Expression of the human beta(3)-adrenergic receptor gene in SK-N-MC cells is under the control of a distal enhancer.

Mechanisms of transcriptional regulation of the human beta(3)-adrenergic receptor were studied using SK-N-MC cells, a human neuroblastoma cell line that expresses beta(3)- and beta(1)-adrenergic receptors endogenously. Deletions spanning different portions of a 7-kb 5'-flanking region of the human beta(3)-adrenergic receptor gene were linked to a luciferase reporter and transfected in SK-N-MC, CV-1, and HeLa cells. Maximal luciferase activity was observed when a 200-bp region located between -6.5 and -6.3 kb from the translation start site was present. This region functioned only in SK-N-MC cells. Electrophoretic mobility shift assays of nuclear extracts from SK-N-MC, CV-1, and HeLa cells using double stranded oligonucleotides spanning different portions of the 200-bp region as probes and transient transfection studies revealed the existence of three cis-acting regulatory elements: A) -6.468 kb-AGGTGGACT--6.458 kb, B) -6.448 kb-GCCTCTCTGGGGAGCAGCTTCTCC-6.428 kb, and C) -6.405 kb-20 repeats of CCTT-6.385 kb. These elements act together to achieve full transcriptional activity. Mutational analysis, antibody supershift, and electrophoretic mobility shift assay competition experiments indicated that element A binds the transcription factor Sp1, element B binds protein(s) present only in nuclear extracts from SK-N-MC cells and brown adipose tissue, and element C binds protein(s) present in both SK-N-MC and HeLa cells. In addition, element C exhibits characteristics of an S1 nuclease-hypersensitive site. These data indicate that cell-specific positive cis-regulatory elements located 6.5 kb upstream from the translation start site may play an important role in transcriptional regulation of the human beta(3)-adrenergic receptor. These data also suggest that brown adipose tissue-specific transcription factor(s) may be involved in the tissue-specific expression of the beta(3)-adrenergic receptor gene.

Regulation of human renin gene promoter activity: a new negative regulatory region determines the responsiveness to TNF alpha.

BACKGROUND: The renin-angiotensin system has been known to regulate blood pressure and body fluid homeostasis. Several lines of evidence have shown that renin gene expression and release are up-regulated by beta-adrenergic stimulation, sodium depletion, and angiotensin converting enzyme inhibition, but down-regulated by cytokines. To further characterize the human renin gene (hREN) promoter structure, its regulation, and to identify an appropriate cell system for study, we examined five cell lines and investigated drug effects on the hREN promoter expression. METHODS: Using the hREN-luciferase reporter gene constructs in the DNA transfection assays, approximately 5 kb of the hREN 5' flanking region was assessed for promoter activity in five different cell lines. Regulation of the hREN promoter activity was investigated using Y-1 adrenal cells that were transfected with the hREN-luciferase DNA and were treated with forskolin, calcium ionophore A23187, phorbol ester, angiotensin II (Ang II), or cytokines. RESULTS: Transient transfection analysis showed that the 5 kb hREN 5' flanking DNA alone was able to confer significant promoter activity in Y-1 adrenal cells. In transfected Y-1 cells, luciferase reporter expression was induced by forskolin, suppressed by the calcium ionophore A23187, and phorbol ester in a dose-dependent manner, but was unaffected by angiotensin II (Ang II). However, when Y-1 reporter cells were transfected with human angiotensin II receptor type 1 (AT1) cDNA, hREN promoter activity was dose-dependently down-regulated by Ang II, which was blockable by losartan, an AT1-selective antagonist. Further studies also showed that hREN promoter activity in Y-1 cells was selectively down-regulated by TNF alpha. Deletion of the hREN promoter sequences between position -3916 and -2822 not only enhanced hREN promoter activity by approximately tenfold, but also caused a failure of down-regulation by TNF alpha. In contrast, neither interleukin (IL)-1 alpha, IL-1 beta, IL-2, nor IL-6 exerted any significant effect. CONCLUSIONS: Together the results suggest that TNF alpha is a negative regulator of the hREN expression in the adrenal cells, and that the TNF alpha responsiveness may be controlled by elements located between the positions -3916 and -2822 of the hREN promoter. Moreover, the Y-1 cell line may provide a valuable model system for studying renin gene regulation.