Prolactin Receptor Antagonist Research Articles

Preparation of Superactive Prolactin Receptor Antagonists.

Most breast cancer deaths are caused by malignant estrogen receptor-positive breast tumors that later recur as metastatic disease. Prolactin (PRL) has been documented as a factor promoting breast cancer development and metastasis. We therefore developed superactive prolactin receptor (PRLR) antagonists aimed at blocking PRL action. We purified 12 novel mutants to homogeneity as monomers, and the most potent antagonist was over 95-fold more active than the previously reported weak antagonist, the mutant Del 1-9 human PRL G129R. This enhanced antagonistic activity resulted mostly from prolonged interaction with the extracellular domain (ECD) of PRLR. All mutants were properly refolded, as indicated by interaction with human PRLR-ECD and by circular dichroism analysis. We then prepared monopegylated variants of the most active mutants to extend their biological half-life in vivo.

Hematopoietic PBX-interacting protein is a novel regulator of mammary epithelial cell differentiation.

Hematopoietic PBX-interacting protein (HPIP, also known as PBXIP1) is an estrogen receptor (ER) interacting protein that regulates estrogen-mediated breast cancer cell proliferation and tumorigenesis. However, its functional significance in the context of mammary gland development is unexplored. Here, we report that HPIP is required for prolactin (PRL)-induced lactogenic differentiation invitro. Molecular analysis of HPIP expression in mice revealed its induced expression at pregnancy and lactation stages of mammary gland. Moreover, PRL is a lactogenic hormone that controls pregnancy as well as lactation and induces Hpip/Pbxip1 expression in a signal transducer and activator of transcription 5a-dependent manner. Using mammary epithelial and lactogenic-competent cell lines, we further show that HPIP plays a regulatory role in PRL-mediated mammary epithelial cell differentiation, which is measured by acini formation, β-casein synthesis, and lipid droplet formation. Further mechanistic studies using pharmacological inhibitors revealed that HPIP modulates PRL-induced β-casein synthesis via phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) activation. This study also identified HPIP as a critical regulator of autocrine PRL signaling as treatment with the PRL receptor antagonist Δ1-9-G129R-hPRL restrained HPIP-mediated PRL synthesis, AKT activation, and β-casein synthesis in cultured HC11 cells. Interestingly, we also uncovered that microRNA-148a (miR-148a) antagonizes HPIP-mediated mammary epithelial cell differentiation. Together, our study identified HPIP as a critical regulator of PRL signaling and revealed a novel molecular circuitry involving PRL, HPIP, PI3K/AKT, and miR-148a that controls mammary epithelial cell differentiation invitro.

Progesterone receptor isoform-dependent cross-talk between prolactin and fatty acid synthase in breast cancer.

Progesterone receptor (PR) isoforms can drive unique phenotypes in luminal breast cancer (BC). Here, we hypothesized that PR-B and PR-A isoforms differentially modify the cross-talk between prolactin and fatty acid synthase (FASN) in BC. We profiled the responsiveness of the FASN gene promoter to prolactin in T47Dco BC cells constitutively expressing PR-A and PR-B, in the PR-null variant T47D-Y cell line, and in PR-null T47D-Y cells engineered to stably re-express PR-A (T47D-YA) or PR-B (T47D-YB). The capacity of prolactin to up-regulate FASN gene promoter activity in T47Dco cells was lost in T47D-Y and TD47-YA cells. Constitutively up-regulated FASN gene expression in T47-YB cells and its further stimulation by prolactin were both suppressed by the prolactin receptor antagonist hPRL-G129R. The ability of the FASN inhibitor C75 to decrease prolactin secretion was more conspicuous in T47-YB cells. In T47D-Y cells, which secreted notably less prolactin and downregulated prolactin receptor expression relative to T47Dco cells, FASN blockade resulted in an augmented secretion of prolactin and up-regulation of prolactin receptor expression. Our data reveal unforeseen PR-B isoform-specific regulatory actions in the cross-talk between prolactin and FASN signaling in BC. These findings might provide new PR-B/FASN-centered predictive and therapeutic modalities in luminal intrinsic BC subtypes.

Prolactin Regulates Pain Responses via a Female-Selective Nociceptor-Specific Mechanism.

SummaryMany clinical and preclinical studies report an increased prevalence and severity of chronic pain among females. Here, we identify a sex-hormone-controlled target and mechanism that regulates dimorphic pain responses. Prolactin (PRL), which is involved in many physiologic functions, induces female-specific hyperalgesia. A PRL receptor (Prlr) antagonist in the hind paw or spinal cord substantially reduced hyperalgesia in inflammatory models. This effect was mimicked by sensory neuronal ablation of Prlr. Although Prlr mRNA is expressed equally in female and male peptidergic nociceptors and central terminals, Prlr protein was found only in females and PRL-induced excitability was detected only in female DRG neurons. PRL-induced excitability was reproduced in male Prlr+ neurons after prolonged treatment with estradiol but was prevented with addition of a translation inhibitor. We propose a novel mechanism for female-selective regulation of pain responses, which is mediated by Prlr signaling in sensory neurons via sex-dependent control of Prlr mRNA translation.

Prolactin Regulates Pain Responses Via a Female-Selective Nociceptor-Specific Mechanism

Many clinical and preclinical studies report an increased prevalence and severity of chronic pain among females. Here, we identify a sex-hormone controlled target and mechanism which regulates dimorphic pain responses. Prolactin (PRL), which is involved in many physiologic functions, induces female-specific hyperalgesia. A PRL receptor (Prlr) antagonist in the hindpaw or spinal cord substantially reduced hyperalgesia in inflammatory models. This effect was mimicked by sensory neuronal ablation of Prlr. Although Prlr mRNA is expressed equally in female and male peptidergic nociceptors and central terminals, Prlr protein was only found in females and PRL-induced excitability was detected only in female DRG neurons. PRL-induced excitability was reproduced in male Prlr+ neurons after prolonged treatment with estradiol but was prevented with addition of a translation inhibitor. We propose a novel mechanism for female-selective regulation of pain responses, which is mediated by Prlr signaling in sensory neurons via sex-dependent control of Prlr mRNA translation.

An in vivo half-life extended prolactin receptor antagonist can prevent STAT5 phosphorylation.

Increasing evidence suggests that signaling through the prolactin/prolactin receptor axis is important for stimulation the growth of many cancers including glioblastoma multiforme, breast and ovarian carcinoma. Efficient inhibitors of signaling have previously been developed but their applicability as cancer drugs is limited by the short in vivo half-life. In this study, we show that a fusion protein, consisting of the prolactin receptor antagonist PrlRA and an albumin binding domain for half-life extension can be expressed as inclusion bodies in Escherichia coli and efficiently refolded and purified to homogeneity. The fusion protein was found to have strong affinity for the two intended targets: the prolactin receptor (KD = 2.3±0.2 nM) and mouse serum albumin (KD = 0.38±0.01 nM). Further investigation showed that it could efficiently prevent prolactin mediated phosphorylation of STAT5 at 100 nM concentration and above, similar to the PrlRA itself, suggesting a potential as drug for cancer therapy in the future. Complexion with HSA weakened the affinity for the receptor to 21±3 nM, however the ability to prevent phosphorylation of STAT5 was still prominent. Injection into rats showed a 100-fold higher concentration in blood after 24 h compared to PrlRA itself.

Suppression of Prolactin Secretion Partially Explains the Antidiabetic Effect of Bromocriptine in ob/ob Mice.

Previous studies have shown that bromocriptine mesylate (Bromo) lowers blood glucose levels in adults with type 2 diabetes mellitus; however, the mechanism of action of the antidiabetic effects of Bromo is unclear. As a dopamine receptor agonist, Bromo can alter brain dopamine activity affecting glucose control, but it also suppresses prolactin (Prl) secretion, and Prl levels modulate glucose homeostasis. Thus, the objective of the current study was to investigate whether Bromo improves insulin sensitivity via inhibition of Prl secretion. Male and female ob/ob animals (a mouse model of obesity and insulin resistance) were treated with Bromo and/or Prl. Bromo-treated ob/ob mice exhibited lower serum Prl concentration, improved glucose and insulin tolerance, and increased insulin sensitivity in the liver and skeletal muscle compared with vehicle-treated mice. Prl replacement in Bromo-treated mice normalized serum Prl concentration without inducing hyperprolactinemia. Importantly, Prl replacement partially reversed the improvements in glucose homeostasis caused by Bromo treatment. The effects of the Prl receptor antagonist G129R-hPrl on glucose homeostasis were also investigated. We found that central G129R-hPrl infusion increased insulin tolerance of male ob/ob mice. In summary, our findings indicate that part of Bromo effects on glucose homeostasis are associated with decrease in serum Prl levels. Because G129R-hPrl treatment also improved the insulin sensitivity of ob/ob mice, pharmacological compounds that inhibit Prl signaling may represent a promising therapeutic approach to control blood glucose levels in individuals with insulin resistance.

Is prolactin a negative neuroendocrine regulator of human skin re-epithelisation after wounding?

Chronic wounds remain a major unmet healthcare challenge, associated with substantial morbidity and economic costs. Therefore, novel treatment strategies and therapeutic approaches need to be urgently developed. Yet, despite the increasingly recognized importance of neurohormonal signaling in skin physiology, the neuroendocrine regulation of cutaneous wound healing has received surprisingly little attention. Human skin, and its appendages, locally express the pleiotropic neurohormone prolactin (PRL), which not only regulates lactation but also hair follicle cycling, angiogenesis, keratinocyte proliferation, and epithelial stem cell functions. Therefore, we examined the effects of PRL in experimentally wounded female human skin organ culture. Overall, this revealed that PRL slightly, but significantly, inhibited epidermal regeneration (reepithelialisation), cytokeratin 6 protein expression and intraepidermal mitochondrial activity (MTCO1 expression), while it promoted keratinocyte terminal differentiation (i.e. involucrin expression) ex vivo. If the current pilot data are confirmed by further studies, PRL may serve as one of the-rarely studied-negative regulators of cutaneous wound healing that control excessive reepithelialisation. This raises the intriguing and clinically relevant question of whether PRL receptor antagonists could actually promote epidermal repair after human skin wounding.

98P - Effect of prolactin receptor antagonist in different cancer models

98P - Effect of prolactin receptor antagonist in different cancer models

Novel reagents for human prolactin research: large-scale preparation and characterization of prolactin receptor extracellular domain, non-pegylated and pegylated prolactin and prolactin receptor antagonist.

To provide new tools for in vitro and in vivo prolactin (PRL) research, novel protocols for large-scale preparation of untagged human PRL (hPRL), a hPRL antagonist (del 1-9-G129R hPRL) that acts as a pure antagonist of hPRL in binding to hPRL receptor extracellular domain (hPRLR-ECD), and hPRLR-ECD are demonstrated. The interaction of del 1-9-G129R hPRL with hPRLR-ECD was demonstrated by competitive non-radioactive binding assay using biotinylated hPRL as the ligand and hPRLR-ECD as the receptor, by formation of stable 1:1 complexes with hPRLR-ECD under non-denaturing conditions using size-exclusion chromatography, and by surface plasmon resonance methodology. In all three types of experiments, the interaction of del 1-9-G129R hPRL was equal to that of unmodified hPRL. Del 1-9-G129R hPRL inhibited the hPRL-induced proliferation of Baf/LP cells stably expressing hPRLR. Overall, the biological properties of del 1-9-G129R hPRL prepared by the protocol described herein were similar to those of the antagonist prepared using the protocol reported in the original study; however, the newly described protocol improved yields by >6-fold. To provide long-lasting hPRL as a new reagent needed for in vivo experiments, we prepared its mono-pegylated analogue and found that pegylation lowers its biological activity in a homologous in vitro assay. As its future use will require the development of a PRL antagonist with highly elevated affinity, del 1-9-G129R hPRL was expressed on the surface of yeast cells. It retained its binding capacity for hPRLR-ECD, and this methodology was shown to be suitable for future development of high-affinity hPRL antagonists using a library of randomly mutated open reading frame of del 1-9-G129R hPRL and selecting high-affinity mutants by yeast surface display methodology.

ATM Is Required for the Prolactin-Induced HSP90-Mediated Increase in Cellular Viability and Clonogenic Growth After DNA Damage.

Prolactin (PRL) acts as a survival factor for breast cancer cells, but the PRL signaling pathway and the mechanism are unknown. Previously, we identified the master chaperone, heat shock protein 90 (HSP90) α, as a prolactin-Janus kinase 2 (JAK2)-signal transducer and activator of transcription 5 (STAT5) target gene involved in survival, and here we investigated the role of HSP90 in the mechanism of PRL-induced viability in response to DNA damage. The ataxia-telangiectasia mutated kinase (ATM) protein plays a critical role in the cellular response to double-strand DNA damage. We observed that PRL increased viability of breast cancer cells treated with doxorubicin or etoposide. The increase in cellular resistance is specific to the PRL receptor, because the PRL receptor antagonist, Δ1-9-G129R-hPRL, prevented the increase in viability. Two different HSP90 inhibitors, 17-allylamino-17-demethoxygeldanamycin and BIIB021, reduced the PRL-mediated increase in cell viability of doxorubicin-treated cells and led to a decrease in JAK2, ATM, and phosphorylated ATM protein levels. Inhibitors of JAK2 (G6) and ATM (KU55933) abolished the PRL-mediated increase in cell viability of DNA-damaged cells, supporting the involvement of each, as well as the crosstalk of ATM with the PRL pathway in the context of DNA damage. Drug synergism was detected between the ATM inhibitor (KU55933) and doxorubicin and between the HSP90 inhibitor (BIIB021) and doxorubicin. Short interfering RNA directed against ATM prevented the PRL-mediated increase in cell survival in two-dimensional cell culture, three-dimensional collagen gel cultures, and clonogenic cell survival, after doxorubicin treatment. Our results indicate that ATM contributes to the PRL-JAK2-STAT5-HSP90 pathway in mediating cellular resistance to DNA-damaging agents.

Internal image anti-idiotypic antibody: A new strategy for the development a new category of prolactin receptor (PRLR) antagonist

Over the past decades, a number of prolactin receptor (PRLR) antagonists have been developed, which can be divided into two categories, PRLR analogue and anti-PRLR antibody. However, until now, there have been no commercially available PRLR antagonists. Here, we described a new approach for the preparation of PRLR antagonist, namely internal image anti-idiotypic antibody strategy. The hybridoma technique was used to generate anti-idiotypic antibodies to PRL. Competitive ELISA, competitive receptor-binding analysis and immunofluorescence assay (IFA) were then used to screen and characterize anti-idiotypic antibodies to PRL. One internal image anti-idiotypic antibody, termed MG7, was obtained. A series of experiments demonstrated that MG7 behaved as a typical internal image anti-idiotypic antibody (Ab2β). MG7 exhibited effective antagonistic activity, which not only inhibited PRL binding to PRLR in a dose-dependent manner but also inhibited PRLR-mediated intracellular signalling. Furthermore, MG7 also blocked Nb2 cell proliferation induced by PRL. The current study suggests that MG7 has the potential application in the PRL/PRLR-related studies in future. In addition, this work also suggests that the internal image anti-idiotypic antibody may represent a novel strategy for the development of PRLR antagonist.

Development of a new anti-prolactin receptor (PRLR) antibody, F56, which can serve as a PRLR antagonist

In this work, we developed a new prolactin receptor (PRLR) antagonist using the hybridoma technique. A series of monoclonal antibodies against prolactin receptor (PRLR) was prepared, from which we characterized and selected one anti-PRLR antibody, F56. Epitome mapping showed that F56 and prolactin (PRL) share a common binding epitope on PRLR, and therefore, F56 could compete with prolactin (PRL) for binding to PRLR. Subsequent experiments indicated that F56 could effectively neutralize PRLR-mediated intracellular signalling molecules, such as signal transducer and activator of transcription (STAT) and extracellular signal-regulated kinase-1 and kinase 2 (ERK1/2), either by endogenously expressed PRLR or in a cell model transfected with PRLR. In addition, further experiments showed that F56 could effectively inhibit PRL-induced cell proliferation. The current study suggests that F56 has potential applications in PRLR-related studies.

Stimulation of prolactin receptor induces STAT-5 phosphorylation and cellular invasion in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in humans and is characterized with poor outcome. In this study, we investigated components of prolactin (Prl) system in cell models of GBM and in histological tissue sections obtained from GBM patients. Expression of Prolactin receptor (PrlR) was detected at high levels in U251-MG, at low levels in U87-MG and barely detectable in U373 cell lines and in 66% of brain tumor tissues from 32 GBM patients by immunohistochemical technique. In addition, stimulation of U251-MG and U87-MG cells but not U373 with Prl resulted in increased STAT5 phosphorylation and only in U251-MG cells with increased cellular invasion. Furthermore, STAT5 phosphorylation and cellular invasion induced in Prl stimulated cells were significantly reduced by using a Prl receptor antagonist that consists of Prl with four amino acid replacements. We conclude that Prl receptor is expressed at different levels in the majority of GBM tumors and that blocking of PrlR in U251-MG cells significantly reduce cellular invasion.

Role of Prolactin Receptors in Lymphangioleiomyomatosis.

Pulmonary lymphangioleiomyomatosis (LAM) is a rare lung disease caused by mutations in the tumor suppressor genes encoding Tuberous Sclerosis Complex (TSC) 1 and TSC2. The protein product of the TSC2 gene is a well-known suppressor of the mTOR pathway. Emerging evidence suggests that the pituitary hormone prolactin (Prl) has both endocrine and paracrine modes of action. Here, we have investigated components of the Prl system in models for LAM. In a TSC2 (+/-) mouse sarcoma cell line, down-regulation of TSC2 using siRNA resulted in increased levels of the Prl receptor. In human LAM cells, the Prl receptor is detectable by immunohistochemistry, and the expression of Prl in these cells stimulates STAT3 and Erk phosphorylation, as well as proliferation. A high affinity Prl receptor antagonist consisting of Prl with four amino acid substitutions reduced phosphorylation of STAT3 and Erk. Antagonist treatment further reduced the proliferative and invasive properties of LAM cells. In histological sections from LAM patients, Prl receptor immuno reactivity was observed. We conclude that the Prl receptor is expressed in LAM, and that loss of TSC2 increases Prl receptor levels. It is proposed that Prl exerts growth-stimulatory effects on LAM cells, and that antagonizing the Prl receptor can block such effects.

The Role of Prolactin in Bone Metastasis and Breast Cancer Cell-Mediated Osteoclast Differentiation.

Metastasis to the bone is a deleterious aspect of breast cancer and is a preferred site that results in bone loss. Hormones such as prolactin (PRL) have not yet been studied for their role in modulating the secondary tumor bone microenvironment. We used quantitative immunohistochemistry with 134 samples of human primary breast cancer and 17 matched primary breast cancers and bone metastases. A Cox proportional hazards regression model was fitted to evaluate the associations between high prolactin receptor (PRLR) expression and time to bone metastasis, adjusting for estrogen receptor status, lymph node status, and chemotherapy status. We assessed osteoclast differentiation, osteoclast size, and measured pit formation in dentine slices. Statistical tests were two-sided. High PRLR expression in the primary breast tumor was associated with a shorter time to metastasis that includes bone (PRLRAQUA Max-per 100 unit hazard ratio = 1.04, 95% confidence interval = 1.00 to 1.07, P = .03). We observed the PRLR in rare samples of bone metastases and matched primary breast cancer. PRL treatment of breast cancer cells induced osteoclast differentiation and bone lysis via secreted factors and was abrogated by a PRLR antagonist (delta1-9-G129R-hPRL). We demonstrated that sonic hedgehog is a PRL-regulated cytokine in breast cancer cells and part of the mechanism that induces osteoclast differentiation. Our evidence indicates that PRL-PRLR can escalate the impact of breast cancer on bone metastasis and that the presence of the PRLR in the tumor microenvironment of breast cancer bone metastasis has the potential to modulate the microenvironment to induce lytic osteoclast formation.

A Neutralizing Prolactin Receptor Antibody Whose In Vivo Application Mimics the Phenotype of Female Prolactin Receptor-Deficient Mice

The prolactin receptor (PRLR) has been implicated in a variety of physiological processes (lactation, reproduction) and diseases (breast cancer, autoimmune diseases). Prolactin synthesis in the pituitary and extrapituitary sites is regulated by different promoters. Dopamine receptor agonists such as bromocriptine can only interfere with pituitary prolactin synthesis and thus do not induce a complete blockade of PRLR signaling. Here we describe the identification of a human monoclonal antibody 005-C04 that blocks PRLR-mediated signaling at nanomolar concentrations in vitro. In contrast to a negative control antibody, the neutralizing PRLR antibody 005-C04 inhibits signal transducer and activator of transcription 5 phosphorylation in T47D cells and proliferation of BaF3 cells stably expressing murine or human PRLRs in a dose-dependent manner. In vivo application of this new function-blocking PRLR antibody reflects the phenotype of PRLR-deficient mice. After antibody administration female mice become infertile in a reversible manner. In lactating dams, the antibody induces mammary gland involution and negatively interferes with lactation capacity as evidenced by reduced milk protein expression in mammary glands and impaired litter weight gain. Antibody-mediated blockade of the PRLR in vivo stimulates hair regrowth in female mice. Compared with peptide-derived PRLR antagonists, the PRLR antibody 005-C04 exhibits several advantages such as higher potency, noncompetitive inhibition of PRLR signaling, and a longer half-life, which allows its use as a tool compound also in long-term in vivo studies. Therefore, we suggest that this antibody will help to further our understanding of the role of auto- and paracrine PRLR signaling in health and disease.

Prolactin potentiates the activity of acid-sensing ion channels in female rat primary sensory neurons

Prolactin (PRL) is a polypeptide hormone produced and released from the pituitary and extrapituitary tissues. It regulates activity of nociceptors and causes hyperalgesia in pain conditions, but little is known the molecular mechanism. We report here that PRL can exert a potentiating effect on the functional activity of acid-sensing ion channels (ASICs), key sensors for extracellular protons. First, PRL dose-dependently increased the amplitude of ASIC currents with an EC50 of (5.89 ± 0.28) × 10−8 M. PRL potentiation of ASIC currents was also pH dependent. Second, PRL potentiation of ASIC currents was blocked by Δ1-9-G129R-hPRL, a PRL receptor antagonist, and removed by intracellular dialysis of either protein kinase C inhibitor GF109203X, protein interacting with C-kinase 1(PICK1) inhibitor FSC-231, or PI3K inhibitor AS605240. Third, PRL altered acidosis-evoked membrane excitability of DRG neurons and caused a significant increase in the amplitude of the depolarization and the number of spikes induced by acid stimuli. Four, PRL exacerbated nociceptive responses to injection of acetic acid in female rats. Finally, PRL displayed a stronger effect on ASIC mediated-currents and nociceptive behavior in intact female rats than OVX female and male rats and thus modulation of PRL may be gender-dependent. These results suggest that PRL up-regulates the activity of ASICs and enhances ASIC mediated nociceptive responses in female rats, which reveal a novel peripheral mechanism underlying PRL involvement in hyperalgesia.

The many faces of prolactin in breast cancer.

Prolactin (PRL) is a neuroendocrine polypeptide hormone primarily produced by the lactotrophs in the anterior pituitary gland of all vertebrates. The physiological role of PRL in mammary glands is relatively certain while its role in breast tumor has been a topic of debate for over 20 years. In this review, the author attempts to briefly summarize the data coming from his laboratory in the past years, focusing on G129R, a PRL receptor (PRLR) antagonist developed by introducing a single amino acid substitution mutation into human PRL (hPRL) at position 129, and a variety of G129R derivatives. The author has proposed two novel ideas for potential use of PRL, not anti-PRL agents, as an adjuvant agent for breast cancer, making it a hormone of many faces.

Prolactin receptor antagonism uncouples lipids from atherosclerosis susceptibility.

The pituitary-derived hormone prolactin has been suggested to stimulate the development of atherosclerosis and cardiovascular disease through its effects on metabolism and inflammation. In this study, we aimed to challenge the hypothesis that inhibition of prolactin function may beneficially affect atherosclerosis burden. Hereto, atherosclerosis-susceptible LDL receptor (Ldlr) knockout mice were transplanted with bone marrow from transgenic mice expressing the pure prolactin receptor antagonist Del1-9-G129R-hPRL or their non-transgenic littermates as control. Recipient mice expressing Del1-9-G129R-hPRL exhibited a decrease in plasma cholesterol levels (-29%; P<0.05) upon feeding a Western-type diet (WTD), which could be attributed to a marked decrease (-47%; P<0.01) in the amount of cholesterol esters associated with pro-atherogenic lipoproteins VLDL/LDL. By contrast, Del1-9-G129R-hPRL-expressing mice did not display any change in the susceptibility for atherosclerosis after 12 weeks of WTD feeding. Both the absolute atherosclerotic lesion size (223 ± 33 × 10(3) μm(2) for Del1-9-G129R-hPRL vs 259 ± 32 × 10(3) μm(2) for controls) and the lesional macrophage and collagen contents were not different between the two groups of bone marrow recipients. Importantly, Del1-9-G129R-hPRL exposure increased levels of circulating neutrophils (+91%; P<0.05), lymphocytes (+55%; P<0.05), and monocytes (+43%; P<0.05), resulting in a 49% higher (P<0.01) total blood leukocyte count. In conclusion, we have shown that prolactin receptor signaling inhibition uncouples the plasma atherogenic index from atherosclerosis susceptibility in Ldlr knockout mice. Despite an associated decrease in VLDL/LDL cholesterol levels, application of the prolactin receptor antagonist Del1-9-G129R-hPRL does not alter the susceptibility for initial development of atherosclerotic lesions probably due to the parallel increase in circulating leukocyte concentrations.