Influence of Obstructive Cholestasis and Sex Hormones on the Ratio of mRNA of Two Alternative Prolactin Receptor Isoforms in Rat Hepatocytes

The effects of obstructive cholestasis and sex hormones on the total content of prolactin receptor mRNA and ratio of mRNAs of its short and long isoforms have been studied in rat hepatocytes. Obstructive cholestasis caused insignificant changes in total content of prolactin receptor mRNA, but the proportion of mRNA of the long isoform increased. Comparison of prolactin receptor mRNA levels in gonadectomized and intact animals revealed opposite effects of male and female sex hormones on total mRNA, but both groups of these hormones increased the proportion of prolactin receptor short form mRNA. Changes in ratio of mRNA of receptor isoforms found in rat hepatocytes under obstructive cholestasis did not depend on levels of sex hormones. Obstructive cholestasis and sex hormones are suggested to regulate the content of long and short prolactin receptor isoforms in hepatocytes independently.     

Prolactin stimulates the proliferation of normal female cholangiocytes by differential regulation of Ca2+-dependent PKC isoforms

Background

Prolactin promotes proliferation of several cells. Prolactin receptor exists as two isoforms: long and short, which activate different transduction pathways including the Ca²⁺-dependent PKC-signaling. No information exists on the role of prolactin in the regulation of the growth of female cholangiocytes. The rationale for using cholangiocytes from female rats is based on the fact that women are preferentially affected by specific cholangiopathies including primary biliary cirrhosis. We propose to evaluate the role and mechanisms of action by which prolactin regulates the growth of female cholangiocytes.

Results

Normal cholangiocytes express both isoforms (long and short) of prolactin receptors, whose expression increased following BDL. The administration of prolactin to normal female rats increased cholangiocyte proliferation. In purified normal female cholangiocytes, prolactin stimulated cholangiocyte proliferation, which was associated with increased [Ca²⁺]_i levels and PKCβ-I phosphorylation but decreased PKCα phosphorylation. Administration of an anti-prolactin antibody to BDL female rats decreased cholangiocyte proliferation. Normal female cholangiocytes express and secrete prolactin, which was increased in BDL rats. The data show that prolactin stimulates normal cholangiocyte growth by an autocrine mechanism involving phosphorylation of PKCβ-I and dephosphorylation of PKCα.

Conclusion

We suggest that in female rats: (i) prolactin has a trophic effect on the growth of normal cholangiocytes by phosphorylation of PKCβ-I and dephosphorylation of PKCα; and (iii) cholangiocytes express and secrete prolactin, which by an autocrine mechanism participate in regulation of cholangiocyte proliferation. Prolactin may be an important therapeutic approach for the management of cholangiopathies affecting female patients.     

Role of sodium/hydrogen exchanger isoform NHE3 in fluid secretion and absorption in mouse and rat cholangiocytes

Na+/H+ exchanger (NHE) isoforms play important roles in intracellular pH regulation and in fluid absorption. The isoform NHE3 has been localized to apical surfaces of epithelia and in some tissues may facilitate the absorption of NaCl. To determine whether the apical isoform NHE3 is present in cholangiocytes and to examine whether it has a functional role in cholangiocyte fluid secretion and absorption, immunocytochemical studies were performed in rat liver with NHE3 antibodies and functional studies were obtained in isolated bile duct units from wild-type and NHE3-/- mice after stimulation with forskolin, using videomicroscopic techniques. Our results indicate that NHE3 protein is present on the apical membranes of rat cholangiocytes and on the canalicular membrane of hepatocytes. Western blots also detect NHE3 protein in rat cholangiocytes and isolated canalicular membranes. After stimulation with forskolin, duct units from NHE3-/- mice fail to absorb the secreted fluid from the cholangiocyte lumen compared with control animals. Similar findings were observed in isolated bile duct units from wild-type mice and rats in the presence of the Na+/H+ exchanger inhibitor 5-(N-ethyl-N-isopropyl)-amiloride. In contrast, we could not demonstrate absorption of fluid from the canalicular lumen of mouse or rat hepatocyte couplets after stimulation of secretion with forskolin. These findings indicate that NHE3 is located on the apical membrane of rat cholangiocytes and that this NHE isoform can function to absorb fluid from the lumens of isolated rat and mouse cholangiocyte preparations.     

Prolactin receptor antagonism in mouse anterior pituitary: effects on cell turnover and prolactin receptor expression

Since anterior pituitary expresses prolactin receptors, prolactin secreted by lactotropes could exert autocrine or paracrine actions on anterior pituitary cells. In fact, it has been observed that prolactin inhibits its own expression by lactotropes. Our hypothesis is that prolactin participates in the control of anterior pituitary cell turnover. In the present study, we explored the action of prolactin on proliferation and apoptosis of anterior pituitary cells and its effect on the expression of the prolactin receptor. To determine the activity of endogenous prolactin, we evaluated the effect of the competitive prolactin receptor antagonist Δ1-9-G129R-hPRL in vivo, using transgenic mice that constitutively and systemically express this antagonist. The weight of the pituitary gland and the anterior pituitary proliferation index, determined by BrdU incorporation, were higher in transgenic mice expressing the antagonist than in wild-type littermates. In addition, blockade of prolactin receptor in vitro by Δ1-9-G129R-hPRL increased proliferation and inhibited apoptosis of somatolactotrope GH3 cells and of primary cultures of male rat anterior pituitary cells, including lactotropes. These results suggest that prolactin acts as an autocrine/paracrine antiproliferative and proapoptotic factor in the anterior pituitary gland. In addition, anterior pituitary expression of the long isoform of the prolactin receptor, measured by real-time PCR, increased about 10-fold in transgenic mice expressing the prolactin receptor antagonist, whereas only a modest increase in the S3 short-isoform expression was observed. These results suggest that endogenous prolactin may regulate its own biological actions in the anterior pituitary by inhibiting the expression of the long isoform of the prolactin receptor. In conclusion, our observations suggest that prolactin is involved in the maintenance of physiological cell renewal in the anterior pituitary. Alterations in this physiological role of prolactin could contribute to pituitary tumor development.     

Loss of growth hormone-dependent characteristics of rat hepatocytes in culture

Summary The liver of rodents is sexually differentiated, i.e. the female liver differs from the male liver. This differentiation is largely controlled by the pattern of growth hormone (GH) secretion. We have attempted to maintain GH-dependent differentiation of cultured rat hepatocytes. We examined the level of alcohol dehydrogenase (ADH) activity, which responds to GH and is higher in female than in male liver, and the estrogen receptor, which is dependent on GH but is present in equal amounts in males and females. ADH activity was maintained in cells from male rats, but fell by 40% in cells from females in medium supplemented with insulin and dexamethasone. The estrogen receptor content of female cells fell dramatically to undetectable levels within 2 d of culture. Extensive supplementation of the medium failed to prevent the decrease in ADH activity in female cells; similarly, the addition of female sex steroids; rat serum; pituitary extracts; rat, human, or bovine GH; or ovine prolactin failed to maintain the enzyme activity. Insulin, dexamethasone, thyroid hormone plus GH or prolactin, or the combination of all five hormones also failed to prevent the loss of estrogen receptors. Short-term cultures of rat hepatocytes, although retaining the liver-specific expression of ADH at the male level, lose GH-dependent expression of the estrogen receptor and stimulation of ADH activity. Supported by grants AA 00081 and AA 06434 from the National Institute on Alcohol Abuse and Alcoholism, Bethesda, MD.     

Glycosylated rat prolactin: isolation and structural characterization.

Isolation of glycosylated 26 kDa rat prolactin and subsequent proper carbohydrate characterization has so far not been reported. In the present work the hormone isoform was isolated to 95% homogeneity by preparative electrophoretic separation on Mini Prep Cell of rat pituitary homogenate. The isoform was then investigated by 2-mercaptoethanol gradient electrophoresis, Cleveland's sequential SDS-PAGE, digestion with endoproteinase Asp-N and N-glycanase. The glycosidic part of the isoform was examined in O-profiling and its monosaccharide composition obtained by FACE and HPAE-PAD analysis. The outcome of the experimental data is: 1) in contrast to unglycosylated 23 kDa rat prolactin, intra-chain S-S bridging is not affected in 26kDa rat prolactin, neither by transiting through a thiol gradient nor in sequential nonreducing/reducing SDS-PAGE; 2) the conformational availability of Asp residues involved in the endoproteinase Asp-N attack is the same in 23- and 26 kDa rat prolactin; the glycan moiety apparently does not cause steric hindrance at this level; 3) no glycosidic N-linkage could be detected, only O-linkage(s); 4) 26 kDa rat prolactin is no glycosyl-phosphaditylinositol-anchored protein; 5) in O-profiling an oligosaccharide chain of Mr +/- 1.4 kDa was recorded; 6) the monosaccharide composition obtained in FACE is peculiar in the sense that next to Fuc, Man, GalNac, GlcNac and NeuAc also Rib was determined; 7) HPAE-PAD analysis identified NeuAc subtypes; 8) in vitro, glycosylation of rat prolactin modulates immune recognition through steric hindrance of the access to the epitope sites.     

Mechanisms of discrimation of the prolatin physiologic action on the liver at the receptor level of hepatocytes and cholangiocytes

The role ofprolactin receptor isoforms in discrimination ofprolactin effects on liver is summarized. The necessity of studying of parameters ofprolactin receptor expression in differentially isolated different types of liver cells is demonstrated. The review is illustrated by data on different regulation by sex hormones and obstructive cholestasis of expression of prolactin receptor isoforms in rat hepatocytes and epithelial cells of bile ducts. The mathematical model is introduced for estimation of intensity of prolactin-induced signal cascades on the basis of experimentally measured parameters of prolactin receptor expression. Some further steps of the investigation of prolactin effects discrimination at the level receptor unit are suggested.     

Ciliated epithelial-specific and regional-specific expression and regulation of the estrogen receptor-beta2 in the fallopian tubes of immature rats: a possible mechanism for estrogen-mediated transport process in vivo

Several ERbeta isoforms have been identified in human and rodent tissues, but it is unclear whether each isoform has distinctly different cellular targeting characteristics and physiological functions. We have investigated the intracellular localization and regulatory patterns for ERbeta isoforms in rat fallopian tubes. Western blot analysis reveals that two ERbeta isoforms corresponding to ERbeta1 and ERbeta2 are expressed in rat fallopian tubes. However, ERbeta2 is the predominant form of ERbeta in this tissue. High-resolution confocal imaging and immunohistochemical analysis provide ample evidence that ERbeta expression is limited almost exclusively to the ciliated epithelial cells, in contrast to ERalpha, which is widely distributed. Furthermore, within the ciliated epithelial cells, ERbeta is colocalized with beta-tubulin IV at stem portion of the cilia. We show that ERbeta2 protein expression is tightly regulated by E(2) or DPN in a time-dependent manner without changes in ERbeta1 expression. These estrogenic effects are inhibited by an ER antagonist, ICI 182,780. In addition, significant alteration of ERbeta immunoreactivity is detected only histologically in the ampullary region. Since the cilia are considered an essential determinant of tubal transport, we further demonstrate that E(2)- or DPN-induced ERbeta2 activation is associated with alterations in tubal protein expression crucial for the regulation of calcium-dependent ciliary beating. Given the coordinated regulation and interaction of ER and progesterone receptor in the cilia, we hypothesize that tubal ERbeta2 may facilitate the estrogen-mediated transport process by processing protein-protein interaction under physiological and/or pathological conditions. We show for the first time that a previously unrecognized localization of ERbeta isoform in rat fallopian tubes can combine with estrogen to individually control the expression of ER beta-isoforms in normal target tissues.     

Biological,immunological and biochemical characterization of cleaved prolactin generated by lactating mammary gland

We have previously shown that rat prolactin is proteolytically cleaved in its loop by peripheral tissues of the rat. Of the tissues examined to date, lactating mammary gland exhibits the highest prolactin-cleaving activity. The objective of this study was to characterize cleaved prolactin, biologically, immunologically and chemically. By modifying an established analytical method, we were able to generate large (μg) amounts of cleaved rat prolactin from cell fractions of rat mammary gland which could then be assayed for biological and immunological activity relative to intact hormone. The cleaved product showed no significant difference relative to the intact rat prolactin when assayed for its ability to compete with ¹²⁵I-labelled ovine prolactin for the prolactin receptor and for its ability to stimulate the proliferation of rat Nb2 lymphoma cells. Cleaved rat prolactin, however, did show a 50–60% reduction in activity relative to intact rat prolactin when assayed by radioimmunoassay. Using Edman degradation and partial amino acid analysis, we determined that the second N-terminus of the cleaved rat prolactin begins at amino acid 149. The divergence of biological and immunological activity produced by proteolytic cleavage in the loop of rat prolactin suggests that biological and immunological sites differ in location. The possible physiological implications of a cleaved rat prolactin molecule generated by target tissue with maintained biological activity and reduced immunological activity are discussed.     

军曹鱼催乳素受体PRLR1基因的克隆及其在不同盐度条件下mRNA表达差异

催乳素受体通过结合催乳素,能调节鱼体渗透压。为研究催乳素受体1(PRLR1)在高盐水体和低盐水体中对军曹鱼(Rachycentron canadum)的渗透调节作用,利用cDNA末端快速扩增(RACE-PCR)技术,获得了军曹鱼PRLR1全长cDNA序列。该基因全长为2629 bp,包含1953 bp的开放阅读框ORF,可编码650个氨基酸。氨基酸序列包含了2个纤维连接蛋白3型结构域(FN3)、保守的WS区和box1。采用qRT-PCR技术,检测不同盐度(10‰、30‰和35‰)条件下鳃、肠、体肾中PRLR1基因mRNA表达情况。结果显示,PRLR1基因在军曹鱼的各个组织中均有表达,其中鳃表达量最高,其次是肌肉、体肾和肠,而在胃、脾、脑和心脏中则微量表达。低盐组、正常组和高盐组中,PRLR1基因的表达量均为鳃最高;肠次之;体肾最低。随着盐度提高,PRLR1基因的鳃、肠和体肾组织表达量变化规律均呈逐步下降趋势。以上结果反映了军曹鱼PRLR1在渗透压器官中的功能差异性,说明PRLR1在军曹鱼渗透压调节上具有重要作用。     

Pinealectomy or light exposure exacerbates biliary damage and liver fibrosis in cholestatic rats through decreased melatonin synthesis

Melatonin, a neuroendocrine hormone synthesized by the pineal gland and cholangiocytes, decreases biliary hyperplasia and liver fibrosis during cholestasis-induced biliary injury via melatonin-dependent autocrine signaling through increased biliary arylalkylamine N-acetyltransferase (AANAT) expression and melatonin secretion, downregulation of miR-200b and specific circadian clock genes. Melatonin synthesis is decreased by pinealectomy (PINX) or chronic exposure to light. We evaluated the effect of PINX or prolonged light exposure on melatonin-dependent modulation of biliary damage/ductular reaction/liver fibrosis. Studies were performed in male rats with/without BDL for 1 week with 12:12 h dark/light cycles, continuous light or after 1 week of PINX. The expression of AANAT and melatonin levels in serum and cholangiocyte supernatant were increased in BDL rats, while decreased in BDL rats following PINX or continuous light exposure. BDL-induced increase in serum chemistry, ductular reaction, liver fibrosis, inflammation, angiogenesis and ROS generation were significantly enhanced by PINX or light exposure. Concomitant with enhanced liver fibrosis, we observed increased biliary senescence and enhanced clock genes and miR-200b expression in total liver and cholangiocytes. In vitro, the expression of AANAT, clock genes and miR-200b was increased in PSC human cholangiocyte cell lines (hPSCL). The proliferation and activation of HHStecs (human hepatic stellate cell lines) were increased after stimulating with BDL cholangiocyte supernatant and further enhanced when stimulated with BDL rats following PINX or continuous light exposure cholangiocyte supernatant via intracellular ROS generation. Conclusion: Melatonin plays an important role in the protection of liver against cholestasis-induced damage and ductular reaction.