Studies on vitamin carrier protiens hormonal induction and modulation of ribflavin carrier protien and retinol

Abstract

STUDIES ON VITAMIN CARRIER PROTEINS: HORMONAL INDUCTION AND MODULATION OF RIBOFLAVIN CARRIER PROTEIN AND RETINOL BINDING PROTEIN IN IMMATURE CHICKS submitted by B. Durga Kumari for the award of the degree of Doctor of Philosophy of the Indian Institute of Science, Bangalore, India. Investigations detailed in this dissertation form a part of a continuing programme of research work in this laboratory on vitamin carrier proteins with particular reference to their isolation and characterization, hormonal induction and modulation, and functional significance during embryonic development. Earlier studies from this laboratory on the kinetics and hormonal modulation of biosynthesis of riboflavin-carrier protein (RCP) in chicken liver have shown gross similarity between estrogen induction of RCP and that of vitellogenin in the avian liver. Since similar RCP is present in the egg white, studies were undertaken to examine the induction and hormonal specificity of RCP synthesis by its presumed locus of production viz., the oviduct. Hitherto, most of the biosynthetic studies on both egg white- and egg yolk proteins have been carried out by immunoprecipitation of these proteins labelled with radiolabelled precursors (for example, vitellogenin in liver and ovalbumin in the oviduct); but a more sensitive method was necessary to measure the minute quantities of RCP in the plasma as well as tissue cytosols of hormone-treated chicks. In view of this, a sensitive and specific radioimmunoassay (RIA) was used to quantify RCP levels in the plasma and oviduct cytosol of chicks during hormonal induction. By using this assay method, RCP concentrations as low as 0.5 ng per ml of plasma or oviduct cytosol could be quantified. During primary stimulation of the immature female birds with estrogen (10 daily injections), there was a rapid increase in both the oviduct weight and the total RCP content after an initial lag period of 2–3 days which presumably represented the period of proliferation and cytodifferentiation of the epithelial cells of the immature oviduct. Either progesterone or testosterone, when given alone during primary stimulation, did not have any influence either on oviduct weight or on its RCP content. Moreover, progesterone given along with estrogen inhibited estrogen-mediated response of the immature oviduct and hence curtailed the increases both in the oviduct weight as well as the total RCP content. Testosterone given along with estrogen stimulated synergistically the estrogen-mediated increase in oviduct weight as well as the total RCP content. However, the RCP content per unit weight of the magnum in these chicks was closely comparable to those in estrogen-treated birds, thereby showing that the increase in total tissue content of RCP is accounted for by enhanced weight of the magnum but not by enhanced concentration per unit weight of the magnum. During secondary stimulation, estrogen given to estrogen-primed chicks resulted in rapid increase of the oviduct weight as well as the total RCP content, without any initial lag period unlike that observed during primary stimulation. Progesterone treatment of such estrogen-primed chicks resulted only in a slight increase in both oviduct weight as well as total RCP content. However, when these RCP values were expressed per mg magnum, the RCP concentration was comparable to that of estrogen-treated birds. This clearly showed that progesterone was relatively ineffective in bringing about an increase in the oviduct weight but, like estrogen, could resume differentiated oviduct cell function in terms of RCP synthesis. When testosterone was given during secondary stimulation to estrogen-primed chicks, there was no increase either in the tissue weight or in the total RCP content of the oviducts. From the above results it would appear that both estrogen and progesterone could support RCP synthesis in differentiated oviduct cells to similar extents while testosterone was ineffective independently in eliciting this response. In contrast, the plasma content, reflecting the hepatic induction of RCP, increased rapidly after an initial lag period during primary stimulation with estrogen. After 10 daily injections of estrogen, RCP content reached a level of about 150 µg/ml of plasma and this was followed by a rapid decline upon cessation of hormone administration, reaching baseline values within 7–8 days of hormone withdrawal. Neither progesterone nor testosterone had any effect on the plasma accumulation of RCP when administered either alone or in combination with estrogen. This testifies to the absolute estrogen specificity of liver RCP synthesis during primary stimulation. Estrogen administration during secondary stimulation to either estrogen-, estrogen plus progesterone-, or estrogen plus testosterone-primed birds resulted in similar rapid increases in the plasma RCP contents without any appreciable lag period. Neither progesterone nor testosterone could substitute for estrogen in this respect during secondary stimulation, unlike the situation with the oviduct. RCP content in both the plasma as well as the oviduct cytosol showed an amplified response during secondary stimulation vis-à-vis during primary stimulation, which is reminiscent of the typical 'memory effect' observed earlier during induction of vitellogenin in the avian and amphibian liver. In vitro incorporation of radiolabelled amino acids into the newly synthesized proteins by the tissue explants was carried out to examine whether the contents of RCP in both the plasma and the oviduct cytosol, measured by RIA, reflect the synthetic rates of RCP in these target tissues. Following estrogen administration during secondary stimulation, there was a rapid increase in the relative rate of RCP synthesis in both liver and oviduct explants, while progesterone was ineffective in stimulating RCP synthesis in liver but induced RCP synthesis in the oviduct more or less to the same extent as estrogen did. The absolute rate of synthesis of RCP represented a significantly higher proportion of enhanced total protein elaboration upon hormone administration. Thus these results confirm the ability of progesterone in inducing RCP synthesis in the estrogen-primed oviduct but not in the liver. These data also confirm the earlier finding that progesterone could replace estrogen in In terms of RCP synthesis in the oviduct during secondary stimulation: In order to examine whether there is any direct correlation between the contents of RCP and its mRNA activity during hormonal stimulation and also to test the possibility of the involvement of a precursor molecule for RCP as postulated earlier, poly(A) RNA was isolated from both the liver and oviduct of hormone- (estrogen or progesterone) treated birds and was translated in vitro in a heterologous cell-free translation system derived from rabbit reticulocytes. In vitro synthesized RCP was immunoprecipitated using specific RCP antibody. When the RCP immunoprecipitates so obtained were analysed on SDS-PAGE, the radioactivity distribution profile on the gel clearly indicated a precursor RCP (pre-RCP) of 38,000 as the primary translation product. When the in vitro translation was carried out in the presence of "stripped" microsomal membranes, this precursor was processed to mature RCP devoid of carbohydrate moiety. Therefore, like other secretory proteins, RCP is also synthesized as pre-RCP which gets processed later on by endoplasmic reticulum membranes. This result also shows that the primary translation products of RCP synthesized in both liver and oviduct of chicks are similar, thereby implying that the gene coding for RCP is the same in both the tissues. Oviduct and liver poly(A)+ RNAs isolated during secondary stimulation showed 2- and 3-fold higher RCP mRNA activities respectively when compared to corresponding activities during primary stimulation. This increase in RCP mRNA activity could either be due to the increase in synthesis of RCP-specific mRNA molecules and/or due to stabilization of cytoplasmic mRNA during secondary stimulation as shown earlier during the induction of vitellogenin, ovalbumin and conalbumin. Poly(A)+ RNA from the livers of progesterone-treated birds did not show any RCP mRNA activity while the oviduct RNA from the same birds showed RCP mRNA activity which was comparable with estrogen-treated birds, thereby confirming the earlier findings that progesterone was ineffective in inducing RCP mRNA activity in the liver but could induce the same in differentiated oviduct, like estrogen. The kinetics of increased elaboration of retinol binding protein (RBP) by the estrogenized chicken liver were also examined with a view to compare and contrast the basic features of hormonal modulation of this constitutive protein with those of an inducible protein like RCP within a single tissue viz., liver. Both RBP and its binding protein in the plasma, prealbumin (PA), have been purified to homogeneity by slightly modified published procedures. To facilitate the biosynthetic studies on these proteins, specific and sensitive RIAs were developed. By using RBP-specific RIA, the kinetics of estrogen-stimulated elaboration of RBP in immature chicks was investigated. After a single injection of the steroid hormone (estrogen), RBP rapidly accumulated in the plasma, without any lag period unlike the case with RCP. There was a 4-fold increase in the plasma RBP at 24 h after the steroid administration. Further, there was no amplification of the response during secondary stimulation. Estrogen did not have any influence on the plasma PA content. This clearly showed that at the doses tested, estrogen specifically enhances RBP but not its binding protein viz., PA. With multiple estrogen injections, there was a sharp 4-fold increase in plasma RBP content which was sustained at that high level as long as the hormone was administered and which started declining once the hormone was withdrawn. This was in sharp contrast to the pattern observed with RCP, which showed a progressive increase throughout the duration of estrogen administration. The circulatory half-life of RBP in the control birds was 4 h as estimated from the measurement of the rate of disappearance of exogenously administered [¹²?I]-labelled RBP. There was no change in the half-life of RBP upon estrogen administration. Simultaneous administration of progesterone with estrogen did not alter the pattern of RBP induction. The antiestrogens, E- and Z-clomiphene citrates, administered 30 min before estrogen, effectively blocked the hormonal influence. The stimulated production of RBP did not exhibit a strict estrogen dose-dependency although a minimal threshold concentration of estrogen, i.e., 1 mg/kg body weight, was found to be essential for eliciting this response. This is in sharp contrast to the situation observed with RCP which showed a progressive increase in its plasma content with the increasing doses of the steroid. While RCP is synthesized in two different tissues viz., chicken liver and oviduct, RBP was found to be synthesized exclusively in the liver. ?-Amanitin or cycloheximide administered along with estrogen severely curtailed the RCP elaboration, showing thereby a requirement for de novo synthesis of both protein and mRNA. In contrast, the inhibition brought about by ?-amanitin in terms of RBP accumulation was only to the extent of 33% at the time of peak accumulation viz., 24 h. This clearly indicated that while the de novo expression of RCP gene has an obligatory requirement for the elaboration of new mRNA and protein intermediates, the early enhancement in transcription to produce RBP-specific mRNA may not be an obligatory requirement for the estrogen-enhanced production of this constitutive protein, i.e., RBP.