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Angiogenic vasa vasorum (VV) expansion plays an essential role in the pathogenesis of hypoxia-induced pulmonary hypertension (PH), a cardiovascular disease. We previously showed that extracellular ATP released under hypoxic conditions is an autocrine/paracrine, the angiogenic factor for pulmonary artery (PA) VV endothelial cells (VVECs), acting via P2Y purinergic receptors (P2YR) and the Phosphoinositide 3-kinase (PI3K)-Akt-Mammalian Target of Rapamycin (mTOR) signaling. To further elucidate the molecular mechanisms of ATP-mediated VV angiogenesis, we determined the profile of ATP-inducible transcription factors (TFs) in VVECs using a TranSignal protein/DNA array. C-Jun, c-Myc, and Foxo3 were found to be upregulated in most VVEC populations and formed nodes connecting several signaling networks. siRNA-mediated knockdown (KD) of these TFs revealed their critical role in ATP-induced VVEC angiogenic responses and the regulation of downstream targets involved in tissue remodeling, cell cycle control, expression of endothelial markers, cell adhesion, and junction proteins. Our results showed that c-Jun was required for the expression of ATP-stimulated angiogenic genes, c-Myc was repressive to anti-angiogenic genes, and Foxo3a predominantly controlled the expression of anti-apoptotic and junctional proteins. The findings from our study suggest that pharmacological targeting of the components of P2YR-PI3K-Akt-mTOR axis and specific TFs reduced ATP-mediated VVEC angiogenic response and may have a potential translational significance in attenuating pathological vascular remodeling.

Hypoadiponectinemia has been widely observed in patients with gestational diabetes mellitus (GDM). To investigate the causal role of hypoadiponectinemia in GDM, adiponectin gene knockout (Adipoq−/−) and wild-type (WT) mice were crossed to produce pregnant mouse models with or without adiponectin deficiency. Adenoviral vector–mediated in vivo transduction was used to reconstitute adiponectin during late pregnancy. Results showed that Adipoq−/− dams developed glucose intolerance and hyperlipidemia in late pregnancy. Increased fetal body weight was detected in Adipoq−/− dams. Adiponectin reconstitution abolished these metabolic defects in Adipoq−/− dams. Hepatic glucose and triglyceride production rates of Adipoq−/− dams were significantly higher than those of WT dams. Robustly enhanced lipolysis was found in gonadal fat of Adipoq−/− dams. Interestingly, similar levels of insulin-induced glucose disposal and insulin signaling in metabolically active tissues in Adipoq−/− and WT dams indicated that maternal adiponectin deficiency does not reduce insulin sensitivity. However, remarkably decreased serum insulin concentrations were observed in Adipoq−/− dams. Furthermore, β-cell mass, but not glucose-stimulated insulin release, in Adipoq−/− dams was significantly reduced compared with WT dams. Together, these results demonstrate that adiponectin plays an important role in controlling maternal metabolic adaptation to pregnancy.

The potential of mesenchymal stromal cells (MSCs) to inhibit anti-tumor immunity is becoming increasingly well recognized, but the precise steps affected by these cells during the development of an anti-tumor immune response remain incompletely understood. Here, we examined how MSCs affect the steps required to mount an effective anti-tumor immune response following administration of adenovirus Fas ligand (Ad-FasL) in the Lewis lung carcinoma (LL3) model. Administration of bone marrow-derived MSCs with LL3 cells accelerated tumor growth significantly. MSCs inhibited the inflammation induced by Ad-FasL in the primary tumors, precluding their rejection; MSCs also reduced the consequent expansion of tumor-specific T cells in the treated hosts. When immune T cells were transferred to adoptive recipients, MSCs impaired, but did not completely abrogate the ability of these T cells to promote elimination of secondary tumors. This impairment was associated with a modest reduction in tumor-infiltrating T cells, with a significant reduction in tumor-infiltrating macrophages, and with a reorganization of the stromal environment. Our data indicate that MSCs in the tumor environment reduce the efficacy of immunotherapy by creating a functional and anatomic barrier that impairs inflammation, T cell priming and expansion, and T cell function-including recruitment of effector cells.

Macrophage infiltration plays an important role in obesity-induced insulin resistance. CCAAT enhancer-binding protein-α (C/EBPα) is a transcription factor that is highly expressed in macrophages. To examine the roles of C/EBPα in regulating macrophage functions and energy homeostasis, macrophage-specific C/EBPα knockout (MαKO) mice were created. Chow-fed MαKO mice exhibited higher body fat mass and decreased energy expenditure despite no change in food intake. However, the obese phenotype disappeared after high-fat (HF) diet feeding. Although there was a transient decrease in insulin sensitivity of chow-fed young MαKO mice, systemic insulin sensitivity was protected during HF-feeding due to preserved insulin sensitivity in skeletal muscle. We also found that C/EBPα-deficient macrophages exhibited a blunted response of cytokine-induced expression of M1 and M2 macrophage markers, suggesting that C/EBPα controls both M1 and M2 polarization. Consistent with decreased exercise capacity, mitochondrial respiration rates and signal pathways for fatty acid oxidation were remarkably reduced in the skeletal muscle of chow-fed MαKO mice. Furthermore, expression levels of inflammatory cytokines were reduced in skeletal muscle of HF-fed MαKO mice. Together, these results imply that C/EBPα is required for macrophage activation, which plays an important role in maintaining skeletal muscle energy metabolism.

Milk lipid is secreted by a unique process, during which triacylglycerol droplets bud from mammary cells coated with an outer bilayer of apical membrane. In all current schemes, the integral protein butyrophilin 1A1 (BTN) is postulated to serve as a transmembrane scaffold, which interacts either with itself or with the peripheral proteins, xanthine oxidoreductase (XOR) and possibly perilipin-2 (PLIN2), to form an immobile bridging complex between the droplet and apical surface. In one such scheme, BTN on the surface of cytoplasmic lipid droplets interacts directly with BTN in the apical membrane without binding to either XOR or PLIN2. We tested these models using both biochemical and morphological approaches. BTN was concentrated in the apical membrane in all species examined and contained mature N-linked glycans. We found no evidence for the association of unprocessed BTN with intracellular lipid droplets. BTN-enhanced green fluorescent protein was highly mobile in areas of mouse milk-lipid droplets that had not undergone post-secretion changes, and endogenous mouse BTN comprised only 0.5-0.7% (w/w) of the total protein, i.e. over 50-fold less than in the milk-lipid droplets of cow and other species. These data are incompatible with models of milk-lipid secretion in which BTN is the major component of an immobile global adhesive complex and suggest that interactions between BTN and other proteins at the time of secretion are more transient than previously predicted. The high mobility of BTN in lipid droplets marks it as a potential mobile signaling molecule in milk.

Hypoxia and HIF-2α-dependent A2A receptor expression and activation increase proliferation of human lung microvascular endothelial cells (HLMVECs). This study was undertaken to investigate the signaling mechanisms that mediate the proliferative effects of A2A receptor. A2A receptor-mediated proliferation of HLMVECs was inhibited by intracellular calcium chelation, and by specific inhibitors of ERK1/2 and PI3-kinase (PI3K). The adenosine A2A receptor agonist CGS21680 caused intracellular calcium mobilization in controls and, to a greater extent, in A2A receptor-overexpressing HLMVECs. Adenoviral-mediated A2A receptor overexpression as well as receptor activation by CGS21680 caused increased PI3K activity and Akt phosphorylation. Cells overexpressing A2A receptor also manifested enhanced ERK1/2 phosphorylation upon CGS21680 treatment. A2A receptor activation also caused enhanced cAMP production. Likewise, treatment with 8Br-cAMP increased PI3K activity. Hence A2A receptor-mediated cAMP production and PI3K and Akt phosphorylation are potential mediators of the A2A-mediated proliferative response of HLMVECs. Cytosolic calcium mobilization and ERK1/2 phosphorylation are other critical effectors of HLMVEC proliferation and growth. These studies underscore the importance of adenosine A2A receptor in activation of survival and proliferative pathways in pulmonary endothelial cells that are mediated through PI3K/Akt and ERK1/2 pathways.

Fas ligand (FasL) gene therapy for cancer has shown promise in rodents; however, its efficacy in higher mammals remains unknown. Here, we used intratumoral FasL gene therapy delivered in an adenovirus vector (Ad-FasL) as neoadjuvant to standard of care in 56 dogs with osteosarcoma. Tumors from treated dogs had greater inflammation, necrosis, apoptosis, and fibrosis at day 10 (amputation) compared to pretreatment biopsies or to tumors from dogs that did not receive Ad-FasL. Survival improvement was apparent in dogs with inflammation or lymphocyte-infiltration scores >1 (in a 3-point scale), as well as in dogs that had apoptosis scores in the top 50th percentile (determined by cleaved caspase-3). Survival was no different than that expected from standard of care alone in dogs with inflammation scores ≤1 or apoptosis scores in the bottom 50th percentile. Reduced Fas expression by tumor cells was associated with prognostically advantageous inflammation, and this was seen only in dogs that received Ad-FasL. Together, the data suggest that Ad-FasL gene therapy improves survival in a subset of large animals with naturally occurring tumors, and that at least in some tumor types like osteosarcoma, it is most effective when tumor cells fail to express Fas.

Aims/hypothesisThe main objective of this study was to investigate whether maternal adiponectin regulates fetal growth through the endocrine system in the fetal compartment.MethodsAdiponectin knockout (Adipoq (-/-) ) mice and in vivo adenovirus-mediated reconstitution were used to study the regulatory effect of maternal adiponectin on fetal growth. Primary human trophoblast cells were treated with adiponectin and a specific peroxisome proliferator-activated receptor α (PPARα) agonist or antagonist to study the underlying mechanism through which adiponectin regulates fetal growth.ResultsThe body weight of fetuses from Adipoq (-/-) dams was significantly greater than that of wild-type dams at both embryonic day (E)14.5 and E18.5. Adenoviral vector-mediated maternal adiponectin reconstitution attenuated the increased fetal body weight induced by maternal adiponectin deficiency. Significantly increased blood glucose, triacylglycerol and NEFA levels were observed in Adipoq (-/-) dams, suggesting that nutrient supply contributes to maternal adiponectin-regulated fetal growth. Although fetal blood IGF-1 concentrations were comparable in fetuses from Adipoq (-/-) and wild-type dams, remarkably low levels of IGF-binding protein 1 (IGFBP-1) were observed in the serum of fetuses from Adipoq (-/-) dams. IGFBP-1 was identified in the trophoblast cells of human and mouse placentas. Maternal fasting robustly increased IGFBP-1 levels in mouse placentas, while reducing fetal weight. Significantly low IGFBP-1 levels were found in placentas of Adipoq (-/-) dams. Adiponectin treatment increased IGFBP-1 levels in primary cultured human trophoblast cells, while the PPARα antagonist, MK886, abolished this stimulatory effect.Conclusions/interpretationThese results indicate that, in addition to nutrient supply, maternal adiponectin inhibits fetal growth by increasing IGFBP-1 expression in trophoblast cells.

Breast cancers expressing hormone receptors for estrogen (ER) and progesterone (PR) represent ~70% of all cases and are treated with both ER-targeted and chemotherapies, with near 40% becoming resistant. We have previously described that in some ER(+) tumors, the resistant cells express cytokeratin 5 (CK5), a putative marker of breast stem and progenitor cells. CK5(+) cells have lost expression of ER and PR, express the tumor-initiating cell surface marker CD44, and are relatively quiescent. In addition, progestins, which increase breast cancer incidence, expand the CK5(+) subpopulation in ER(+)PR(+) breast cancer cell lines. We have developed models to induce and quantitate CK5(+)ER(-)PR(-) cells, using CK5 promoter-driven luciferase (Fluc) or green fluorescent protein (GFP) reporters stably transduced into T47D breast cancer cells (CK5Pro-GFP or CK5Pro-Luc). We validated the CK5Pro-GFP-T47D model for high-content screening in 96-well microplates and performed a pilot screen using a focused library of 280 compounds from the National Institutes of Health clinical collection. Four hits were obtained that significantly abrogated the progestin-induced CK5(+) cell population, three of which were members of the retinoid family. Hence, this approach will be useful in discovering small molecules that could potentially be developed as combination therapies, preventing the acquisition of a drug-resistant subpopulation.

Chronic hepatitis C virus (HCV) infection greatly increases the risk for type 2 diabetes and nonalcoholic steatohepatitis; however, the pathogenic mechanisms remain incompletely understood. Here we report gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK) transcription and associated transcription factors are dramatically up-regulated in Huh.8 cells, which stably express an HCV subgenome replicon. HCV increased activation of cAMP response element-binding protein (CREB), CCAAT/enhancer-binding protein (C/EBPβ), forkhead box protein O1 (FOXO1), and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) and involved activation of the cAMP response element in the PEPCK promoter. Infection with dominant-negative CREB or C/EBPβ-shRNA significantly reduced or normalized PEPCK expression, with no change in PGC-1α or FOXO1 levels. Notably, expression of HCV nonstructural component NS5A in Huh7 or primary hepatocytes stimulated PEPCK gene expression and glucose output in HepG2 cells, whereas a deletion in NS5A reduced PEPCK expression and lowered cellular lipids but was without effect on insulin resistance, as demonstrated by the inability of insulin to stimulate mobilization of a pool of insulin-responsive vesicles to the plasma membrane. HCV-replicating cells demonstrated increases in cellular lipids with insulin resistance at the level of the insulin receptor, increased insulin receptor substrate 1 (Ser-312), and decreased Akt (Ser-473) activation in response to insulin. C/EBPβ-RNAi normalized lipogenic genes sterol regulatory element-binding protein-1c, peroxisome proliferator-activated receptor γ, and liver X receptor α but was unable to reduce accumulation of triglycerides in Huh.8 cells or reverse the increase in ApoB expression, suggesting a role for increased lipid retention in steatotic hepatocytes. Collectively, these data reveal an important role of NS5A, C/EBPβ, and pCREB in promoting HCV-induced gluconeogenic gene expression and suggest that increased C/EBPβ and NS5A may be essential components leading to increased gluconeogenesis associated with HCV infection.

Adiponectin enhances mitochondrial biogenesis and oxidative metabolism in skeletal muscle. This study aimed to investigate the underlying mechanisms through which adiponectin induces mitochondrial biogenesis in skeletal muscle. Mitochondrial contents, expression, and activation status of p38 mitogen-activated protein kinase (MAPK) and PPARγ coactivator 1α (PGC-1α) were compared between skeletal muscle samples from adiponectin gene knockout, adiponectin-reconstituted, and control mice. Adenovirus-mediated adiponectin and MAPK phosphatase-1 (MKP1) overexpression were used to verify the relationship of MKP1 and PGC-1α in adiponectin-enhanced mitochondrial biogenesis using cultured C2C12 myotubes and PGC-1α knockout mice. An inhibitory effect of adiponectin on MKP1 gene expression was observed in mouse skeletal muscle and cultured C2C12 myotubes. Overexpression of MKP1 attenuated adiponectin-enhanced mitochondrial biogenesis, with significantly decreased PGC-1α expression and p38 MAPK phosphorylation. Although in vivo adiponectin overexpression reduced MKP1 protein levels, the stimulative effects of adiponectin on mitochondrial biogenesis vanished in skeletal muscle of PGC-1α knockout mice. Therefore, our study indicates that adiponectin enhances p38 MAPK/PGC-1α signaling and mitochondrial biogenesis in skeletal muscle by suppressing MKP1 expression.

Cytoplasmic lipid droplets (CLD) in mammary epithelial cells undergo secretion by a unique membrane envelopment process to produce milk lipids. Adipophilin (ADPH/Plin2), a member of the perilipin/PAT family of lipid droplet-associated proteins, is hypothesized to mediate CLD secretion through interactions with apical plasma membrane elements. We found that the secretion of CLD coated by truncated ADPH lacking the C-terminal region encoding a putative four-helix bundle structure was impaired relative to that of CLD coated by full-length ADPH. We used homology modeling and analyses of the solution and membrane binding properties of purified recombinant ADPH C terminus to understand how this region possibly mediates CLD secretion. Homology modeling supports the concept that the ADPH C terminus forms a four-helix bundle motif and suggests that this structure can form stable membrane bilayer interactions. Circular dichroism and protease mapping studies confirmed that the ADPH C terminus is an independently folding α-helical structure that is relatively resistant to urea denaturation. Liposome binding studies showed that the purified C terminus binds to phospholipid membranes through electrostatic dependent interactions, and cell culture studies documented that it localizes to the plasma membrane. Collectively, these data provide direct evidence that the ADPH C terminus forms a stable membrane binding helical structure that is important for CLD secretion. We speculate that interactions between the four-helix bundle of ADPH and membrane phospholipids may be an initial step in milk lipid secretion.

OBJECTIVE Adiponectin is an adipocyte-derived hormone that sensitizes insulin and improves energy metabolism in tissues. This study was designed to investigate the direct regulatory effects of adiponectin on lipid metabolism in adipocytes. RESEARCH DESIGN AND METHODS Basal and hormone-stimulated lipolysis were comparatively analyzed using white adipose tissues or primary adipocytes from adiponectin gene knockout and control mice. To further study the underlying mechanisms through which adiponectin suppresses lipolysis, cultured 3T3-L1 adipocytes and adenovirus-mediated gene transduction were used. RESULTS Significantly increased lipolysis was observed in both adiponectin gene knockout mice and primary adipocytes from these mice. Hormone-stimulated glycerol release was inhibited in adiponectin-treated adipocytes. Adiponectin suppressed hormone-sensitive lipase activation without altering adipose triglyceride lipase and CGI-58 expression in adipocytes. Moreover, adiponectin reduced protein levels of the type 2 regulatory subunit RIIα of protein kinase A by reducing its protein stability. Ectopic expression of RIIα abolished the inhibitory effects of adiponectin on lipolysis in adipocytes. CONCLUSIONS This study demonstrates that adiponectin inhibits lipolysis in adipocytes and reveals a novel function of adiponectin in lipid metabolism in adipocytes.

Since transgenes were first cloned into recombinant adenoviruses almost 30 years ago, a variety of viral vectors have become important tools in genetic research. Viruses adeptly transport genetic material into eukaryotic cells, and replacing all or part of the viral genome with genes of interest or silencing sequences creates a method of gene expression modulation in which the timing and location of manipulations can be specific. The hypothalamo-neurohypophyseal system (HNS), consisting of the paraventricular (PVN) and supraoptic (SON) nuclei in the hypothalamus, regulates fluid balance homeostasis and is highly plastic, yet tightly regulated by extracellular fluid (ECF) osmolality and volume. Its reversible plasticity and physiological relevance make it a good system for studying interactions between gene expression and physiology. Here, four viral vectors were compared for their ability to transduce magnocellular neurosecretory neurons (MNCs) of the SON in adult rats. The vectors included an adenovirus, a lentivirus (HIV) and two serotypes of adeno-associated viruses (AAV5 and AAV2). Though adenovirus and AAV2 vectors have previously been used to transduce SON neurons, HIV and AAV5 have not. All four vectors transduced MNCs, but the AAV vectors were the most effective, transducing large numbers of MNCs, with minimal or no glial transduction. The AAV vectors were injected using a convection enhanced delivery protocol to maximize dispersal through the tissue, resulting in the transduction of neurons throughout the anterior to posterior length of the SON (∼1.5mm). AAV5, but not AAV2, showed some selectivity for SON neurons relative to those in the surrounding hypothalamus.

We have made improvements to E1-deleted adenovirus (Ad) transducing vectors that both substantially reduce the innate inflammatory response provoked by the virus in BALB/c mouse ears and increase the duration of expression of the GFP transgene in BALB/c mouse liver. These improvements result from testing the hypothesis that induction of strong innate responses is primarily a result of the powerful enhancer contained within the strong CMV promoter activating expression of Ad genes retained within the vector. A DNA fragment containing four CTCF-binding sites, which was expected to act as a chromatin insulator, was introduced 5′, 3′, or both 5′ and 3′ of a CMV-GFP cassette in an attempt to reduce activation of Ad gene expression by the enhancer. The presence of this sequence in any of the configurations led to reduction of the innate immune response, as assayed by mouse ear swelling, to the low level induced by a virus deleted for the E1 region and carrying no introduced sequence. In addition, the duration of GFP expression in the liver more than doubled. The prolonged GFP expression indicates that GFP does not play the limiting role in shutting down vector expression. The CTCF-binding sequence introduced appears to act as a chromatin insulator in Ad DNA, but position-independence of the elements in reducing the innate immune response indicate unanticipated complexities in the mechanism by which Ad vectors induce innate immune responses.

E1-deleted adenovirus (FG Ad) transducing vectors are limited for use in vivo by their induction of strong innate and adaptive inflammatory responses. We have examined the contribution of the transgene cassette, particularly the foreign promoter driving transgene expression, in the induction of innate inflammation using a mouse ear model in which swelling is measured as a sensitive surrogate marker of the total innate inflammatory response. The commonly used cytomegalovirus major immediate early (CMV) promoter led to high-level swelling that was independent of transgene expression, while the Rous sarcoma virus and human ubiquitin C promoters led to intermediate levels of swelling and the Ad E1A promoter or no promoter led to equally low levels of swelling. Significant swelling was induced by a virus in which the E1A promoter directed pIX expression, supporting the possibility that activation of expression of Ad genes retained in the vector plays an important role in the inflammatory response. Taken together, our findings support the idea that strong foreign promoters likely play the limiting role in the induction of innate and adaptive immune responses that limit the duration of transgene expression after transduction by FG Ad vectors.

Despite advancements in therapies developed for the treatment of cancer, patient prognosis and mortality rates have improved minimally, and metastasis remains the primary cause of cancer mortality worldwide. An underlying mechanism promoting metastasis in many types of cancer is epithelial-mesenchymal transition (EMT). Here the authors report a novel 3D model of EMT and metastatic breast cancer suitable for high-throughput screening (HTS) drug discovery. The primary assay incorporates the expression of the prognostic biomarker vimentin, as a luciferase reporter of EMT, in basil-like/triple-negative MDA-MB-231 breast carcinoma spheroids. Using this model, the authors developed a number of known antitumor agents as control modulators of EMT. U0126, PKC412, PF2341066, dasatinib, and axitinib downregulated vimentin expression by 70% to 90% as compared to untreated spheroids. Counterassays were developed to measure spheroid viability and the invasive potential of MDA-MB-231 spheroids after small-molecule treatment and used to confirm hits from primary screening. Finally, the authors conducted a pilot screen to validate this model for HTS using a purified library of marine secondary metabolites. From 230 compounds screened, they obtained a Z' score of 0.64, indicative of an excellent assay, and confirmed 4 hits, including isonaamidine B, papuamine, mycalolide E, and jaspamide. This HTS model demonstrates the potential to identify small-molecule modulators of EMT that could be used to discover novel antimetastatic agents for the treatment of cancer.

The survival of naive T cells is compromised in the absence of molecules encoded by the major histocompatibility complex (MHC) while antigen-experienced T cells survive. We hypothesized that survival pressures in an in vivo, MHC-deficient environment would permit enrichment of less frequent antigen-experienced autoreactive cells at the expense of the majority of antigen naive T cells. To test this hypothesis, we generated MHC class I- and class II-deficient mice in NOD and C57Bl/6 (B6) backgrounds, and examined the capacity of adoptively transferred autoimmune-prone NOD T cells, or non-autoimmune prone naive B6 T cells, respectively, to reject transplanted wild-type pancreatic islets or transplantable tumors in the MHC-deficient mice. In the MHC-deficient environment, CD4 T cells acquired self-hostile properties (islet rejection and tumor invasion) that were independent from their genetic propensity for autoreactivity, while CD8 T cells required appropriate prior exposure to antigen in order to survive and function (reject tumor) in this environment; however, disengagement of Tob1, a negative regulator of proliferation, led to a reverse phenotype with regard to persistence of CD4 and CD8 T cells in the MHC-deficient environment. Our data suggest that self-peptide/MHC interactions have dual roles to facilitate survival and restrain autoreactivity, thus acting as integral components of an intrinsic network of negative regulation that maintains tolerance.

Mammary epithelial cells secrete lipids by an envelopment process that produces lipid droplets coated by membranes derived from the plasma membrane and possibly secretory vesicles. This secretion process, which resembles viral budding, is hypothesized to be mediated by specific interactions between molecules on the surface of intracellular lipids and membrane elements of the cell. Multiple lines of evidence indicate that milk lipid secretion occurs through a tripartite complex between the integral transmembrane protein, butyrophilin (BTN); the soluble metabolic enzyme, xanthine oxidoreductase (XOR); and the lipid droplet surface protein, adipophilin (ADPH). However, topological evidence from freeze-fracture replica immunolabelling (FRIL) challenge this model and suggests that milk lipid secretion is mediated by butyrophilin alone. Advances in our understanding of the molecular, structural, and functional properties of these proteins now make it possible to understand the physiological functions of each of these molecules in detail and to identify the specific molecular determinants that mediate milk lipid secretion.

Prolactin (PRL) and glucocorticoids act synergistically to stimulate transcription of the beta-casein milk protein gene. Signal transducer and activator of transcription 5 (Stat5) mediates PRL-dependent trans-activation, and glucocorticoid potentiation occurs through cross talk between glucocorticoid receptor (GR) and Stat5 at the beta-casein promoter. In the mouse, progesterone withdrawal leads to terminal differentiation and secretory activation of the mammary gland at parturition, indicating progesterone's role in repressing milk protein gene expression during pregnancy. To investigate the mechanism of the inhibitory action of progesterone, experiments were performed with cell culture systems reconstituted to express progesterone receptor (PR), the PRL receptor/Stat5 signaling pathway, and GR, enabling evaluation of PR, GR, and Stat5 interactions at the beta-casein promoter. With COS-1, normal murine mammary gland, HC-11, and primary mammary epithelial cells, progestin-PR directly repressed the PRL receptor/Stat5a signaling pathway's mediation of PRL-induced beta-casein transcription. Progestin-PR also inhibited glucocorticoid-GR enhancement of PRL induced trans-activation of beta-casein. Inhibition depended on a functional PR DNA binding domain and specific PR-DNA interactions at the beta-casein promoter. Chromatin immunoprecipitation assays in HC-11 cells revealed recruitment of PR and Stat5a to the beta-casein promoter by progestin or PRL, respectively. Recruitment was disrupted by cotreatment with progestin and PRL, suggesting a mutual interference between activated PR and Stat5a. Without PRL, progestin-PR also recruited Stat5a to the beta-casein promoter, suggesting that recruitment of an unactivated form of Stat5a may contribute to inhibition of beta-casein by progesterone. These results define a negative cross talk between PR and Stat5a/GR that may contribute to the physiological role of progesterone to repress lactogenic hormone induction of the beta-casein gene in the mammary gland during pregnancy.

This study investigated how maternal overnutrition and obesity regulate expression and activation of proteins that facilitate lipid transport in the placenta. To create a maternal overnutrition and obesity model, primiparous C57BL/6 mice were fed a high-fat (HF) diet throughout gestation. Fetuses from HF-fed dams had significantly increased serum levels of free fatty acid and body fat. Despite no significant difference in placental weight, lipoprotein lipase (LPL) protein levels and activity were remarkably elevated in placentas from HF-fed dams. Increased triglyceride content and mRNA levels of CD36, VLDLr, FABP3, FABPpm, and GPAT2 and -3 were also found in placentas from HF-fed dams. Although both peroxisome proliferator–activated receptor-γ (PPARγ) and CCAAT/enhancer binding protein-α protein levels were significantly increased in placentas of the HF group, only PPARγ exhibited a stimulative effect on LPL expression in cultured JEG-3 human trophoblasts. Maternal HF feeding remarkably decreased SIRT1 expression in placentas. Through use of an SIRT1 activator and inhibitor and cultured trophoblasts, an inhibitory effect of SIRT1 on LPL expression was demonstrated. We also found that SIRT1 suppresses PPARγ expression in trophoblasts. Most importantly, inhibition of PPARγ abolished the SIRT1-mediated regulatory effect on LPL expression. Together, these results indicate that maternal overnutrition induces LPL expression in trophoblasts by reducing the inhibitory effect of SIRT1 on PPARγ.

Despite the utility of recombinant adenoviral vectors in basic research, their therapeutic promise remains unfulfilled. Most engineered adenoviral vectors use a heterologous promoter to transcribe a foreign gene. We show that adenoviruses containing the cytomegalovirus immediate-early promoter induce the expression of the proapoptotic cellular protein TAp73 via the cyclin-dependent kinase-retinoblastoma protein-E2F pathway in murine embryonic fibroblasts. Cells transduced with these vectors also expressed high levels of the adenoviral E4-orf6/7 and E2A proteins. By contrast, adenoviruses containing the ubiquitin C promoter failed to elicit these effects. E4-orf6/7 is necessary and sufficient for increased TAp73 expression, as shown by using retrovirus-mediated E4-orf6/7 expression and adenovirus with the E4-orf6/7 gene deleted. Activation of TAp73 likely occurs via E4-orf6/7-induced dimerization of E2F and subsequent binding to the inverted E2F-responsive elements within the TAp73 promoter. In addition, adenoviral vectors containing the cytomegalovirus immediate-early promoter, but not the ubiquitin C promoter, cooperated with chemotherapeutic agents to decrease cellularity in vitro. In contrast to murine embryonic fibroblasts, adenoviruses containing the ubiquitin C promoter, but not the cytomegalovirus immediate-early promoter, induced both E4-orf6/7 and TAp73 in human foreskin fibroblasts, emphasizing the importance of cellular context for promoter-dependent effects. Because TAp73 is important for the efficacy of chemotherapy, adenoviruses that increase TAp73 expression may enhance cancer therapies by promoting apoptosis. However, such adenoviruses may impair the long-term survival of transduced cells during gene replacement therapies. Our findings reveal previously unknown effects of foreign promoters in recombinant adenoviral vectors and suggest means to improve the utility of engineered adenoviruses by better controlling their impact on viral and cellular gene expression.

The spatiotemporal regulation of cAMP can generate microdomains just beneath the plasma membrane where cAMP increases are larger and more dynamic than those seen globally. Real-time measurements of cAMP using mutant cyclic nucleotide-gated ion channel biosensors, pharmacological tools and RNA interference (RNAi) were employed to demonstrate a subplasmalemmal cAMP signaling module in living cells. Transient cAMP increases were observed upon stimulation of HEK293 cells with prostaglandin E1. However, pretreatment with selective inhibitors of type 4 phosphodiesterases (PDE4), protein kinase A (PKA) or PKA/A-kinase anchoring protein (AKAP) interaction blocked an immediate return of subplasmalemmal cAMP to basal levels. Knockdown of specific membrane-associated AKAPs using RNAi identified gravin (AKAP250) as the central organizer of the PDE4 complex. Co-immunoprecipitation confirmed that gravin maintains a signaling complex that includes PKA and PDE4D. We propose that gravin-associated PDE4D isoforms provide a means to rapidly terminate subplasmalemmal cAMP signals with concomitant effects on localized ion channels or enzyme activities.

Valproic acid (VPA) has been used for the treatment of epilepsy and bipolar disorders for more than 30 yr. Obesity and insulin resistance are common side effects of VPA treatment. Adiponectin is an adipocyte-derived protein that plays an important role in controlling insulin sensitivity and glucose homeostasis. In this report, we examined the effects of VPA on adiponectin gene expression in C57BL/6J mice and in differentiated 3T3-L1 adipocytes. VPA treatment significantly decreased adiponectin protein and mRNA levels in both mice and 3T3-L1 adipocytes. The adipocyte study showed that VPA inhibited adiponectin gene expression in a dose- and time-dependent manner. Repression of adiponectin expression by VPA occurred at the transcription level and correlated with inhibition of histone deacetylase activity. Therapeutic concentrations of VPA increased overall histone acetylation and increased adiponectin promoter-driven luciferase expression in fibroblasts, but decreased adiponectin promoter activity in differentiated 3T3-L1 adipocytes. VPA treatment decreased adipogenic transcription factor CCAAT/enhancer binding protein-alpha (C/EBPalpha) levels and binding of C/EBPalpha to the adiponectin promoter without altering the levels of peroxisome proliferator-activated receptor-gamma and steroid regulatory element binding protein-1. Furthermore, VPA did not suppress adiponectin gene expression in C/EBPalpha gene-deficient adipocytes that stably expressed exogenous peroxisome proliferator-activated receptor-gamma2. Together, these results demonstrate that histone deacetylase inhibitor VPA suppresses adiponectin gene expression in mature adipocytes. The study also provides evidence that diminished C/EBPalpha protein level and decreased binding at the adiponectin promoter mediate the inhibitory effects of VPA on adiponectin gene transcription.

The site-specific insertion of an unnatural amino acid into proteins in vivo via nonsense suppression has resulted in major advances in recent years. The ability to incorporate two different unnatural amino acids in vivo would greatly increase the scope and impact of unnatural amino acid mutagenesis. Here, we show the concomitant suppression of an amber and an ochre codon in a single mRNA in mammalian cells by importing a mixture of aminoacylated amber and ochre suppressor tRNAs. This result provides a possible approach to site-specific insertion of two different unnatural amino acids into any protein of interest in mammalian cells. To our knowledge, this result also represents the only demonstration of concomitant suppression of two different termination codons in a single gene in vivo.

Previous studies using mannose-binding lectin (MBL) and complement C4–deficient mice have suggested that the lectin pathway (LP) is not required for the development of inflammatory arthritis in the collagen Ab–induced arthritis (CAIA) model. MBL, ficolins and collectin-11 are key LP pattern recognition molecules that associate with three serine proteases—MASP-1, MASP-2, and MASP-3—and with two MBL-associated proteins designated sMAP and MBL-associated protein of 44kDA (MAp44). Recent studies have shown that MAp44, an alternatively spliced product of the MASP-1/3 gene, is a competitive inhibitor of the binding of the recognition molecules to all three MASPs. In these studies, we examined the effect of treatment of mice with adenovirus (Ad) programmed to express human MAp44 (AdhMAp44) on the development of CAIA. AdhMAp44 and Ad programming GFP (AdGFP) expression were injected i.p. in C57BL/6 wild type mice prior to the induction of CAIA. AdhMAp44 significantly reduced the clinical disease activity (CDA) score by 81% compared with mice injected with AdGFP. Similarly, histopathologic injury scores for inflammation, pannus, cartilage and bone damage, as well as C3 deposition in the cartilage and synovium, were significantly reduced by AdhMAp44 pretreatment. Mice treated with AdmMAp44, programming expression of mouse MAp44, also showed significantly decreased CDA score and histopathologic injury scores. In addition, administration of AdhMAp44 significantly diminished the severity of Ross River virus–induced arthritis, an LP-dependent model. Our study provides conclusive evidence that an intact complement LP is essential to initiate CAIA, and that MAp44 may be an appropriate treatment for inflammatory arthritis.

We hypothesized that cAMP response element-binding protein (CREB) could function as a molecular determinant of smooth muscle cell fate. In arterial sections from the systemic and pulmonary circulation, CREB content was high in proliferation-resistant medial subpopulations of smooth muscle cells and low in proliferation-prone regions. In vessels from neonatal calves exposed to chronic hypoxia, CREB content was depleted and smooth muscle cell (SMC) proliferation was accelerated. Induction of quiescence by serum deprivation in culture led to increased CREB content. Highly proliferative SMC in culture were observed to have low CREB content. Exposure to proliferative stimuli such as hypoxia or platelet-derived growth factor decreased SMC CREB content. Assessment of CREB gene transcription by nuclear run-on analysis and transcription from a CREB promoter-luciferase construct indicate that CREB levels in SMC are in part controlled at the level of transcription. Overexpression of wild type or constitutively active CREB in primary cultures of SMC arrested cell cycle progression. Additionally, expression of constitutively active CREB decreased both proliferation and chemokinesis. Consistent with these functional properties, active CREB decreased the expression of multiple cell cycle regulatory genes, as well as genes encoding growth factors, growth factor receptors, and cytokines. Our data suggest a unique mode of cellular phenotype determination at the level of the nuclear content of CREB.

Most adenovirus transducing vectors have the cytomegalovirus major immediate-early (CMV) or the Rous sarcoma virus long terminal repeat (RSV) promoter driving expression of the transgene. Both of these promoters are highly active in transfection and transduction assays in 293 cells, in which transducing vectors are constructed and grown, and in HeLa cells. The CMV promoter exhibits rapid activation while the RSV promoter exhibits a lag prior to the onset of viral DNA replication in transduction assays. While the use of very strong promoters facilitates expression of the transgene, high-level expression of certain gene products hinders virus construction and growth. For such genes, the use of the adenovirus type 5 E1A promoter offers advantages. The E1A promoter exhibits modest activity in HeLa cells after transfection or transduction, but very little activity in 293 cells, suggesting that the E1A promoter would permit construction and growth of vectors encoding deleterious gene products that could not be constructed with the CMV and RSV promoters. This idea was tested through attempts to construct viruses encoding the immunoglobulin loop 6 and transmembrane regions of the prostaglandin F2α receptor regulatory protein (FPRP), a product that inhibits adenovirus vector construction for reasons that are not clear. Only the E1A promoter permitted construction and growth of the transducing vector encoding the fragment of FPRP.

cAMP, the classical second messenger, regulates many diverse cellular functions. The primary effector of cAMP signals, protein kinase A, differentially phosphorylates hundreds of cellular targets. Little is known, however, about the spatial and temporal nature of cAMP signals and their information content. Thus, it is largely unclear how cAMP, in response to different stimuli, orchestrates such a wide variety of cellular responses. Previously, we presented evidence that cAMP is produced in subcellular compartments near the plasma membrane, and that diffusion of cAMP from these compartments to the bulk cytosol is hindered. Here we report that a uniform extracellular stimulus initiates distinct cAMP signals within different cellular compartments. By using cyclic nucleotide-gated ion channels engineered as cAMP biosensors, we found that prostaglandin E 1 stimulation of human embryonic kidney cells caused a transient increase in cAMP concentration near the membrane. Interestingly, in the same time frame, the total cellular cAMP rose to a steady level. The decline in cAMP levels near the membrane was prevented by pretreatment with phosphodiesterase inhibitors. These data demonstrate that spatially and temporally distinct cAMP signals can coexist within simple cells.

In utero gene therapy (IUGT) offers the promise of treating a wide variety of genetic diseases before the development of disease manifestations. The most convenient and potentially easiest method of targeting the fetus is through injection into the amniotic cavity. For long-term correction of genetic defects, retroviral vectors have great potential as a tool for gene therapy strategies. However, retroviral vectors are limited by growth to low titers. In an attempt to increase the amount of vector particles delivered and assess the potential of intraamniotic administration, we injected a retroviral vector producer cell line encoding the lacZ gene into the amniotic fluid of a nonhuman primate model. After birth the infants were analyzed for vector-mediated transduction. Two of four fetuses were successfully transduced, with transgene expression detected in the esophagus, trachea, and stomach. In some sections of tissue, nearly 100% of the cells lining the lumen of these tissues were positive for transduction. Although successful, the limited number of tissues in which transduction was observed led to an in vitro analysis of the effects of amniotic fluid (AF). The presence of amniotic fluid inhibited transduction by 99%. AF affected both the transducing activity of the vector and the health of the packaging cells. The negative effects of AF were gestational age dependent; greater inhibition was observed from AF collected at later stages of pregnancy. The fact that transduction was successful despite these negative effects indicates that this approach is a promising strategy for gene therapy.

Phosphodiesterases (PDEs) catalyze the hydrolysis of the second messengers cAMP and cGMP. However, little is known about how PDE activity regulates cyclic nucleotide signals in vivo because, outside of specialized cells, there are few methods with the appropriate spatial and temporal resolution to measure cyclic nucleotide concentrations. We have previously demonstrated that adenovirus-expressed, olfactory cyclic nucleotide–gated channels provide real-time sensors for cAMP produced in subcellular compartments of restricted diffusion near the plasma membrane (Rich, T.C., K.A. Fagan, H. Nakata, J. Schaack, D.M.F. Cooper, and J.W. Karpen. 2000. J. Gen. Physiol. 116:147–161). To increase the utility of this method, we have modified the channel, increasing both its cAMP sensitivity and specificity, as well as removing regulation by Ca2+-calmodulin. We verified the increased sensitivity of these constructs in excised membrane patches, and in vivo by monitoring cAMP-induced Ca2+ influx through the channels in cell populations. The improved cAMP sensors were used to monitor changes in local cAMP concentration induced by adenylyl cyclase activators in the presence and absence of PDE inhibitors. This approach allowed us to identify localized PDE types in both nonexcitable HEK-293 and excitable GH4C1 cells. We have also developed a quantitative framework for estimating the KI of PDE inhibitors in vivo. The results indicate that PDE type IV regulates local cAMP levels in HEK-293 cells. In GH4C1 cells, inhibitors specific to PDE types I and IV increased local cAMP levels. The results suggest that in these cells PDE type IV has a high Km for cAMP, whereas PDE type I has a low Km for cAMP. Furthermore, in GH4C1 cells, basal adenylyl cyclase activity was readily observable after application of PDE type I inhibitors, indicating that there is a constant synthesis and hydrolysis of cAMP in subcellular compartments near the plasma membrane. Modulation of constitutively active adenylyl cyclase and PDE would allow for rapid control of cAMP-regulated processes such as cellular excitability.

The current, static methodologies for measuring cyclic AMP (cAMP) may underestimate its regulatory properties. Here, we have exploited the Ca2+-conducting properties of cyclic nucleotide-gated (CNG) channels to measure cAMP in live cells, in response to various stimuli. We placed a mutated CNG channel with high sensitivity to cAMP in adenovirus to maximize and render facile its expression in numerous cell types. The ready, continuous nature of the readout contrasted with the traditional approach, which yielded similar static information, but lacked any continuous or interactive qualities. It seems fair to predict that this readily adopted approach will broaden the perception of cAMP signaling.

3T3-L1 cells have been used as a model to study the differentiation and physiology of adipocytes. Exogenous expression of proteins in these cells offers the prospect of understanding the protein's function(s) in adipose tissue. Viral vectors, in particular, adenovirus, have proven to be a powerful means for introduction of genes into many cell types. However, we have previously shown that 3T3-L1 cells are inefficiently transduced by adenovirus (Orlicky, D. J., and J. Schaack. 2001. J. Lipid Res. 42: 460–466). To overcome the inefficient transduction, we have stably introduced the gene-encoding coxsackie and adenovirus receptor (CAR), which was modified by deletion of the region encoding the cytoplasmic tail, into 3T3-L1 cells. 3T3-L1 CARΔ1 cells are transduced approximately 100-fold more efficiently than parental 3T3-L1 cells. 3T3-L1 CARΔ1 cells should prove to be a useful tool for examination of exogenous protein expression in fat cells. —Orlicky, D. J., J. DeGregori, and J. Schaack. Construction of stable coxsackievirus and adenovirus receptor-expressing 3T3-L1 cells.

Aims/hypothesisAdiponectin is an adipocyte-derived hormone that plays an important role in energy homeostasis. The main objective of this study was to investigate whether or not adiponectin regulates brown adipose tissue (BAT) activation and thermogenesis.MethodsCore body temperatures (CBTs) of genetic mouse models were monitored at room temperature and during cold exposure. Cultured brown adipocytes and viral vector-mediated gene transduction were used to study the regulatory effects of adiponectin on Ucp1 gene expression and the underlying mechanisms.ResultsThe CBTs of adiponectin knockout mice (Adipoq(-/-)) were significantly higher than those of wild type (WT) mice both at room temperature and during the cold (4°C) challenge. Conversely, reconstitution of adiponectin in Adipoq(-/-) mice significantly blunted β adrenergic receptor agonist-induced thermogenesis of interscapular BAT. After 10 days of intermittent cold exposure, Adipoq(-/-) mice exhibited higher UCP1 expression and more brown-like structure in inguinal fat than WT mice. Paradoxically, we found that the anti-thermogenic effect of adiponectin requires neither AdipoR1 nor AdipoR2, two well-known adiponectin receptors. In sharp contrast to the anti-thermogenic effects of adiponectin, AdipoR1 and especially AdipoR2 promote BAT activation. Mechanistically, adiponectin was found to inhibit Ucp1 gene expression by suppressing β3-adrenergic receptor expression in brown adipocytes.Conclusions/interpretationThis study demonstrates that adiponectin suppresses thermogenesis, which is likely to be a mechanism whereby adiponectin reduces energy expenditure.

In nonexcitable cells, we had previously established that Ca2+-sensitive adenylyl cyclases, whether expressed endogenously or heterologously, were regulated exclusively by capacitative Ca2+ entry (Fagan, K. A., Mahey, R. and Cooper, D. M. F. (1996) J. Biol. Chem. 271, 12438–12444; Fagan, K. A., Mons, N., and Cooper, D. M. F. (1998) J. Biol. Chem. 273, 9297–9305). Relatively little is known about how these enzymes are regulated by Ca2+ in excitable cells, where they predominate. Furthermore, no effort has been made to determine whether the prominent voltage-gated Ca2+ entry, which typifies excitable cells, overwhelms the effect of any capacitative Ca2+ entry that may occur. In the present study, we placed the Ca2+-stimulable, adenylyl cyclase type VIII in an adenovirus vector to optimize its expression in the pituitary-derived GH4C1 cell line. In these cells, a modest degree of capacitative Ca2+ entry could be discerned in the face of a dramatic voltage-gated Ca2+ entry. Nevertheless, both modes of Ca2+ entry were equally efficacious at stimulating adenylyl cyclase. A striking release of Ca2+from intracellular stores, triggered either by ionophore or thyrotrophin-releasing hormone, was incapable of stimulating the adenylyl cyclase. It thus appears as though the intimate colocalization of adenylyl cyclase with capacitative Ca2+ entry channels is an intrinsic property of these molecules, regardless of whether they are expressed in excitable or nonexcitable cells.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the appearance of intracytoplasmic inclusions called Lewy bodies (LB) in dopamine neurons in the substantia nigra and the progressive loss of these neurons. Recently, mutations in the alpha-synuclein gene have been identified in early-onset familial PD, and alpha-synuclein has been shown to be a major component of LB in all patients. Yet, the pathophysiological function of alpha-synuclein remains unknown. In this report, we have investigated the toxic effects of adenovirus-mediated alpha-synuclein overexpression on dopamine neurons in rat primary mesencephalic cultures and in a rat dopaminergic cell line - the large T-antigen immortalized, mesencephalon-derived 1RB3AN27 (N27). Adenovirus-transduced cultures showed high-level expression of alpha-synuclein within the cells. Overexpression of human mutant alpha-synuclein (Ala(53)Thr) selectively induced apoptotic programmed cell death of primary dopamine neurons as well as N27 cells. The mutant protein also potentiated the neurotoxicity of 6-hydroxydopamine (6-OHDA). By contrast, overexpression of wild-type human alpha-synuclein was not directly neurotoxic but did increase cell death after 6-OHDA. Overexpression of wild-type rat alpha-synuclein had no effect on dopamine cell survival or 6-OHDA neurotoxicity. These results indicate that overexpression of human mutant alpha-synuclein directly leads to dopamine neuron death, and overexpression of either human mutant or human wild-type alpha-synuclein renders dopamine neurons more vulnerable to neurotoxic insults.

Cyclic AMP is a ubiquitous second messenger that coordinates diverse cellular functions. Current methods for measuring cAMP lack both temporal and spatial resolution, leading to the pervasive notion that, unlike Ca2+, cAMP signals are simple and contain little information. Here we show the development of adenovirus-expressed cyclic nucleotide–gated channels as sensors for cAMP. Homomultimeric channels composed of the olfactory α subunit responded rapidly to jumps in cAMP concentration, and their cAMP sensitivity was measured to calibrate the sensor for intracellular measurements. We used these channels to detect cAMP, produced by either heterologously expressed or endogenous adenylyl cyclase, in both single cells and cell populations. After forskolin stimulation, the endogenous adenylyl cyclase in C6-2B glioma cells produced high concentrations of cAMP near the channels, yet the global cAMP concentration remained low. We found that rapid exchange of the bulk cytoplasm in whole-cell patch clamp experiments did not prevent the buildup of significant levels of cAMP near the channels in human embryonic kidney 293 (HEK-293) cells expressing an exogenous adenylyl cyclase. These results can be explained quantitatively by a cell compartment model in which cyclic nucleotide–gated channels colocalize with adenylyl cyclase in microdomains, and diffusion of cAMP between these domains and the bulk cytosol is significantly hindered. In agreement with the model, we measured a slow rate of cAMP diffusion from the whole-cell patch pipette to the channels (90% exchange in 194 s, compared with 22–56 s for substances that monitor exchange with the cytosol). Without a microdomain and restricted diffusional access to the cytosol, we are unable to account for all of the results. It is worth noting that in models of unrestricted diffusion, even in extreme proximity to adenylyl cyclase, cAMP does not reach high enough concentrations to substantially activate PKA or cyclic nucleotide–gated channels, unless the entire cell fills with cAMP. Thus, the microdomains should facilitate rapid and efficient activation of both PKA and cyclic nucleotide–gated channels, and allow for local feedback control of adenylyl cyclase. Localized cAMP signals should also facilitate the differential regulation of cellular targets.

Adenoviruses offer great potential as gene therapy agents but are limited by the strong inflammatory response that occurs in response to the recombinant virus. Since the degree of inflammation correlates in part with the potential of the viral vector for replication, we constructed a preterminal protein (pTP) deletion mutant adenovirus type 5 vector, Ad5 dl 308 ΔpTP β-gal, that is replication incompetent due to deletion of the pTP gene and that has the E1 genes replaced by the Escherichia coli lacZ reporter gene under the control of the cytomegalovirus major immediate-early promoter. This virus was compared with a first-generation, replication-defective adenovirus vector, Ad5 dl 308β-gal, that is isogenic except that it contains a wild-type pTP gene. To examine transduction efficiency and induction of inflammation, we developed a novel system involving intradermal injection of BALB/c mouse ears. Mouse ears can be accurately measured to determine the degree of edema as an indirect measurement of inflammation. Edema and inflammation were induced in a dose- and time-dependent manner by both viruses and correlated well. LacZ activity correlated inversely with edema and inflammation. The pTP-defective vector Ad5 dl 308 ΔpTP β-gal transduced mouse ears much more efficiently and induced edema and inflammatory cell infiltration approximately 10-fold less efficiently than the first-generation vector Ad5 dl 308β-gal. The diminished inflammatory response and increased efficiency of transduction observed with Ad5 dl 308 ΔpTP β-gal indicate its promise as a gene therapy agent for other tissues. The results also demonstrate that the mouse ear model offers potential for the study of adenovirus-induced inflammation because of the ready access of the ears, the relative ease of continuous measurement, and the sensitivity to adenovirus transducing vectors.

The finding that many human melanomas express distinct antigens that can be recognised by specific cytolytic T lymphocytes (CTL) implies that immunotherapeutic strategies against this cancer might prove effective. The ex vivo delivery of a tumour-associated antigen to autologous cells and the subsequent re-administration of these cells to the patient might prove effective in boosting the T cell immune response. Recombinant human adenoviral vectors provide an efficient delivery system and have many advantages over other viral and non-viral delivery vehicles. Infection of a panel of human melanoma cell lines by AdCMVMAGE-1, a novel recombinant adenovirus which incorporates the full-length MAGE-1 cDNA, was shown to induce production of high levels of MAGE-1 protein. Incubation of transduced HLA-A1 expressing melanoma cell lines with 2 anti-MAGE-1.A1 CTL clones resulted in specific recognition and lysis of target cells, indicating that the exogenous MAGE-1 protein was processed and presented in a normal manner. Furthermore, quantitative analyses demonstrated a correlation between the efficiency of transduction and the proportion of cells lysed. Importantly for future clinical trials, stimulation of peripheral blood lymphocytes (PBLs) from a melanoma patient by AdCMVMAGE-1-transduced autologous cells resulted in the generation of specific CTLs against the MAGE-1 antigen. Together, our data emphasize the utility of adenoviruses as vaccination vehicles and highlight the potential efficacy of this approach for the treatment of melanoma. Int. J. Cancer 72:1045–1055, 1997. © 1997 Wiley-Liss, Inc.

Hypoxia-inducible transcription factors HIF-1α and HIF-2α can contribute to pulmonary hypertension and vascular remodeling, but their mechanisms remain unknown. This study investigated the role of HIF-1α and HIF-2α in pulmonary artery endothelial and smooth muscle cells. The exposure of human pulmonary artery endothelial cells (HPAECs) to hypoxia (10% O2 or 5% O2) increased proliferation over 48 hours, compared with cells during normoxia (21% O2). The adenovirus-mediated overexpression of HIF-2α that is transcriptionally active during normoxia (mutHIF-2α) increased HPAEC proliferation, whereas the overexpression of HIF-1α, which is transcriptionally active during normoxia (mutHIF-1α), exerted no effect. The knockdown of HIF-2α decreased proliferation during both hypoxia and normoxia. Both HIFs increased migration toward fibrinogen, used as a chemoattractant. In an angiogenesis tube formation assay, mutHIF-2α–transduced cells demonstrated increased tube formation, compared with the mutHIF-1α–transduced cells. In addition, the tubes formed in HIF-2α–transduced cells were more enduring than those in the other groups. In human pulmonary artery smooth muscle cells (HPASMCs), chronic exposure to hypoxia increased proliferation, compared with cells during normoxia. For HPASMCs transduced with adenoviral HIFs, HIF-1α increased proliferation, whereas HIF-2α exerted no such effect. Thus, HIF-1α and HIF-2α exert differential effects in isolated cells of the human pulmonary vasculature. This study demonstrates that HIF-2α plays a predominant role in the endothelial growth pertinent to the remodeling process. In contrast, HIF-1α appears to play a major role in pulmonary smooth muscle growth. The selective targeting of each HIF in specific target cells may more effectively counteract hypoxic pulmonary hypertension and vascular remodeling.

Milk lipid is secreted by a unique process, during which triacylglycerol droplets bud from mammary cells coated with an outer bilayer of apical membrane. In all current schemes, the integral protein butyrophilin 1A1 (BTN) is postulated to serve as a transmembrane scaffold, which interacts, either with itself, or with the peripheral proteins, xanthine oxidoreductase (XOR) and possibly perilipin-2 (PLIN2), to form an immobile bridging complex between the droplet and apical surface. In one such scheme, BTN on the surface of cytoplasmic lipid droplets interacts directly with BTN in the apical membrane without binding to either XOR or PLIN2. We tested these models using both biochemical and morphological approaches. BTN was concentrated in the apical membrane in all species examined and contained mature N-linked glycans. We found no evidence for the association of unprocessed BTN with intracellular lipid droplets. BTN-enhanced-green-fluorescent-protein was highly mobile in areas of mouse milk-lipid droplets that had not undergone post-secretion changes, and endogenous mouse BTN comprised only 0.5–0.7%, (w/w) of the total protein, i.e., over fifty-fold less than in the milk-lipid droplets of cow and other species. These data are incompatible with models of milk-lipid secretion in which BTN is the major component of an immobile global adhesive complex and suggest that interactions between BTN and other proteins at the time of secretion are more transient than previously predicted. The high mobility of BTN in lipid droplets, mark it as a potential mobile signaling molecule in milk.

Fibroblasts derived from the cerebral cortex of an aged Bonnet monkey (Macaca radiata) were utilized to express recombinant cDNAs encoding rat glial-cell-line-derived neurotrophic factor (GDNF) and human prepro brain-derived neurotrophic factor (BDNF) by lipofection. The cells showed stable expression and secretion of biologically active proteins. Conditioned medium from fibroblasts expressing BDNF or GDNF increased the number of surviving mesencephalic tyrosine-hydroxylase-immunoreactive neurons after 7 days in culture. The trophic effects of BDNF and GDNF were examined at two different plating densities of embryonic mesencephalic cells. At 50 000 cells/cm2 plating density, treatment of the mesencephalic cultures with BDNF-conditioned medium increased the number of tyrosine-hydroxylase-immunoreactive neurons by about 40% compared with vector-transfected control. At the same plating density, GDNF-conditioned medium increased the number of surviving tyrosine-hydroxylase-immunoreactive neurons above the vector-transfected control by 30%. When the tissue was plated at a higher density, viz., 75 000 cells/cm2, the number of tyrosine-hydroxylase-immunoreactive neurons increased by 41% with BDNF-conditioned medium, and by 56% with GDNF-conditioned medium above vector-transfected controls. Conditioned medium from cells secreting GDNF was also found to reduce the number of apoptotic tyrosine-hydroxylase-immunoreactive cells by 50%.

293 cell lines that inducibly express high levels of adenovirus type 5 precursor terminal protein (pTP) under the control of a tetracycline-dependent promoter were constructed. To construct the cell lines expressing pTP, 293 cells were stably transfected with a plasmid encoding the tetracycline repressor/VP16 transactivator protein (tTA) using selection with hygromycin. Cell lines that expressed high levels of tTA activity were then stably transfected with plasmids in which pTP expression is directed by the tTA-dependent promoter from either a cDNA or a modified genomic construct using selection with G418. Cell lines that expressed high, inducible levels of pTP efficiently complemented a temperature-sensitive pTP mutant virus for growth and plaque formation at the nonpermissive temperature.