B A White

A simple technique for the simultaneous measurement of relative levels of a specific mRNA in numerous small samples of animal cells or tissue is described. The technique involves denaturation of cytoplasmic preparations, followed by dotting of up to 96 samples onto a single sheet of nitrocellulose, hybridization with a 32P-labeled cDNA plasmid, autoradiography, and scanning. By analyzing cytoplasmic preparations instead of purified RNA, manipulations of multiple samples prior to analysis is minimized. Experiments with a clonal line of rat pituitary tumor (GH3) cells showed that this technique can be employed to follow the induction by Ca2+ of prolactin mRNA sequences, employing cytoplasm prepared from as little as 2.5 x 10(4) cells. The specificity of the technique for prolactin mRNA was shown by employing GC cells, a GH3 cell variant lacking detectable prolactin mRNA sequences. Experiments with cultured rat hemipituitaries showed that the prolactin mRNA present in cytoplasm corresponding to as little as 1/100 of a pituitary could be readily detected. This technique is quite simple, requires very small amounts of cells or tissue, and permits the simultaneous analysis of multiple samples. Hence, it should be quite useful for studies with various experimental systems of the regulation of specific mRNA levels.

In a concerted analysis of the genes encoding three mouse steroid hydroxylases, we identified and characterized a transcriptional regulatory protein, designated steroidogenic factor 1 (SF-1), that contributes to the coordinate expression in adrenocortical cells. SF-1, an orphan member of the nuclear receptor family, binds to PyCAAGGPyCPu motifs upstream of the steroid hydroxylases to regulate their expression. In the present study, we extend these findings by examining the role of SF-1 in regulation of the rat P450 aromatase gene in gonadal tissues. The 5'-flanking region of the rat aromatase gene was isolated by a polymerase chain reaction-based approach, using primers corresponding to the 5'- and 3'-ends of a published aromatase sequence. DNA sequence analysis revealed three differences between our sequence and the previously published sequence, including a 44-base pair (bp) insertion. Moreover, the transcription initiation site, as determined by primer extension analysis, differed from that previously proposed. The new transcription initiation site is located 23 bp 3' of a putative TATA box. When a revised rat sequence was compared to that of the human aromatase PII promoter by BEST-FIT analysis, a region of about 300 bp was identified that was 80% conserved between the two promoters. A potential SF-1 site, CCAAGGTCA, was identified at position -82 within this region. An oligonucleotide probe containing this putative SF-1 site was used in gel mobility shift assays. Consistent with previous studies, a specific complex was observed with nuclear extracts from gonadal steroidogenic tissues but was absent with nuclear extracts from nonsteroidogenic tissues. The role of SF-1 in this steroidogenic cell-specific complex was next addressed more directly. Bacterial extracts containing an SF-1-glutathione S-transferase fusion protein interacted specifically with the putative SF-1 site, and polyclonal antisera against SF-1-glutathione S-transferase specifically abolished the complex formed with nuclear extracts from rat ovaries or R2C rat Leydig tumor cells. Finally, the aromatase SF-1 element increased expression of an SV40 promoter/luciferase construct in transient transfection experiments in a steroidogenic cell-selective manner. Collectively, these studies implicate SF-1 in the regulation of steroid hydroxylase gene expression in nonadrenal tissues, significantly extending previous studies in adrenocortical cells.

Addition of CaCl2 (0.4 mM) to GH3 cells incubated in a serum-free, Ca2+-free medium increased relative prolactin synthesis (prolactin synthesis/total protein synthesis) 7- to 150-fold by 3-4 days, but had no effect on relative growth hormone synthesis. Half-maximal and maximal stimulation of relative prolactin synthesis occurred at about 0.07 and 0.2 mM CaCl2, respectively. Neither MgCl2 (0.4 mM) nor KCl 0.4 mM) affected relative prolactin synthesis. 32P-labeled rat prolactin and growth hormone cDNA plasmids were employed to measure by RNA gel blot hybridization changes in cytoplasmic levels of the corresponding mRNA sequences. Both prolactin mRNA sequence levels and translatable prolactin mRNA increased in parallel with the CaCl2-induced increase in relative prolactin synthesis. CaCl2 increased prolactin mRNA sequences by 7- to 200-fold, without affecting growth hormone mRNA sequence levels. Dexamethasone (100 nM) decreased prolactin mRNA sequences in cells incubated with CaCl2, antagonized the CaCl2-induced increases in prolactin mRNA sequences independently of the CaCl2 concentration.

The regulation of prolactin RNA sequences by Ca2+, epidermal growth factor (EGF), and thyrotropin-releasing hormone (TRH), singly and in combination, was studied in GH3 cells incubated in a chemically defined medium. Ca2+ increased all large prolactin RNA sequences in the nucleus, thus suggesting a transcriptional site of action. A recently developed cytoplasmic dot hybridization procedure was employed to study regulation of cytoplasmic prolactin mRNA. Ca2+ alone stimulated prolactin mRNA with a lag of between 3 and 9 h and yielded a sizable final increase (14- to 18-fold) of prolactin mRNA. With EGTA present to chelate any residual Ca2+, prolactin mRNA was not detectably increased by TRH, showing that TRH requires Ca2+ to stimulate prolactin mRNA. Under these conditions, prolactin mRNA was only slightly (4-fold) increased by EGF. Either peptide plus an optimal concentration of Ca2+ yielded a 60- to 70-fold increase in prolactin mRNA, showing that both EGF and TRH regulate prolactin mRNA synergistically with Ca2+.