vHNF1 and HNF1 are two nuclear proteins that bind to an essential element in the promoter proximal sequences of albumin and of many other liver‐specific genes. HNF1 predominates in hepatocytes but is absent in dedifferentiated hepatoma cells. These cells contain vHNF1 but fail to express most of the liver traits. In the present work we have isolated cDNA clones for vHNF1 and found that it is a homeoprotein homologous to HNF1 in regions important for DNA binding. Unexpectedly, vHNF1 transactivated the albumin promoter in transfection experiments. Like the HNF1 mRNA, the vHNF1 message was found in kidney, liver and intestine although in different proportions. The fact that vHNF1 and HNF1 readily form heterodimers in vitro and the biochemical characterization of vHNF1/HNF1 heterodimers in nuclear extracts of kidney, liver and several cell lines, strongly argue that such heterodimers exist in vivo. Our results raise the possibility that heterodimerization between homeoproteins could be a common phenomenon in higher eukaryotes, which may have implications in the regulatory network sustained between these factors.

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We have previously shown that the immunosuppressant cyclosporine A (CsA) increases the activity, the protein level, and the steady-state levels of the mRNA of the endothelial nitric-oxide synthase (eNOS) gene in bovine aortic endothelial cells (BAEC). We have now investigated the mechanisms responsible for these effects. Preincubation with an inhibitor of RNA polymerase II abol- Whereas cyclosporine A (CsA) 1 still stands as a cornerstone therapy for immunosuppression, its use encompasses serious side effects among which nephrotoxicity and hypertension are often encountered. The pathogenesis of their appearance has been investigated in clinical studies and animal models, but a complete understanding is still elusive. The partial reversibility of these effects suggests that the underlying disturbance has a functional component.Acute nephrotoxicity is associated with renal vasoconstriction and a reduced glomerular filtration rate. A significant amount of data support that the vasoconstrictor peptide, endothelin-1, is involved in this response (1, 2). Furthermore, transforming growth factor ␤ (TGF-␤) has been proposed as a link between CsA-enhanced endothelin synthesis and many of the pathological fibrotic lesions observed with chronic CsA toxicity (3).Although endothelin and other paracrine vasoconstrictors have been identified as mediators of the hypertensive response (1, 4, 5) associated with CsA toxicity, a less clear picture has emerged regarding the potential dysfunction of the L-arginine-NO-cGMP pathway. Several reports have described that there is a defect in the relaxation of blood vessels in response to endothelial agonists, but the precise step of diminished NO synthesis, enhanced degradation, or receptor abnormality has not been established (6 -8). In previous work, using endothelial cells in culture treated with CsA, we found that NO synthesis is not only not reduced but moderately enhanced (9). These observations have been also confirmed in rats (10), in healthy volunteers, in whom venous antecubital infusion of CsA significantly decreased forearm blood flow (11), and recently in patients treated with CsA after heart transplantation (12). These data indicate that nitric oxide may constitute an important regulatory mechanism that protects against CsA-associated vasoconstriction in vivo. In support of this hypothesis we found that the expression of the eNOS gene is increased in bovine aortic endothelial cells (BAEC) treated with CsA (12-14). We have now investigated the mechanisms by which eNOS is upregulated by CsA. EXPERIMENTAL PROCEDURESCell Culture and Reagents-BAEC were isolated from thoracic aortas using previously described methods (15). Phenotype characterization was based on their typical cobblestone appearance and uniform uptake of fluorescent acetylated low density lipoprotein. Cells were maintained in RPMI 1640 supplemented with 10% calf serum, 2 mM glutamine, 100 units/ml penicillin, and 100 g/ml streptomycin in an atmosphere of 95% O 2 and 5% CO 2 . Experiments were performed on confluent ...