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The Egr-1 promoter contains information for constitutive and inducible expression in transgenic mice¹

JO C. TSAI^*, LIXIN LIU^*, BRIAN C. COOLEY, MARIA R. DICHIARA, JAMES N. TOPPER and WILLIAM C. AIRD^*2

^* Department of Molecular Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215, USA;
Department of Orthopaedic Surgery, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA; and
Department of Medicine, Stanford University School of Medicine, Stanford, California 94305, USA

²Correspondence: Beth Israel Deaconess Medical Center, Molecular Medicine, RW-663, 330 Brookline Ave., Boston MA 02215, USA. E-mail: waird{at}caregroup.harvard.edu

SPECIFIC AIMS

The overall aim of this study was to determine whether the Egr-1 promoter contains information for appropriate expression in vivo. To that end, transgenic mice were generated with a construct containing 1200 bp of the mouse Egr-1 promoter coupled to nuclear localized LacZ and analyzed for reporter gene expression under both basal and inducible conditions.

PRINCIPAL FINDINGS

The Egr-1 transgene is expressed in a subset of organs and cell types

A total of 9 founder lines were generated with the Egr-1-lacZ construct. Of these 9 lines, 3 revealed LacZ expression. Whole mount staining of all three lines revealed similar patterns of transgene expression. LacZ staining was detected at the ostia of aortic tributaries, in blood vessels of the brain and heart, and in the liver. In contrast, there was no LacZ staining in whole mounts of the lung or spleen. Tissue sections were obtained from two of the lines and revealed similar patterns of ß-galactosidase activity. In sections of the brain, the X-Gal reaction product was present within a subset of neurons and in a subpopulation of endothelial cells and vascular smooth muscle cells lining the large arteries of the subarachnoid and subpial space (Fig. 1a , b ,, c ). In the heart, expression was detected in a minority of endothelial cells lining the capillaries of the myocardial wall and the large coronary arteries (Fig. 1d , e ). The X-Gal reaction product was also detected in occasional cardiomyocytes and vascular smooth muscle cells. ß-galactosidase activity in the liver appeared to be confined to hepatocytes (Fig. 1f , g , h ). Despite the seemingly organized, mosaic pattern of ß-galactosidase activity in liver whole mounts, LacZ-positive hepatocytes were found to be randomly scattered throughout the three acinar zones. Reporter gene expression was not detected in tissue sections of the lung (Fig. 1i ), skeletal muscle (Fig. 1j ), kidney (Fig. 1k ), or spleen (Fig. 1l ).

View larger version (155K): [in this window] [in a new window]   Figure 1. LacZ staining of tissue sections from adult Egr-1-lacZ mice. Tissue cryosections were obtained from adult Egr-1-lacZ-#20 mice and processed for LacZ staining. In sections of the brain, the X-Gal reaction product was detected in endothelial cells (a, b) and smooth muscle cells (b, artery on the left) of the subarachnoid and subpial blood arteries as well as a subset of neurons in the cerebral cortex (c). In the heart, expression was present within a minority of endothelial cells within the coronary arteries and myocardial capillaries (d, low power; e, high power). The reporter gene was expressed in a subpopulation of hepatocytes (f and g, low power; h, high power). There was no detectable ß-galactosidase activity in the lung (i), skeletal muscle (j), kidney (k), or spleen (l). The faint blue stain in these latter organs represents background endogenous ß-galactosidase activity.

The Egr-1 transgene is expressed in a pattern similar to that of the endogenous gene

Immunofluorescent studies were used to compare the distribution of the transgene with that of the endogenous Egr-1 gene.

Both the LacZ antigen and the endogenous Egr-1 were present in a minority of endothelial cells within the capillaries and coronary arteries of the heart. In addition, Egr-1 was detected in a small number of cardiomyocytes and vascular smooth muscle cells of the heart. In the liver, the Egr-1 and LacZ gene products were present in a subpopulation of hepatocytes as well as occasional sinusoidal endothelial cells. In the cortex of the brain, Egr-1 and LacZ were expressed in endothelial cells of the superficial arteries and in a subpopulation of neurons within the cortex and hippocampus. Immunodetectable Egr-1, but not LacZ, was rarely found in endothelial cells of pulmonary and splenic arteries. The presence of Egr-1 in these vascular beds was far less common than in the heart or brain.

The Egr-1 transgene is induced after partial hepatectomy

To determine whether the Egr-1 transgene contained information for response to proliferative signals, partial hepatectomies were carried out in Egr-1-lacZ transgenic mice. Since liver specimens were harvested at the time of the hepatectomy and at some time after hepatectomy, each animal had its own prehepatectomy control. LacZ mRNA was significantly induced 24 and 48 h after partial hepatectomy, whereas Egr-1 transcripts were increased at 24 h and normalized by 48 h. The discrepancy between LacZ and Egr-1 response at 48 h may reflect differences in the half-life of the respective messages. The X-Gal reaction product was upregulated in posthepatectomy whole mounts (Fig. 2A , panels a, b). LacZ staining of posthepatectomy liver sections revealed increased intensity of ß-galactosidase activity in hepatocytes and increased number of LacZ-positive hepatocytes (Fig. 2B , panels c–f). The LacZ-positive cells appeared to be randomly distributed throughout the hepatic nodules. Immunofluorescent studies of posthepatectomy liver sections revealed increased levels of endogenous Egr-1 protein in hepatocytes (Fig. 2C ). Finally, in quantitative assays, ß-galactosidase activity was induced between two- and fivefold 1–3 days after partial hepatectomy (Fig. 2B ). In contrast, partial hepatectomy did not induce endogenous ß-galactosidase activity in the liver of nontransgenic control mice between 4 h and 3 days. These findings demonstrate that the response of the Egr-1 gene during liver regeneration is mediated by transcriptional control elements in the 5' flanking region of the gene.

View larger version (152K): [in this window] [in a new window]   Figure 2. Expression of LacZ in response to partial hepatectomy. A) LacZ staining of a 48 h posthepatectomy whole mount liver specimen (b) is compared with its prehepatectomy control (a). LacZ staining of liver sections from prehepatectomy (c, low power; e, high power) and 24 h posthepatectomy (d, low power; f, high power) specimens show increased intensity of staining as well as increased numbers of LacZ-positive hepatocytes in the posthepatectomy livers. These specimens were processed and stained in parallel. B) ß-Galactosidase activity of posthepatectomy specimens was compared to the prehepatectomy controls and expressed as fold induction relative to levels of expression in the prehepatectomy liver. Each bar represents the average of duplicate samples from an individual animal. The results were obtained from Egr-1-lacZ-#20 mice with the exception of a single Egr-1-lacZ-#9 mouse (*).

CONCLUSIONS AND SIGNIFICANCE

Using immunofluorescent assays, we have shown that the endogenous Egr-1 gene is constitutively expressed in endothelial cells, vascular smooth muscle cells, cardiomyocytes, neuronal cells and hepatocytes of the adult mouse. A striking feature of the present study was the marked heterogeneity of Egr-1 expression. For example, in sections of the myocardial wall, only a minority of microvascular endothelial cells and cardiomyocytes contained immunoreactive Egr-1 protein. There are several possible explanations for the heterogeneous distribution of Egr-1. Some subpopulations of cells may be genetically preprogrammed to express the Egr-1 gene. In certain lineages such as cardiomyocytes and neurons, Egr-1 levels may correlate with the state of cellular differentiation. Finally, variation in Egr-1 levels among neighboring cells may reflect differences in signal input at the level of the microenvironment.

Under in vitro conditions, the Egr-1 gene is induced by a wide variety of extracellular signals. Environmental regulation of Egr-1 has been shown to be mediated by five serum response elements (SREs) in the 5' flanking region. However, the role of these SREs or other upstream transcriptional control elements in mediating physiological expression of the Egr-1 gene has yet to be determined.

In the current report, we show that the 1200 bp Egr-1 promoter directs expression in subsets of endothelial cells, cardiomyocytes, hepatocytes and neurons in transgenic mice. Importantly, the spatial distribution of the LacZ reporter gene correlated with that of the endogenous gene. In serial sections, Egr-1-postive and LacZ-positive cells were derived from the same cell populations. Moreover, similar to the endogenous gene, the reporter gene was expressed in a minority of cells within the various lineages.

To test whether the Egr-1 promoter contained information for environmental induction and temporal regulation, we used a partial hepatectomy model. The loss of hepatic tissue provides a powerful stimulus for cellular proliferation. During this process, Egr-1 and other immediate early genes are rapidly activated. In the present report, we have demonstrated the partial hepatectomy response is transduced by sequences within the 1200 bp promoter region. Reporter gene activity and mRNA were increased above baseline after the procedure and remained elevated for 72 h. In liver sections stained with X-Gal, there was an increase both in the number of expressing hepatocytes and in the degree of staining per hepatocyte. In other words, in response to partial hepatectomy, nonexpressing cells were recruited to express the transgene. These findings suggest that the 1200 bp promoter does indeed contain information for appropriate temporal regulation of the Egr-1 gene.

The Egr-1 transgene represents a novel molecular tool for studying immediate early gene regulation. The results of the present study support the conclusion derived from a myriad of in vitro studies, namely, that the 1200 bp promoter region directs basal expression and is responsible for transducing extracellular signals. The transgenic mouse may be used to study the in vivo response of the promoter to other environmental modifications that are known to induce the endogenous Egr-1 gene, including ischemia/hypoxia, epileptic seizures, visual stimulation, and endothelial cell injury. Moreover, the LacZ reporter gene may serve as a useful marker in studies designed to dissect the pathways that mediate partial hepatectomy-induced expression of Egr-1 and other immediate early genes. Finally, the present investigation lays an important foundation for delineating the cis-regulatory elements in mediating inducible gene expression in vivo. By generating transgenic mice that harbor deletant promoter-reporter gene constructs, we will be in a position to determine the relative role of the SREs and other transcriptional control elements in mediating physiological expression in constitutive and inducible states.

View larger version (13K): [in this window] [in a new window]   Scheme 1. No caption available.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.99-1072fje

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