0013-7227/92/1313-1017$03.00/0 Endocrinology Copyright 0 1992 by The Endocrine

Vol. 131, No. 3

Printed

Society

Posttranscriptional Gene in IM-9-P3

Regulation of the Human Cells by Retinoic Acid

BIRGIT GELLERSEN, RITA KEMPF, AND GABRIEL E. DrMATTIA

STEFAN

HARTUNG,

ANNETTE

in U.S.A.

Prolactin

BONHOFF,

Institute for Hormone and Fertility Research, 2000 Hamburg 54, Grandweg 64, Germany; and the Eukaryotic Regulatory Biology Program and Center for Molecular Genetics (G.E.D.), University of California-San Diego School of Medicine, La Jolla, California 92093-0648 ABSTRACT The IM-9-P3 family of cell lines, which are derived from the Blymphoblastoid IM-9 cell line, transcribe the human PRL (hPRL) gene by utilization of the decidual-type promoter and provide a model to study factors controlling extrapituitary expression of the hPRL gene. Here we describe regulation of hPRL gene expression in members of the IM-9-P3 family by retinoic acid (RA). When cells were incubated in medium supplemented with fetal calf serum that had been treated with dextran-coated charcoal, the addition of RA caused a Z-fold stimulation of hPRL secretion in the low hPRL-producing clone IMg-P31 and the moderate producer IM-g-P32 (EDao, 0.53 and 0.13 nM, respectively), but not in the high hPRL-producing IM-g-P33 clone. Secretion from the RA-responsive cell lines increased steadily over the first 24 h of exposure and remained elevated for several days. The concomitant increase in hPRL mRNA steady state levels was not due to enhanced transcription of the hPRL gene, as assessed by nuclear

run-on experiments, but, rather, to message stabilization. In RA-treated IM-g-P32 cells, the half-life of hPRL mRNA was significantly increased from 9 to 22 h. The transcripts were found to be preferentially associated with membrane-bound polysomes, thus being available for the secretorv oathwav. When we studied the exnression of notential transducers of-the RAsignal, namely the RA receptor SubtypeshRARol, -A and -y and cellular RA-binding protein, we did not detect hRARr or cellular RA-binding protein transcripts in the hPRL-negative clone IM-9-P6 or the hPRL-oositive clones IM-9-P31. IM-9-P32. and IM-9P33. hRARa was equally expressed in all cell hnes and not regulated by RA, whereas hRARp was differentially expressed and controlled by RA. This receptor subtype was absent from hPRL-negative members of the IM-9-P family, strongly induced by RA in the RA-responsive IM-g-P31 and IM-g-P32 cell lines via rapid transcriptional up-regulation, and only slightly induced in the R&resistant I-M-9-P33celliine, suggesting a function in mediation of the effect of RA on hPRL gene expression. (Endocrinology 131: 1017-1025,1992)

T

The focus of this study is retinoic acid (RA), the active metabolite of vitamin A in most biological processes,which plays a pivotal role in the regulation of growth and differentiation of many epithelial and mesenchymal cell types (reviewed in Refs. 11-13). The cloning of a number of intracellular retinoid-binding proteins has shed light on the pathway by which the RA signal is transduced. At least three subtypes of RA receptors (hRARLu,hRAR/3, and hRARy) have been identified that are structurally related to the steroid/ thyroid hormone receptor family of transcription factors (1416). The function of cellular RA-binding protein (CRABP) is stiI1 speculative; it might modulate the concentration of free RA in the cell or direct RA metabolism/catabolism (reviewed in Ref. 17). The direct transcriptional effect of RA-RAR complexes has been confirmed by the identification of a number of specific RA response elements @ARES), for example in the hRARP gene (18, 19) and the laminin-Bl gene (20). Expression of the rat GH gene can be regulated by RA via the thyroid hormone response element, with which the RAR has been shown to interact (21, 22). In addition to its direct effect on the transcription of various genes, RA can function posttranscriptionally to modulate the steady state level of a particular message(23, 24). We show in this paper that hPRL gene expression is controlled by RA. When we exposed cells of the IM-9-P3 series to the retinoid, we observed a stimulation of hPRL expression in IM-9-P31 and IM-9-P32 cells, but not in IM9-P33 cells. We identified the site of action of RA to be posttranscriptional and investigated the possible role of the

ISSUE -specific expression of the human PRL (hPRL) gene is directed by two different promoter regions. In the pituitary lactotroph, transcription of the hPRL geneyields a mature transcript of 1 kilobase (kb) (1, 2), whereas uterine decidua and myometrium hPRL mRNAs are approximately 150 nucleotides longer (3, 4) due to the transcription of an additional 5’-noncoding exon (exon la) located about 6 kb up-stream of the pituitary exon 1 (5, 6). We have described a set of cell lines, the IM-9-P family, that ectopically produce hPRL by utilization of this alternative decidual-type cap site (5, 7). Cloning of the IM-9-P cell line, a spontaneousvariant of the IM-9 B-lymphoblastoid cell line (8), led to the establishment of cell lines that produce undetectable (IM-9-P6), low (IM-9-P31), moderate (IM-9-P32), or high (IM-9-P33) amounts of hPRL (9). These unique cell lines provide us with a model system with which to study the mechanismsregulating transcription of the hPRL gene from the uterinespecific cap site. Much like decidual stromal cells, cells of the IM-9-P3 serieshave proven refractory to most of the classical hormonal regulators of pituitary hPRL expression (10). Clearly, modulation of uterine hPRL gene expressionis under the control of a different set of factors, which may be linked to the tissue-remodelling process occurring during uterine decidualization. Within this concept we have tested a number of factors that might be responsible for the activation and regulation of uterine hPRL gene expression. Received March 30, 1992. Address all correspondence and requests for reprints to: Dr. Birgit Geilemen, Institute for Hormone and Fertility Research, Grandweg 64, W-2000 Hamburg 54, Germany. 1017

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 24 November 2015. at 21:50 For personal use only. No other uses without permission. . All rights reserved.

RA REGULATION RAR subtypes and CRABP in establishing or -resistant phenotype.

OF hPRL GENE

a RA-responsive

Materials and Methods Cell culture The human B-lymphoblast cell line IM-9 (CCL 159, American Type Culture Collection, Rockville, MD); the hPRL-oroducina clonal derivatives IM-9-P31, Ih&9-P32, and Il\i-9-P33; and the hP

Posttranscriptional regulation of the human prolactin gene in IM-9-P3 cells by retinoic acid.

The IM-9-P3 family of cell lines, which are derived from the B-lymphoblastoid IM-9 cell line, transcribe the human PRL (hPRL) gene by utilization of t...
2MB Sizes 0 Downloads 0 Views