Jeffrey M. Leiden

An Integrated Stress Response Regulates Amino Acid Metabolism and Resistance to Oxidative Stress

Eukaryotic cells respond to unfolded proteins in their endoplasmic reticulum (ER stress), amino acid starvation, or oxidants by phosphorylating the alpha subunit of translation initiation factor 2 (eIF2alpha). This adaptation inhibits general protein synthesis while promoting translation and expression of the transcription factor ATF4. Atf4(-/-) cells are impaired in expressing genes involved in amino acid import, glutathione biosynthesis, and resistance to oxidative stress. Perk(-/-) cells, lacking an upstream ER stress-activated eIF2alpha kinase that activates Atf4, accumulate endogenous peroxides during ER stress, whereas interference with the ER oxidase ERO1 abrogates such accumulation. A signaling pathway initiated by eIF2alpha phosphorylation protects cells against metabolic consequences of ER oxidation by promoting the linked processes of amino acid sufficiency and resistance to oxidative stress.

Immune responses to transgene-encoded proteins limit the stability of gene expression after injection of replication-defective adenovirus vectors.

The use of replication–defective adenoviruses (RDAd) for human gene therapy has been limited by host immune responses that result in transient recombinant gene expression in vivo. It remained unclear whether these immune responses were directed predominantly against viral proteins or, alternatively, against foreign transgene–encoded proteins. In this report, we have compared the stability of recombinant gene expression in adult immunocompetent mice following intramuscular (i.m.) injection with identical RDAd encoding self (murine) or foreign (human) erythropoietin. Our results demonstrate that immune responses directed against foreign transgene–encoded proteins are the major determinants of the stability of gene expression following i.m. injection of RDAd. Moreover, we demonstrate long–term recombinant gene expression in immunocompetent animals following a single i.m. injection of RDAd encoding a self protein. These findings are important for the design of future preclinical and clinical gene therapy trials.

Cytostatic gene therapy for vascular proliferative disorders with a constitutively active form of the retinoblastoma gene product

Vascular smooth muscle cell (SMC) proliferation in response to injury is an important etiologic factor in vascular proliferative disorders such as atherosclerosis and restenosis after balloon angioplasty. The retinoblastoma gene product (Rb) is present in the unphosphorylated and active form in quiescent primary arterial SMCs, but is rapidly inactivated by phosphorylation in response to growth factor stimulation in vitro. A replication-defective adenovirus encoding a nonphosphorylatable, constitutively active form of Rb was constructed. Infection of cultured primary rat aortic SMCs with this virus inhibited growth factor-stimulated cell proliferation in vitro. Localized arterial infection with the virus at the time of balloon angioplasty significantly reduced SMC proliferation and neointima formation in both the rat carotid and porcine femoral artery models of restenosis. These results demonstrate the role of Rb in regulating vascular SMC proliferation and suggest a gene therapy approach for vascular proliferative disorders associated with arterial injury.

Adenovirus-mediated over-expression of the cyclin/cyclin-dependent kinase inhibitor, p21 inhibits vascular smooth muscle cell proliferation and neointima formation in the rat carotid artery model of balloon angioplasty.

Vascular smooth muscle cell (VSMC) proliferation after arterial injury is important in the pathogenesis of a number of vascular proliferative disorders, including atherosclerosis and restenosis after balloon angioplasty. Thus, a better understanding of the molecular mechanisms underlying VSMC proliferation in response to arterial injury would have important therapeutic implications for patients with atherosclerotic vascular disease. The p21 protein is a negative regulator of mammalian cell cycle progression that functions both by inhibiting cyclin dependent kinases (CDKs) required for the initiation of S phase, and by binding to and inhibiting the DNA polymerase delta co-factor, proliferating cell nuclear antigen (PCNA). In this report, we show that adenovirus-mediated over-expression of human p21 inhibits growth factor-stimulated VSMC proliferation in vitro by efficiently arresting VSMCs in the G1 phase of the cell cycle. This p21-associated cell cycle arrest is associated both with significant inhibition of the phosphorylation of the retinoblastoma gene product (Rb) and with the formation of complexes between p21 and PCNA in VSMCs. In addition, we demonstrate that localized arterial infection with a p21-encoding adenovirus at the time of balloon angioplasty significantly reduced neointimal hyperplasia in the rat carotid artery model of restenosis. Taken together, these studies demonstrate the important role of p21 in regulating Rb phosphorylation and cell cycle progression in VSMC, and suggest a novel cytostatic gene therapy approach for restenosis and related vascular proliferative disorders.

The Ets-1 Transcription Factor Is Required for the Development of Natural Killer Cells in Mice

In this report we have investigated the role of the Ets-1 transcription factor in the differentiation of the NK cell lineage in mice. Splenic NK cells express high levels of Ets-1. Ets-1-deficient mice produced by gene targeting developed mature erythrocytes, monocytes, neutrophils, and T and B lymphocytes. However, spleens from the Ets-1-deficient mice contained significantly reduced numbers of natural killer (NK) cells, and splenocytes from these mice lacked detectable cytolytic activity against NK cell targets in vitro. Moreover, unlike wild-type animals, Ets-1-deficient mice developed tumors following subcutaneous injection of NK-susceptible RMA-S cells. These NK cell defects could not be correlated with defects in the expression of IL-12, IL-15, and IL-18 or the IL-2 or IL-15 receptors. Thus, Ets-1 defines a novel transcriptional pathway that is required for the development of the NK cell lineage in mice.

Human GATA-3: a lineage-restricted transcription factor that regulates the expression of the T cell receptor alpha gene.

In addition to its role in the recognition of foreign antigens, the T cell receptor (TCR) alpha gene serves as a model system for studies of developmentally‐regulated, lineage‐specific gene expression in T cells. TCR alpha gene expression is restricted to cells of the TCR alpha/beta+ lineage, and is controlled by a T cell‐specific transcriptional enhancer located 4.5 kb 3′ to the C alpha gene segment. The TCR alpha enhancer contains four nuclear protein binding sites called T alpha 1‐T alpha 4. In this report we describe the identification and characterization of a novel human cDNA, hGATA‐3 that binds to the T alpha 3 element of the human TCR alpha enhancer. hGATA‐3 contains a zinc finger domain that is highly related to the DNA‐binding domain of the erythroid‐specific transcription factor, GATA‐1, and binds to a region of T alpha 3 that contains a consensus GATA binding site (AGATAG). Northern blot analyses of hematopoietic cell lines demonstrate that hGATA‐3 is expressed exclusively in T cells. Overexpression of hGATA‐3 in HeLa cells or human B cells specifically activated transcription from a co‐transfected reporter plasmid containing two copies of the T alpha 3 binding site located upstream of the minimal SV40 promoter. Taken together these results demonstrate that hGATA‐3 is a novel lineage‐specific hematopoietic transcription factor that appears to play an important role in regulating the T cell‐specific expression of the TCR alpha gene.

Critical Roles for Transcription Factor GATA-3 in Thymocyte Development

The transcription factor GATA-3 is expressed at every stage of thymic development, but its role in thymocyte differentiation is unknown. The fact that RAG chimeric animals lacking GATA-3 cannot generate early thymocytes from common lymphoid progenitors has thus far precluded investigation of the function of GATA-3 in the thymus. To address this, we generated mice deficient in GATA-3 at early and late stages of thymic differentiation. Our studies revealed that GATA-3 is involved in beta selection and is indispensable for single-positive CD4 thymocyte development. Thus, our data demonstrate that the coordinated and regulated expression of GATA-3 at each stage of thymic development is critical for the generation of mature T cells.

Regulation of cell-type-specific interleukin-2 receptor alpha-chain gene expression: potential role of physical interactions between Elf-1, HMG-I(Y), and NF-kappa B family proteins.

The interleukin 2 receptor alpha-chain (IL-2R alpha) gene is rapidly and potently induced in T cells in response to mitogenic stimuli. Previously, an inducible enhancer between nucleotides -299 and -228 that contains NF-kappa B and CArG motifs was identified. We now report the characterization of a second essential positive regulatory element located between nucleotides -137 and -64 that binds Elf-1 and HMG-I(Y). This element had maximal activity in lymphoid cells, paralleling the cell type specificity of Elf-1 expression. Transcription from the IL-2R alpha promoter was inhibited when either the Elf-1 or the HMG-I(Y) binding site was mutated. Coexpression of both proteins activated transcription of the -137 to -64 element in COS-7 cells. Elf-1 physically associated with HMG-I and with NF-kappa B p50 and c-Rel in vitro, suggesting that protein-protein interactions might functionally coordinate the actions of the upstream and downstream positive regulatory elements. This is the first report of a physical interaction between an Ets family member and NF-kappa B family proteins. These findings provide significant new insights into the protein-protein and protein-DNA interactions that regulate cell-type-specific and inducible IL-2R alpha gene expression and also have implications for other genes regulated by Elf-1 and NF-kappa B family proteins.

Efficient and Stable Transduction of Cardiomyocytes After Intramyocardial Injection or Intracoronary Perfusion With Recombinant Adeno-Associated Virus Vectors

BACKGROUND The delivery of recombinant genes to cardiomyocytes holds promise for the treatment of a variety of cardiovascular diseases. Previous gene transfer approaches that used direct injection of plasmid DNA or replication-defective adenovirus vectors have been limited by low transduction frequencies and transient transgene expression due to immune responses, respectively. In this report, we have tested the feasibility of using intramyocardial injection or intracoronary infusions of recombinant adeno-associated virus (rAAV) vectors to program transgene expression in murine cardiomyocytes in vivo. METHODS AND RESULTS We constructed an rAAV containing the LacZ gene under the transcriptional control of the cytomegalovirus (CMV) promoter (AAVCMV-LacZ). We then injected 1x10(8) infectious units (IU) of this virus into the left ventricular myocardium of adult CD-1 mice. Control hearts were injected with the AdCMV-LacZ adenovirus vector. Hearts harvested 2, 4, and 8 weeks after AAVCMV-LacZ injection demonstrated stable beta-galactosidase (beta-gal) expression in large numbers of cardiomyocytes without evidence of myocardial inflammation or myocyte necrosis. In contrast, the AdCMV-LacZ-injected hearts displayed transient beta-gal expression, which was undetectable by 4 weeks after injection. Explanted C57BL/6 mouse hearts were also perfused via the coronary arteries with 1.5x10(9) IU of AAVCMV-LacZ and assayed 2, 4, and 8 weeks later for beta-gal expression. beta-Gal expression was detected in <1% of cardiomyocytes at 2 weeks after perfusion but was detected in up to 50% of cardiomyocytes 4 to 8 weeks after perfusion. CONCLUSIONS Direct intramyocardial injection or coronary artery perfusion with rAAV vectors can be used to program stable transgene expression in cardiomyocytes in vivo. rAAV appears to represent a useful vector for the delivery of therapeutic genes to the myocardium.

Impaired cardiomyocyte relaxation and diastolic function in transgenic mice expressing slow skeletal troponin I in the heart

1 To assess the specific functions of the cardiac isoform of troponin I (cTnI), we produced transgenic mice that expressed slow skeletal troponin I (ssTnI) specifically in cardiomyocytes. Cardiomyocytes from these mice displayed quantitative replacement of cTnI with transgene‐encoded ssTnI. 2 The ssTnI transgenic mice were viable and fertile and did not display increased mortality or detectable cardiovascular histopathology. They exhibited normal ventricular weights and heart rates. 3 Permeabilized transgenic cardiomyocytes demonstrated an increased Ca2+ sensitivity of tension and a lack of contractile responsiveness to cAMP‐dependent protein kinase (PKA). Isolated cardiomyocytes from transgenic mice had normal velocities of unloaded shortening but unlike wild‐type controls exhibited no enhancement of the velocity of shortening in response to treatment with isoprenaline. Transgenic cardiomyocytes exhibited greater extents of shortening than non‐transgenic cardiomyocytes at baseline and after treatment with isoprenaline. 4 The rates of rise of intracellular [Ca2+] and the peak amplitudes of the intracellular [Ca2+] transients were similar in transgenic and wild‐type myocytes. However, the half‐time of intracellular [Ca2+] decay was significantly greater in the transgenic myocytes. This change in decay of intracellular [Ca2+] was correlated with an increase in the re‐lengthening time of the transgenic cells. 5 These changes in cardiomyocyte function in vitro were manifested in vivo as impaired diastolic function both at baseline and after stimulation with isoprenaline. 6 Thus, cTnI has important roles in regulating the Ca2+ sensitivity of cardiac myofibrils and controlling cardiomyocyte relaxation and cardiac diastolic function. cTnI is also required for the normal responsiveness of cardiomyocytes to β‐adrenergic receptor stimulation.