345 Long isoform of prolactin receptor predominates in rat intrahepatic bile ducts and further increases under obstructive cholestasis R L Bogorad, T Y Ostroukhova, A N Orlova, P M Rubtsov1 and O V Smirnova Laboratory of Endocrinology, Faculty of Biology, Lomonosov Moscow State University, Vorob’ev Hills, 119992, Moscow, Russian Federation 1Laboratory of Hormones and Receptors, Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilova Str., 119991, Moscow, Russian Federation (Requests for offprints should be addressed to R L Bogorad; Email: [email protected]) Abstract Prolactin participates in the regulation of liver function. bromocriptine-treated animals. Common bile-duct However, prolactin receptor (PrlR) expression and its ligation was used as the experimental model of regulation have been described only for hepatocytes. In cholestasis. Our results demonstrate that the expression this study, we investigated the expression and regulation pattern and regulation of PrlR isoforms is totally of PrlR isoforms in the other important intrahepatic different in cholangiocytes compared with hepato- cellular compartment: the biliary epithelial cells, or cytes: (1) mature rat cholangiocytes express low levels cholangiocytes. Our aim was to determine whether of PrlR, while it is very high in hepatocytes, (2) only prolactin should be considered as a potential regulator the long isoform is detected in cholangiocytes, while of cholangiocyte function under normal and pathologi- the short isoform predominates in hepatocytes and (3) cal conditions. Cholangiocytes and hepatocytes were PrlR levels in cholangiocytes are induced by obstructive differentially isolated from rat liver. PrlR expression was cholestasis, but not by sex hormones or prolactin, while analysed at the mRNA level by isoform-specific it is the opposite in hepatocytes. From these data, the semiquantitative PCR, and at the protein level by actions of prolactin on liver are anticipated to exhibit immunostaining of liver sections. Hormonal regulation strong cell-type specificity in both normal and of PrlR expression was evaluated by comparing pathological conditions. intact rats with gonadectomized, pituitary-grafted or Journal of Endocrinology (2006) 188, 345–354 Introduction STAT activation, resulting from formation of short and long PrlR heterodimers (Perrot-Applanat et al. 1997). Several prolactin receptor (PrlR) isoforms have been However, short PrlR is also involved in transduction of identified in mammals. In rat, they have been shown to be specific signals, including activation of serine/threonine produced via alternative splicing of PrlR pre-mRNA: the protein kinase cascades and interaction with 17-hydroxy- formation of long and short isoforms result from alterna- steroid dehydrogenase (Das & Vonderhaar 1995, Duan tive inclusion of proximal exon 10 or distal exon 11 into et al. 1997, Nikelainen et al. 1998). the mature mRNA, respectively (Bole-Feysot et al. 1998). Liver is very specific compared with other organs with These receptor isoforms differ in length and structure of respect to PrlR expression: the level of PrlR is higher than their intracellular domain, which affects signalling prop- in most other tissues, and this mainly involves the short erties. Thus, tissue sensitivity and responsiveness to pro- isoform since the long PrlR is 10% of total (Nagano & lactin depend on both the level of total PrlR expression Kelly 1994). The total level of PrlR in hepatocytes is and the ratio of short to long isoforms. regulated by sex hormones: oestrogens increase and Prolactin signalling is initiated by formation of a com- androgens decrease the content of PrlR mRNA and plex involving one prolactin molecule and two molecules protein (Jolicoeur et al. 1989, Sakaguchi et al. 1994). As in of PrlR. The long PrlR isoform activates various signalling other tissues, prolactin was shown to be another positive cascades, among which Janus kinase/signal transduction regulator of PrlR expression in the liver (Baxter & and activators of transcription (JAK/STAT) is considered Zaltsman 1984, Bogorad et al. 2000). Although PrlR to be the central pathway (Bole-Feysot et al. 1998). The expression is well documented, the actions of prolactin in short PrlR isoform exerts dominant-negative effects on liver remain poorly understood (Bole-Feysot et al. 1998). Journal of Endocrinology (2006) 188, 345–354 DOI: 10.1677/joe.1.06468 0022–0795/06/0188–345 2006 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org Downloaded from Bioscientifica.com at 09/26/2021 09:48:06AM via free access 346 R L BOGORAD and others · Prolactin receptors in rat cholangiocytes Relatively few disembodied data were published regard- according to Alpini et al. (1989). In order to elevate the ing prolactin effects on hepatocyte proliferation (Picolleti circulating prolactin level some rats were subjected to et al. 1997) as well as on expression of a number of transplantation of two donor pituitaries into kidney hepatocyte proteins (Bole-Feysot et al. 1998, Liu et al. capsule. The completeness of graft acceptance was deter- 1992). No liver phenotype has been reported in prolactin- mined for each animal by autopsy. This well-characterized or PrlR-knockout mice (Horseman et al. 1997, Ormandy impact increases the prolactin level approximately 5-fold, et al. 1997), which does not exclude compensatory actions to 55 ng/ml (Garcia et al. 2003). In 2 days recipient by other cytokine receptors. animals underwent CBDL. Although liver consists essentially of hepatocytes, Bromocriptine was injected daily after CBDL as 50% another important cell compartment involves intrahepatic ethanol suspension of bromocriptine pills (Gedeon biliary epithelial cells, referred to as cholangiocytes. These Richter, Budapest, Hungary; 10 µg/kg animal weight) to cells are organized in a three-dimensional network of decrease the level of circulating prolactin. Vehicle was ducts, which implies interactions with other hepatic cell injected into control animals. This treatment decreases types. Despite their relatively small number, cholangio- the prolactin level 10-fold, to approximately 1 ng/ml cytes play an essential role in liver functions, including bile (Bogorad et al. 2000). formation and secretion (Alpini et al. 1994b). The pro- The tissues were subjected to simultaneous isolation of portion and activity of these two cell types can change bile-duct units and hepatocytes or fixed in 4% paraform dramatically depending on the conditions. During hepatic and used for immunohistochemical staining. diseases, such as obstructive cholestasis or jaundice, All animal groups were compared with the correspond- the bile tree grows intensively, and the proportion of ing sham-operated group to control for non-specific cholangiocytes can be increased to up to 20% of total effects. The preliminary reverse transcriptase (RT)-PCR liver cells (Alpini et al. 1994a). The responsiveness of experiments did not reveal any influence of sham opera- cholangiocytes to various regulatory factors is also mark- tions on PrlR mRNA level or ratio of PrlR isoforms in edly enhanced under cholestatic conditions (Alpini et al. cholangiocytes (one-way analysis of variance (ANOVA), 1994b, Alvaro et al. 2000b). Because prolactin participates P>0·6; data not shown). in the regulation of water–ion balance and secretory processes (Bole-Feysot et al. 1998), its involvement in the regulation of cholangiocyte function is expected. How- Immunohistochemistry and cytophotometry ever, since evidence is still lacking that these cells express Tissues were embedded in paraplast and sliced into 3 µm PrlR, any effect of prolactin under normal or pathological sections. Immunohistochemical staining of sections with states remains speculative. These issues were addressed in anti-PrlR antibodies (clone U5; provided by Dr V Goffin our study. We report the profile of expression on both and Dr P Kelly, INSERM, Paris, France) and cyto- long and short PrlR isoforms in rat liver, under normal photometry were done as described previously (Bogorad and cholestatic conditions. We also provide some clues on et al. 2000). One hundred and fifty cells on two tissue their regulation by sex hormones and prolactin. The sections of each animal were measured for the intensity of unexpected conclusion of our investigations is that the PrlR-positive immunostaining, which is represented as expression of PrlR isoforms and their regulation in optical density (OD). cholangiocytes and hepatocytes are dramatically different, and in many cases opposite. Simultaneous isolation of bile-duct units and hepatocytes In some experiments, the rat livers were subjected to Materials and Methods isolation of hepatocytes and cholangiocytes as intrahepatic bile-duct units (Mennone et al. 1995). Briefly, the liver Animals was perfused by Ca2+-/Mg2+-free Hanks’ balanced salts All animals were housed on standard rodent chow and solution (all reagents and enzymes for cell isolation and tap water ad libitum and received humane care, the reagents for tissues preparations were from Sigma) study protocols complied with the Faculty of Biology’s containing 0·7 mM EDTA, then perfused through the (Lomonosov Moscow State University) guidelines. portal vein by collagenase (0·05%) in Hanks’ balanced salts Intact mature 12-week-old male and female rats as well solution with 40 mM HEPES and 5 mM CaCl2. Residual as immature rats were used to determine the parameters of portal tissue was mechanically dissociated