Leptin, Ciliary Neurotrophic Factor, Leukemia Inhibitory Factor and In ...

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and the gp130-cytokines ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF) and interleukin-6 (IL-6), with the aim of discussing their role in the ...

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Leptin, Ciliary Neurotrophic Factor, Leukemia Inhibitory Factor and Interleukin-6: Class-I Cytokines Involved in the Neuroendocrine Regulation of the Reproductive Function E. Dozio1, M. Ruscica2, E. Galliera1, M.M. Corsi1,3 and P. Magni2,* 1

Department of Human Morphology and Biomedical Sciences “Citta' Studi”, Università degli Studi di Milano, Milan, Italy; 2Department of Endocrinology, Pathophysiology and Applied Biology, Centre of Excellence on Neurodegenerative Diseases and Inter-University Centre for Research on Reproductive Health, Università degli Studi di Milano, Milan, Italy; 3Laboratory of Biothecnological Applications, IRCCS Istituto Ortopedico Galeazzi, Milan, Italy Abstract: Class-I cytokines represent a large group of molecules involved in different physiological processes including host defence, immune regulation, food intake, energy metabolism and, relevant for this review, reproduction. In this latter respect, here, we focus the attention on four of these molecules, specifically leptin, ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF) and interleukin-6 (IL-6). These cytokines present similar three-dimensional fold structure, interact with related class-I receptors, which are expressed in the same regions (i.e., hypothalamus), and activate similar intracellular pathways. Leptin and CNTF share functional similarities, by acting at hypothalamic and pituitary levels, and their receptors are colocalized in the arcuate and paraventricular nuclei of the hypothalamus. For both these molecules, no effect on GnRH migration has been described. LIF has also been shown to affect gonadotropin secretion and here we present the novel observation that it is also able to stimulate GnRH secretion in vitro. Moreover, in the mouse, LIF is prenatally expressed in nasal regions where GnRH neurons originate and start their migration, and in vitro it stimulates intrinsic cell motility and directional migration. The role of the prototypical cytokine, IL-6, on the GnRH-LH axis is not fully clear and additional information seem necessary to better clarify this aspect. In conclusion, the data here discussed suggest that this family of cytokines appears to participate to the complex control of the reproductive function by affecting the development and function of the hypothalamus-pituitary system at different ontogenic times and anatomical sites.

Keywords: Leptin, CNTF, LIF, IL-6, GnRH neurons, GT1-7 cells, GN11 cells, reproduction. CLASS-I CYTOKINES Class-I cytokines represent a large group of molecules involved in different physiological processes, including host defence, immune regulation, food intake, energy metabolism and reproduction. All class-I cytokines described to date are characterized by limited primary sequence identity, while are all fold into a bundle of four alpha-helices [1, 2] and signal via related receptors that share molecular signatures and activate similar intracellular pathways [3, 4]. A unique aspect of class-I cytokines is that the four alpha-helices are arranged in a ‘up-up-down-down’ fashion as a result of the antiparallel orientation of two consecutive pairs of helices and, in most of these cytokines, this bundle fold is stabilized by up to three disulphide bridges. On the basis of the length of their alpha-helices, these cytokines are subdivided into ‘long chain’ or ‘short chain’ cytokines [5]. Growth hormone, prolactin, leptin, erythropoietin and the glycoprotein 130 (gp130)-cytokines are made up of 170-250 amino acids and belong to the ‘long chain’ group; cytokines like interleukin (IL)-2, IL-3, IL-4 and IL-13 typically do not exceed 160 amino acids and represent the ‘short chain’ group [6]. *Address correspondence to this author at the Department of Endocrinology, Pathophysiology and Applied Biology, Center of Excellence on Neurodegenerative Diseases, Università degli Studi di Milano, via G. Balzaretti, 9, 20133 Milano, Italy; Tel: ++39-02-5031.8229; Fax: ++39-02-5031.8204; E-mail: [email protected] 1389-2037/09 $55.00+.00

In the present review we will focus our attention on four cytokines of the ‘long chain’ group and specifically leptin and the gp130-cytokines ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF) and interleukin-6 (IL-6), with the aim of discussing their role in the neuroendocrine regulation of the reproductive function. THE KEY ROLE OF GONADOTROPIN-RELEASING HORMONE NEURONS IN THE REPRODUCTIVE FUNCTION Reproduction is the key function for the propagation of the species and requires a correct development and maturation of the gonads, as well as of the neuroendocrine control exerted by the action of gonadotropin-releasing hormone (GnRH)-secreting neurons. The hypothalamic decapeptide GnRH is the key hormone in the control of the reproductive function. The initiation of the reproductive cascade in all mammalian species requires the completion, during embryonic life, of a developmental program consisting in the migration of the GnRH neurons, from the olfactory placode, along olfactory nerves and through the nasal compartment, into the septal-hypothalamic region [7, 8]. Once the final destination is reached, they undergo differentiation and axonal elongation to make contacts with other hypothalamic neurons and the pituitary portal vessels in the median eminence and start releasing GnRH in a pulsatile fashion to © 2009 Bentham Science Publishers Ltd.

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modulate gonadotropin secretion. An impaired migration of GnRH neurons might represent a cause of clinical disorders, such as Kallmann’s syndrome and hypogonadotropic hypogonadism [9-11]. The mechanisms underlying the migration of GnRH neurons are still not fully understood, although several factors have been described (reviewed in [12, 13]). GnRH neurons receive inputs from numerous central neurotransmitters, as well as peripheral modulators, which regulate gonadotropin secretion through a GnRH-dependent pathway. Some central molecules play a direct role in the regulation of GnRH pulsatility (like  aminobutyric acid, glutamate and neuropeptide Y (NPY)), whereas others, specifically peripheral molecules, provide permissive signals affecting both directly and indirectly, via interneuronal circuits, GnRH neurons activity [14-16]. Among the central factors regulating GnRH neurons, NPY has extensively been studied and is considered a major neuromodulatory link among nutrition and reproduction at the central nervous system (CNS) level. NPY is a 36-amino acid peptide, belonging to the pancreatic polypeptide family, which is very abundant in the CNS and, in particular, in the hypothalamus [17], where it is synthesized primarily in perikariya of the arcuate nucleus and then transported via axonal projections to several hypothalamic nuclei such as the lateral hypothalamic area and the paraventricular, dorsomedial and ventromedial nuclei [18, 19]. In the medial preoptic area, NPY neurons come in close contact with GnRH neurons and may provide direct input to both cell bodies producing GnRH in the preoptic area and their nerve terminals in the median eminence [20]. Since the majority of hormonal circulating signals reaching the brain (like sex steroids and leptin) have been found to directly affect the NPY hypothalamic system, the peptide has been proposed as a potential link among peripheral signals and GnRH neurons [21, 22]. It has been shown that continuous uninterrupted NPY receptor activation results in suppression of gonadotropin secretion and in experimental conditions that upregulate NPY synthesis and release the reproductive function is also impaired [23]. Thus, it is possible that signals which down-regulate the NPY system may positively affect the hypothalamic regulation of reproduction. In recent years, major progress in understanding the molecular signals and physiological mechanisms responsible for the control of reproductive function has been made. Furthermore, the discovery of leptin and the subsequent characterization of its key reproductive actions opened the way for the identification of other local and peripheral signals involved in such function. LEPTIN The hypothalamus is the center of integration of two major determinants of species survival: energy metabolism and reproductive function. Proper function of the gonadotropic axis, and hence reproductive capacity, is gated by metabolic and nutritional factors. A major breakthrough in the characterization of the mechanisms for the integrated control of reproduction and metabolism took place in 1994, with the cloning of the adipocyte-borne hormone, leptin. Leptin is a 16 kDa protein secreted by the white adipose tissue, and thus defined as an adipokine, in proportion to the amount of body

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energy stores and functions as satiety factor in the regulation of body weight [24, 25]. Soon after its identification, leptin was shown to function as a pleiotropic mediator in a wide range of neuroendocrine systems and was demonstrated to be a permissive metabolic signal for the reproductive function [26-28]. To reach the hypothalamus, leptin needs to be transported across the blood-brain barrier (BBB) via a transport system (principally represented by the short isoform of the leptin receptor (OB-R) OB-Ra) with limited capacity and high saturability, thus representing a potential regulatory site for leptin resistance [29, 30]. Congenital leptin deficiency has been associated with hypogonadotropic hypogonadism and infertility. The reproductive aspects of the phenotype of either leptin-deficient humans or mice can be corrected by the administration of leptin, demonstrating the role played by this adipokine in this form of hypogonadism [26, 31, 32]. Leptin exerts its effects by interacting with specific single membrane-spanning domain receptors. The multiple forms of OB-R, which have been identified in different murine and human tissues, include a long intracellular domain form (OB-Rb), a group of short intracellular domain forms (OBRa, OB-Rc) and a soluble form (OB-Re), lacking the transmembrane domain and representing a circulating binding protein [33, 34]. OB-Rb is the only isoform with both the protein motifs necessary for activation of the Janus kinase 2 (Jak) and signal transducer and activators of transcription 3 (STAT3) pathway. Although the Jak2/STAT3 pathway has been considered the major signalling mechanism activated by OB-Rb, mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3-K) have also been implicated in OB-Rb signalling [35]. Different studies have demonstrated that leptin can directly act in both the hypothalamus and the pituitary to stimulate GnRH and gonadotropin release, respectively. Neuroendocrine mechanisms through which leptin influences GnRH neuronal activity have not been completely elucidated. OB-R is abundantly expressed in the hypothalamus [36] and we observed that the OB-Rb isoform is expressed in immortalized GnRH-secreting neurons GT1-7 cells, which release GnRH after leptin stimulation [37]. Moreover, leptin has been shown to enhance gonadotropin liberation by acting directly at the pituitary level and/or through hypothalamic sites of action, which resulted in a dose-related increase in luteinizing hormone (LH) and, to a lesser extent, folliclestimulating hormone (FSH) release [38]. The direct effect of leptin on GnRH-secreting GT1-7 neurons completes and integrates those data indicating the possibility that leptin may indirectly modulate the secretion of GnRH at the hypothalamic levels. Thus, the net effect of leptin on this process would derive from the direct modulation of GnRH-secreting nerve terminals present in the median eminence, as well as of interneurons producing transmitters, such as NPY, proopiomelanocortin-derived peptides and norepinephrine [23, 38]. Although a direct effect of leptin in the regulation of GnRH secretion has been observed by our group in GT1-7 cells, double-labelling studies conducted in rodents [39] and higher primates [40] have failed to demonstrate the expression of OB-R on GnRH neurons. As the GT1-7 and similar GT1-1 cells are clonal cell lines which retain many of the properties of GnRH neurons [41], a discrepancy from the in vitro and in vivo studies has been observed. However, we

Leptin, Ciliary Neurotrophic Factor

cannot exclude the possibility that a small population of GnRH neurons expresses the OB-R and mediates the direct stimulatory influence of leptin on the neurohormone. At the moment, whether leptin exerts direct effects on GnRH secretion seems to be controversial and no additional information is available to better clarify this point. GN11 cells, an in vitro model of GnRH neurons which retains the properties of immature and migrating GnRH neurons [42], were also utilized by our group to evaluate the chemotactic properties of different molecules by means of the Boyden chamber assay. Relative to leptin, due to the lack of the specific receptor subunits (personal observation, Dozio et al.) the cytokine did not display any chemotactic activity. These data indicate that the effects of this molecule, even if it shares similar receptor subunits with other cytokines, requires the presence of OB-R. GP130 CYTOKINES: RECEPTOR ACTIVATION AND SIGNAL TRANSDUCTION The family of cytokines signalling through the common receptor subunit gp130 comprises IL-6, IL-11, LIF, CNTF, oncostatin M (OSM), cardiotrophin-1 and cardiotrophin-1like cytokine, which are also known as IL-6-family of cytokines. The receptors involved in IL-6-type cytokines signalling are type I membrane proteins (extracellular N-terminus, one transmembrane domain) with the exception of the CNTF receptor alpha (CNTFR) which is linked to the plasma membrane by a glycosylphosphatidylinositol anchor. The first event in signaling activation is the ligand-induced homo or heterodimerization of signal-transducing receptor subunits. All IL-6-type cytokines recruit gp130 to their receptor complexes and signal via gp130 alone or in combination with LIF receptor beta (LIFR) or OSM receptor. Among these cytokines, CNTF and LIF signal via gp130LIFR heterodimers, but only CNTF first binds to the specific CNTFR subunit which is not involved in the signaltransduction pathway, while IL-6, after binding to the specific IL-6 receptor alpha (IL-6R) subunit, induces gp130 homodimerization. These cytokines utilize tyrosine kinases of the Jak family which activate STAT proteins. In addition, other SH2 domain-containing proteins bind and link cytokine signaling to the MAPK pathway. Other signaling pathway, such as the PI3-K, may also be activated in some cell types (see [3] for a review). CILIARY NEUROTROPHIC FACTOR (CNTF) CNTF is a 22 kDa protein synthesized by Schwann cells in the periphery [43] and by neurons and astrocytes in the CNS [44]. CNTF expression and synthesis is dramatically increased by trauma, inflammation and ischemia [45-47]. The cytokine may be either locally synthesized or produced outside the CNS and may enter the central structures by crossing the BBB via a saturable transport system [48]. CNTF receptor (CNTF-R) is composed of one ligand binding subunit CNTF, attached to the outer surface of the cell membrane by a glycosylphosphatidylinositol linkage and two signal transducing subunits, LIFR and gp130 [49]. CNTF-Rs are found in the arcuate and paraventricular nuclei of the hypothalamus, two areas involved in the regulation of

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neuroendocrine functions, and the specific binding subunit CNTFR has been shown to colocalize with OB-R in the same nuclei [50]. In this connection, it is worth noting that CNTF and leptin, by interacting with the same neurons in the hypothalamus, have been reported to share a number of biological effects and, in particular, both cytokines appeared to exert facilitatory effects on the reproductive axis. CNTF has been shown to prevent the fall in LH that accompanies fasting [51]. In addition, intracerebroventricular injection of anti-CNTF antibody in normal fed ovariectomized rats attenuates the prolactin and LH surges, whereas CNTF administration to fasted rats significantly restores the plasma levels of these hormones [52]. Moreover, CNTF has been shown to increase hypothalamic GnRH release from medial basal hypothalamic explants harvested from proestrous female rats and to cause a dose-dependent inhibition of GnRHstimulated LH secretion from dispersed anterior pituitary cells and perifused anterior pituitary fragments from proestrous female with no effect on basal LH release [53]. Recently, using GT1-7 neurons, we observed that the permissive action of CNTF on reproductive processes involves also a direct control on GnRH release [54]. However, there is also the possibility that CNTF may activate indirect mechanisms involving additional hypothalamic neural circuits and some neurotransmitters, such as neuropeptide Y (NPY) [51], which may involve either direct action on GnRH neurons, or indirectly through other orexigenic peptide systems, such as opioids and galanin, also known to influence GnRH secretion [23]. Relative to GnRH migration, due to the lack of the specific receptor subunits on the GN11 model system (personal observation, Dozio et al.) CNTF did not display any chemotactic activity, indicating that the effects of this molecule, even if it shares similar receptor subunits with other cytokines, are mediated by the interaction with the specific binding subunit [55]. LEUKEMIA INHIBITORY FACTOR (LIF) LIF is a secreted glycoprotein with a range of molecular weight forms, from 38 to 67 kDa, resulting from differential glycosylation of a protein of approximately 20 kDa [56]. LIF acts through the LIF cell-surface receptor complex (LIF-R), consisting of two different subunits, the specific low affinity LIFR and the gp130. After binding to LIFR, LIF induces a conformational change followed by the heterodimerization of LIFR and gp130 [57, 58] which resulted in a high affinity receptor complex able to activate the intracellular signaling pathways Jaks/STAT, MAPK and the PI3-K/protein kinase B/Akt [59-61]. LIF has been described as a pleiotropic cytokine since it was able to exert various effects on different cell types. Some studies indicated that LIF participates to the complex control of the reproductive function principally by exerting peripheral actions, like stimulation of the proliferation of primordial germ cells, spermatocyte differentiation [62] and reduction of testosterone synthesis by Leydig cells [63]. Furthermore, LIF expression has been observed in uterine tissue with a major role in the regulation and preimplantation development of the mammalian embryo, and in implantation and early pregnancy [64, 65]. Recently, we demonstrated that LIF was also involved in the central regulation of reproductive function by its ability to modulate the migration of GnRH neurons, which is a fundamental

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process for the development of normal reproductive function. Specifically, LIF has been shown to stimulate the migration of GN11 cells, playing a general role in stimulating the intrinsic cell motility (chemokinesis) other than the directional migration (chemotaxis). All the main signaling pathways known to be coupled to LIF-R (Jaks/STAT, MAPK and the PI3-K/ Akt) were independently activated by LIF in this cell line and each of these gave a contribution to LIFinduced migration. Moreover, we reported that LIF is prenatally expressed in nasal regions where GnRH neurons originate and start their migration, suggesting the potential for a significant action of this cytokine as a chemokinetic factor to increase GnRH cell motility [55]. These data integrated those of previous studied which observed that LIF mRNA is expressed in adult brain as well as in other peripheral organs like liver, thymus, spleen and gut [66]. Moreover, LIF expression has been shown to be developmentally modulated in neural tissue and LIF binding sites were found in both the developing and adult rat nervous system [67]. A saturable transport system across the BBB has also been described for LIF. This system appears to be different from that utilized by CNTF, which does not cause any significant inhibition of the entry of LIF into the brain [48, 68]. By utilizing GT1-7 cells, we report here that at nanomolar concentrations, LIF may also affect the reproductive function by directly stimulating GnRH release (Fig. 1). In particular, exposure of GT1-7 cells for 30 min to different LIF concentration (10-12 to 10-9 M) resulted in increased GnRH release at the dose of 10-10 M. Probably at higher doses the lack of effect might result from down-regulation of the receptor and/or of the signaling pathways, similar to that reported for leptin [37]. It is worth noting that such cytokine represents a molecule able to modulate the reproductive function at different levels, acting not only during embryonic GnRH migration, but also in the adult age as a permissive molecule on GnRH release, and LH and prolactin secretion by the pituitary [69]. It is also possible that LIF may regulate the function of some hypothalamic interneurons, as NPY neurons, although no specific data relative to LIF effect on these targets are available to better clarify this point. Anyway, there are some reports which indicated that in the peripheral nervous system LIF suppresses NPY expression both in vitro and in vivo and that in the CNS (specifically in the hippocampus) NPY is controlled by LIF [70-73].

Fig. (1). Effect of LIF treatment on GnRH release by GT1-7 cells. GT1-7 cells (kindly provided by Dr. R. I. Weiner, Reproductive Endocrinology Center, University of California, San Francisco, CA) were grown at 37°C in a humidified CO2 incubator in Dulbecco's Minimum Essential Medium (DMEM; Biochrom, Berlin, Germany) supplemented with 1 mmol/L sodium pyruvate, 100 g/mL streptomycin, 100 U/mL penicillin, 2 mmol/L-glutamine and 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY) in 24-wells plates at the density of 0.15*106 cells/well. After 3-4 days, cells were incubated with different LIF concentrations (10-12 to 10-9 M) for 30 min. At the end of the incubation time, the culture medium was collected, boiled for 10 min and centrifuged for 5 min at 1000 rpm and GnRH accumulated into the culture medium was measured by radioimmunoassay (RIA) using a commercially GnRH RIA kit by Peninsula (San Carlos, CA). LIF treatment induced a significant stimulation of GnRH release at the concentration of 10-10 M. Statistical analysis was performed using the Prism statistical analysis package (GraphPad Software, San Diego, CA). Differences between treatment groups were evaluated by ANOVA, followed by post hoc Dunnett’s test. Data are expressed as mean ± SD (n = 6). *P < 0.05 vs. control.

INTERLEUKIN-6 IL-6 is produced by a variety of cells including monocytes, fibroblast, endothelial cells, astrocytes, microglia and neurons [74, 75] and it has been found to be expressed in the hypothalamus [76]. Although IL-6 can cross the BBB by a saturable transport system to reach the CNS, only a small amount represents the intact cytokine due to its extensive degradation in the brain [77]. Thus, the relative contribution of peripheral IL-6 actions in the CNS is not fully clear and also the reports concerning the role of IL-6 on the GnRH-LH system seem to be controversial. Feleder et al. observed a stimulatory effect of IL-6 on GnRH release by utilizing the preoptic mediobasal hypothalamic area dissected from male rats and indicated that antagonism at IL-6-R potentiated the inhibitory effect of lipopolysaccharide on GnRH secretion, as a consequence of the lack of the stimulatory effect of IL-6

on GnRH [78]. Another work by Yamaguchi et al. confirmed the stimulatory role of IL-6 on GnRH release by utilizing a primary culture system of hypothalamic neurons producing GnRH; in particular, they observed that IL-6 stimulated the release of GnRH in a dose- and time-dependent manner and that this effect was significantly blocked by an anti-IL-6 antiserum [79]. Differently from these works, Watanobe observed that the direct infusion of IL-6 into the medial preoptic area was without effect on GnRH release, even at the highest concentration infused. Since no IL-6-R has been demonstrated on GnRH neurons, it was hypothesized that the observed effects of this molecule on GnRH release could be obtained via indirect mechanism. Differently from the other cytokines previously described, IL-6 did not seem to interfere with hypothalamic NPY secretion; therefore, it

Leptin, Ciliary Neurotrophic Factor

was hypothesized that this cytokine may either inhibit cellfiring rates or prevent NPY synthesis directly or attenuate the postsynaptic effects of NPY or affect different hypothlamic mediators [80]. Here we report the novel observation that GT1-7 and GN11 cells express IL-6R mRNA, indicating that also a direct effect may be possible (Fig. 2). IL-6 actions seem to be controversial also relative to LH secretion. Although one in vivo study reported that central administration of IL-6 was inhibitory to LH secretion [81], other reports demonstrated no effects of IL-6 [82-84]. Similarly, the reported actions of IL-6 on pituitary LH release in vitro vary from stimulatory [85] to inhibitory [86] or no effect [82]. In particular, the observation by Russell et al. [86] that IL-6 did not affect LH release from dispersed anterior pituitary cells from male or proestrous female rats, but significantly suppressed GnRH-stimulated LH release from male, but not proestrus female dispersed pituitaries, suggests the existence of gender-specific differences. These inconsistencies may also derive from the differences in the experimental protocols employed (including in vivo vs. in vitro conditions) and the species and gender of animals used, and may also derive from the use of homologous vs. heterologous cytokines for administration [84]. Presently, whether IL-6 may affect the GnRH-LH axis is not clear and additional information seem necessary to better clarify this point.

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SUMMARY AND CONCLUSIONS The class-I cytokines here considered represent a group of molecules which display considerable overlap in their spectrum of biological actions. The reasons of these similarities appear to be linked to the similar three-dimensional fold structure, the interaction with related class-I receptors, the expression of these receptors in the same regions (i.e. hypothalamus), and the activation of similar intracellular signaling cascade. Specifically, for gp130 cytokines this redundancy is explained by the shared use of the gp130 receptor subunit that funnels the intracellular response along the same signaling cascade. In fact, gp130-knockout mice present an embryonically lethal phenotype, probably due to the loss of signaling capability for a large group of molecules [87]. Besides the overlap in receptor conformation, the ability to activate the same intracellular pathways seems also to be important. Leptin signals via a non-gp130 receptor type; anyway OB-R, similarly to the gp130 receptors, does not display intrinsic kinase activity, but requires the activation of the Jak proteins and activates the same intracellular pathways. Relative to leptin, CNTF, LIF and IL-6, in the in vitro model GT1-7 cells, we observed the expression of the receptors for all these cytokines and we described the ability of leptin, CNTF and LIF to modulate GnRH release in the same range of concentrations. Differently from LIF, whose function as pro-migratory agent was observed in GN11, leptin and

Fig. (2). Reverse transcription-polymerase chain reaction (RT-PCR) analysis of the gene expression of IL-6R, in GT1-7 cells, GN11 cells and in mouse hypothalamus (hyp, positive control) (mw, molecular weight markers). Control RNA (pAW109) is a control reagent provided with the RT-PCR kit. After RNA extraction with the phenol-chloroform method, using the Tri Reagent solution (Sigma-Aldrich, Milan, Italy), 1 g total RNA, quantified after an initial DNAase digestion step using the Deoxyribonuclease I kit (Sigma-Aldrich), was reverse transcribed using a commercially available kit and a DNA thermal cycler (both from Applera, Milan, Italy), at 25°C/10 min, then 42°C/60 min and a final hold at 85°C/5 min. PCR amplification was performed using 50 ng of cDNA as a template and appropriate oligonucleotides (Invitrogen, Milan, Italy) (forward: 5'-TGCCAACCTTGTGGTATCAGCC-3'; reverse: 5'-TGAAGACACAGAGAAGCAATCC-3' [88]) in 25 L of a premixed ready-to-use solution containing TaqDNA Polymerase (GoTaq Green Master Mix, Promega Italia, Milan, Italy). The PCR reaction included an initial cDNA denaturating step (95°C/5min) followed by 40 cycles (94°C/30 sec+55°C/30 sec+72°C/45 sec) and a final step incubation at 72°C for 7 min. Amplification products were separated by 2% agarose gel electrophoresis and detected by ethidium bromide fluorescence on a UV transilluminator. The identity of the PCR products has been confirmed by sequencing (IFOM, Milan, Italy).

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Table 1.

Dozio et al.

IL-6 Related Cytokines Participate to the Central Control of Reproductive Function Acting at Different Ontogenic Times and Anatomical Sites LEPTIN

CNTF

LIF

IL-6

NE

NE

yes §§

NE

Expression of receptors in GN11 cells

no (PO)

no (PO)

yes §§

yes +

Effect on GN11 cell migration

no (PO)

no (PO)

yes §§

NE

yes *

yes °

yes °

yes °°°

NE

yes °°

yes §

yes §§§

Expression of receptors in GT1-7 cells

yes **

yes ***

yes ***

yes +

Effect on GnRH secretion

yes **

yes ***

yes +

yes ^ /no ^^

Expression along the GnRH migratory route

Enter BBB CNS expression

NE: not evaluated Data presented in this table were collected from: PO: Dozio et al., personal observation; * [29]; ** [37]; *** [54]; ° [48, 68]; °° [44]; °°° [77]; § [66]; §§ [55]; §§§ [76]; ^ [78]; ^^ [84]; + present study.

CNTF did not display any chemotatic activity due to the lack of the specific receptor subunits in these cells, indicating that the effects of these molecules, even if they share similar receptors, are mediated by the interaction with the specific binding subunit, whose expression probably depends on the developmental stage. The role of IL-6 in this process has not been fully clarified so far.

LH

= Luteinizing hormone

MAPK

= Mitogen-activated protein kinase

NPY

= Neuropeptide Y

OB-R

= Leptin receptor

OSM

= Oncostatin M

In conclusion, the available data indicate that IL-6related cytokines appear to participate to the complex control of the reproductive function at different ontogenic times and anatomical sites, with both central (development and function of the hypothalamus-pituitary system) and peripheral actions (Table 1).

PI3-K

= Phosphatidylinositol-3 kinase

STAT

= Signal transducer and activator of transcription

ACKNOWLEDGEMENT

REFERENCES [1] [2]

This work was supported in part by grants from the University of Milan (PUR 2008). ABBREVIATIONS BBB

= Blood brain barrier

CNTF

= Ciliary neurotrophic factor

CNTF-R = Ciliary neurotrophic factor receptor CNTFR = Ciliary neurotrophic factor receptor alpha

[3] [4]

[5]

FSH

= Follicle-stimulating hormone

[6]

GnRH

= Gonadotropin-releasing hormone

[7]

gp130

= Glycoprotein 130

Jak

= Janus kinase

IL

= Interleukin

IL-6-R

= Interleukin-6 receptor

IL-6R

= Interleukin-6 receptor alpha

LIF

= Leukemia inhibitory factor

LIF-R

= Leukemia inhibitory factor receptor

LIFR

= Leukemia inhibitory factor receptor beta

[8]

[9]

[10]

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Leptin, Ciliary Neurotrophic Factor

[11]

[12]

[13] [14]

[15]

[16] [17]

[18]

[19]

[20]

[21] [22]

[23] [24]

[25] [26]

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Revised: May 01, 2009

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