversed with a genetic switch. These mice will be useful for understanding gene-expression changes that accompany the reversal of hypercholesterolemia and atherosclerosis

The chemokine SDF-1 and its receptor CXCR4 control cell migration in the immune and nervous systems. Recent studies in zebrafish have shown that SDF-1 and CXCR4 also guide the migration of germ cells and sensory organs of the lateral line

Rho family GTPases are important regulators of epithelial tight junctions (TJs); however, little is known about how the GTPases themselves are controlled during TJ assembly and function. We have identified and cloned a canine guanine nucleotide exchange factor (GEF) of the Dbl family of proto-oncogenes that activates Rho and associates with TJs. Based on sequence similarity searches and immunological and functional data, this protein is the canine homologue of human GEF-H1 and mouse Lfc, two previously identified Rho-specific exchange factors known to associate with microtubules in nonpolarized cells. In agreement with these observations, immunofluorescence of proliferating MDCK cells revealed that the endogenous canine GEF-H1/Lfc associates with mitotic spindles. Functional analysis based on overexpression and RNA interference in polarized MDCK cells revealed that this exchange factor for Rho regulates paracellular permeability of small hydrophilic tracers. Although overexpression resulted in increased size-selective paracellular permeability, such cell lines exhibited a normal overall morphology and formed fully assembled TJs as determined by measuring transepithelial resistance and by immunofluorescence and freeze-fracture analysis. These data indicate that GEF-H1/Lfc is a component of TJs and functions in the regulation of epithelial permeability.

Active repression of protein synthesis protects cells against protein malfolding during endoplasmic reticulum stress, nutrient deprivation and oxidative stress. However, long-term adaptation to these conditions requires synthesis of new stress-induced proteins. Phosphorylation of the alpha-subunit of translation initiation factor 2 (eIF2alpha) represses translation in diverse stressful conditions. GADD34 is a stress-inducible regulatory subunit of a holophosphatase complex that dephosphorylates eIF2alpha, and has been hypothesized to play a role in translational recovery. Here, we report that GADD34 expression correlated temporally with eIF2alpha dephosphorylation late in the stress response. Inactivation of both alleles of GADD34 prevented eIF2alpha dephosphorylation and blocked the recovery of protein synthesis, normally observed late in the stress response. Furthermore, defective recovery of protein synthesis markedly impaired translation of stress-induced proteins and interfered with programmed activation of stress-induced genes in the GADD34 mutant cells. These observations indicate that GADD34 controls a programmed shift from translational repression to stress-induced gene expression, and reconciles the apparent contradiction between the translational and transcriptional arms of cellular stress responses