Transcriptional Regulation of α_1b Adrenergic Receptors (α_1bAR) by Nuclear Factor 1 (NF1): a Decline in the Concentration of NF1 Correlates with the Downregulation of α_1bAR Gene Expression in Regenerating Liver

Abstract

The 5 upstream region from ؊490 to ؊540 (footprint II) within the dominant P2 promoter of the rat ␣ 1b adrenergic receptor (␣ 1b AR) gene is recognized by a sequence-specific DNA-binding protein (B. Gao, M. S. Spector, and G. Kunos, J. Biol. Chem. 270:5614-5619, 1995). This protein, detectable in Southwestern (DNAprotein) blots of crude nuclear extracts as 32-and 34-kDa bands, has been purified 6,000-fold from rat livers by DEAE-Sepharose, heparin-Sepharose, and DNA affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and UV cross-linking of the purified protein indicated the same molecular mass as that in crude extracts. Methylation interference analysis revealed strong contact with a TTGGCT hexamer and weak contact with a TGGCGT hexamer in the 3 and 5 portions of footprint II, respectively. Nucleotide substitutions within these hexamers significantly reduced protein binding to footprint II and the promoter activity of P2 in Hep3B cells. The purified protein also bound to the nuclear factor 1 (NF1)/CTF consensus sequence, albeit with lower affinity. Gel mobility supershift and Western blotting (immunoblotting) analyses using an antibody against the NF1/CTF protein identified the purified 32-and 34-kDa polypeptides as NF1 or a related protein. Cotransfection into Hep3B cells or primary rat hepatocytes of cDNAs of the NF1-like proteins NF1/L, NF1/X, and NF1/Red1 resulted in a three-to fivefold increase in transcription directed by wild-type P2 but not by the mutated P2. Partial hepatectomy markedly decreased the levels of NF1 in the remnant liver and its binding to P2, which paralleled declines in the rate of transcription of the ␣ 1b AR gene and in the steady-state levels of its mRNA. These observations indicate that NF1 activates transcription of the rat ␣ 1b AR gene via interacting with its P2 promoter and that a decline in the expression of NF1 is one of the mechanisms responsible for the reduced expression of the ␣ 1b AR gene during liver regeneration.The ␣ 1b adrenergic receptor (␣ 1b AR) is a G-protein-coupled receptor that mediates the acute metabolic effects of catecholamines in the liver and is also involved as a comitogen in the regenerative response after the loss or injury of liver tissue (13,14,35,36). Expression of the ␣ 1b AR gene in the rat liver is controlled by hormonal and developmental factors as well as by conditions associated with hepatocyte dedifferentiation (8,33,35,36,49). Such regulation has been shown to occur at the transcriptional level under many of these conditions, such as after partial hepatectomy (36), during primary culturing of rat hepatocytes (28), and in response to glucocorticoids (53), cycloheximide (25), phorbol esters (26), and thyrotropin and cyclic AMP (31). In order to understand the molecular mechanisms involved in the transcription of the ␣ 1b AR gene under a variety of physiological and pathological conditions, we have cloned the rat ␣ 1b AR gene and identified its multiple promoters and the cis-acting elements in its regulatory domai...

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