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Abstract-The signaling cascade elicited by angiotensin II (Ang II) resembles that characteristic of a growth factor, and recent evidence indicates transactivation of epidermal growth factor receptor (EGF-R) by G protein-coupled receptors. Here, we report the involvement of EGF-R in Ang II-induced synthesis of fibronectin and transforming growth factor-␤ (TGF-␤) in cardiac fibroblasts. Ang II stimulated fibronectin mRNA levels dose dependently, with a maximal increase (Ϸ5-fold) observed after 12 hours of incubation. Fibronectin synthesis induced by Ang II or calcium ionophore was completely abolished by tyrosine kinase inhibitors and intracellular Ca 2ϩ chelating agents. Ang II-induced fibronectin mRNA was not affected by protein kinase C inhibitors or protein kinase C depletion, whereas specific inhibition of EGF-R function by a dominant negative EGF-R mutant and tyrphostin AG1478 abolished induction of fibronectin mRNA. We isolated the rat fibronectin gene, including the 5Ј-flanking region, and found that the activator protein-1 (AP-1) binding site present in the promoter region was responsible for the Ang II responsiveness of this gene. A gel retardation assay revealed the binding of nuclear protein to the AP-1 site, which was supershifted with anti-c-fos and anti-c-jun but not anti-activating transcription factor (ATF)-2 antibodies. Conditioned medium from Ang II-treated cells contained TGF-␤ bioactivity, and addition of neutralizing TGF-␤ antibody modestly (46%) inhibited induction of fibronectin. Ang II-induced synthesis of TGF-␤ was also abolished by inhibition of EGF-R function. The effect of TGF-␤ was exerted by stabilizing fibronectin mRNA without affecting the promoter activity and required de novo protein synthesis. We concluded that Ang II-induced expression of fibronectin and TGF-␤ is mediated by downstream signaling of EGF-R transactivated by Ca 2ϩ -dependent tyrosine kinase and that Ang II-induced fibronectin mRNA expression is regulated by 2 different mechanisms, which are transcriptional control by binding of the c-fos/c-jun complex to the AP-1 site and posttranscriptional control by mRNA stabilization due to autocrine or paracrine effects of TGF-␤. Thus, this study suggests that the action of Ang II on extracellular matrix formation should be interpreted in association with the EGF-R signaling cascade. (Circ Res. 1999;84:1073-1084.) Key Words: angiotensin II receptor Ⅲ angiotensin II type 1 receptor Ⅲ angiotensin II type 2 receptor Ⅲ angiotensin II Ⅲ epidermal growth factor receptor C ardiac fibroblasts isolated from neonatal rat hearts have abundant high-affinity angiotensin II (Ang II) receptors, which are classified pharmacologically as belonging to the Ang II type 1 receptor (AT 1 ) subtype. 1,2 These cells have been used to examine AT 1 -mediated signaling 2-4 and extracellular matrix remodeling in heart failure. 5 AT 1 stimulation was found to stimulate DNA synthesis and cell proliferation 1 and also to increase the synthesis of extracellular matrix proteins, 4,6 which suggests that cardi...

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The human embryonal carcinoma cell line NEC14 can be induced to differentiate by the addition of 10(-2)M N,N'-hexamethylene-bis-acetamide (HMBA). A subtractive cDNA library specific to undifferentiated NEC14 cells was constructed using oligo(dT)30-Latex and polymerase chain reaction (PCR). The method was designed to improve the efficiency of subtraction and the enrichment of cDNA clones corresponding to low abundance mRNAs. The single strand of cDNA was made from mRNA prepared from the HMBA-treated NEC14 cells using an oligo(dT)30 primer covalently linked to Latex particles. After removal of the mRNA template by heat-denaturation and centrifugation, the subtractive hybridization was carried out between the cDNA-oligo(dT)30-Latex and mRNA from untreated NEC14 cells. Unhybridized mRNA collected by centrifugation was hybridized repeatedly to the cDNA-oligo(dT)30-Latex and subtractive mRNA was converted to cDNA. The subtractive cDNA was then amplified by PCR and cloned into pBluescript II KS-. The cDNA library thus constructed consisted of approximately 10,000 independent clones with cDNA inserts of 1.7 Kb on average. Differential hybridization of these transformants indicated that approximately 3% of them contained cDNA inserts specific to the undifferentiated EC cells, some of which were derived from low abundance mRNAs.

The guanine insertion enzyme from Escherichia coli catalyzes exchange of guanine located at the first position of the anticodon of tRNA with radioactive guanine (N. Okada and S. Nishimura, unpublished data). tRNA isolated from various tumors, including slowly growing Morris hepatoma 7794A, incorporated considerable guanine with E. coli guanine insertion enzyme, whereas tRNA isolated from all normal tissues so far tested, except regenerating rat liver, incororated scarcely any. In the rat ascites hepatoma AH7974, the guanine was mostly incorporated into minor isoaccepting species of tRNA^sP that contained the guanine residue instead of Q base in the first position of the anticodon. This is a sensitive and easy method for identifying unique tRNA species in tumor tissues.Many new isoaccepting tRNA species have been found in particular tissues, cells at various stages of differentiation, tumor tissues, transformed cells, and cells grown under different culture conditions (for reviews, see refs. 1-3). Various techniques have been used in attempts to detect unique tRNA species in tumor cells, and it has been found that the amount of methylated nucleosides in tRNA and the activities of tRNA methylases are generally high in tumor tissues (3, 4). However, analyses of modified nucleosides using total unfractionated tRNA have given results dependent upon the tumor tissues examined (5). Recently, Kuchino and Borek (6) demonstrated that the tRNAPhe that specifically appeared in Novikoff hepatoma and Ehrlich ascites tumor cells contains 1-methylguanine, unlike tRNAPhe in normal tissues. The new tRNAPhe species that appears in some tumor tissues is due to lack of modified base Y in the position next to the anticodon (7,8). A new isoaccepting species of tRNA has often been detected in tumor cells by analyzing changes in the chromatographic profile of amino acid acceptor activity (3). However, new tRNA cannot always be detected in this way because the elution position of tRNA may be influenced by several factors. In addition, most methods used previously require a large quantity of material and pure species of tRNA, which are difficult to obtain from tumor tissues.In this paper, we report a method for detecting unique tRNA species specifically present in tumor cells. We previously reported that the guanine insertion enzyme from rabbit reticulocytes, discovered by Farkas (9), specifically catalyzes the exchange of modified base Q in Escherichia coli tRNA with guanine without breaking the polynucleotide chain (10). A guanine insertion enzyme has also been found in other organisms, such as E. coli (N. Okada and S. Nishimura, unpublished data) and Ehrlich ascites tumor cells (11), and an extensively purified preparation from E. coli catalyzed exchange of guanine with guanine, but not of Q base in tRNA with guanine (N.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fac...