Julia L. Cook

Nrf2, a Cap’n’Collar Transcription Factor, Regulates Induction of the Heme Oxygenase-1 Gene

Stress response elements, which mediate induction of the mouse heme oxygenase-1 (HO-1) gene by several agents, resemble the binding site for the activator protein-1 (Jun/Fos), Maf, and Cap’n’Collar/basic leucine zipper (CNC-bZIP) families of proteins. In L929 fibroblasts, significant activation of an HO-1 enhancer-reporter fusion gene was observed only with the CNC-bZIP class of proteins with Nrf2 exhibiting the highest level oftrans-activation, between 25- and 30-fold. To further examine the role of this factor in HO-1 gene regulation, a dominant-negative mutant, Nrf2M, was generated and conditionally expressed in L929 cells. The mutant protein was detected in cytoplasmic and nuclear fractions but did not affect cell growth. Under conditions of Nrf2M overexpression, HO-1 mRNA accumulation in response to heme, cadmium, zinc, arsenite, andtert-butylhydroquinone was inhibited by 85–95%. In contrast, overexpression of a dominant-negative mutant of c-Jun decreased L929 cell growth but did not inhibit HO-1 gene activation. Nrf2 does not homodimerize, but CNC-bZIP·small Maf protein heterodimers and Nrf2·Jun protein complexes are proposed to function as trans-activators. Co-expression of Jun proteins or p18, however, had no significant affect or inhibited Nrf2-mediated trans-activation. Taken together, these results implicate Nrf2 in the induction of the HO-1 gene but suggest that the Nrf2 partner in this function is a factor other than p18 or Jun proteins.

Exogenous administration of heme oxygenase-1 by gene transfer provides protection against hyperoxia-induced lung injury

Heme oxygenase-1 (HO-1) confers protection against a variety of oxidant-induced cell and tissue injury. In this study, we examined whether exogenous administration of HO-1 by gene transfer could also confer protection. We first demonstrated the feasibility of overexpressing HO-1 in the lung by gene transfer. A fragment of the rat HO-1 cDNA clone containing the entire coding region was cloned into plasmid pAC-CMVpLpA, and recombinant adenoviruses containing the rat HO-1 cDNA fragment Ad5-HO-1 were generated by homologous recombination. Intratracheal administration of Ad5-HO-1 resulted in a time-dependent increase in expression of HO-1 mRNA and protein in the rat lungs. Increased HO-1 protein expression was detected diffusely in the bronchiolar epithelium of rats receiving Ad5-HO-1, as assessed by immunohistochemical studies. We then examined whether ectopic expression of HO-1 could confer protection against hyperoxia-induced lung injury. Rats receiving Ad5-HO-1, but not AdV-betaGal, a recombinant adenovirus expressing Escherichia coli beta-galactosidase, before exposure to hyperoxia (>99% O2) exhibited marked reduction in lung injury, as assessed by volume of pleural effusion and histological analyses (significant reduction of edema, hemorrhage, and inflammation). In addition, rats receiving Ad5-HO-1 also exhibited increased survivability against hyperoxic stress when compared with rats receiving AdV-betaGal. Expression of the antioxidant enzymes manganese superoxide dismutase (Mn-SOD) and copper-zinc superoxide dismutase (CuZn-SOD) and of L-ferritin and H-ferritin was not affected by Ad5-HO-1 administration. Furthermore, rats treated with Ad5-HO-1 exhibited attenuation of hyperoxia-induced neutrophil inflammation and apoptosis. Taken together, these data suggest the feasibility of high-level HO-1 expression in the rat lung by gene delivery. To our knowledge, we have demonstrated for the first time that HO-1 can provide protection against hyperoxia-induced lung injury in vivo by modulation of neutrophil inflammation and lung apoptosis.

The intracrine hypothesis: An update

The intracellular actions of peptide hormones, growth factors, as well as of extracellular-signaling enzymes and DNA-binding proteins, either within target cells or within their cells of synthesis has been called intracrine action. Although these intracrine moieties are structurally diverse, they share certain characteristics of synthesis and function. This has given rise to the development of a theory of intracrine action which permits testable predictions to be made regarding the functioning of these peptides/proteins. Here the intracrine hypothesis is briefly described and then recent experimental findings which bear on predictions made earlier on the basis of the theory are discussed. These findings provide new support for the intracrine hypothesis.

Review: Lessons from in vitro studies and a related intracellular angiotensin II transgenic mouse model

In the classical renin-angiotensin system, circulating ANG II mediates growth stimulatory and hemodynamic effects through the plasma membrane ANG II type I receptor, AT1. ANG II also exists in the intracellular space in some native cells, and tissues and can be upregulated in diseases, including hypertension and diabetes. Moreover, intracellular AT1 receptors can be found associated with endosomes, nuclei, and mitochondria. Intracellular ANG II can function in a canonical fashion through the native receptor and also in a noncanonical fashion through interaction with alternative proteins. Likewise, the receptor and proteolytic fragments of the receptor can function independently of ANG II. Participation of the receptor and ligand in alternative intracellular pathways may serve to amplify events that are initiated at the plasma membrane. We review historical and current literature relevant to ANG II, compared with other intracrines, in tissue culture and transgenic models. In particular, we describe a new transgenic mouse model, which demonstrates that intracellular ANG II is linked to high blood pressure. Appreciation of the diverse, pleiotropic intracellular effects of components of the renin-angiotensin system should lead to alternative disease treatment targets and new therapies.

The mitochondrial component of intracrine action

In recent years the actions of intracellular-acting, extracellular signaling proteins/peptides (intracrines) have become increasingly defined. General principles of intracrine action have been proposed. Mitochondria represent one locus of intracrine action, and thus far, angiotensin II, transforming growth factor-beta, growth hormone, atrial natriuretic peptide, Wnt 13, stanniocalcin, other renin-angiotensin system components, and vascular endothelial-derived growth factor, among others, have been shown to be mitochondria-localizing intracrines. The implications of this mitochondrial intracrine biology are discussed.

Transgenic mice expressing an intracellular fluorescent fusion of angiotensin II demonstrate renal thrombotic microangiopathy and elevated blood pressure

We have generated transgenic mice that express angiotensin II (ANG II) fused downstream of enhanced cyan fluorescent protein, expression of which is regulated by the mouse metallothionein promoter. The fusion protein, which lacks a secretory signal, is retained intracellularly. In the present study, RT-PCR, immunoblot analyses, whole-animal fluorescent imaging, and fluorescent microscopy of murine embryonic fibroblasts confirm expression of the fusion protein in vivo and in vitro. The transgene is expressed in all tissues tested (including brain, heart, kidney, liver, lung, and testes), and radioimmunoassay of plasma samples obtained from transgenic mice indicate no increase in circulating ANG II over wild-type levels, consistent with intracellular retention of the transgene product. Kidneys from transgenic and corresponding wild-type littermates were histologically evaluated, and abnormalities in transgenic mice consistent with thrombotic microangiopathy were observed; microthrombosis was frequently observed within the glomerular capillaries and small vessels. In addition, systolic and diastolic blood pressures, measured by telemetry (n = 8 for each group), were significantly higher in transgenic mice compared with wild-type littermates. Blood pressure of line A male transgenic mice was 125 + or - 1.7 over 97 + or - 1.6 compared with 109 + or - 1.7 over 83 + or - 1.4 mmHg in wild-type littermates (systolic over diastolic). In summary, overexpression of an intracellular fluorescent fusion protein of ANG II correlates with elevated blood pressure and kidney pathology. This transgenic model may be useful to further explore the intracellular renin-angiotensin system and its implication in abnormal kidney function and hypertension.

The Basis of an Intracrine Pharmacology

Intracrines are extracellular signaling peptide factors that can act in the intracellular space after either internalization or retention in the cells that synthesize them. They are structurally diverse and include hormones, growth factors, enzymes, DNA‐binding proteins, and other peptide moieties. We have suggested principles of intracrine action and have applied those principles to forms of cellular and tissue differentiation, hormonal responsiveness, and memory. Moreover, recent findings make clear that some currently available pharmaceuticals act via the alteration of intracrine function. Thus, the beginnings of an intracrine pharmacology are at hand and we here review principles applicable to the design of such agents. The intracrine pharmacology of the renin‐angiotensin system, angiogenesis, and stem cell development is discussed.

Temporal changes in gene expression following cryogenic rat brain injury

Expression of 18 genes was examined at 8 different time points between 1 h and 28 days following cryogenic rat brain injury. The genes include thymidine kinase (TK), p53 tumor suppressor, c-fos, renin, myelin basic protein (MBP), proteolipid protein (PLP), transferrin, transferrin receptor, platelet-derived growth factor A (PDGF A), platelet-derived growth factor B (PDGF B), platelet-derived growth factor receptor alpha (PDGF alpha receptor), platelet-derived growth factor receptor beta (PDGF beta receptor), glial fibrillary acidic protein (GFAP), transforming growth factor-beta 1 (TGF-beta 1), basic fibroblast growth factor (bFGF), fibroblast growth factor receptor-1 (FGF-R1), insulin-like growth factor-1 (IGF-1), and somatostatin. Time courses of gene expression were determined for RNAs derived from hippocampus and cortex. Genes were divided into categories based upon those in which statistically significant changes in expression were first observed at or before 24 h (early genes) and those in which changes were first observed at or after 72 h (late genes). In the present model, many genes demonstrate elevated RNA levels in the cortex prior to hippocampus, following injury. RNAs transcribed from late genes tend to be elevated concurrently in cortex and hippocampus.

Mechanisms of Disease: intracrine physiology in the cardiovascular system

The field of intracrine physiology attempts to codify the biological actions of intracrines—extracellular signaling proteins or peptides that also operate in the intracellular space, either because they are retained in their cells of synthesis or because they have been internalized by a target cell. Intracrines are structurally diverse; hormones, growth factors, DNA-binding proteins and enzymes can all display intracrine functionality. Here, we review the role of intracrines in the heart and vasculature, including the intracrine actions of renin–angiotensin-system components in cardiac pathology, dynorphin B in cardiac development, and a variety of factors in pathologic and therapeutic angiogenesis. We argue that principles of intracrine physiology can inform our understanding of important pathologic processes such as left ventricular hypertrophy, diabetic cardiomyopathy and arrythmogenesis, and can aid the development of more-effective therapeutic interventions in cardiovascular disease.

Senescence, apoptosis, and stem cell biology: the rationale for an expanded view of intracrine action

Some extracellular-signaling peptides also at times function within the intracellular space. We have termed these peptides intracrines and have argued that intracrine function is associated with a wide variety of peptides/proteins including hormones, growth factors, cytokines, enzymes, and DNA-binding proteins among others. Here we consider the possibility that intracrines participate in the related phenomena of senescence, apoptosis, and stem cell regulation of tissue biology. Based on this analysis, we also suggest that the concept of intracrine action be expanded to include possible regulatory peptide transfer via exosomes/microvesicles and possibly by nanotubes. Moreover, the process of microvesicular and nanotube transfer of peptides and other biologically relevant molecules, which we inclusively term laterality, is explored. These notions have potentially important therapeutic implications, including implications for the therapy of cardiovascular disease.