Endocannabinoids affect the reproductive functions in teleosts and amphibians.

Following the discovery in the brain of the bonyfish Fugu rubripes of two genes encoding for type 1 cannabinoid receptors (CB1A and CB1B), investigations on the phylogeny of these receptors have indicated that the cannabinergic system is highly conserved. Among the multiple functions modulated by cannabinoids/endocannabinoids through the CB1 receptors one of the more investigated is the mammalian reproduction. Therefore, since studies performed in animal models other than mammals might provide further insight into the biology of these signalling molecules, the major aim of the present paper was to review the comparative data pointing toward the endocannabinoid involvement in the reproductive control of non-mammalian vertebrates, namely bonyfish and amphibians. The expression and distribution of CB1 receptors were investigated in the CNS and gonads of two teleosts, Pelvicachromis pulcher and Carassius auratus as well as in the anuran amphibians Xenopus laevis and Rana esculenta. In general the large diffusion of neurons targeted by cannabinoids in both fish and amphibian forebrain indicate endocannabinoids as pivotal local messengers in several neural circuits involved in either sensory integrative activities, like the olfactory processes (in amphibians) and food response (in bonyfish), or neuroendocrine machinery (in both). By using immunohistochemistry for CB1 and GnRH-I, the codistribution of the two signalling molecules was found in the fish basal telencephalon and preoptic area, which are key centers for gonadotropic regulation in all vertebrates. A similar topographical codistribution was observed also in the septum of the telencephalon in Rana esculenta and Xenopus laevis. Interestingly, the double standard immunofluorescence on the same brain section, aided with a laser confocal microscope, showed that in anurans a subset of GnRH-I neurons exhibited also the CB1 immunostaining. The fact that CB1-LI-IR was found indeed in the FSH gonadotrophs of the Xenopus pituitary gland and CB1 receptors together with the fatty acid amide hydrolase, the degradative enzyme of the endocannabinoid anandamide, were demonstrated in both bonyfish and frog gonads, strongly suggests that endocannabinoids are involved in central and peripheral gonadotropic functions of teleosts and amphibians.

Changes of gonadal CB1 cannabinoid receptor mRNA in the gilthead seabream, Sparus aurata, during sex reversal.

Two cannabinoid receptor-like genes (CB1-like), named CB1A and CB1B, have been isolated in teleost fish, specifically in the puffer fish, Fugu rubripes. However, information on the physiological roles, such as the control of reproduction and development in fish is still scarce. Therefore, the aim of the present study was to investigate the presence of CB1-like mRNA in the gonads of a marine teleost species, the gilthead seabream, Sparus aurata, a hermaphrodite species in which the gonadal tissues first develop as testes, and then as functional ovary. We isolated an 890 bp fragment (GenBank accession number ); that corresponded to the open reading frame of the teleost CB1 receptor gene, encoding for the central portion of the protein, which was aligned with the other bony fish sequence. Using "in situ" hybridization, CB1-like mRNA was localized in both mature and sex-reversing gonads, and relative changes in CB1-like expression levels were detected through semi-quantitative RT-PCR. In the mature testis and in the testicular part of the sex-reversing gonad, CB1 expression levels were found to be much higher compared to the ovarian portion. This suggests that the CB1 signaling is likely involved in the process of testicular regression of the S. aurata, but its actual role has yet to be determined.

The endocannabinoid system in the brain of Carassius auratus and its possible role in the control of food intake.

Cannabinoid receptors and the endocannabinoids anandamide and 2-arachidonoylglycerol have been suggested to regulate food intake in several animal phyla. Orthologs of the mammalian cannabinoid CB(1) and CB(2) receptors have been identified in fish. We investigated the presence of this endocannabinoid system in the brain of the goldfish Carassius auratus and its role in food consumption. CB(1)-like immunoreactivity was distributed throughout the goldfish brain. The prosencephalon showed strong CB(1)-like immunoreactivity in the telencephalon and the inferior lobes of the posterior hypothalamus. Endocannabinoids were detected in all brain regions of C. auratus and an anandamide-hydrolysing enzymatic activity with features similar to those of mammalian fatty acid amide hydrolase was found. Food deprivation for 24 h was accompanied by a significant increase of anandamide, but not 2-arachidonoylglycerol, levels only in the telencephalon. Anandamide caused a dose-dependent effect on food intake within 2 h of intraperitoneal administration to satiated fish and significantly enhanced or reduced food intake at low (1 pg/g body weight) or intermediate (10 pg/g) doses, respectively, the highest dose tested (100 pg/g) being inactive. We suggest that endocannabinoids might variously contribute to adaptive responses to food shortage in fish.

CB1 cannabinoid receptors in amphibian spinal cord: relationships with some nociception markers.

The role of cannabinoids in spinal analgesia has so far been investigated in mammals and the interactions between cannabinoid receptors and markers involved in nociception have been described in the rat spinal cord. An endocannabinoid system is well developed also in the amphibian brain. However, the anatomical substrates of pain modulation have been scarcely investigated in anamniotes, neither is there reference to such a role for cannabinoids in lower vertebrates. In the present paper we employed multiple cytochemical approaches to study the distribution of CB1 cannabinoid receptors and their morphofunctional relationships with some nociception markers (i.e. Substance P, nitric oxide synthase, GABA and mu opioid receptors) in the spinal cord of the anuran amphibian Xenopus laevis. We found a co-distribution of CB1 receptors with the aforementioned signaling molecules, as well as a more limited cellular co-localization, in the dorsal and central fields of the spinal cord. These regions correspond to the mammalian laminae I-IV and X, respectively, areas strongly involved in spinal analgesia. Comparison of these results with those previously obtained in the mammalian spinal cord, reveals a number of similarities between the two systems and suggests that cannabinoids might participate in the control of pain sensitivity also in the amphibian spinal cord.

Environmental estrogens and reproductive biology in amphibians.

In this paper, the effects of an estrogenic compound, 4-nonyl-phenol (NP), on the amphibians Rana esculenta and Triturus carnifex are described together with those on sexual differentiation in Xenopus laevis. NP increased plasma vitellogenin in male frogs and newts in a dose-related manner; moreover, inhibitory effects on gonadotropin and prolactin (PRL) secretion by pituitary were found together with an elevation of plasma androgens. NP treatment also caused a remarkable increase in number of prolactin-immunolabeled cells, suggesting that xenoestrogen might induce, at least in the newt pituitary, a PRL accumulation possibly due to a reduction of the hormone release. In addition, both NP and bisphenol A caused feminization by increasing the percentage of female phenotypes in X. laevis, and the in vivo effects were more pronounced than those of estradiol-17beta.

Relationships between CB1 cannabinoid receptors and pituitary endocrine cells in Xenopus laevis: an immunohistochemical study.

The distribution of the cannabinoid CB1 receptor and its relationships with individual endocrine cell types were investigated by immunohistochemistry in the anterior lobe of the Xenopus adenohypophysis. By use of a specific primary antibody raised in rabbit against the amino terminus of the rat CB1, we have found numerous CB1-like-immunoreactive cells distributed throughout all of the pituitary anterior lobe with the exception of the ventrocranial area adjacent to the median eminence of the neurohypophysis. Aided by both double-immunostaining on consecutive serial sections and double-simultaneous immunofluorescence on the same section of the gland, the CB1-like immunoreactivity was compared to some specific hormone immunoreactive cells. CB1 labelings were mainly codistributed, and even colocalized, with lactotrophs and thyrotrophs. Gonadotrophs containing CB1 receptors were also observed. In contrast, corticotrophs, which are located mainly in the ventrocranial pole of the anterior lobe, were generally devoid of CB1. Since nerve terminals immunoreactive to the CB1 antibody were observed within the vascular zone of the median eminence, the possibility that endocannabinoids are involved in the control of some secretory activities of Xenopus pituitary, either indirectly via hypothalamic neurosecretory mechanisms or directly on the pituitary cells, was envisaged. In particular, the present study suggests the occurrence of a direct cannabinergic modulation of the prolactin, gonadotrophin, and thyrotrophin secretions through the CB1 receptor.

Role of coactivators and corepressors in steroid and nuclear receptor signaling: potential markers of tumor growth and drug sensitivity.

Nuclear receptors regulate target gene expression in response to steroid and thyroid hormones, retinoids, vitamin D and other ligands. These ligand-dependent transcription factors function by contacting various nuclear cooperating proteins, called coactivators and corepressors, which mediate local chromatin remodeling as well as communication with the basal transcriptional apparatus. Nuclear receptors and their coregulatory proteins play a role in cancer and other diseases, one leading example being the estrogen receptor pathway in breast cancer. Coregulators are often present in limiting amounts in cell nuclei and modifications of their level of expression and/or structure lead to alterations in nuclear receptor functioning, which may be as pronounced as a complete inversion of signaling, i.e. from stimulating to repressing certain genes in response to an identical stimulus. In addition, hemizygous knock-out of certain coactivator genes has been demonstrated to produce cancer-prone phenotypes in mice. Thus, assessment of coactivator and corepressor expression and structure in tumors may turn out to be essential to determine the role of nuclear receptors in cancer and to predict prognosis and response to therapy.

Hepatocyte growth factor is a regulator of monocyte-macrophage function.

Hepatocyte growth factor (HGF) is a potent paracrine mediator of stromal/epithelial interactions, which is secreted as a matrix-associated inactive precursor (pro-HGF) and locally activated by tightly controlled urokinase cleavage. It induces proliferation and motility in epithelial and endothelial cells, and plays a role in physiological and pathological processes involving invasive cell growth, such as angiogenesis and parenchymal regeneration. We now report that HGF induces directional migration and cytokine secretion in human monocytes. Monocyte activation by endotoxin and IL-1beta results in the up-regulation of the HGF receptor expression and in the induction of cell-associated pro-HGF convertase activity, thus enhancing cell responsiveness to the factor. Furthermore, we provide evidence for the secretion of biologically active HGF by activated monocytes, implying an autocrine stimulation. Altogether, these data indicate that monocyte function is modulated by HGF in a paracrine/autocrine manner, and provide a new link between stromal environment and mononuclear phagocytes.

AP-2 transcription factors in the regulation of ERBB2 gene transcription by oestrogen.

Transcription of the ERBB2 oncogene is repressed by oestrogen in human breast cancer cells. We show that a 218 bp fragment of the human ERBB2 gene promoter is responsive to oestrogen in transient transfection in ZR75.1 and SKBR.3 cells when the oestrogen receptor is expressed. Deletion analysis of this fragment shows that a sequence located at the 5' end, which is known to mediate ERBB2 overexpression in breast cancer, is also responsible for the oestrogen response. This sequence binds AP-2 transcription factors and appears functionally identical to an element of the oestrogen-dependent enhancer described in the first intron of human ERBB2. We observed that oestrogen treatment down-regulates expression of AP-2 proteins but does not affect the DNA binding activity of AP-2. Constitutive expression of AP-2beta or AP-2gamma, but not AP-2alpha, abrogates the estrogenic repression. Our results demonstrate that AP-2 transcription factors are implicated in the oestrogenic regulation of ERBB2 gene expression and suggest a complex interplay involving the different AP-2 isoforms and other unidentified factors.

Hepatocyte growth factor is a coupling factor for osteoclasts and osteoblasts in vitro.

Hepatocyte growth factor (HGF), also known as scatter factor, is a powerful motogen, mitogen, and morphogen produced by cells of mesodermal origin, acting on epithelial and endothelial cells. Its receptor is the tyrosine kinase encoded by the c-MET protooncogene. We show that the HGF receptor is expressed by human primary osteoclasts, by osteoclast-like cell lines, and by osteoblasts. In both cell lineages, HGF stimulation triggers the receptor kinase activity and autophosphorylation. In osteoclasts, HGF receptor activation is followed by increase in intracellular Ca2+ concentration and by activation of the pp60c-Src kinase. HGF induces changes in osteoclast shape and stimulates chemotactic migration and DNA replication. Osteoblasts respond to HGF by entering the cell cycle, as indicated by stimulation of DNA synthesis. Interestingly, osteoclasts were found to synthesize and secrete biologically active HGF. These data strongly suggest the possibility of an autocrine regulation of the osteoclast by HGF and a paracrine regulation of the osteoblast by the HGF produced by the osteoclast.

The HIV-1 nef protein interferes with phosphatidylinositol 3-kinase activation 1.

nef is a human immunodeficiency virus (HIV) gene encoding a 27-kDa myristoylated protein with structural features of a signal transducing molecule, but whose functions are largely unknown. We studied the interactions of Nef with the signal transduction pathways triggered by the platelet-derived growth factor (PDGF) receptor. The association of phosphatidylinositol (PI) 3-kinase with the activated receptor was severely impaired by nef expression. Conversely, PDGF-induced receptor tyrosine phosphorylation, binding to phospholipase C-gamma and to Ras-GAP were not modified. Microtubule-associated protein kinase activation and intracellular calcium influx in response to PDGF were either unaffected or only slightly enhanced. Nef significantly reduced the proliferative response to the growth factor, while the chemotactic response was unchanged. These data show that Nef affects selectively the PI 3-kinase signaling pathway and suggest that this interference results in some of the HIV adverse effects on host cell functions.

Hepatocyte growth factor induces proliferation and differentiation of multipotent and erythroid hemopoietic progenitors.

Hepatocyte growth factor (HGF) is a mesenchymal derived growth factor known to induce proliferation and "scattering" of epithelial and endothelial cells. Its receptor is the tyrosine kinase encoded by the c-MET protooncogene. Here we show that highly purified recombinant HGF stimulates hemopoietic progenitors to form colonies in vitro. In the presence of erythropoietin, picomolar concentrations of HGF induced the formation of erythroid burst-forming unit colonies from CD34-positive cells purified from human bone marrow, peripheral blood, or umbilical cord blood. The growth stimulatory activity was restricted to the erythroid lineage. HGF also stimulated the formation of multipotent CFU-GEMM colonies. This effect is synergized by stem cell factor, the ligand of the tyrosine kinase receptor encoded by the c-KIT protooncogene, which is active on early hemopoietic progenitors. By flow cytometry analysis, the receptor for HGF was found to be expressed on the cell surface in a fraction of CD34+ progenitors. Moreover, in situ hybridization experiments showed that HGF receptor mRNA is highly expressed in embryonic erythroid cells (megaloblasts). HGF mRNA was also found to be produced in the embryonal liver. These data show that HGF plays a direct role in the control of proliferation and differentiation of erythroid progenitors, and they suggest that it may be one of the long-sought mediators of paracrine interactions between stromal and hemopoietic cells within the hemopoietic microenvironment.