Hernandez LL, Gregerson KA, Horseman ND. Mammary gland serotonin regulates parathyroid hormone-related protein and other bonerelated signals. Am J Physiol Endocrinol Metab 302: E1009-E1015, 2012. First published February 7, 2012 doi:10.1152/ajpendo.00666.2011.-Breast cells drive bone demineralization during lactation and metastatic cancers. A shared mechanism among these physiological and pathological states is endocrine secretion of parathyroid hormone-related protein (PTHrP), which acts through osteoblasts to stimulate osteoclastic bone demineralization. The regulation of PTHrP has not been accounted for fully by any conventional mammotropic stimuli or tumor growth factors. Serotonin (5-HT) synthesis within breast epithelial cells is induced during lactation and in advancing breast cancer. Here we report that serotonin deficiency (knockout of tryptophan hydroxylase-1) results in a reduction of mammary PTHrP expression during lactation, which is rescued by restoring 5-HT synthesis. 5-HT induced PTHrP expression in lactogen-primed mammary epithelial cells from either mouse or cow. In human breast cancer cells 5-HT induced both PTHrP and the metastasis-associated transcription factor Runx2/Cbfa1. Based on receptor expression and pharmacological evidence, the 5-HT2 receptor type was implicated as being critical for induction of PTHrP and Runx2. These results connect 5-HT synthesis to the induction of bone-regulating factors in the normal mammary gland and in breast cancer cells.5-hydroxytryptamine; lactation; osteoblast; prolactin; RANK ligand; RUNX2/CBFA1 A KEY FUNCTION OF THE MAMMARY GLANDS is to regulate the mobilization of calcium from bone. During lactation women and other mammals lose a significant portion of bone mass, which is restored after lactation ceases (2,8,26,54). Failure to mobilize bone calcium extraction at the onset of lactation causes hypocalcemia in dairy cows, leading to a severe convulsive syndrome referred to as periparturient paresis or "milk fever" (17, 35). To drive calcium mobilization, the mammary glands become endocrine organs and secrete parathyroid hormone-related peptide (PTHrP) into the bloodstream (9,27,46,50,51,53,54). PTHrP was originally discovered as the factor responsible for humoral hypercalcemia of malignancy and is secreted from a variety of advanced soft-tissue cancers (5,8,28,47). The NH 2 -terminal portion of PTHrP is similar to that of parathyroid hormone (PTH) and acts via the type 1 PTH receptors (PTH1R) to induce the receptor activator of NF-B ligand (RANKL) (34).PTHrP is undetectable in the circulation except during lactation, in advanced metastastic disease, or in patients with hyperprolactinemia (5,6,9,24,42,48). Despite obvious correlations with states of elevated prolactin (PRL), PRL did not induce PTHrP in conventional cell cultures of mammary epithelium (29, 52), and our laboratory has done numerous experiments that confirmed that PRL does not induce PTHrP in mammary cells by a direct mechanism (unpublished results).A previous study showed that serotonin (5...

Medications that perturb serotonin balance dysregulate lactation, and the effects are consistent with those predicted by the physiological effects of intramammary 5-HT bioactivity. Mothers taking serotonergic drugs may need additional support to achieve their breastfeeding goals.

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The physiology of mood regulation in the postpartum is poorly understood despite the fact that postpartum depression (PPD) is a common pathology. Serotonergic mechanisms and their dysfunction are widely presumed to be involved, which has led us to investigate whether lactation induces changes in central or peripheral serotonin (5-HT) systems and related affective behaviors. Brain sections from lactating (day 10 postpartum) and age-matched nulliparous (non-pregnant) C57BL/6J mice were processed for 5-HT immunohistochemistry. The total number of 5-HT immunostained cells and optical density were measured. Lactating mice exhibited lower immunoreactive 5-HT and intensity in the dorsal raphe nucleus when compared with nulliparous controls. Serum 5-HT was quantified from lactating and nulliparous mice using radioimmunoassay. Serum 5-HT concentrations were higher in lactating mice than in nulliparous controls. Affective behavior was assessed in lactating and non-lactating females ten days postpartum, as well as in nulliparous controls using the forced swim test (FST) and marble burying task (MBT). Animals were treated for the preceding five days with a selective serotonin reuptake inhibitor (SSRI, citalopram, 5mg/kg/day) or vehicle. Lactating mice exhibited a lower baseline immobility time during the FST and buried fewer marbles during the MBT as compared to nulliparous controls. Citalopram treatment changed these behaviors in lactating mice with further reductions in immobility during the FST and decreased marble burying. In contrast, the same regimen of citalopram treatment had no effect on these behaviors in either non-lactating postpartum or nulliparous females. Our findings demonstrate changes in both central and peripheral 5-HT systems associated with lactation, independent of pregnancy. They also demonstrate a significant interaction of lactation and responsiveness to SSRI treatment, which has important implications in the treatment of PPD. Although recent evidence has cast doubt on the effectiveness of SSRIs, these results support their therapeutic use in the treatment of PPD.

The role that opiate peptides play in suckling-induced prolactin (PRL) release was examined in 10-day post-partum lactating rats. The opiate receptor antagonist naloxone (NAL) suppressed suckling-induced PRL release in a dose-dependent manner and a large dose abolished the response. These results suggest either that opiate neurons are situated in the neuronal pathway mediating this neuroendocrine response, or alternatively, that opiate neurons are situated such that they can modulate neuronal transmission in this pathway. It is suggested that NAL blocks a tonic, inhibitory β-endorphinergic input to the tuberoinfundibular dopaminergic (TIDA) neurons, hence, NAL administration in effect stimulates the TIDA neurons and in this way overrides the suckling response. Intravenous, bolus administration of β-en-dorphin (β-END) produced a PRL response that was similar to the suckling response in terms of latency of onset and duration while the magnitude of the β-END-induced response was 2-fold greater than that produced by the suckling stimulus. NAL abolished β-END-induced PRL release at a much lower dose than that required to inhibit suckling-induced PRL release. This suggests that the neural mediation of the suckling response involves a mechanism in addition to the one inhibited by opiate receptor blockade.

Dopamine (DA) is a physiological regulator of PRL secretion, exerting tonic inhibitory control. DA activates an inward rectifier K(+) (IRK) channel in rat lactotropes, causing membrane hyperpolarization and inhibition of Ca(2+)-dependent action potentials. Both the activation of this effector K(+) channel and the inhibition of PRL release are mediated by D(2)-type receptor activation and pertussis toxin- sensitive G proteins. To study the molecular basis of this physiologically relevant channel, a homology-based PCR approach was employed to identify members of the IRK channel family expressed in the anterior pituitary gland. Nondegenerate primers corresponding to regions specific for IRK channels known to be G protein activated (GIRKs; gene subfamily Kir 3.0) were synthesized and used in the PCR with reverse transcribed female rat anterior pituitary messenger RNA as the template. PCR products of predicted sizes for Kir 3.1, 3.2, and 3.4 were consistently observed by ethidium bromide staining after 16 amplification cycles. The identities of the products were confirmed by subcloning and sequencing. Expression of each of these gene products in anterior pituitary was confirmed by Northern blot analysis. Functional analysis of the GIRK proteins was performed in the heterologous expression system, Xenopus laevis oocytes. Macroscopic K(+) currents were examined in oocytes injected with different combinations of Kir 3.0 complementary RNA (cRNA) and G protein subunit (beta(1)gamma(2)) cRNA. The current-voltage relationships demonstrated strong inward rectification for each individual and pairwise combination of GIRK channel subunits. Oocytes coinjected with any pair of GIRK subunit cRNA exhibited significantly larger inward K(+) currents than oocytes injected with only one GIRK channel subtype. Ligand-dependent activation of only one of the GIRK combinations (GIRK1 and GIRK4) was observed when channel subunits were coexpressed with the D(2) receptor in Xenopus oocytes. Dose-response data fit to a Michaelis-Menten equation gave an apparent K(d) similar to that for DA binding in anterior pituitary tissue. GIRK1 and GIRK4 proteins were coimmunoprecipitated from anterior pituitary lysates, confirming the presence of native GIRK1/GIRK4 oligomers in this tissue. These data indicate that GIRK1 and GIRK4 are excellent candidate subunits for the D(2)-activated, G protein-gated channel in pituitary lactotropes, where they play a critical role in excitation-secretion coupling.

A variety of fundamental differences have evolved in the physiology of the human and rodent prolactin (PRL) systems. The PRL gene in humans and other primates contains an alternative promoter, 5.8 kbp upstream of the pituitary transcription start site, which drives expression of PRL in "extrapituitary" tissues, where PRL is believed to exert local, or paracrine, actions. Several of these extrapituitary PRL tissues serve a reproductive function (eg, mammary gland, decidua, prostate, etc), consistent with the hypothesis that local PRL production may be involved in, and required for, normal reproductive physiology in primates. Rodent research models have generated significant findings regarding the role of PRL in reproduction. Specifically, disruption (knockout) of either the PRL gene or its receptor causes profound female reproductive defects at several levels (ovaries, preimplantation endometrium, mammary glands). However, the rodent PRL gene differs significantly from the human, most notably lacking the alternative promoter. Understanding of the physiological regulation and function of extrapituitary PRL has been limited by the absence of a readily accessible experimental model, because the rodent PRL gene does not contain the alternative promoter. To overcome these limitations, we have generated mice that have been "humanized" with regard to the structural gene and tissue expression of PRL. Here, we present the characterization of these animals, demonstrating that the human PRL transgene is responsive to known physiological regulators both in vitro and in vivo. More importantly, the expression of the human PRL transgene is able to rescue the reproductive defects observed in mouse PRL knockout (mPRL(-)) females, validating their usefulness in studying the function or regulation of this hormone in a manner that is relevant to human physiology.

Serotonin is a homeostatic regulator of the mammary gland during lactation. The contribution of mammary-derived serotonin to circulating serum serotonin concentrations was previously unknown. We have developed mice with mammary-specific disruptions of tryptophan hydroxylase 1 (Tph1) or low-density lipoprotein receptor-related protein 5 (Lrp5) that are induced during late pregnancy and lactation via use of the whey acidic protein (WAP)-Cre cre-lox system. Our objective was to characterize dams with a lactation- and mammary-specific disruption of Lrp5 (WAP-Cre × Lrp5 FL/FL) or Tph1 (WAP-Cre × Tph1 FL/FL). Milk yield and pup weights were recorded throughout lactation. Dams were euthanized on d10 postpartum and mammary glands and duodenal tissue were harvested. WAP-Cre × Lrp5 FL/FL dams had elevated serotonin concentrations in both the mammary gland and circulation compared to controls. In contrast, WAP-Cre × Tph1 FL/FL dams had decreased mammary gland and serum serotonin concentrations compared to controls. Alveolar morphology, milk yield, and pup weights were similar. Mammary-derived serotonin makes a significant contribution to circulating serotonin concentrations during lactation, with no effect on milk yield or alveolar morphology. These transgenic models can and should be confidently used in future lactation studies to further elucidate the contribution of serotonin to the maintenance of lactation.