Laura J. Mauro

Vasoactive intestinal peptide is a hypothalamic prolactin-releasing neuropeptide in the turkey (Meleagris gallopavo).

The hypothesis that vasoactive intestinal peptide (VIP) functions as a hypothalamic prolactin (PRL)-releasing peptide in the turkey was tested by determining the effects of hypothalamic VIP immunoneutralization and pituitary VIP receptor blockade on hypothalamic extract (HE)-induced PRL secretion from dispersed anterior pituitaries. Incubation of cells with porcine VIP (pVIP; 0.5 or 10 nM) significantly stimulated PRL secretion. This effect was inhibited in a dose-related manner by 1-hr preincubation of pVIP with a VIP antisera (A/S; 1:500-1:50,000). Likewise, HE (0.3 equivalent)-stimulated PRL secretion was inhibited by preincubation with VIP A/S (P less than 0.0001). A 96-98% reduction in PRL secretion was obtained from cells cultured with HE, that was previously incubated with 1/500 dilution of antiserum. Pretreatment of pituitary cells for 15 min with [4Cl-D-Phe6,Leu17] VIP, a VIP receptor antagonist (10(-5) M), significantly depressed the PRL response to 0.5 nM VIP (9.9 +/- 0.5 micrograms/500,000 cells vs 4.9 +/- 0.1 micrograms/500,000 cells; 22.4 +/- 0.9 micrograms/500,000 cells vs 14.7 +/- 0.4 micrograms/500,000 cells) or 0.3 eq HE (8.8 +/- 0.6 micrograms/500,000 cells vs 5.2 +/- 0.2 micrograms/500,000 cells; 15.3 +/- 0.3 micrograms/500,000 cells vs 8.2 +/- 0.2 micrograms/500,000 cells). These results suggest that hypothalamic stimulation of PRL secretion appears to be mediated by receptors specific for VIP and that VIP is an endogenous hypothalamic PRL-releasing peptide in the turkey.

Effects of reproductive status, ovariectomy, and photoperiod on vasoactive intestinal peptide in the female turkey hypothalamus

Vasoactive intestinal peptide (VIP) appears to be a physiologically relevant prolactin (PRL)-releasing factor during the avian reproductive cycle, yet little is known of the factors involved in modulating the hypothalamic concentrations of this neuropeptide. A heterologous chicken VIP radioimmunoassay was developed to examine the effects of reproductive status, ovariectomy, and photoperiod on hypothalamic VIP immunoreactivity in the female turkey. VIP concentrations were highest in the median eminence/infundibular nuclear complex (ME/INF) relative to other subregions of the hypothalamus and changed only in this region during the reproductive cycle. Quiescent, nonphotostimulated hens subjected to stimulatory photoperiod exhibited a 1.6-fold increase in VIP in the ME/INF (quiescent 59.9 +/- 6.0 vs photostimulated 95.8 +/- 7.1 pg/microgram protein). An additional 2-fold increase in ME/INF VIP concentrations was observed in laying hens (183.0 +/- 28.5 pg/microgram protein). Coincident increases in plasma PRL were also observed. In contrast, during incubation and the photorefractory stage, a dissociation between hypothalamic VIP and plasma PRL occurred. No changes were observed in VIP in incubating hens, yet a 6-fold increase in PRL was noted, compared to layers. In addition, ME/INF VIP concentrations exhibited no change during the photorefractory stage, whereas a 28-fold decrease in plasma PRL occurred. VIP concentrations in the ME/INF of laying hens were unaffected by ovariectomy, whereas exposure to short photoperiod reduced VIP by 44%. The inhibitory effects of short photoperiod could not be reversed by administration of exogenous steroids, while steroid treatment reduced VIP concentrations by 45% in the ovariectomized hens. These results provide additional correlative evidence for a modulatory role of VIP in PRL secretion and suggest that the expression of this neuropeptide in the INF may serve as a neural link between photoperiodic mechanisms and PRL release during the avian reproductive cycle.

Progesterone action in breast, uterine, and ovarian cancers.

Progesterone and progesterone receptors (PRs) are essential for the development and cyclical regulation of hormone-responsive tissues including the breast and reproductive tract. Altered functions of PR isoforms contribute to the pathogenesis of tumors that arise in these tissues. In the breast, progesterone acts in concert with estrogen to promote proliferative and pro-survival gene programs. In sharp contrast, progesterone inhibits estrogen-driven growth in the uterus and protects the ovary from neoplastic transformation. Progesterone-dependent actions and associated biology in diverse tissues and tumors are mediated by two PR isoforms, PR-A and PR-B. These isoforms are subject to altered transcriptional activity or expression levels, differential crosstalk with growth factor signaling pathways, and distinct post-translational modifications and cofactor-binding partners. Herein, we summarize and discuss the recent literature focused on progesterone and PR isoform-specific actions in breast, uterine, and ovarian cancers. Understanding the complexity of context-dependent PR actions in these tissues is critical to developing new models that will allow us to advance our knowledge base with the goal of revealing novel and efficacious therapeutic regimens for these hormone-responsive diseases.

Melanopsin expression in dopamine-melatonin neurons of the premammillary nucleus of the hypothalamus and seasonal reproduction in birds

Melanopsin (OPN4) is a photoreceptive molecule regulating circadian systems in mammals. Previous studies from our laboratory have shown that co-localized dopamine-melatonin (DA-MEL) neurons in the hypothalamic premammillary nucleus (PMM) are putatively photosensitive and exhibit circadian rhythms in DAergic and MELergic activities. This study investigates turkey OPN4x (tOPN4x) mRNA distribution in the hypothalamus and brainstem, and characterizes its expression in PMM DA-MEL neurons, using in situ hybridization (ISH), immunocytochemistry (ICC), double-label ISH/ICC, and real time-PCR. The mRNA encoding tOPN4x was found in anatomically discrete areas in or near the hypothalamus and the brainstem, including nucleus preopticus medialis (POM), nucleus septalis lateralis (SL), PMM and the pineal gland. Double ICC, using tyrosine hydroxylase (TH, the rate limiting enzyme in DA synthesis)-and OPN4x antibodies, confirmed the existence of OPN4x protein in DA-MEL neurons. Also, tOPN4x mRNA expression was verified with double ISH/ICC using tOPN4x mRNA and TH immunoreactivity. PMM and pineal gland tOPN4x mRNA expression levels were diurnally high during the night and low during the day. A light pulse provided to short day photosensitive hens during the photosensitive phase at night significantly down-regulated tOPN4x expression. The expression level of tOPN4x mRNA in PMM DA-MEL neurons of photorefractory hens was significantly lower as compared with that of short or long day photosensitive hens. The results implicate tOPN4x in hypothalamic PMM DA-MEL neurons as an important component of the photoreceptive system regulating reproductive activity in temperate zone birds.

Enhanced vasoactive intestinal peptide-induced prolactin secretion from anterior pituitary cells of incubating turkeys (Meleagris gallopavo).

During incubation, female turkeys exhibit elevated circulating prolactin (PRL) which may be the result of enhanced pituitary responsiveness to vasoactive intestinal peptide (VIP). This hypothesis was tested by comparison of spontaneous and porcine VIP-induced PRL secretion from anterior pituitary cells of hens in various reproductive conditions. The effect of VIP and luteinizing hormone releasing hormone (LHRH), alone and in combination, on luteinizing hormone (LH) secretion was also examined. Incubation with pVIP (10(-10) to 10(-6) M) significantly stimulated PRL secretion at all incubation times tested (1-5 hr). This increase was greatest in cells from incubating hens, with those from laying, photorefractory, and quiescent (nonphotostimulated) hens secreting successively less PRL. These responses were obtained when spontaneous PRL secretions were compared. VIP induced approximately a similar 1.5-fold increase in LH secretion, in all reproductive groups. Also, VIP enhanced LHRH-induced LH secretion (1.2- to 1.6-fold; P less than 0.0001). It is concluded that PRL secretion in vitro by pituitary cells from turkey hens in various reproductive stages reflects the circulating levels of PRL at these stages.

Tyrosine Phosphatases as Regulators of Skeletal Development and Metabolism

The protein tyrosine kinases (PTK) and the protein tyrosine phosphatases (PTPs) are enzymes which play an integral role in tyrosine phosphorylation‐dependent signaling cascades. By catalyzing the phosphorylation and dephosphorylation of cellular proteins, these enzymes direct the steady‐state levels of specific phosphoproteins and ultimately dictate the functional state of all cells. The importance of this type of signaling in the skeleton is accepted but poorly understood. The contribution of the PTKs to signaling events in bone has been well studied but, in contrast, the regulation by PTPs is poorly defined. The recent identification of 107 genes within the human genome which encode members of the PTP superfamily emphasizes the need to consider the importance of these proteins in skeletal tissue. In this prospective, we will summarize the present state of our knowledge regarding the function of this enzyme superfamily, illustrating its relevance to the development and maintenance of the skeleton and highlighting future directions that should improve our understanding of these critical signaling molecules.

Photoreceptive oscillators within neurons of the premammillary nucleus (PMM) and seasonal reproduction in temperate zone birds

The pathway for light transmission regulating the reproductive neuroendocrine system in temperate zone birds remains elusive. Based on the evidence provided from our studies with female turkeys, it is suggested that the circadian clock regulating reproductive seasonality is located in putatively photosensitive dopamine-melatonin (DA-MEL) neurons residing in the premammillary nucleus (PMM) of the caudal hypothalamus. Melanopsin is expressed by these neurons; a known photopigment which mediates light information pertaining to the entrainment of the clock. Exposure to a gonad stimulatory photoperiod enhances the activity of the DAergic system within DA-MEL neurons. DAergic activity encoding the light information is transmitted to the pars tuberalis, where thyroid-stimulating hormone, beta (TSHβ) cells reside, and induces the release of TSH. TSH stimulates tanycytes lining the base of the third ventricle and activates type 2 deiodinase in the ependymal which enhances triiodothyronine (T3) synthesis. T3 facilitates the release of gonadotropin-releasing hormone-I which stimulates luteinizing hormone/follicle stimulating hormone release and gonad recrudescence. These data taken together with the findings that clock genes are rhythmically expressed in the PMM where DA-MEL neurons are localized imply that endogenous oscillators containing photoreceptors within DA-MEL neurons are important in regulating the DA and MEL rhythms that drive the circadian cycle controlling seasonal reproduction.

Reciprocal regulation of extracellular signal regulated kinase 1/2 and mitogen activated protein kinase phosphatase-3

Mitogen activated protein kinase phosphatase-3 (MKP-3) is a putative tumor suppressor. When transiently overexpressed, MKP-3 dephosphorylates and inactivates extracellular signal regulated kinase (ERK) 1/2. Little is known about the roles of endogenous MKP-3, however. We previously showed that MKP-3 is upregulated in cell lines that express oncogenic Ras. Here we tested the roles of endogenous MKP-3 in modulating ERK1/2 under conditions of chronic stimulation of the Ras/Raf/MEK1/2/ERK1/2 pathway by expression of oncogenic Ras. We used two cell lines: H-ras MCF10A, breast epithelial cells engineered to express H-Ras, and DLD-1, colon cancer cells that express endogenous Ki-Ras. First, we found that MKP-3 acts in a negative feedback loop to suppress basal ERK1/2 when oncogenic Ras stimulates the Ras/Raf/MEK1/2/ERK1/2 cascade. ERK1/2 was required to maintain elevated MKP-3, indicative of a negative feedback loop. Accordingly, knockdown of MKP-3, via siRNA, increased ERK1/2 phosphorylation. Second, by using siRNA, we found that MKP-3 helps establish the sensitivity of ERK1/2 to extracellular activators by limiting the duration of ERK1/2 phosphorylation. Third, we found that the regulation of ERK1/2 by MKP-3 is countered by the complex regulation of MKP-3 by ERK1/2. Potent ERK1/2 activators stimulated the loss of MKP-3 within 30 min due to an ERK1/2-dependent decrease in MKP-3 protein stability. MKP-3 levels recovered within 120 min due to ERK1/2-dependent resynthesis. Preventing MKP-3 resynthesis, via siRNA, prolonged ERK1/2 phosphorylation. Altogether, these results suggest that under the pressure of oncogenic Ras expression, MKP-3 reins in ERK1/2 by serving in ERK1/2-dependent negative feedback pathways.

Photoperiodic modulation of clock gene expression in the avian premammillary nucleus

The premammillary nucleus (PMM) has been shown to contain a daily endogenous dual‐oscillation in dopamine (DA)/melatonin (MEL) as well as c‐fos mRNA expression that is associated with the daily photo‐inducible phase of gonad growth in turkeys. In the present study, the expression of clock genes (Bmal1, Clock, Cry1, Cry2, Per2 and Per3) in the PMM was determined under short (8 : 16 h light/dark cycle) and long (16 : 8 h light/dark cycle) photoperiods relative to changes associated with the diurnal rhythm of DA and MEL. Constant darkness (0 : 24 h light/dark cycle) was used to assess the endogenous response of clock genes. In addition, light pulses were given at zeitgeber time (ZT) 8, 14 and 20 to ascertain whether clock gene expression is modulated by light pulse stimulation and therefore has a daily phase‐related response. In the PMM, the temporal clock gene expression profiles were similar under short and long photoperiods, except that Per3 gene was phase‐delayed by approximately 16 h under long photoperiod. In addition, Cry1 and Per3 genes were light‐induced at ZT 14, the photosensitive phase for gonad recrudescence, whereas the Clock gene was repressed. Gene expression in established circadian pacemakers, the visual suprachiasmatic nucleus (vSCN) and the pineal, was also determined. Clock genes in the pineal gland were rhythmic under both photoperiods, and were not altered after light pulses at ZT 14, which suggests that pineal clock genes may not be associated with the photosensitive phase and reproductive activities. In the vSCN, clock gene expression was phase‐shifted depending on the photoperiod, with apexes at night under short day length and during the day under long day length. Furthermore, light pulses at ZT 14 induced the Per2 gene, whereas it repressed the Bmal1 gene. Taken together, the changes in clock gene expression observed within the PMM were unique compared to the pineal and vSCN, and were induced by long photoperiod and light during the daily photosensitive phase; stimuli that are also documented to promote reproductive activity. These results show that Cry1 and Per3 are involved in the photic response associated with the PMM neuronal activation and are coincident with an essential circadian mechanism (photosensitive phase) controlling the reproductive neuroendocrine system.

Secretion of MCP-1 and other paracrine factors in a novel tumor-bone coculture model

BackgroundThe bone-tumor microenvironment encompasses unique interactions between the normal cells of the bone and marrow cavity and the malignant cells from a primary or metastasized cancer. A multitude of paracrine factors within this microenvironment such as the growth factor, TGF-β, and the chemokine, MCP-1, are secreted by many of these cell types. These factors can act in concert to modulate normal and malignant cell proliferation, malignant cell migration and invasion and, often, mediate bone cancer pain. Although many valuable in vitro and in vivo models exist, identifying the relevant paracrine factors and deciphering their interactions is still a challenge. The aim of our study is to test an ex vivo coculture model that will allow monitoring of the expression, release and regulation of paracrine factors during interactions of an intact femur explant and tumor cells.MethodsIntact or marrow-depleted neonatal mouse femurs and select murine and human sarcoma or carcinoma cell lines were incubated singly or in coculture in specialized well plates. Viability of the bone and cells was determined by immunohistochemical stains, microscopy and marrow cytopreps. Secretion and mRNA expression of paracrine factors was quantitated by ELISA and real-time RT-PCR.ResultsCompartments of the bone were optimally viable for up to 48 h in culture and tumor cells for up to 4 days. Bone was the major contributor of TGF-β and MMP2 whereas both bone and sarcoma cells secreted the chemokine MCP-1 in cocultures. Synergistic interaction between the femur and sarcoma resulted in enhanced MCP-1 secretion and expression in cocultures and was dependent on the presence of the hematopoietic component of the bone as well as other bone cells. In contrast, coculturing with breast carcinoma cells resulted in reduction of TGF-β and MCP-1 secretion from the bone.ConclusionThese studies illustrate the feasibility of this model to examine paracrine interactions between intact bone and tumor cells. Further study of unique regulation of MCP-1 secretion and signaling between these cell types in different types of cancer will be possible using this simulated microenvironment.