Faculty Bibliography

BACKGROUND: Clinical use of cancer gene therapy has been prevented by the inability to deliver high levels of local transgene expression with acceptable host toxicity. The authors' laboratory has developed an ex vivo technique to genetically modify free flaps to deliver immunotherapy locally without systemic toxicity. METHODS: Superficial inferior epigastric flaps were dissected in Fischer rats, perfused with a viral vector expressing the antitumor interleukin-12 (IL-12) for 1 hour, and re-anastomosed. Beneath the flaps was a bolus of 1 x 10(6) beta-human chorionic gonadotropin-secreting MADB-106 tumor cells. Tumor growth was monitored using beta-human chorionic gonadotropin levels (secreted by the tumor) and size. IL-12 expression in tissue was assessed by enzyme-linked immunosorbent assay. Tumor inflammatory infiltrate was assessed using immunohistologic staining (CD8 and CD161) and enzyme-linked immunosorbent assay (interferon-gamma). Serum levels of liver enzymes and histologic analysis were used to assess systemic toxicity. RESULTS: IL-12 expression was confirmed in the flap and surrounding tissue. The rate of tumor growth in the IL-12-treated group was significantly suppressed compared with the control group (p < 0.001). Liver enzyme levels remained normal, and histological evaluation of the liver, lung, and spleen revealed no evidence of inflammation in the treated group. CONCLUSIONS: Using genetically modified free flaps, the authors were able to deliver IL-12 directly into the local environment of a tumor and suppress its growth without eliciting toxic systemic effects. This technique could provide valuable adjuvant treatment after oncologic surgery for soft-tissue cancers, with the transduced flap reconstructing the defect and supplying a therapeutic agent to the resected tumor bed.

PURPOSE: Beta-adrenergic receptor (AR) antagonists are frequently prescribed ophthalmic drugs, yet previous investigations into how catecholamines affect corneal wound healing have yielded conflicting RESULTS: With the use of an integrated pharmacologic and genetic approach, the authors investigated how the beta-AR impacts corneal epithelial healing. METHODS: Migratory rates of cultured adult murine corneal epithelial (AMCE) cells and in vivo corneal wound healing were examined in beta2-AR(+/+) and beta2-AR(-/-) mice. Signaling pathways were evaluated by immunoblotting. results. The beta-AR agonist isoproterenol decreased AMCE cell migratory speed to 70% of untreated controls, and this was correlated with a 0.60-fold decrease in levels of activated phospho-ERK (P-ERK). Treatment with the beta-AR antagonist (timolol) increased speed 33% and increased P-ERK 2.4-fold (P < 0.05). The same treatment protocols had no effect on AMCE cells derived from beta2-AR(-/-) mice; all treatment groups showed statistically equivalent migratory speeds and ERK phosphorylation. In beta2-AR(+/+) animals, the beta-AR agonist (isoproterenol) delayed the rate of in vivo corneal wound healing by 79%, whereas beta-AR antagonist (timolol) treatment increased the rate of healing by 16% (P < 0.05) compared with saline-treated controls. In contrast, in the beta2-AR(-/-) mice, all treatment groups demonstrated equivalent rates of wound healing. Additionally, murine corneal epithelial cell expressed the catecholamine-synthesizing enzyme tyrosine hydroxylase and detectable levels of epinephrine (184.5 pg/mg protein). CONCLUSIONS: The authors provide evidence of an endogenous autocrine catecholamine signaling pathway dependent on an intact beta2-AR for the modulation of corneal epithelial wound repair

The hair follicle contributes cells to the interfollicular epidermis after wounding, but the functional role of these cells has not been resolved. To address this question, Langton et al. (this issue, 2008) take advantage of the Edaradd mutant mouse, which lacks hair follicles on its tail. They discover an initial sluggish response of the hairless tail epidermis to wounding that is rapidly compensated for by recruitment of epidermal cells from outside the normally responsive area. This suggests that the hair follicle is important but not necessary for normal wound healing

The transcriptional activation of CHOP (C/EBP-homologous protein) by amino acid deprivation involves ATF2 and ATF4 binding at the amino acid response element within the promoter. In this report, we investigate the role of JDP2 (Jun Dimerization Protein 2) in the amino acid control of CHOP transcription following amino acid starvation. Our results show that JDP2 binds to the CHOP AARE in unstimulated cells and that its binding decreases following amino acid starvation. We demonstrate that JDP2 acts as a repressor and suggest that it could be functionally associated with HDAC3 to inhibit CHOP transcription.

Ablation of germ-line precursor cells in Caenorhabditis elegans extends lifespan by activating DAF-16, a forkhead transcription factor (FOXO) repressed by insulin/insulin-like growth factor (IGF) signaling (IIS). Signals from the gonad might thus regulate whole-organism aging by modulating IIS. To date, the details of this systemic regulation of aging by the reproductive system are not understood, and it is unknown whether such effects are evolutionarily conserved. Here we report that eliminating germ cells (GCs) in Drosophila melanogaster increases lifespan and modulates insulin signaling. Long-lived germ-line-less flies show increased production of Drosophila insulin-like peptides (dilps) and hypoglycemia but simultaneously exhibit several characteristics of IIS impedance, as indicated by up-regulation of the Drosophila FOXO (dFOXO) target genes 4E-BP and l (2)efl and the insulin/IGF-binding protein IMP-L2. These results suggest that signals from the gonad regulate lifespan and modulate insulin sensitivity in the fly and that the gonadal regulation of aging is evolutionarily conserved

Tissue ischemia promotes vasculogenesis through chemokine-induced recruitment of bone marrow-derived endothelial progenitor cells (EPCs). Diabetes significantly impairs this process. Because hyperglycemia increases reactive oxygen species in a number of cell types, and because many of the defects responsible for impaired vasculogenesis involve HIF1-regulated genes, we hypothesized that HIF1 function is impaired in diabetes because of reactive oxygen species-induced modification of HIF1alpha by the glyoxalase 1 (GLO1) substrate methylglyoxal. Decreasing superoxide in diabetic mice by either transgenic expression of manganese superoxide dismutase or by administration of an superoxide dismutase mimetic corrected post-ischemic defects in neovascularization, oxygen delivery, and chemokine expression, and normalized tissue survival. In hypoxic fibroblasts cultured in high glucose, overexpression of GLO1 prevented reduced expression of both the EPC mobilizing chemokine stromal cell-derived factor-1 (SDF-1) and of vascular epidermal growth factor, which modulates growth and differentiation of recruited EPCs. In hypoxic EPCs cultured in high glucose, overexpression of GLO1 prevented reduced expression of both the SDF-1 receptor CXCR4, and endothelial nitric-oxide synthase, an enzyme essential for EPC mobilization. HIF1alpha modification by methylglyoxal reduced heterodimer formation and HIF1alpha binding to all relevant promoters. These results provide a basis for the rational design of new therapeutics to normalize impaired ischemia-induced vasculogenesis in patients with diabetes

Physiological mineralization in growth plate cartilage is highly regulated and restricted to terminally differentiated chondrocytes. Since mineralization occurs in the extracellular matrix, we asked whether major extracellular matrix components (collagens) of growth plate cartilage are directly involved in regulating the mineralization process. Our findings show that types II and X collagen interacted with cell surface expressed annexin V. These interactions led to a stimulation of annexin V-mediated Ca2+ influx resulting in an increased intracellular Ca2+ concentration, [Ca2+]i, and ultimately increased alkaline phosphatase activity and mineralization of growth plate chondrocytes. Consequently, stimulation of these interactions (ascorbate to stimulate collagen synthesis, culturing cells on type II collagen-coated dishes, or overexpression of full-length annexin V) resulted in increase of [Ca2+]i, alkaline phosphatase activity and mineralization of growth plate chondrocytes, whereas inhibition of these interactions (3,4-dehydro-L-proline to inhibit collagen secretion, K-201, a specific annexin channel blocker, overexpression of N-terminus deleted mutant annexin V that does not bind to type II collagen and shows reduced Ca2+ channel activities) decreased [Ca2+]i, alkaline phosphatase activity and mineralization. In conclusion, the interactions between collagen and annexin V regulate mineralization of growth plate cartilage. Since annexin V is upregulated during pathological mineralization events of articular cartilage, it is possible that these interactions also regulate pathological mineralization

Phospholamban (PLN) regulates cardiac contractility by modulation of sarco(endo)plasmic reticulum calcium ATPase (SERCA) activity. While PLN and SERCA1a, an isoform from skeletal muscle, have been structurally characterized in great detail, direct information about the conformation of PLN in complex with SERCA has been limited. We used solid-state NMR (ssNMR) spectroscopy to deduce structural properties of both the A 36F 41A 46 mutant (AFA-PLN) and wild-type PLN (WT-PLN) when bound to SERCA1a after reconstitution in a functional lipid bilayer environment. Chemical-shift assignments in all domains of AFA-PLN provide direct evidence for the presence of two terminal alpha helices connected by a linker region of reduced structural order that differs from previous findings on free PLN. ssNMR experiments on WT-PLN show no significant difference in binding compared to AFA-PLN and do not support the coexistence of a significantly populated dynamic state of PLN after formation of the PLN/SERCA complex. A combination of our spectroscopic data with biophysical and biochemical data using flexible protein-protein docking simulations provides a structural basis for understanding the interaction between PLN and SERCA1a.

Hepatic secretion of apolipoprotein-B (apoB), the major protein of atherogenic lipoproteins, is regulated through posttranslational degradation. We reported a degradation pathway, post-ER pre secretory proteolysis (PERPP), that is increased by reactive oxygen species (ROS) generated within hepatocytes from dietary polyunsaturated fatty acids (PUFA). We now report the molecular processes by which PUFA-derived ROS regulate PERPP of apoB. ApoB exits the ER; undergoes limited oxidant-dependent aggregation; and then, upon exit from the Golgi, becomes extensively oxidized and converted into large aggregates. The aggregates slowly degrade by an autophagic process. None of the oxidized, aggregated material leaves cells, thereby preventing export of apoB-lipoproteins containing potentially toxic lipid peroxides. In summary, apoB secretory control via PERPP/autophagosomes is likely a key component of normal and pathologic regulation of plasma apoB levels, as well as a means for remarkably late-stage quality control of a secreted protein

Clathrin-coated vesicles are vehicles for intracellular trafficking in all nucleated cells, from yeasts to humans. Many studies have demonstrated their essential roles in endocytosis and cellular signalling processes at the plasma membrane. By contrast, very few of their non-endocytic trafficking roles are known, the best characterized being the transport of hydrolases from the Golgi complex to the lysosome. Here we show that clathrin is required for polarity of the basolateral plasma membrane proteins in the epithelial cell line MDCK. Clathrin knockdown depolarized most basolateral proteins, by interfering with their biosynthetic delivery and recycling, but did not affect the polarity of apical proteins. Quantitative live imaging showed that chronic and acute clathrin knockdown selectively slowed down the exit of basolateral proteins from the Golgi complex, and promoted their mis-sorting into apical carrier vesicles. Our results demonstrate a broad requirement for clathrin in basolateral protein trafficking in epithelial cells.