The Transforming Growth Factor Alpha Gene Family Is Involved in the Neuroendocrine Control of Mammalian Puberty SR Ojeda, YJ Ma and F Rage

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REVIEW ARTICLE: A TRIBUTE TO SM McCANN The transforming growth factor alpha gene family is involved in the neuroendocrine control of mammalian puberty SR Ojeda, YJ Ma and F Rage

Division of Neuroscience, Oregon Regional Primate Research Center, 505 NW 185th Avenue, Beaverton, OR 97006, USA

The concept is proposed that the central control of mammalian female puberty requires the interactive participation of neuronal networks and glial cells of the astrocytic lineage. Accord- ing to this concept neurons and astrocytes control the pubertal process by regulating the secretory activity of those neurons that secrete luteinizing hormone-releasing hormone (LHRH). LHRH, in turn, governs sexual development by stimulating the secretion of pituitary gonadotropins. Astrocytes affect LHRH neuronal function via a cell–cell signaling mechanism involving several growth factors and their corresponding receptors. Our laboratory has ident- iﬁed two members of the epidermal growth factor/transforming growth factor (EGF/TGF␣) family as components of the glial-neuronal interactive process that regulates LHRH secretion. Transforming growth factor alpha (TGF␣) and its distant congener neu-differentiation factor, NDF, are produced in hypothalamic astrocytes and stimulate LHRH release via a glial interme- diacy. The actions of TGF␣ and NDF on hypothalamic astrocytes involve the interactive activation of their cognate receptors and the synergistic effect of both ligands in stimulating the

glial release of prostaglandin E2 (PGE2). In turn, PGE2 acts directly on LHRH neurons to stimulate LHRH release. A variety of experimental approaches has led to the conclusion that both TGF␣ and NDF are physiological components of the central mechanism controlling the initiation of female puberty. Keywords: glial cells; hypothalamus; growth factors; sexual development; astrocytes; cell–cell communication; LHRH neurons

The onset of mammalian female puberty requires the ronal network is well-documented, recent studies have functional activation of a specialized group of hypo- raised the possibility that glial cells may also play a thalamic neurons that produce luteinizing hormone- role in the control of LHRH neuronal activity.9 Ana- releasing hormone (LHRH), the neurohormone govern- tomical evidence demonstrates that the glial ensheath- ing sexual maturation and reproductive function. ment of LHRH neurons in both primates and rodents Elucidation of the mechanisms controlling LHRH neu- is substantial,10,11 the main difference between species ronal function is critical for the understanding of the being a more prominent glial apposition to LHRH cellular and molecular processes underlying sexual perikarya in primates.10 Glial apposition of LHRH development and adult reproductive capacity. It is well nerve terminals in the median eminence of the hypo- accepted that transsynaptic inputs, both excitatory1,2 thalamus is, however, similarly abundant in both rats and inhibitory,3 contribute to the central regulation of and primates. The median eminence contains astro- LHRH secretion. A combination of a decrease in trans- cytes and modified ependymoglial cells known as tany- synaptic inhibitory tone and an increase in excitatory cytes.12 These specialized glial cells line the ventral inputs to LHRH neurons is thought to be instrumental portion of the third ventricle and send their processes in bringing about the initiation of puberty. The main towards the endothelial wall of the portal blood vascul- excitatory inputs affecting LHRH secretion include ature, establishing along their way intimate contact neuronal systems that utilize norepinephrine (NE),4 with LHRH nerve terminals.12,13 More intriguingly, sex neuropeptide Y (NPY)1 and excitatory amino acids.2,5,6 steroids appear to modulate this structural arrange- The inhibitory neurotransmitters, on the other hand, ment, as gonadectomy decreases the space between are gamma aminobutyric acid (GABA)3,7 and opioid LHRH nerve terminals and the basal lamina, presum- peptides.8 ably due to retraction of the glial cells.14 An obvious While this transsynaptic regulation of the LHRH neu- interpretation of these structural changes is that they increase the access of LHRH nerve endings to the portal vessels. The finding that about 50% of glial cells in 15 Correspondence: SR Ojeda, DVM, Division of Neuroscience, Ore- the median eminence have estrogen receptors further gon Regional Primate Research Center, 505 NW 185th Avenue, supports the notion of a direct action of estrogen on Beaverton, OR 97006, USA. E-mail: ojedasȰohsu.edu hypothalamic astrocytes.16 As glial-neuronal synaptoid Glial growth factors and LHRH secretion SR Ojeda et al 356 specializations are commonly seen in the median emi- logical blockade of EGFR tyrosine kinase activity36 tar- nence,12 this region appears to be a primary site where geted to the median eminence delays the onset of glial cells influence LHRH neuronal function. In this puberty,23 suggesting that activation of EGFR in this article, we will discuss some of the recent results region of the hypothalamus is a physiological compo- obtained by our laboratory concerning the potentially nent of the central mechanism underlying the acqui- important role played by glial cells in the regulation of sition of reproductive competence. In another study, LHRH secretion. we asked the question as to whether focal activation In a series of reports, we documented the concept of TGF␣ secretion would be able to stimulate LHRH that glia-derived transforming growth factor alpha neuronal activity, and thus accelerate the onset of (TGF␣), a mitogenic polypeptide member of the epider- puberty. To produce this focal increase we used two mal growth factor (EGF) family,17–19 plays an important gene delivery systems. In one of them, fibroblasts were role in the process by which astrocytes affect LHRH stably transfected with a retroviral construct in which secretory activity.16,20–24 More recently, we obtained expression of the human TGF␣ gene was directed by evidence for the involvement of other members of the the phosphoglycerate kinase promoter, which is consti- EGF/TGF␣ family in this process. Neuregulins, also tutively active in most mammalian cells. The other known as heregulins25 or—in the case of the rat—as delivery system consisted of a plasmid construct in neu-differentiation factor (NDF),26 are alternatively which the human TGF␣ gene was placed under the spliced products of a single gene.26,27 Our studies control of a mouse MT promoter. Since this promoter showed that NDF is produced in hypothalamic astro- is activated by heavy metals, TGF␣ expression can be cytes and facilitates LHRH release through a mech- increased several-fold by simply exposing the cells car-

anism involving stimulation of glial PGE2 release and rying the construct to zinc chloride. facilitation of TGF␣ action on astrocytes (Ma et al, The genetically modified fibroblasts were then unpublished). implanted into different hypothalamic sites of juvenile The biological actions of TGF␣ and NDF are initiated female rats and the onset of puberty was evaluated. To by binding to membrane-spanning receptors endowed activate a circadian expression of the TGF␣ gene con- with protein kinase activity for cellular signaling.28–32 trolled by the MT promoter, animals bearing these cells The receptor for TGF␣ (which also recognizes EGF, were injected once daily with zinc chloride. The amphiregulins and other related members of the results demonstrated that cells carrying either con- family) is a 170-kDa glycosylated protein known as the struct were able to accelerate the onset of puberty only EGF receptor (EGFR). EGFR has an extracellular when they were grafted in the immediate vicinity of domain containing the amino acid motifs essential for LHRH neurons. The effect appears to be more robust ligand recognition, a single transmembrane domain, when the cells were located in contact with LHRH and an intracellular domain endowed with tyrosine nerve terminals in the median eminence of the hypo- kinase activity. The receptors for NDF, known as HER- thalamus.37 Thus, these experiments demonstrate that 3 (also named ERBB-3)29,31 and HER-4,30 are related to a localized activation of TGF␣ production can acceler- EGFR. An additional related receptor, known as HER- ate puberty, as long as the activation occurs within the 2 (neu) receptor,32,33 appears to function as an auxiliary confines of the LHRH neuron microenvironment. subunit for the high affinity binding of TGF␣/EGF to As indicated before, TGF␣ is not the only member of EGFR and NDF/neuregulins to HER-3 and HER-4.34 the EGF family that participates in the control of LHRH In the hypothalamus of both rats23 and nonhuman secretion. In very recent in vitro experiments we found primates,20 the TGF␣ gene and its protein product are that glia-derived NDF enhances the stimulatory action predominantly expressed in astroglial cells of certain of TGF␣ on LHRH release via activation of an HER- nuclei, such as the suprachiasmatic nucleus, arcuate 2/HER-4 receptor complex. Blockade of NDF signal nucleus and dorsomedial nucleus.23 Both TGF␣ mRNA transduction via an antisense oligonucleotide, which and protein are also abundant in tanycytes and glial disrupts the synthesis of HER-2, also attenuated the cells of the median eminence, the level of expression effect of TGF␣ on LHRH release indicating that the increasing significantly around the time of puberty.20,23 TGF␣/EGFR and NDF/HER ligand-receptor systems Importantly, EGFR is also present in the median emi- function in a tightly coordinated manner to regulate nence,20,22 indicating the involvement of a TGF␣-EGFR neuroendocrine reproductive function. interaction in neuroendocrine regulation. That TGF␣ The stimulatory effects of TGF␣ on LHRH neuronal may be involved in the control of LHRH neuronal activity are not exerted directly, as LHRH neurons do activity was demonstrated by the finding that TGF␣ is not contain EGFR.22 Instead, TGF␣ acts on hypothal- able to stimulate the release of LHRH from median emi- amic astrocytes, which are endowed with EGFR,16,38 nence fragments in a dose-related manner.35 Several and respond to the peptide with an increase in TGF␣ ␣ 16 39 observations suggest that TGF -induced activation of gene expression and PGE2 release. The conditioned EGFR contributes to the normal initiation of mam- medium from hypothalamic astrocytes treated with malian female puberty. For instance, overexpression of TGF␣ stimulates LHRH release from an LHRH-produc- TGF␣ in transgenic mice carrying a human TGF␣ gene ing cell line, which expresses no EGFR and is unre- ␣ 38 under the control of a metallothionein (MT) promoter sponsive to TGF itself. That glial PGE2 is, at least in (which is activated by heavy metals, such as Zn2+) part, responsible for this stimulatory effect is suggested 21 induces precocious puberty. In contrast, pharmaco- by the finding that blockade of PGE2 action by immu- Glial growth factors and LHRH secretion SR Ojeda et al 357

Figure 1 Glial–neuronal interactive mechanisms proposed to participate in the central regulation of mammalian puberty. While neuronal networks associated with LHRH neurons act transsynaptically [1] to regulate LHRH secretion, they can also affect astrocytic function via specific receptors located on glial cells [2]. Excitatory amino acids exemplify this regulatory influence as they can affect glial production of growth factors via metabotropic receptors. Astrocytes affect LHRH secretion through both direct actions on LHRH neurons (for instance, fibroblast growth factor),38 and indirect paracrine/autocrine mechanisms [3] leading to the formation and release of neuroactive molecules able to directly affect LHRH release, such as prostaglan- 41 din E2 [4]. PGE2 is also able to affect astrocytic function. Growth factors and other neuroactive substances produced by astrocytes may also influence the transsynaptic communication between LHRH neurons and its associate neuronal circuitry [5]. noneutralization of the prostaglandin in the con- Acknowledgements ditioned medium abolishes the increase in LHRH induced by this medium.39 This work was supported by NIH grants HD25123, P30 In addition to TGF␣ itself, glial expression of the Population Center Grant HD18185, and RR00163 for TGF␣ gene appears to be upregulated by gonadal ster- the operation of the ORPRC. oids (estrogen) and neuronal inputs (excitatory amino 16,40 acids), suggesting that in addition to the above out- References lined glial–neuronal signaling pathway mediated by growth factors and eicosanoids, there is a neuron-to- 1 Crowley WR, Parker SL, Sahu A, Kalra SP. Interacting transmem- glia signaling mechanism mediated by excitatory neu- brane signals regulating GnRH and LH secretion. In: Plant TM, Lee PA (eds). 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