The 5'region of the rat phosphoenolpyruvate carboxykinase gene confers pH sensitivity to chimeric genes expressed in renal and liver cell lines capable of expressing PEPCK

The 5' flanking regions of the rat phosphoenolpyruvate carboxykinase gene were used to form chimeric gene constructs with the human growth hormone gene. These constructs were transfected into several renal and one liver cell line and the production of growth hormone (HGH) measured by immunoassay. Cyclic-AMP and glucocorticoid responsiveness of HGH production was observed in all cell lines. In two lines, the rat NRK52E renal epithelial line and the rat H4IIE hepatoma cell line, both capable of expressing PEPCK, lowering extracellular pH increased HGH production several fold. Comparison of hormone and pH effect on cells transfected with a thymidine kinase promoter-HGH chimera indicated that the PEPCK 5' flanking region effects were specific. Thus, part of the pH responsiveness of the PEPCK gene in vivo may be attributed to properties of the 5' flanking regions.     

Interaction of a tissue-specific factor with an essential rat growth hormone gene promoter element.

Rat growth hormone (rGH) gene expression is normally restricted to the anterior pituitary. As a model of this tissue specificity, we compared the transient expression of an rGH-chloramphenicol acetyltransferase (CAT) hybrid gene in rGH-producing rat pituitary tumor (GC) cells and in non-rGH-producing rat fibroblast (rat-2) cells. Deletion analysis of the rGH portion of this hybrid gene demonstrated that DNA sequences within 140 base pairs 5' to the rGH gene were sufficient for correct cell type-specific expression. Deletion of an additional 35 base pairs of the rGH 5'-flanking DNA resulted in a loss of expression of the transfected hybrid gene and correlated with the interaction of a putative trans-acting factor with this region of the rGH promoter. This factor was detectable by DNase I footprinting in a crude nuclear extract from GC cells but not from rat-2 cells. Site-directed mutagenesis of the footprint region caused complete loss of expression of a hybrid gene containing 530 base pairs 5' to the rGH gene. Thus, the interaction of this factor, which we term GC2, is likely to be essential for the tissue-specific expression of the rGH gene.     

Sequences required for cell-type specific thyroid hormone regulation of rat growth hormone promoter activity

We have located sequences within the rat growth hormone (rGH) promoter region which are required for pituitary cell-type specific responsiveness to T3 (thyroid hormone, 3,5,3'-L-triiodothyronine). Transient transfections with a series of plasmids containing as few as 202 nucleotides upstream of the start site of the rat growth hormone mRNA showed specific induction by T3 in rat pituitary cell lines. Both the magnitude and the kinetics of this response were similar to those of the endogenous rGH gene, showing a strong early induction followed by a decline in T3 effect. Deletion of an additional 19 base pairs (to -183 relative to the start site) eliminated this induction. Plasmids containing sequences up to -237 or -202 showed significant promoter activity but no T3 responsiveness in transfections of mouse fibroblasts or monkey kidney cells. The presence of high affinity nuclear T3 binding proteins was demonstrated in both cell types. These results show that sequences between -183 and -202 are required for pituitary cell specific T3 regulation of the rGH promoter. The lack of T3 responsiveness in non-pituitary cells suggests that such regulation may be mediated by factors present in pituitary cells and absent in other cells.     

Isolation and characterization of rat cDNA clones for two distinct thyroid hormone receptors

Two distinct v-erbA-related cDNA clones representing the products of different genes were isolated from a rat liver cDNA library. The first, rc-erbA-alpha, was 82% identical to v-erbA and encoded a polypeptide with a calculated molecular mass of 45,000 daltons. This cDNA clone arises from the same gene product as a v-erbA-related cDNA isolated from rat brain by Thompson et al. (Thompson, C. C., Weinberger, C., Lebo, R., and Evans, R. (1987) Science 237, 1610-1614). The second cDNA clone, rc-erbA-beta, was 76% identical to v-erbA and encoded a polypeptide with a calculated molecular mass of 52,000 daltons. Both rc-erbA-alpha and rc-erbA-beta translational products bound 3,5,3'-triiodo-L-thyronine with affinities equal to each other (Kd approximately equal to 0.4 nM) and comparable to the nuclear thyroid hormone receptor extracted from rat liver. The relative affinities of a series of thyroid hormone analogs for both translational products were also identical. In various tissues and cell lines, the relative levels of rc-erbA-beta RNA, but not rc-erbA-alpha RNA, correlated with measurements of nuclear 3,5,3'-triiodo-L-thyronine binding sites. Based on this correlation, we suggest that rc-erbA-beta may encode the "classical" nuclear thyroid hormone receptor, whereas rc-erbA-alpha may encode an isoreceptor species with differing functional properties.     

Analysis of photoaffinity label derivatives to probe thyroid hormone receptor in human fibroblasts, GH1 cells, and soluble receptor preparations

The regulation of growth hormone gene expression by thyroid hormone in cultured GH1 cells is mediated by a chromatin-associated receptor. We have previously described a photoaffinity label derivative of 3,5,3'-triiodo-L-thyronine (L-T3) in which the alanine side chain was modified to form N-2-diazo-3,3,3-trifluoropropionyl-L-T3 (L-[125I]T3-PAL). On exposure to 254 nm UV light, L-[125I]T3-PAL generates a carbene which covalently modifies two thyroid hormone receptor forms in intact GH1 cells; an abundant 47,000 Mr species and a less abundant 57,000 Mr form. We have now synthesized similar photoaffinity label derivatives of 3,5,3',5'-tetraiodo-L-thyronine (L-T4) and 3,3',5'-triiodo-L-thyronine (L-rT3). Both compounds identify the same receptor forms in intact cells and in nuclear extracts in vitro as L-[125I]T3-PAL. Labeling by L-[125I]rT3-PAL was low and consistent with the very low occupancy of receptor by L-rT3. Underivatized L-[125I]T3 and L-[125I]T4 labeled the same receptor forms at 254 nm but at a markedly lower efficiency than their PAL derivatives. In contrast, N-bromoacetyl-L-[125I]T3, a chemical affinity labeling agent, did not derivatize either receptor form in vitro. The relative efficiency of coupling to receptor at 254 nm was L-[125I]T4-PAL greater than L-[125I]T3-PAL greater than L-[125I]T4 greater than L-[125I]T3. Although L-[125I]T4-PAL has a lower affinity for receptor than L-[125I]T3-PAL, its coupling efficiency was 5-10-fold higher. This suggests that the alanine side chain of L-[125I]T4-PAL is positioned in the ligand binding region near a residue which is efficiently modified by photoactivation. With L-[125I]T4-PAL we were able to identify three different molecular weight receptor species in human fibroblast nuclei.