Faculty Bibliography

There is a growing body of evidence that suggests that amino acids play an important role in controlling gene expression, but the cell specificity of the amino-acid-mediated regulation of gene expression in mammals remains unknown. Using a model of muscle cells (C2C12) at two stages of differentiation, i.e. myoblasts and myotubes, we employed transcriptional profiling to show that amino-acid deficiency does not regulate the same set of gene in differentiated and non-differentiated cells. Furthermore, in myotubes, the GCN2 pathway is not activated by amino-acid starvation due to an amino-acid supply from intracellular proteolysis associated with a low GCN2 expression.

p202, an interferon-inducible protein that belongs to an interferon-inducible p200 family, was highly induced in the course of osteogenesis of pluripotent C2C12 cells and the chondrogenesis of C3H10T1/2 cells. Differential expression of p202 is probably due, at least in part, to the transactivation of the p202 gene by Smad transcription factors. Overexpressing p202 inhibited, whereas lowering p202 via a siRNA approach enhanced, chondrocyte differentiation. In contrast, overexpression of p202 enhanced, whereas knockdown of p202 inhibited, osteoblast differentiation. Molecular mechanism studies revealed that p202 and parathyroid hormone-related peptide (PTHrP) formed a positive feedback loop, since (1) overexpressing p202 markedly enhanced whereas knocking down p202 suppressed the expression of PTHrP; and (2) p202 expression was increased in growth plate chondrocytes of PTHrP receptor transgenic mouse embryos; however, its expression was reduced in PTHrP knockout mouse embryos. Taken together, our findings demonstrate that p202 protein is a novel, important mediator of chondrogenic and osteogenic differentiation

We reported earlier that an interferon-inducible p204 protein serves as a cofactor of Cbfa1 and promotes osteogenesis. Here we establish that p204 demonstrates prominent expression in growth plate chondrocytes. It is differentially expressed in the course of bone morphogenetic protein-2-triggered chondrocyte differentiation of pluripotent C3H10T1/2 cells. This expression is probably due to the activation of p204 gene by Cbfa1 and repression by Sox5 transcription factor. Cbfa1 and Sox5 bind to the 5'-flanking regulatory region of p204 gene at their consensus binding elements. Overexpression of p204 accelerates chondrocyte hypertrophy, as revealed by enhanced expression of type X Collagen and matrix metalloproteinase-13; however, knockdown of p204 via an siRNA approach abolishes hypertrophic chondrocyte differentiation. p204 acts as a cofactor of Cbfa1 in chondrocyte hypertrophy: (1) overexpression of p204 augments, whereas suppression of p204 decreases, the Cbfa1-dependent transactivation of a Collagen X-specific reporter gene; (2) p204 enhances Cbfa1-mediated chondrocyte hypertrophy; and (3) p204 associates with Cbfa1 in chondrocyte differentiation. In addition, altered expression of p204 in chondrocyte hypertrophy was accompanied by altered levels of Indian hedgehog (IHH) and parathyroid hormone/parathyroid hormone-related peptide receptor-1 (PTHR1). Collectively, p204 is a novel regulator of chondrocyte differentiation by (1) acting as a coactivator of Cbfa1 and (2) affecting IHH/PTPrP signaling.Cell Death and Differentiation advance online publication, 18 July 2008; doi:10.1038/cdd.2008.112

The T-cell-derived, pleiotropic cytokine interferon (IFN)-gamma is believed to play a key regulatory role in immune-mediated demyelinating disorders of the central nervous system, including multiple sclerosis and experimental autoimmune encephalomyelitis. Our previous work has demonstrated that the endoplasmic reticulum (ER) stress response modulates the response of oligodendrocytes to this cytokine. The ER stress response activates the pancreatic ER kinase, which coordinates an adaptive program known as the integrated stress response by phosphorylating translation initiation factor 2alpha (eIF2alpha). In this study, we found that growth arrest and DNA damage 34 (GADD34), a stress-inducible regulatory subunit of a phosphatase complex that dephosphorylates eIF2alpha, was selectively up-regulated in myelinating oligodendrocytes in mice that ectopically expressed IFN-gamma in the central nervous system. We also found that a GADD34 mutant strain of mice displayed increased levels of phosphorylated eIF2alpha (p-eIF2alpha) in myelinating oligodendrocytes when exposure to IFN-gamma, as well as diminished oligodendrocyte loss and hypomyelination. Furthermore, treatment with salubrinal, a small chemical compound that specifically inhibits protein phosphatase 1(PP1)-GADD34 phosphatase activity, increased the levels of p-eIF2alpha and ameliorated hypomyelination and oligodendrocyte loss in cultured hippocampal slices exposed to IFN-gamma. Thus, our data provide evidence that an enhanced integrated stress response could promote oligodendrocyte survival in immune-mediated demyelination diseases

Gap junction redistribution and reduced expression, a phenomenon termed gap junction remodeling (GJR), is often seen in diseased hearts and may predispose towards arrhythmias. We have recently shown that short-term pacing in the mouse is associated with changes in connexin43 (Cx43) expression and localization, but not with increased inducibility into sustained arrhythmias. We hypothesized that short-term pacing, if imposed on murine hearts with decreased Cx43 abundance, could serve as a model for evaluating the electrophysiologic effects of GJR. We paced wildtype (normal Cx43 abundance) and heterozygous Cx43 knockout mice (Cx43(+/-), 66% mean reduction in Cx43) for six hours at 10-15% above their average sinus rate. We investigated the electrophysiologic effects of pacing on the whole animal using programmed electrical stimulation, and in isolated ventricular myocytes with patch clamp studies. Cx43(+/-) myocytes had significantly shorter action potential durations (APD) and increased steady state and inward rectifier potassium currents (Iss and IK1, respectively) compared to wildtype littermate cells. In Cx43(+/-) hearts, pacing resulted in significant prolongation of ventricular effective refractory period and action potential duration, and significant diminution of Iss compared to unpaced Cx43(+/-) hearts. However, these changes were not seen in paced wildtype mice. These data suggest that Cx43 abundance plays a critical role in regulating currents involved in myocardial repolarization and their response to pacing. Our study may aid in understanding how dyssynchronous activation of diseased, Cx43-deficient myocardial tissue can lead to electrophysiologic changes which may contribute to the worsened prognosis often associated with pacing in the failing heart. Key words: Connexin43, ventricular myocytes, mouse, gap junction

Magnetic resonance (MR) imaging is becoming a pivotal diagnostic method to identify and characterize vulnerable atherosclerotic plaques. We previously reported a reconstituted high-density lipoprotein (rHDL) nanoparticle platform enriched with Gd-based amphiphiles as a plaque-specific MR imaging contrast agent. Further modification can be accomplished by inserting targeting moieties into this platform to potentially allow for improved intraplaque macrophage uptake. Since studies have indicated that intraplaque macrophage density is directly correlated to plaque vulnerability, modification of the rHDL platform may allow for better detection of vulnerable plaques. In the current study we incorporated a carboxyfluoresceine-labeled apolipoprotein E-derived lipopeptide, P2fA2, into rHDL. The in vitro macrophage uptake and in vivo MR efficacy were demonstrated using murine J774A.1 macrophages and the apolipoprotein E knock-out (apoE(-/-)) mouse model of atherosclerosis. The in vitro studies indicated enhanced association of murine macrophages to P2fA2 enriched rHDL (rHDL-P2A2) nanoparticles, relative to rHDL, using optical techniques and MR imaging. The in vivo studies showed a more pronounced and significantly higher signal enhancement of the atherosclerotic wall 24 h after the 50 micromol Gd/kg injection of rHDL-P2A2 relative to administration of rHDL. The normalized enhancement ratio for atherosclerotic wall of rHDL-P2A2 contrast agent injection was 90%, while that of rHDL was 53% 24 h post-injection. Confocal laser scanning microscopy revealed that rHDL-P2A2 nanoparticles co-localized primarily with intraplaque macrophages. The results of the current study confirm the hypothesis that intraplaque macrophage uptake of rHDL may be enhanced by the incorporation of the P2fA2 peptide into the modified HDL particle

High density lipoprotein (HDL) is an important natural nanoparticle that may be modified for biomedical imaging purposes. Here we developed a novel technique to create unique multimodality HDL mimicking nanoparticles by incorporation of gold, iron oxide, or quantum dot nanocrystals for computed tomography, magnetic resonance, and fluorescence imaging, respectively. By including additional labels in the corona of the particles, they were made multifunctional. The characteristics of these nanoparticles, as well as their in vitro and in vivo behavior, revealed that they closely mimic native HDL.