Multiple Regulation of Prolactin Receptor Gene Expression in Rat Liver

Multiple Regulation of Prolactin Receptor Gene Expression in Rat Liver Christine Jolicoeur, Jean-Marie Boutin, Hiroaki Okamura, Sally Raguet, Jean Djiane, and Paul A. Kelly Unite d'Endocrinologie Moleculaire Institut National de la Recherche Agronomique, CRJJ (J.D.) 78350 Jouy-en-Josas, France factor have been reported (8-12), the precise role of PRL in liver remains to be determined. Rat hepatic PRL-R is hormonally regulated. GH has been shown to positively regulate PRL-R levels (1315). PRL itself exerts a control on its receptor, inducing up- or down-regulation depending on the concentration and duration of exposure to PRL (15-19). Gonadal steroids are also major regulators of PRL-R expression. The number of receptors is very low in livers of young animals, but increases 7-fold in females during sexual maturation, while levels are low or undetectable in males during the corresponding period (20, 21). In females, receptor levels fluctuate during the estrous cycle (22), decrease after ovariectomy (22, 23), and increase during pregnancy (20). In males, castration results in an increase in the number of hepatic PRLbinding sites (24). In both sexes, PRL-R levels are strongly stimulated by estrogens (25) and reduced by androgens(13, 26). The variations in PRL-R expression could be regulated at the transcriptional, posttranscriptional, or translational level. Few studies have been completed on regulation of receptor gene expression. Coordinate regulation of distinct insulin receptor mRNA species by glucocorticoid has been reported in rat Fao hepatoma cells (27). Multiple mRNA forms encoding the human progesterone receptor are synchronously regulated by estrogen and progesterone in breast cancer cells (28). Down-regulation of estrogen and glucocorticoid receptors is associated with a reduction in mRNA levels (29, 30). In the current study we present the steady state PRL-R mRNA levels in female rat liver as a function of age and in response to estrogen administration and compare them with the number of PRL-R. These results suggest that there are multiple levels of regulation of PRL-R synthesis in rat liver. Sex steroids are major regulators of PRL receptor expression in rat liver. Using a probe encoding the rat PRL receptor we have studied receptor mRNA levels in female rat liver during ontogeny and in response to estrogen treatment. Steady state mRNA levels were determined by Northern blot and densitometric analysis. Messenger RNA levels have been compared to the number of binding sites, which was assessed by Scatchard analysis of [125l]ovine PRL binding in membrane preparations. Our results show that steady state mRNA and binding levels of PRL receptors are both regulated by development and estrogens, but that binding does not exactly parallel mRNA levels. From the developmental stages of prepuberty to adult, receptor numbers increase 8fold, whereas mRNA levels increase 3-fold. Estrogen treatment stimulates receptor levels 6-fold, but mRNA levels are only increased 3-fold. These results suggest that PRL receptor gene expression in rat liver is regulated at the transcriptional or posttranscriptional level as well as at the translational level. (Molecular Endocrinology 3: 895-900, 1989) INTRODUCTION PRL is encoded by a member of the GH/PRL/placental lactogen gene family. Recently, receptors for GH (1) and PRL (2) have been cloned and shown to form a new receptor gene family. The PRL receptor (PRL-R) is a small protein (41 K) (2-6) that is widely distributed in a number of tissues (7). The liver is the tissue with the highest PRL binding in the rat. Although specific effects on ornithine decarboxylase, somatomedin, synlactin, insulin-like growth factor I, and a liver lactogenic RESULTS Ontogenesis of PRL-R Expression in Female Rat Liver 0888-8809/89/0895-0900$02.00/0 Molecular Endocrinology Copyright © 1989 by The Endocrine Society The number of PRL-R and receptor mRNA levels have been determined in rat liver at various ages: in fetus, 895 Laboratory of Molecular Endocrinology McGill University Royal Victoria Hospital (J.-M.B., H.O., S.R., P.A.K.) Montreal, Quebec, Canada H3A 1A1 MOL ENDO-1989 896 Vol 3 No. 5 newborn fetus II 21 days PRL-R Expression in Liver from Estrogen-Treated Female Rat Liver The effects of administration of a long-acting estrogen on hepatic PRL-R mRNA and receptor numbers were studied in adult female rat liver. Estradiol valerate was injected sc on day 0, and the animals were killed 1,3, 5, and 7 days after initiation of treatment. Poly(A)+ mRNA and microsomes were prepared from livers of four to eight animals at each time point. Figure 4 shows Northern blot analysis of PRL-R mRNA in estrogentreated rat liver, while Fig. 5 illustrates the Scatchard analysis of binding on the corresponding days of treatment. In Fig. 6, the quantitation of mRNA levels as well as the number of PRL-binding sites are shown. Relative abundance of PRL-R mRNA is low in nontreated animals (day 0), and no significant change is visible on day 1. There is a marked increase in PRL-R mRNA abundance between days 1-3, after which levels remain elevated until the end of the study. As was true for the developmental studies, the induction of receptor number did not directly parallel steady state mRNA levels. Although there is a similar lag period, a greater increase occurs in the number of PRL-binding sites than in mRNA levels from days 1-3. Steady state mRNA levels stabilized between days 3-7, while receptor numbers increased between days 1-7. DISCUSSION The effects of sex steroids on PRL-binding sites in liver are well established (20-22, 25, 26, 32). In this report we present for the first time a comparison between the PRL-R mRNA level and the number of PRL-R at various ages and in response to estrogen treatment. PRL-R mRNA in liver was studied by Northern blot analysis, and the number of PRL-R in the corresponding samples was determined by Scatchard analysis. It should be clear that comparisons are not made between absolute values at each time point, but, rather, between relative steady state mRNA and binding levels. The number of PRL-binding sites does not exactly parallel PRL-R mRNA levels during ontogeny in normal 40 days 70 days IT Fig. 1. Northern Blot Analysis of PRL-R mRNA in Female Rat Liver at Different Ages Each lane represents mRNA from individual rats. Ten micrograms of poly(A)+ RNA were loaded in each well. The blot was hybridized at high stringency (see Materials and Methods) with a complete 1.6-kb F3 RNA probe and a 300-bp B1 RNA probe. The x-ray film was exposed for 16 h without an intensifying screen. The length of PRL-R mRNA is 2.2 kb, and that of B1 mRNA is 1.5 kb. newborn, and 21-, 40-, and 70-day-old females. At least four independent samples were measured at each stage. Each sample was divided for PRL-R mRNA and hormone binding measurements. Figure 1 shows the Northern blot analysis of PRL-R mRNA in female rat liver as a function of a (...truncated)