Prolactin Receptor Antagonist Research Articles

Development of new prolactin analogs acting as pure prolactin receptor antagonists.

Prolactin (PRL) promotes tumor growth, as recently highlighted by the spontaneous appearance of prostate hyperplasia and mammary neoplasia in PRL transgenic mice. Increasing experimental evidence argues for the involvement of autocrine PRL in this process. Human (h)PRL receptor antagonists have been developed to counteract these undesired proliferative actions of PRL. However, all PRL receptor antagonists obtained to date exhibit partial agonism, limiting their therapeutic use as full antagonists. This is the case for the first generation antagonists (the prototype of which is G129R-hPRL) that we developed ten years ago, which display antagonistic activity in some, but not all in vitro bioassays, and fail to inhibit PRL activity in transgenic mice expressing this analog. We recently developed new human PRL antagonists devoid of agonistic properties, and therefore able to act as pure antagonists. This was demonstrated using several in vitro bioassays, including assays able to detect extremely low levels of receptor activation. These new compounds also act as pure antagonists in vivo, as demonstrated by their ability to competitively inhibit PRL-triggered signaling cascades in various target tissues (liver, mammary gland and prostate). Finally, using transgenic mice specifically expressing PRL in the prostate, which have constitutively activated signaling cascades and prostate hyperplasia, these new PRL analogs are able to completely revert PRL-activated events to basal levels. These second generation antagonists are good candidates to be used as inhibitors of the growth-promoting actions of hPRL.

The Brain Prolactin System: Involvement in Stress Response Adaptations in Lactation

This review focuses on prolactin as a potential candidate for the regulation of emotional and neuroendocrine stress responses in the brain. In particular, we summarise evidence for a brain prolactin receptor-mediated anxiolytic action both in female and male rats, and for inhibitory actions on the reactivity of the hypothalamic-pituitary-adrenal (HPA) axis and the neurohypophysial oxytocin system. These physiological functions of the brain prolactin system are especially relevant in the peripartum period, as an attenuation of behavioural and neuroendocrine stress responses has been described during pregnancy and lactation. At this time, there is an increase in brain prolactin receptor expression and binding, and an increase in hypothalamic prolactin gene expression. In the absence of a selective prolactin receptor antagonist, complementary approaches including chronic intracerebral infusion of prolactin, and antisense targeting of the long form of the brain prolactin receptor were used to investigate the actions of prolactin. The hypothesis of a brain prolactin system activated in the peripartum period which contributes to the adaptive changes in stress responsiveness in order to support reproductive functions is strongly emphasised.

Central Infusions of the Recombinant Human Prolactin Receptor Antagonist, S179D-PRL, Delay the Onset of Maternal Behavior in Steroid-Primed, Nulliparous Female Rats**This work was supported by USPHS Grants R01-HD-19789 and K05-MH-01374 (to R.S.B.) and funds provided by the Cancer Federation and Sensus Drug Development Corp. (to A.M.W.).

The expression of maternal behavior in the newly parturient rat is under endocrine regulation. Blocking endogenous PRL secretion with bromocriptine delays the normal rapid expression of maternal care shown toward foster young in steroid-primed virgin female rats. The recent development of the PRL receptor antagonist S179D-PRL, a mutant of human PRL in which the serine residue at the 179 position is replaced with aspartate, provides a potentially useful tool to examine the role of PRL in neural processing. In the present report, three experiments were conducted that examined the effects of this PRL antagonist on the induction of maternal behavior. In each experiment, ovariectomized, nulliparous rats were treated sequentially with SILASTIC capsules implanted sc with progesterone (days 1-11) and estradiol (days 11-17), a treatment that stimulates a rapid onset of maternal behavior in virgin rats. On day 11, females were implanted with Alzet miniosmotic pumps connected to cannulae directed unilaterally at the lateral ventricle (Exp 1) or bilaterally at the medial preoptic area (MPOA; Exp 2 and 3). Pumps contained either doses of S179D-PRL (0.115 or 1.15 mg/ml; Exp 1 and 2), wild-type human PRL (1.15 mg/ml; Exp 3), or the saline vehicle (Exp 1-3). Testing for maternal behavior began on day 12, a day after pump insertion, and animals were tested daily for 6 days. Latencies to contact, retrieve, and group foster test young were recorded. Administration of both the high and low doses of S179D-PRL infused into the lateral ventricle (Exp 1) or MPOA (Exp 2) significantly delayed the onset of maternal behavior. In contrast, MPOA infusions of the control hormone, wild-type human PRL, in Exp 3 did not delay the onset of maternal behavior. These findings support the concept that the effects of S179D-PRL are caused by its actions as a PRL receptor antagonist rather than by a nonspecific effect of the protein. Overall, these results demonstrate the effectiveness of S179D-PRL acting at the level of the central nervous system (and, more specifically, within the MPOA) to regulate maternal behavior, a PRL-mediated response.

Chapter 18 Lactogenic hormone regulation of maternal behavior

Biological factors can profoundly affect a mother's response to her young. For example, it is well known that the hormones of pregnancy act on the maternal brain to stimulate the spontaneous onset of maternal behavior at parturition. Studies in the rat have provided an excellent model to investigate maternal behavior in mammals, since maternal behavior in rats is easily observable and readily quantifiable and it is well-documented that the endocrine state of gestation helps to bring about the onset of maternal behavior around the time of birth. The same response in virgin animals requires a number of days of constant exposure to pups before maternal-like behaviors emerge. To date, research has established that the steroid hormones, estradiol and progesterone, and the lactogenic hormones, prolactin and the placental lactogens, act in concert to stimulate maternal behavior in the pregnant female. Treatment of adult, virgin rats with these hormones can stimulate a rapid onset of maternal care. In the present chapter experiments are described that demonstrate key roles for prolactin and placental lactogens in the onset of maternal behavior. Central sites of action of prolactin and placental lactogens, including the medial preoptic area, appear to be involved in stimulating the onset of maternal care. Other studies are discussed which support the involvement of the prolactin receptor in the endocrine regulation of maternal behavior using prolactin receptor antagonist and 'knock-out' models in rats and mice, respectively. Overall, these studies indicate that during pregnancy the endocrine system primes the mother's brain so that the new mother displays appropriate and successful behaviors toward her newborn at parturition.

Phosphorylation of bovine prolactin eliminates luteotropic activity in the rat

Phosphorylated and nonphosphorylated prolactins were isolated from bovine pituitaries and their luteotropic activity determined in female rats. Three groups of rats in day 1 of diestrus were treated ip twice daily for three days with 0.25 mg of either prolactin preparation or vehicle. Rats were sacrificed each day of treatment. Serum progesterone concentrations of the groups receiving vehicle or phosphorylated prolactin were similar and the vaginal cytology of these animals indicated that phosphorylated bovine prolactin (bPRL) treatment had not prolonged diestrus. Treatment with nonphosphorylated bPRL significantly increased serum progesterone concentration and the vaginal cytology indicated a diestrus prolonged for up to 4 days. Nonphosphorylated and phosphorylated bPRLs were cleared from the blood at similar rates after ip injection. In vitro receptor binding studies demonstrated that phosphorylated bPRL did not bind the ovarian prolactin receptor. Nonphosphorylated, but not phosphorylated, bPRL competed with radiolabeled bovine hormone for occupancy of rat ovarian prolactin receptors. These data are the first to test the activities of phosphorylated bPRL in vivo and indicate; 1) nonphosphorylated bPRL is luteotropic, 2) phosphorylated bPRL is neither luteotropic nor a prolactin receptor agonist or antagonist and 3) phosphorylated bPRL is not dephosphorylated in vivo rapidly enough to provide sufficient biologicallyactive bPRL to maintain luteal function.

Development of recombinant human prolactin receptor antagonists by molecular mimicry of the phosphorylated hormone.

Previous studies have demonstrated that naturally phosphorylated PRL antagonizes the growth-promoting effects of unmodified PRL in two different PRL-responsive cell lines. In this study our aim was to produce a molecular mimic of phosphorylated PRL by substituting a fairly bulky, negatively charged amino acid (glutamate or aspartate) for the normally phosphorylated serine [serine 179 in human PRL (hPRL)]. In addition, because of the marked effect of phosphorylation on biological activity, we investigated the importance of the unmodified serine in the growth-promoting activity of PRL. hPRL complementary DNA was obtained from the American Type Culture Collection and subcloned into pT7-SCII after site-directed mutagenesis using the deoxyuridine approach. Proteins were expressed in Escherichia coli BL21 (DE3) and were primarily found in inclusion bodies. Agonist and antagonist activities of each serine 179 mutant were assessed using the Nb2 bioassay. Compared with standard hPRL, the recombinant wild-type was more active in the Nb2 assay, attesting to both the absence, or low level, of endotoxin contamination in preparations from these cells and the appropriate folding of the molecule. The aspartate and glutamate mutants had no intrinsic agonist activity, but both antagonized the growth-promoting activity of wild-type PRL, with the aspartate mutant proving to be a very effective antagonist. Two hundred picograms per ml of the aspartate mutant negated 75% of the growth response to 400 pg/ml wild-type PRL. When serine 179 was mutated to alanine or valine, mutant PRLs with 0% and 14% of the biological activity of wild-type PRL, respectively, were produced. These results demonstrate 1) that molecular mimicry of the phosphorylated hormone does produce a PRL antagonist, and 2) that the serine at position 179 is crucial to the growth-promoting activity of PRL. The aspartate mutant can now be used to study many aspects of the physiology of PRL.

Development of Recombinant Human Prolactin Receptor Antagonists by Molecular Mimicry of the Phosphorylated Hormone

Development of Recombinant Human Prolactin Receptor Antagonists by Molecular Mimicry of the Phosphorylated Hormone

Prolactin receptor antagonists.

Most prolactin (PRL) in the circulation is produced by the pituitary. However, a wide variety of traditional target tissues of the hormone have also been shown to produce their own prolactin. The amount produced per cell is low, but may well be sufficient for autocrine/paracrine activity. Although dopamine agonists allow one to study the target tissue effects of pituitary PRL, other agents, such as PRL receptor (PRLR) antagonists, are needed to analyze autocrine/paracrine loops. With PRLR antagonists, it should be possible to dissect out the role of extrapituitary prolactin in both the normal physiology, and the pathophysiology of various tissues. In tissues where the locally produced PRL may promote disease, such antagonists have the potential to be important therapeutics. This article briefly, but critically, reviews current understanding of PRL-receptor interactions and initial signaling, and describes the development of both growth hormone (GH) and PRL antagonists within that context. In the final section, results with a very potent PRL antagonist further one theme of the article, which is whether the simple receptor dimerization model explains all signal transduction following PRLR binding.

Prolactin Receptor Antagonists That Inhibit the Growth of Breast Cancer Cell Lines

We investigated the mechanism of action of the human prolactin (hPRL) receptor on four different breast cancer cell lines, T-47D, MCF-7, BT-474, and SK-BR3, that express elevated levels of the receptor compared with normal cells. Cells treated with human growth hormone (hGH), which binds and activates the hPRL receptor, exhibited bell-shaped dose-response growth curves consistent with the sequential dimerization mechanism proposed for the hPRL receptor (Fuh, G., Colosi, P., Wood, W.I., and Wells, J.A. (1993) J. Biol. Chem. 268, 5376-5381). Growth stimulation was enhanced by Zn2+, which preferentially increases the affinity of hGH for the hPRL receptor. Furthermore, receptor-selective variants of hGH that bind the hPRL receptor but not the hGH receptor were agonistic, providing additional support that specific binding to the hPRL receptor can stimulate these breast cancer cells to grow. On this basis we produced variants of hGH and human placental lactogen (hPL) that were potential antagonists because they bind but do not dimerize the hPRL receptor. The hPL-based antagonist was less potent than the hGH-based antagonist toward the growth of MCF-7 cells, consistent with the lower affinity of hPL for hPRL receptor than for hGH. However, the hPL-based antagonist was more potent than the hGH antagonist for BT-474 cells. Antibodies to the hPRL receptor inhibited growth of FDC-P1 cells transfected with the hPRL receptor; these also inhibited MCF-7 cells and T47D cells but not BT-474 cells. A unique feature of BT-474 cells was found when screening its cDNA revealed the presence of a novel alternative splice of the hPRL receptor that codes for the soluble extracellular domain; this may explain these differential inhibitory effects. These studies provide further molecular insight into the potential role of the hPRL receptor in breast cancer and demonstrate that hPRL receptor antagonists can inhibit the growth of breast cancer cells.

Mechanism-based design of prolactin receptor antagonists.

The mechanism of action of two forms of the prolactin (PRL) receptor was studied using analogs of human growth hormone (hGH). At low concentrations (approximately 1 pM), hGH binds and stimulates proliferation of Nb2 cells containing the 391-residue PRL receptor as well as murine lymphoid FDC-P1 cells transfected with the 591-residue hPRL receptor. However, at high concentrations (approximately 70 microM) hGH inhibits proliferation of both these cell lines. Such a "bell-shaped" hormone response curve was observed for hGH stimulation of the hGH receptor (Fuh, G., Cunningham, B.C., Fukunaga, R., Nagata, S., Goeddel, D. V., and Wells, J.A. (1992) Science 256, 1677-1680) and is consistent with the sequential formation of an active hormone-(receptor)2 complex in which hGH binds through a first site (Site 1) to a first receptor and then through a second site (Site 2) to a second receptor. By analogy to hGH activation of the hGH receptor, we find that hGH variants that are mutated in Site 1 or Site 2 are greatly reduced as agonists. Similarly, only Site 2 mutants are potent antagonists of either hGH or hPRL stimulated cell proliferation. These and other data support the notion that hGH and hPRL activate the PRL receptor by sequential dimerization and provide a rational basis for the design of potent antagonists to the prolactin receptor.