kDa Prolactin Research Articles

FRI314 Prolactinoma And Macroprolactin; A Diagnostic Dilemma

Abstract Disclosure: A. Farooqi: None. S. Dewan: None. J. Jun: None. Prolactin is a 199 amino acid, 23 (Kilo Dalton) kDa protein secreted by pituitary lactotroph cells. The two other major forms of prolactin present in circulation are big prolactin, the dimeric 40-60 kDa prolactin and big-big prolactin or macroprolactin, 100-150 kDa complexes of 23 kDa prolactin and IgG autoantibodies. Hypothalamic dopamine is the primary regulator of prolactin secretion through its inhibitory effect. Prolactinoma accounts for ∼40% of pituitary tumors with clinical presentations of hypogonadotropic hypogonadism and tumor-related mass effects. A 77-year-old female with a past medical history of hypertension and hyperlipidemia was admitted after a mechanical fall. She underwent a computed tomography (CT) of the brain which incidentally showed a pituitary mass. Magnetic resonance imaging (MRI) of pituitary showed a homogenously enhanced 3.0 x 3.5 x 2.8 cm mass centered in the Sella turcica, invading the supra-seller cistern, left cavernous sinus, and orbital apex abutting the optic chiasm and optic tracts. The patient did not report headache, nausea, vomiting, visual changes, or abnormal milky nipple discharge. Physical examination was unremarkable without any neurologic deficit. Formal ophthalmology evaluation showed intact extra ocular movements, visual field-testing using octopus 24-2 and Humphrey test showed few missed points, though not suggestive of pituitary related loss. Serum prolactin was measured at 1432 ng/ml (reference range, 0-25). The presence of macroprolactin was considered due to the absence of clinical features of hyperprolactinemia. The monomeric prolactin was found to be 68.7% (<40%) at 867.5 ng/ml of the total prolactin at 1259.7 ng/ml with 31.3 % of other prolactin forms are present. Evaluation for other pituitary functions was unremarkable with thyroid stimulating hormone (TSH) of 0.72 uIU/ml (0.49-4.76l), morning serum cortisol of 52.9 ug/dl (6.7-22.4), and insulin like growth factor (IGF-1) 73 ng/ml (20-214). Cabergoline 0.5 mg twice weekly was prescribed. This case illustrates a menopausal state as a probable explanation for lack of clinical features of hyperprolactinemia with macro adenoma while macroprolactin should be considered. Presentation: Friday, June 16, 2023

The Relevant Participation of Prolactin in the Genesis and Progression of Gynecological Cancers.

Prolactin (PRL) is a hormone produced by the pituitary gland and multiple non-pituitary sites, vital in several physiological processes such as lactation, pregnancy, cell growth, and differentiation. However, PRL is nowadays known to have a strong implication in oncogenic processes, making it essential to delve into the mechanisms governing these actions. PRL and its receptor (PRLR) activate a series of effects such as survival, cellular proliferation, migration, invasion, metastasis, and resistance to treatment, being highly relevant in developing certain types of cancer. Because women produce high levels of PRL, its influence in gynecological cancers is herein reviewed. It is interesting that, other than the 23 kDa PRL, whose mechanism of action is endocrine, other variants of PRL have been observed to be produced by tumoral tissue, acting in a paracrine/autocrine manner. Because many components, including PRL, surround the microenvironment, it is interesting to understand the hormone’s modulation in cancer cells. This work aims to review the most important findings regarding the PRL/PRLR axis in cervical, ovarian, and endometrial cancers and its molecular mechanisms to support carcinogenesis.

A 60 kDa prolactin variant secreted by cervical cancer cells modulates apoptosis and cytokine production.

Prolactin (PRL) is associated with different types of cancer, such as cervical cancer. Recombinant PRL has antiapoptotic effect on cervical cancer cells, and it can also induce cytokine production on macrophages. A 60kDa variant of PRL is produced by cervical cancer cells. The aim of the present study was to evaluate this variant's bioactivity, to test its effect on cervical cancer cell apoptosis, and to assess its ability to induce cytokine production on THP-1 macrophages. First, 60kDa PRL was isolated and used to stimulate Nb2 cells. Later, apoptosis was measured after exposure to 60kDa PRL. Finally, cytokines were measured on THP-1 stimulated supernatants. Our results show that 60kDa PRL increased Nb2 cell proliferation. Apoptosis was decreased after stimuli with 60kDa PRL in cervical cancer cells. IL-1β and TNF-α are produced by THP-1 macrophages after stimuli. These results suggest that 60kDa PRL produced by cervical cancer cells is able to reduce apoptosis in HeLa, SiHa and C-33A cells and induce IL-1β and TNF-α production by THP-1 macrophages.

The prolactin receptor is expressed in rheumatoid arthritis and psoriatic arthritis synovial tissue and contributes to macrophage activation

Objectives. Prolactin (PRL) is a lactation-inducing hormone with immunomodulatory properties and is found at elevated levels in the serum of patients with RA and other rheumatic diseases. The PRL receptor (PRLR) has been shown to be expressed by macrophages in atherosclerotic plaques. The aim of this study was to examine PRLR expression by synovial macrophages and its role in the regulation of macrophage activation.Methods. Serum monomeric 23 kDa PRL levels were measured in 119 RA patients using a fluoroimmunometric assay. PRLR expression was assessed in synovial tissue of 91 RA, 15 PsA and 8 OA patients by immunohistochemistry and digital image analysis. Double IF was used to identify PRLR-expressing cells. The effects of PRL on monocyte-derived macrophage gene expression were examined by quantitative real-time PCR and ELISA.Results. Serum PRL levels were similar in female and male RA patients. Median (interquartile range) PRLR expression was significantly higher (P < 0.05) in RA and PsA synovial tissue compared with OA. PRLR colocalized with synovial CD68+ macrophages and von Willebrand factor+ endothelial cells. In vitro, PRLR was prominently expressed in IFN-γ-and IL-10-polarized macrophages compared with other polarizing conditions. PRL by itself had negligible effects on macrophage gene expression, but cooperated with CD40L and TNF to increase expression of pro-inflammatory genes including IL-6, IL-8 and IL-12β.Conclusions. Synovial PRLR expression is enhanced in patients with inflammatory arthritis compared with OA, and PRL cooperates with other pro-inflammatory stimuli to activate macrophages. These results identify PRL and PRLR as potential new therapeutic targets in inflammatory arthritis.

New insights in prolactin: pathological implications.

Prolactin is a hormone that is mainly secreted by lactotroph cells of the anterior pituitary gland, and is involved in many biological processes including lactation and reproduction. Animal models have provided insights into the biology of prolactin proteins and offer compelling evidence that the different prolactin isoforms each have independent biological functions. The major isoform, 23 kDa prolactin, acts via its membrane receptor, the prolactin receptor (PRL-R), which is a member of the haematopoietic cytokine superfamily and for which the mechanism of activation has been deciphered. The 16 kDa prolactin isoform is a cleavage product derived from native prolactin, which has received particular attention as a result of its newly described inhibitory effects on angiogenesis and tumorigenesis. The discovery of multiple extrapituitary sites of prolactin secretion also increases the range of known functions of this hormone. This Review summarizes current knowledge of the biology of prolactin and its receptor, as well as its physiological and pathological roles. We focus on the role of prolactin in human pathophysiology, particularly the discovery of the mechanism underlying infertility associated with hyperprolactinaemia and the identification of the first mutation in human PRLR.

Regulation of blood vessels by prolactin and vasoinhibins.

Prolactin (PRL) stimulates the growth of new blood vessels (angiogenesis) either directly through actions on endothelial cells or indirectly by upregulating proangiogenic factors like vascular endothelial growth factor (VEGF). Moreover, PRL acquires antiangiogenic properties after undergoing proteolytic cleavage to vasoinhibins, a family of PRL fragments (including 16kDa PRL) with potent antiangiogenic, vasoconstrictive, and antivasopermeability effects. In view of the opposing actions of PRL and vasoinhibins, the regulation of the proteases responsible for specific PRL cleavage represents an efficient mechanism for controlling blood vessel growth and function. This review briefly describes the vascular actions of PRL and vasoinhibins, and addresses how their interplay could help drive biological effects of PRL in the context of health and disease.

Vasoinhibins, N-terminal mouse prolactin fragments, participate in mammary gland involution

Vasoinhibins are a family of peptides that act on endothelial cells to suppress angiogenesis and promote apoptosis-mediated vascular regression. Vasoinhibins include the N-terminal fragments from prolactin (PRL), GH, and placental lactogen. One of the vasoinhibins, the N-terminal PRL fragment of 16 kDa, is generated by the lysosomal representative protease cathepsin D (Cath D). Because the normal growth and involution of the mammary gland (MG) are profoundly affected by the expansion and regression of blood vessels and also because PRL stimulates the growth and differentiation of MG, we proposed that intact PRL produced during lactation contributes to MG angiogenesis and increased blood flow, whereas during involution, the N-terminal PRL fragment would have proapoptotic effects on mammary epithelial cells (MECs). Therefore, we investigated the production of the N-terminal PRL fragment and its direct effect on the MG. Mouse PRL (mPRL) was proteolytically cleaved by Cath D between amino acids 148 and 149. N-terminal PRL fragment and Cath D expression increased during MG involution. Furthermore, incubation of MG fragments and MCF7 with recombinant 16 kDa mPRL revealed a proapoptotic effect in MECs. Ectopic mPRL in MECs was cleaved to 16 kDa PRL by Cath D in the MG lysosomal fraction. The majority of PRL derived from pituitary gland was cleaved to 16 kDa PRL in culture medium. Therefore, N-terminal PRL fragment increases during the involution period, has a proapoptotic effect on MECs, and is mainly generated by secreted Cath D in the extracellular space of MG.

Opposing roles of Akt and STAT3 in the protection of the maternal heart from peripartum stress

Peripartum cardiomyopathy (PPCM) is a pregnancy-associated cardiomyopathy in previously healthy women. Mice with a cardiomyocyte-restricted deletion of signal transducer and activator of transcription-3 (STAT3, CKO) develop PPCM. PI3K-Akt signalling is thought to promote cardiac hypertrophy and protection during pregnancy. We evaluated the role of activated Akt signalling in the maternal heart postpartum. CKO mice were bred to mice harbouring an Akt transgene, specifically expressed in cardiomyocytes (CAkt(tg)) generating CKO; CAkt(tg), CAkt(tg), CKO, and wild-type sibling mice. CAkt(tg) and CKO;CAkt(tg) female mice developed PPCM with systolic dysfunction. Both genotypes displayed cardiac hypertrophy and lower capillary density, showed increased phosphorylation of p66 Src homology 2 domain containing protein and FoxO3A, and reduced expression of manganese superoxide dismutase as well as increased cathepsin D activity and increased miR-146a levels [indicative for generation of the anti-angiogenic 16 kDa prolactin (PRL)]. Cardiac inflammation and fibrosis was accelerated in CKO;CAkt(tg) and associated with high postpartum mortality. The PRL blocker, bromocriptine (BR), prevented heart failure and the decrease in capillary density in CKO;CAkt(tg) and CAkt(tg) mice. BR attenuated high mortality, up-regulation of CCL2, and cardiac inflammation as well as fibrosis in CKO;CAkt(tg). PRL infusion induced cardiac inflammation in CKO;CAkt(tg) independent of pregnancy. In neonatal rat cardiomyocytes, PRL and interferon γ (IFNγ) induced the expression of CCL2 via activation of Akt. Postpartum Akt activation is detrimental for the peripartum heart as it lowers anti-oxidative defence and in combination with low STAT3 conditions, accelerate cardiac inflammation and fibrosis. PRL and its cleaved 16 kDa form are central for Akt-induced PPCM as indicated by the protection from the disease by PRL blockade.

Phenotyping and outcome on contemporary management in a German cohort of patients with peripartum cardiomyopathy

Peripartum cardiomyopathy (PPCM) is a life-threatening heart disease developing towards the end of pregnancy or in the months following delivery in previously healthy women in terms of cardiac disease. Enhanced oxidative stress and the subsequent cleavage of the nursing hormone Prolactin into an anti-angiogenic 16 kDa subfragment emerged as a potential causal factor of the disease. We established a prospective registry with confirmed PPCM present in 115 patients (mean baseline left ventricular ejection fraction, LVEF: 27 ± 9 %). Follow-up data (6 ± 3 months) showed LVEF improvement in 85 % and full recovery in 47 % while 15 % failed to recover with death in 2 % of patients. A positive family history of cardiomyopathy was present in 16.5 %. Pregnancy-associated hypertension was associated with a better outcome while a baseline LVEF ≤ 25 % was associated with a worse outcome. A high recovery rate (96 %) was observed in patients obtaining combination therapy with beta-blocker, angiotensin-converting enzyme (ACE) inhibitors/angiotensin-receptor-blockers (ARBs) and bromocriptine. Increased serum levels of Cathepsin D, the enzyme that generates 16 kDa Prolactin, miR-146a, a direct target of 16 kDa Prolactin, N-terminal-pro-brain-natriuretic peptide (NT-proBNP) and asymmetric dimethylarginine (ADMA) emerged as biomarkers for PPCM. In conclusion, low baseline LVEF is a predictor for poor outcome while pregnancy-induced hypertensive disorders are associated with a better outcome in this European PPCM cohort. The high recovery rate in this collective is associated with a treatment concept using beta-blockers, ACE inhibitors/ARBs and bromocriptine. Increased levels of Cathepsin D activity, miR-146a and ADMA in serum of PPCM patients support the pathophysiological role of 16 kDa Prolactin for PPCM and may be used as a specific diagnostic marker profile.Electronic supplementary materialThe online version of this article (doi:10.1007/s00395-013-0366-9) contains supplementary material, which is available to authorized users.

Circulating microparticles as indicators of peripartum cardiomyopathy

Peripartum cardiomyopathy (PPCM) is associated with high mortality and morbidity. Endothelial damage involving cathepsin-D to form a 16 kDa prolactin (PRL) peptide is pathogenetically relevant. Inhibiting PRL peptide with bromocriptine has yielded promising results. We investigated whether microparticles (MPs) can be quantified in serum as markers for diagnosis and treatment effects in PPCM. Patients with PPCM were compared with age-matched healthy post-partum women (PPCTR), healthy pregnant women (PCTR), healthy non-pregnant women (NPCTR), patients with ischaemic cardiomyopathy (ICM), patients with stable coronary artery disease (CAD) and healthy controls (HCTR). Peripartum cardiomyopathy treated with bromocriptine (PPCM-BR) and with PPCM without bromocriptine-treatment as control (PPCM-BRCTR) were compared. Microparticles were determined by flow cytometry. Endothelial MPs (EMPs) were elevated in PPCM compared with PPCTR, PCTR, and NPCTR, each P< 0.001. They were significantly elevated compared with ICM, CAD, and HCTR (P< 0.001). Pregnancy (PCTR) exhibited only slight increases vs. ICM, CAD, NPCTR, and HCTR. The increase in PPCM was due to an increase of activated but not apoptotic EMPs. Platelet-derived microparticles were highly increased in PPCM compared with ICM (P< 0.001) but 9.3 ± 4.4-fold compared with CAD (P< 0.001). In NPCTR (P< 0.001) compared with NPCTR, the increase was 5.9 ± 1.7-fold (P< 0.001). Microparticles generated from monocytes (MMPs) were increased 2.4 ± 1.8-fold in PPCM compared with PCTR (P< 0.001) and 4.8 ± 3.6-fold compared with CAD (P< 0.001), whereas leucocyte MPs (LMPs) were not significantly elevated. Endothelial microparticles were significantly reduced in PPCM treated additionally with bromocriptine compared with PPCM treated only with heart failure therapy (P< 0.001). Microparticle profiles may in long-term distinguish PPCM from normal pregnancy, heart failure, and vascular diseases and might be a diagnostic marker related to the pathomechanism of PPCM.

Bromocriptine as a new therapeutic agent for peripartum cardiomyopathy

Peripartum cardiomyopathy (PPCM) is a poorly understood, rare disorder in which left ventricular systolic dysfunction and symptoms of heart failure occur between the last month of pregnancy and the first 5 months postpartum. Recent data suggest that uncontrolled oxidative stress leads to the activation of the prolactin cleaving enzyme cathepsin D that in turn leads to an increase in a cleaved 16 kDa prolactin. This cleaved form that has an angiostatic and proapoptotic role appears to drive the disease by adversely impacting the endothelium and cardiomyocyte. Bromocriptine that reduces the prolactin production by dopamine agonist actions may improve outcomes in patients with peripartum cardiomyopathy by eliminating the cleaved form of prolactin despite the activation of the cleaving enzyme. In limited case reports and proof of concept studies use of bromocriptine in the early stages has been shown to improve outcomes in patients with peripartum cardiomyopathy. However, larger randomized control study is still awaited.

N-terminal prolactin-derived fragments, vasoinhibins, are proapoptoptic and antiproliferative in the anterior pituitary.

The anterior pituitary is under a constant cell turnover modulated by gonadal steroids. In the rat, an increase in the rate of apoptosis occurs at proestrus whereas a peak of proliferation takes place at estrus. At proestrus, concomitant with the maximum rate of apoptosis, a peak in circulating levels of prolactin is observed. Prolactin can be cleaved to different N-terminal fragments, vasoinhibins, which are proapoptotic and antiproliferative factors for endothelial cells. It was reported that a 16 kDa vasoinhibin is produced in the rat anterior pituitary by cathepsin D. In the present study we investigated the anterior pituitary production of N-terminal prolactin-derived fragments along the estrous cycle and the involvement of estrogens in this process. In addition, we studied the effects of a recombinant vasoinhibin, 16 kDa prolactin, on anterior pituitary apoptosis and proliferation. We observed by Western Blot that N-terminal prolactin-derived fragments production in the anterior pituitary was higher at proestrus with respect to diestrus and that the content and release of these prolactin forms from anterior pituitary cells in culture were increased by estradiol. A recombinant preparation of 16 kDa prolactin induced apoptosis (determined by TUNEL assay and flow cytometry) of cultured anterior pituitary cells and lactotropes from ovariectomized rats only in the presence of estradiol, as previously reported for other proapoptotic factors in the anterior pituitary. In addition, 16 kDa prolactin decreased forskolin-induced proliferation (evaluated by BrdU incorporation) of rat total anterior pituitary cells and lactotropes in culture and decreased the proportion of cells in S-phase of the cell cycle (determined by flow cytometry). In conclusion, our study indicates that the anterior pituitary production of 16 kDa prolactin is variable along the estrous cycle and increased by estrogens. The antiproliferative and estradiol-dependent proapoptotic actions of this vasoinhibin may be involved in the control of anterior pituitary cell renewal.

Abstract P4-03-02: Establishing a Relationship between Breast Cancer, Prolactin and Altered Fat Metabolism

Abstract Despite decades of research, breast cancer remains one of the most common cancers world-wide. Mammary carcinoma has been associated with a high-fat diet, and its rate in overweight post-menopausal women is up to 50% higher than in normal-weight women. The current study examined breast cancer as a metabolic disease in the context of altered fat catabolism, with a focus on examining the effects of prolactin (PRL) on the adenosine 5'-monophosphate-activated protein kinase (AMPK) energy sensing pathway that culminates with carnitine palmitoyl transferase 1 (CPT1), an enzyme that shuttles long-chain fatty acids into the mitochondrial matrix for beta oxidation. When a cell has high energy demands or is stressed, AMPK activation leads to either increased glucose uptake or the phosphorylation of acetyl-CoA carboxylase (ACC), resulting in a reduction in malonyl-CoA levels that lift an allosteric inhibition on CPT1, in turn leading to an increase in CPT1 enzyme activity. Conversely, increased malonyl-CoA levels, together with increased fatty acid synthase activity, favor lipogenesis. PRL has been shown to affect fat metabolism in adipocytes by altering malonyl-CoA levels, and we therefore examined whether CPT1 expression was altered in breast cancer cells in response to treatment with recombinant human PRL. The metabolic effects of this hormone, which is normally produced in breast tissue and has been epidemiologically linked with breast cancer, were investigated in a normal human breast epithelial cell line (184B5) and in breast cancer cells. PRL up-regulated CPT1 expression at both the mRNA and protein levels in cancer cells, but not in 184B5 cells. Of note, compared to MCF-7 or MDA-MB-231 cells, T47D cells expressed the highest levels of CPT1. The 85 kDa PRL receptor isoform was also highly expressed in this cell line compared to 184B5 or MCF-7 cells. Furthermore, PRL dose-dependently increased CPT1 enzyme activity in T47D cells, also inducing the phosphorylation of both the AMPKα catalytic subunit at threonine 172 and ACC. In T47D cells treated with AMPKα siRNA, transient knock-down of the catalytic AMPK subunit correlated with a similar knock-down in CPT1 mRNA levels. CPT1 levels in the knock-down cells could not be restored by treatment with PRL, suggesting that the PRL-mediated effect requires activation of the AMPK pathway. We also demonstrated that treatment with PRL leads to the phosphorylation of TAK1 and LKB1, two kinases that are known to activate AMPKα. It is known that PRL contributes to breast cancer by inducing cell proliferation, survival, motility, and angiogenesis. Our work proposes a novel role for this hormone in mammary carcinogenesis through its effect on fat metabolism via the induction of the AMPK pathway. Elucidating how breast cancer cells differentially produce and utilize energy compared to normal breast epithelial cells may lead to the development of new therapies for the treatment or improved management of this complex disease. (Research supported by Canadian Institutes of Health Research.) Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-03-02.

La prolactine et son fragment 16 kDa dans les tissus de mammifères

Prolactin, owing to its origins, actions and molecular forms, is an ubiquitous and pleiotropic hormone. Indeed prolactin, initially thought to be essentially synthesized in the hypophysis, is also produced by several tissues in mammals. It is involved in more than 300 different biological activities, such as reproduction, developmental immunity and behaviour. It is also described under several molecular forms resulting from co- or post-translational modifications and enzymatic cleavage. Among these, the 16 kDa form, derived from native prolactin, has received particular attention because of its inhibitory effect on angiogenesis. Recent results have suggested an important role of tissue enzymes in the production of this form in several tissues (retina, myocardium and mammary gland). The cleavage leading to the production of 16 kDa prolactin may occur outside the cells, in the interstitial medium and therefore in the vicinity of blood capillaries. This process implies tissue-specific mechanisms of regulation. A better knowledge of the location of the cleavage and of the regulation of these activities of the cleaving enzymes is now essential for controlling the processes. This knowledge will allow a better understanding of the relationships between some pathologies (cardiomyopathy, pre-eclampsia, retinopathy) and modification of the production of the anti-angiogenic form of prolactin.

16-kDa Prolactin Reduces Angiogenesis, but Not Growth of Human Breast Cancer Tumors In Vivo

Prolactin (PRL) is a peptide hormone necessary for normal growth and development of the human breast. In addition, high levels of PRL in plasma correlate with increased risk of breast cancer, especially among postmenopausal women. Several isoforms of PRL exist in human circulation, including a 16 kDa isoform that is an N-terminal fragment of the full-length 23 kDa PRL. 16 kDa PRL has been shown to be anti-angiogenic in vitro and in vivo, and to reduce formation of tumors from prostate, colon and melanoma cancer cell lines. Here we explore the effect of 16 kDa PRL expression in vitro and in vivo using two breast cancer cell line models (MCF-7 and MDA-MB-231) and also the HCT-116 colon cancer cell line. In all three cell lines, 16 kDa PRL expression inhibited cell proliferation in vitro compared to empty vector controls. In vivo results were markedly different between the two types of cell lines. HCT-116 cells expressing 16 kDa PRL exhibited reduced vascularization and tumor formation, consistent with published results. The breast cancer cell lines expressing 16 kDa PRL also exhibited inhibition of angiogenesis in vivo but no reduction in tumor size or formation. These results suggest that the effects of 16 kDa PRL on tumor formation may vary across tissue types. The unique sensitivity of breast cancer to PRL as a mitogen and/or additional factors in the mammary gland environment (e.g. local hormone/mitogen concentration) may play a dominant role in tumor formation in vivo, thus outweighing the anti-angiogenesis effects and in vitro reduction in cell proliferation induced by 16 kDa PRL.

A Cathepsin D-Cleaved 16 kDa Form of Prolactin Mediates Postpartum Cardiomyopathy

Postpartum cardiomyopathy (PPCM) is a disease of unknown etiology and exposes women to high risk of mortality after delivery. Here, we show that female mice with a cardiomyocyte-specific deletion of stat3 develop PPCM. In these mice, cardiac cathepsin D (CD) expression and activity is enhanced and associated with the generation of a cleaved antiangiogenic and proapoptotic 16 kDa form of the nursing hormone prolactin. Treatment with bromocriptine, an inhibitor of prolactin secretion, prevents the development of PPCM, whereas forced myocardial generation of 16 kDa prolactin impairs the cardiac capillary network and function, thereby recapitulating the cardiac phenotype of PPCM. Myocardial STAT3 protein levels are reduced and serum levels of activated CD and 16 kDa prolactin are elevated in PPCM patients. Thus, a biologically active derivative of the pregnancy hormone prolactin mediates PPCM, implying that inhibition of prolactin release may represent a novel therapeutic strategy for PPCM.

Prolactin-like hormone in the nematode Trichinella spiralis larvae

Expression of prolactin (PRL) or prolactin-like hormone has been reported in invertebrates. We investigated the larval phase of Trichinella spiralis: (a) to express 23 kDa PRL, (b) to define its localization and (c) to test its possible biological activity. Immunostaining in isolated larvae demonstrated positive material to 23 kDa PRL by all along the stichosome, specifically in the stichocytes. Homogenized immunoblot larvae showed a 23 kDa protein band. To assess PRL release and its biological activity, larvae were incubated in culture medium and the excretory/secretory products were analyzed by the Nb2 cells bioassay. A cellular growth equivalent until 10 nM PRL and using antibody against 23 kDa PRL, the growth was blocked. In conclusion our result provides evidence that PRL-like hormone is expressed and secreted by the larvae of T. spiralis.

Matrix metalloproteases from chondrocytes generate an antiangiogenic 16 kDa prolactin

The 16 kDa N-terminal fragment of prolactin (16K-prolactin) is a potent antiangiogenic factor. Here, we demonstrate that matrix metalloproteases (MMPs) produced and secreted by chondrocytes generate biologically functional 16K-prolactin from full-length prolactin. When incubated with human prolactin at neutral pH, chondrocyte extracts and conditioned medium, as well as chondrocytes in culture, cleaved the Ser155-Leu156 peptide bond in prolactin, yielding - upon reduction of intramolecular disulfide bonds - a 16 kDa N-terminal fragment. This 16K-prolactin inhibited basic fibroblast growth factor (FGF)-induced endothelial cell proliferation in vitro. The Ser155-Leu156 site is highly conserved, and both human and rat prolactin were cleaved at this site by chondrocytes from either species. Conversion of prolactin to 16K-prolactin by chondrocyte lysates was completely abolished by the MMP inhibitors EDTA, GM6001 or 1,10-phenanthroline. Purified MMP-1, MMP-2, MMP-3, MMP-8, MMP-9 and MMP-13 cleaved human prolactin at Gln157, one residue downstream from the chondrocyte protease cleavage site, with the following relative potency: MMP-8 > MMP-13 > MMP-3 > MMP-1= MMP-2 > MMP-9. Finally, chondrocytes expressed prolactin mRNA (as revealed by RT-PCR) and they contained and released antiangiogenic N-terminal 16 kDa prolactin (detected by western blot and endothelial cell proliferation). These results suggest that several matrix metalloproteases in cartilage generate antiangiogenic 16K-prolactin from systemically derived or locally produced prolactin.

Cathepsin D released by lactating rat mammary epithelial cells is involved in prolactin cleavage under physiological conditions.

The 16 kDa prolactin fragment arises from partial proteolysis of the native 23 kDa prolactin pituitary hormone. The mammary gland has been involved in this processing, although it has not been clarified whether it occurs in stroma or epithelial cells or extracellularly. Also, the processing enzyme has not been defined yet. Here we show that the incubation medium of stroma-deprived mammary acini from lactating rat contains an enzymatic activity able to cleave, in a temperature- and time-dependent fashion, the 23 kDa prolactin to generate a 16 kDa prolactin detectable under reducing conditions. This cleavage was not impaired in the presence of hirudin, a thrombin inhibitor, but strongly weakened in the presence of pepstatin A, a cathepsin D inhibitor. Cathepsin D immuno-depletion abolished the capability of acini-conditioned medium to cleave the 23 kDa prolactin. Brefeldin A treatment of acini, a condition that largely abolished the apical secretion of milk proteins, did not impair the secretion of the enzymatically active single chain of cathepsin D. These results show that mature cathepsin D from endosomes or lysosomes is released, likely at the baso-lateral site of mammary epithelial cells, and that a cathepsin D-dependent activity is required to effect, under physiological conditions, the cleavage of 23 kDa prolactin in the extracellular medium. This is the first report demonstrating that cathepsin D can perform a limited proteolysis of a substrate at physiological pH outside the cell.

Roles of prolactin and related members of the prolactin/growth hormone/placental lactogen family in angiogenesis.

Prolactin, growth hormone and placental lactogen are members of a family of polypeptide hormones which share structural similarities and biological activities. Numerous functions have been attributed to these hormones, among which stand out their recently discovered effects on angiogenesis, the process by which new blood vessels are formed from the pre-existing microvasculature. Prolactin, growth hormone and placental lactogen, along with two non-classical members of the family, proliferin and proliferin-related protein, can act both as circulating hormones and as paracrine/autocrine factors to either stimulate or inhibit various stages of the formation and remodeling of new blood vessels, including endothelial cell proliferation, migration, protease production and apoptosis. Such opposing actions can reside in similar but independent molecules, as is the case of proliferin and proliferin-related protein, which stimulate and inhibit angiogenesis respectively. The potential to exert opposing effects on angiogenesis can also reside within the same molecule as the parent protein can promote angiogenesis (i.e. prolactin, growth hormone and placental lactogen), but after proteolytic processing the resulting peptide fragment acquires anti-angiogenic properties (i.e. 16 kDa prolactin, 16 kDa growth hormone and 16 kDa placental lactogen). The unique properties of the peptide fragments versus the full-length molecules, the regulation of the protease responsible for specific protein cleavage, the selective expression of specific receptors and their associated signal transduction pathways are issues that are being investigated to further establish the precise contribution of these hormones to angiogenesis under both physiological and pathological situations. In this review article, we summarize the known and speculative issues underlying the effects of the prolactin, growth hormone and placental lactogen family of proteins on angiogenesis, and address important remaining enigmas in this field of research.