Comparative analysis of Krüppel-like factors expression in the retinas of zebrafish and mice during development and after injury.

The Krüppel-like factors (KLFs) have emerged as important transcriptional regulators of various cellular processes, including neural development. Some of them have been described as intrinsic factors involved in axon regeneration in the central nervous system (CNS) of vertebrates. Zebrafish are known for their ability to regenerate several tissues in adulthood, including the CNS, a capability lost during vertebrate evolution and absent in adult mammals. The role that KLFs could play in this differential ability remains unknown. Therefore, in this study, we analyzed the endogenous response of certain KLFs implicated in axon regeneration (KLFs 6, 7, 9, and 13) during retina development and after axon injury. The results showed that the expression of Klfs 6, 7, and 13 decreases in the developing retina of mice but not in zebrafish, while the mRNA levels of Klf9 strongly increase in both species. The response to injury was further analyzed using optic nerve crush (ONC) as a model of lesion. Our analysis during the acute phase (hours) demonstrated an induction of Klfs 6 and 7 expression exclusively in the zebrafish retina, while Klfs 9 and 13 mRNA levels increased in both species. Further analysis of the chronic response (days) showed that mRNA levels of Klf6 transiently increase in the retinas of both zebrafish and mice, whereas those of Klf7 decrease later after optic nerve injury. In addition, the analysis revealed that the expression of Klf9 decreases, while that of Klf13 increases in the retinas of zebrafish in response to optic nerve injury but remains unaltered in mice. Altogether, these findings support the hypothesis that KLFs may play a role in the differential axon regeneration abilities exhibited by fish and mice.

Differential peptide-dependent regulation of growth hormone (GH): A comparative analysis in pituitary cultures of reptiles, birds, and mammals.

Growth hormone (GH) is a pituitary protein that exerts pleiotropic roles in vertebrates. The mechanisms regulating GH synthesis and secretion are finely controlled by hypothalamic neuropeptides and other factors. These processes have been considerably studied in mammals but are still poorly understood in other groups. To better understand the pituitary GH regulation during vertebrate phylogeny, we compared the effects of incubating several peptides on cultures of ex-vivo pituitary fragments obtained from representative specimens of reptiles (iguana), birds (chicken) and mammals (rat). The peptides used were: growth hormone-releasing hormone (GHRH), thyrotropin-releasing hormone (TRH), pituitary adenylate cyclase-activating polypeptide (PACAP), ghrelin, gonadotropin-releasing hormone (GnRH), and somatostatin (SST). In rat pituitary cultures, GH secretion was stimulated by GHRH and TRH, while gh mRNA expression was increased by GHRH and PACAP. In the case of chicken pituitaries, GH release was promoted by GHRH, ghrelin, PACAP, and GnRH, although the latter two had a dual effect since at a shorter incubation time they decreased GH secretion; in turn, gh mRNA expression was significantly stimulated by TRH, PACAP, and GnRH. The most intense effects were observed in iguana pituitary cultures, where GH secretion was significantly augmented by GHRH, PACAP, TRH, ghrelin, and GnRH; while gh mRNA expression was stimulated by GHRH, TRH, and PACAP, but inhibited by ghrelin and SST. Also, in the three species, SST was able to block the GHRH-stimulated GH release. Furthermore, it was found that the expression of Pou1f1 mRNA was increased with greater potency by GHRH and PACAP in the iguana, than in chicken or rat pituitary cultures. Additionally, in-silico analysis of the gh gene promoter structures in the three species showed that the reptilian promoter has more Pit-1 consensus binding sites than their avian and mammalian counterparts. Taken together, results demonstrate that pituitary peptide-mediated GH regulatory mechanisms are differentially controlled along vertebrate evolution.

KLF13 Regulates the Activity of the GH-Induced JAK/STAT Signaling by Targeting Genes Involved in the Pathway.

The Krüppel-like factor 13 (KLF13) has emerged as an important transcription factor involved in essential processes of the central nervous system (CNS). It predominantly functions as a transcriptional repressor, impacting the activity of several signaling pathways with essential roles in the CNS, including the JAK/STAT pathway, which is the canonical mediator of growth hormone (GH) signaling. It is now recognized that GH has important actions as a neurotrophic factor. Therefore, we analyzed the effects of KLF13 on the activity of the JAK/STAT signaling pathway in the hippocampus-derived cell line HT22. Results showed that KLF13 directly regulates the expression of several genes involved in the JAK-STAT pathway, including Jak1, Jak2, Jak3, and Socs1, by associating with their proximal gene promoters. In addition, it was found that in KLF13-deficient HT22 neurons, the expression of Jak1, Stat3, Socs1, Socs3, and Igf1 was dysregulated, exhibiting mRNA levels that went up to 7-fold higher than the control cell line. KLF13 displayed a differential effect on the GH-induced JAK/STAT pathway activity, decreasing the STAT3 branch while enhancing the STAT5 branch. In KLF13-deficient HT22 cells, the activity of the STAT3 branch was enhanced, mediating the GH-dependent augmented expression of the JAK/STAT output genes Socs1, Socs3, Igf1, and Bdnf. Furthermore, GH treatment increased both the nuclear content of KLF13 and Klf13 mRNA levels, suggesting that KLF13 could be part of the mechanisms that maintain the homeostatic state of this pathway. These findings support the notion that KLF13 is a regulator of JAK/STAT activity.

Gonadotropin-releasing hormone and growth hormone act as anti-inflammatory factors improving sensory recovery in female rats with thoracic spinal cord injury.

The potential for novel applications of classical hormones, such as gonadotropin-releasing hormone (GnRH) and growth hormone (GH), to counteract neural harm is based on their demonstrated neurotrophic effects in both in vitro and in vivo experimental models and a growing number of clinical trials. This study aimed to investigate the effects of chronic administration of GnRH and/or GH on the expression of several proinflammatory and glial activity markers in damaged neural tissues, as well as on sensory recovery, in animals submitted to thoracic spinal cord injury (SCI). Additionally, the effect of a combined GnRH + GH treatment was examined in comparison with single hormone administration. Spinal cord damage was induced by compression using catheter insufflation at thoracic vertebrae 10 (T10), resulting in significant motor and sensory deficits in the hindlimbs. Following SCI, treatments (GnRH, 60 µg/kg/12 h, IM; GH, 150 µg/kg/24 h, SC; the combination of both; or vehicle) were administered during either 3 or 5 weeks, beginning 24 h after injury onset and ending 24 h before sample collection. Our results indicate that a chronic treatment with GH and/or GnRH significantly reduced the expression of proinflammatory (IL6, IL1B, and iNOS) and glial activity (Iba1, CD86, CD206, vimentin, and GFAP) markers in the spinal cord tissue and improved sensory recovery in the lesioned animals. Furthermore, we found that the caudal section of the spinal cord was particularly responsive to GnRH or GH treatment, as well as to their combination. These findings provide evidence of an anti-inflammatory and glial-modulatory effect of GnRH and GH in an experimental model of SCI and suggest that these hormones can modulate the response of microglia, astrocytes, and infiltrated immune cells in the spinal cord tissue following injury.

Growth Hormone (GH) Crosses the Blood-Brain Barrier (BBB) and Induces Neuroprotective Effects in the Embryonic Chicken Cerebellum after a Hypoxic Injury.

Several motor, sensory, cognitive, and behavioral dysfunctions are associated with neural lesions occurring after a hypoxic injury (HI) in preterm infants. Growth hormone (GH) expression is upregulated in several brain areas when exposed to HI conditions, suggesting actions as a local neurotrophic factor. It is known that GH, either exogenous and/or locally expressed, exerts neuroprotective and regenerative actions in cerebellar neurons in response to HI. However, it is still controversial whether GH can cross the blood-brain barrier (BBB), and if its effects are exerted directly or if they are mediated by other neurotrophic factors. Here, we found that in ovo microinjection of Cy3-labeled chicken GH resulted in a wide distribution of fluorescence within several brain areas in the chicken embryo (choroid plexus, cortex, hypothalamus, periventricular areas, hippocampus, and cerebellum) in both normoxic and hypoxic conditions. In the cerebellum, Cy3-GH and GH receptor (GHR) co-localized in the granular and Purkinje layers and in deep cerebellar nuclei under hypoxic conditions, suggesting direct actions. Histological analysis showed that hypoxia provoked a significant modification in the size and organization of cerebellar layers; however, GH administration restored the width of external granular layer (EGL) and molecular layer (ML) and improved the Purkinje and granular neurons survival. Additionally, GH treatment provoked a significant reduction in apoptosis and lipoperoxidation; decreased the mRNA expression of the inflammatory mediators (TNFα, IL-6, IL-1ß, and iNOS); and upregulated the expression of several neurotrophic factors (IGF-1, VEGF, and BDNF). Interestingly, we also found an upregulation of cerebellar GH and GHR mRNA expression, which suggests the existence of an endogenous protective mechanism in response to hypoxia. Overall, the results demonstrate that, in the chicken embryo exposed to hypoxia, GH crosses the BBB and reaches the cerebellum, where it exerts antiapoptotic, antioxidative, anti-inflammatory, neuroprotective, and neuroregenerative actions.

Neuroprotective and Regenerative Effects of Growth Hormone (GH) in the Embryonic Chicken Cerebral Pallium Exposed to Hypoxic-Ischemic (HI) Injury.

Prenatal hypoxic−ischemic (HI) injury inflicts severe damage on the developing brain provoked by a pathophysiological response that leads to neural structural lesions, synaptic loss, and neuronal death, which may result in a high risk of permanent neurological deficits or even newborn decease. It is known that growth hormone (GH) can act as a neurotrophic factor inducing neuroprotection, neurite growth, and synaptogenesis after HI injury. In this study we used the chicken embryo to develop both in vitro and in vivo models of prenatal HI injury in the cerebral pallium, which is the equivalent of brain cortex in mammals, to examine whether GH exerts neuroprotective and regenerative effects in this tissue and the putative mechanisms involved in these actions. For the in vitro experiments, pallial cell cultures obtained from chick embryos were incubated under HI conditions (<5% O2, 1 g/L glucose) for 24 h and treated with 10 nM GH, and then collected for analysis. For the in vivo experiments, chicken embryos (ED14) were injected in ovo with GH (2.25 µg), exposed to hypoxia (12% O2) for 6 h, and later the pallial tissue was obtained to perform the studies. Results show that GH exerted a clear anti-apoptotic effect and promoted cell survival and proliferation in HI-injured pallial neurons, in both in vitro and in vivo models. Neuroprotective actions of GH were associated with the activation of ERK1/2 and Bcl-2 signaling pathways. Remarkably, GH protected mature neurons that were particularly harmed by HI injury, but was also capable of stimulating neural precursors. In addition, GH stimulated restorative processes such as the number and length of neurite outgrowth and branching in HI-injured pallial neurons, and these effects were blocked by a specific GH antibody, thus indicating a direct action of GH. Furthermore, it was found that the local expression of several synaptogenic markers (NRXN1, NRXN3, GAP-43, and NLG1) and neurotrophic factors (GH, BDNF, NT-3, IGF-1, and BMP4) were increased after GH treatment during HI damage. Together, these results provide novel evidence supporting that GH exerts protective and restorative effects in brain pallium during prenatal HI injury, and these actions could be the result of a joint effect between GH and endogenous neurotrophic factors. Also, they encourage further research on the potential role of GH as a therapeutic complement in HI encephalopathy treatments.

Krüppel-Like Factors 9 and 13 Block Axon Growth by Transcriptional Repression of Key Components of the cAMP Signaling Pathway.

Krüppel-like factors (KLFs) are zinc finger transcription factors implicated in diverse biological processes, including differentiation of neural cells. The ability of mammalian neurons to elongate axons decreases during postnatal development in parallel with a decrease in cAMP, and increase in expression of several Klf genes. The paralogous KLFs 9 and 13 inhibit neurite outgrowth, and we hypothesized that their actions are mediated through repression of cAMP signaling. To test this we used the adult mouse hippocampus-derived cell line HT22 engineered to control expression of Klf9 or Klf13 with doxycycline, or made deficient for these Klfs by CRISPR/Cas9 genome editing. We also used primary hippocampal cells isolated from wild type, Klf9 -/- and Klf13 -/- mice. Forced expression of Klf9 or Klf13 in HT22 changed the mRNA levels of several genes involved with cAMP signaling; the predominant action was gene repression, and KLF13 influenced ∼4 times more genes than KLF9. KLF9 and KLF13 repressed promoter activity of the protein kinase a catalytic subunit alpha gene in transfection-reporter assays; KLF13, but not KLF9 repressed the calmodulin 3 promoter. Forskolin activation of a cAMP-dependent promoter was reduced after forced expression of Klf9 or Klf13, but was enhanced in Klf gene knockout cells. Forced expression of Klf9 or Klf13 blocked cAMP-dependent neurite outgrowth in HT22 cells, and axon growth in primary hippocampal neurons, while Klf gene knockout enhanced the effect of elevated cAMP. Taken together, our findings show that KLF9 and KLF13 inhibit neurite/axon growth in hippocampal neurons, in part, by inhibiting the cAMP signaling pathway.

Molecular Mechanisms for Krüppel-Like Factor 13 Actions in Hippocampal Neurons.

Krüppel-like factors (KLFs) play key roles in nervous system development and function. Several KLFs are known to promote, and then maintain neural cell differentiation. Our previous work focused on the actions of KLF9 in mouse hippocampal neurons. Here we investigated genomic targets and functions of KLF9's paralog KLF13, with the goal of understanding how these two closely related transcription factors influence hippocampal cell function, proliferation, survival, and regeneration. We engineered the adult mouse hippocampus-derived cell line HT22 to control Klf13 expression with doxycycline. We also generated HT22 Klf13 knock out cells, and we analyzed primary hippocampal cells from wild type and Klf13-/- mice. RNA sequencing showed that KLF13, like KLF9, acts predominantly as a transcriptional repressor in hippocampal neurons and can regulate other Klf genes. Pathway analysis revealed that genes regulated by KLF13 are involved in cell cycle, cell survival, cytoarchitecture regulation, among others. Chromatin-streptavidin sequencing conducted on chromatin isolated from HT22 cells expressing biotinylated KLF13 identified 9506 genomic targets; 79% were located within 1-kb upstream of transcription start sites. Transfection-reporter assays confirmed that KLF13 can directly regulate transcriptional activity of its target genes. Comparison of the target genes of KLF9 and KLF13 found that they share some functions that were likely present in their common ancestor, but they have also acquired distinct functions during evolution. Flow cytometry showed that KLF13 promotes cell cycle progression, and it protects cells from glutamate-induced excitotoxic damage. Taken together, our findings establish novel roles and molecular mechanisms for KLF13 actions in mammalian hippocampal neurons.

The Paralogous Krüppel-like Factors 9 and 13 Regulate the Mammalian Cellular Circadian Clock Output Gene Dbp.

An intricate transcription-translation feedback loop (TTFL) governs cellular circadian rhythms in mammals. Here, we report that the zinc finger transcription factor Krüppel-like factor 9 (KLF9) is regulated by this TTFL, it associates in chromatin at the core circadian clock and clock-output genes, and it acts to modulate transcription of the clock-output gene Dbp. Our earlier genome-wide analysis of the mouse hippocampus-derived cell line HT22 showed that KLF9 associates in chromatin with Per1, Per3, Dbp, Tef, Bhlhe40, Bhlhe41, Nr1d1, and Nr1d2. Of the 3514 KLF9 peaks identified in HT22 cells, 1028 contain E-box sequences to which the transcriptional activators CLOCK and BMAL1 may bind, a frequency significantly greater than expected by chance. Klf9 mRNA showed circadian oscillation in synchronized HT22 cells, mouse hippocampus, and liver. At the clock-output gene Dbp, KLF9 exhibited circadian rhythmicity in its association in chromatin in HT22 cells and hippocampus. Forced expression of KLF9 in HT22 cells repressed basal Dbp transcription and strongly inhibited CLOCK+BMAL1-dependent transcriptional activation of a transfected Dbp reporter. Mutational analysis showed that this action of KLF9 depended on 2 intact KLF9-binding motifs within the Dbp locus that are in close proximity to E-boxes. Knockout of Klf9 or the paralogous gene Klf13 using CRISPR/Cas9 genome editing in HT22 cells had no effect on Dbp expression, but combined knockout of both genes strongly impaired circadian Dbp mRNA oscillation. Like KLF9, KLF13 also showed association in chromatin with clock- and clock-output genes, and forced expression of KLF13 inhibited the actions of CLOCK+BMAL1 on Dbp transcription. Our results suggest novel and partly overlapping roles for KLF9 and KLF13 in modulating cellular circadian clock output by a mechanism involving direct interaction with the core TTFL.

Neuroprotective Effects of Growth Hormone (GH) and Insulin-Like Growth Factor Type 1 (IGF-1) after Hypoxic-Ischemic Injury in Chicken Cerebellar Cell Cultures.

It has been reported that growth hormone (GH) and insulin-like growth factor 1 (IGF-1) exert protective and regenerative actions in response to neural damage. It is also known that these peptides are expressed locally in nervous tissues. When the central nervous system (CNS) is exposed to hypoxia-ischemia (HI), both GH and IGF-1 are upregulated in several brain areas. In this study, we explored the neuroprotective effects of GH and IGF-1 administration as well as the involvement of these endogenously expressed hormones in embryonic chicken cerebellar cell cultures exposed to an acute HI injury. To induce neural damage, primary cultures were first incubated under hypoxic-ischemic (<5% O2, 1g/L glucose) conditions for 12 h (HI), and then incubated under normal oxygenation and glucose conditions (HI + Ox) for another 24 h. GH and IGF-1 were added either during or after HI, and their effect upon cell viability, apoptosis, or necrosis was evaluated. In comparison with normal controls (Nx, 100%), a significant decrease of cell viability (54.1 ± 2.1%) and substantial increases in caspase-3 activity (178.6 ± 8.7%) and LDH release (538.7 ± 87.8%) were observed in the HI + Ox group. On the other hand, both GH and IGF-1 treatments after injury (HI + Ox) significantly increased cell viability (77.2 ± 4.3% and 72.3 ± 3.9%, respectively) and decreased both caspase-3 activity (118.2 ± 3.8% and 127.5 ± 6.6%, respectively) and LDH release (180.3 ± 21.8% and 261.6 ± 33.9%, respectively). Incubation under HI + Ox conditions provoked an important increase in the local expression of GH (3.2-fold) and IGF-1 (2.5-fold) mRNAs. However, GH gene silencing with a specific small-interfering RNAs (siRNAs) decreased both GH and IGF-1 mRNA expression (1.7-fold and 0.9-fold, respectively) in the HI + Ox group, indicating that GH regulates IGF-1 expression under these incubation conditions. In addition, GH knockdown significantly reduced cell viability (35.9 ± 2.1%) and substantially increased necrosis, as determined by LDH release (1011 ± 276.6%). In contrast, treatments with GH and IGF-1 stimulated a partial recovery of cell viability (45.2 ± 3.7% and 53.7 ± 3.2%) and significantly diminished the release of LDH (320.1 ± 25.4% and 421.7 ± 62.2%), respectively. Our results show that GH, either exogenously administered and/or locally expressed, can act as a neuroprotective factor in response to hypoxic-ischemic injury, and that this effect may be mediated, at least partially, through IGF-1 expression.

Growth Hormone Neuroprotection Against Kainate Excitotoxicity in the Retina is Mediated by Notch/PTEN/Akt Signaling.

Purpose: In the retina, growth hormone (GH) promotes axonal growth, synaptic restoration, and protective actions against excitotoxicity. Notch signaling pathway is critical for neural development and participates in the retinal neuroregenerative process. We investigated the interaction of GH with Notch signaling pathway during its neuroprotective effect against excitotoxic damage in the chicken retina. Methods: Kainate (KA) was used as excitotoxic agent and changes in the mRNA expression of several signaling markers were determined by qPCR. Also, changes in phosphorylation and immunoreactivity were determined by Western blotting. Histology and immunohistochemistry were performed for morphometric analysis. Overexpression of GH was performed in the quail neuroretinal-derived immortalized cell line (QNR/D) cell line. Exogenous GH was administered to retinal primary cell cultures to study the activation of signaling pathways. Results: KA disrupted the retinal cytoarchitecture and induced significant cell loss in several retinal layers, but the coaddition of GH effectively prevented these adverse effects. We showed that GH upregulates the Notch signaling pathway during neuroprotection leading to phosphorylation of the PI3K/Akt signaling pathways through downregulation of PTEN. In contrast, cotreatment of GH with the Notch signaling inhibitor, DAPT, prevented its neuroprotective effect against KA. We identified binding sites in Notch1 and Notch2 genes for STAT5. Also, GH prevented Müller cell transdifferentiation and downregulated Sox2, FGF2, and PCNA after cotreatment with KA. Additionally, GH modified TNF receptors immunoreactivity suggesting anti-inflammatory actions. Conclusions: Our data indicate that the neuroprotective effects of GH against KA injury in the retina are mediated through the regulation of Notch signaling. Additionally, anti-inflammatory and antiproliferative effects were observed.

Upregulation of GH, but not IGF1, in the hippocampus of the lactating dam after kainic acid injury.

Lactation embodies a natural model of morphological, neurochemical, and functional brain plasticity. In this reproductive stage, the hippocampus of the female is less sensitive to excitotoxins in contrast to nulliparity. Growth hormone (GH) and insulin-like growth factor 1 (IGF1) are known to be neuroprotective in several experimental models of brain lesion. Here, activation of the GH-IGF1 pituitary-brain axis following kainic acid (7.5 mg/kg i.p. KA) lesion was studied in lactating and nulliparous rats. Serum concentrations of GH and IGF1 were uncoupled in lactation. Compared to virgin rats, the basal concentration of GH increased up to 40% but IGF1 decreased 58% in dams, and only GH increased further after KA treatment. In the hippocampus, basal expression of GH mRNA was higher (2.8-fold) in lactating rats than in virgin rats. GH mRNA expression in lactating rats increased further after KA administration in the hippocampus and in the hypothalamus, in parallel to GH protein concentration in the hippocampus of KA-treated lactating rats (43% vs lactating control), as detected by Western blot and immunofluorescence. Except for the significantly lower mRNA concentration in the liver of lactating rats, IGF1 expression was not altered by the reproductive condition or by KA treatment in the hippocampus and hypothalamus. Present results indicate upregulation of GH expression in the hippocampus after an excitotoxic lesion, suggesting paracrine/autocrine actions of GH as a factor underlying neuroprotection in the brain of the lactating dam. Since no induction of IGF1 was detected, present data suggest a direct action of GH.

Characterization and distribution of GHRH, PACAP, TRH, SST and IGF1 mRNAs in the green iguana.

The somatotropic axis (SA) regulates numerous aspects of vertebrate physiology such as development, growth, and metabolism and has influence on several tissues including neural, immune, reproductive and gastric tract. Growth hormone (GH) is a key component of SA, it is synthesized and released mainly by pituitary somatotrophs, although now it is known that virtually all tissues can express GH, which, in addition to its well-described endocrine roles, also has autocrine/paracrine/intracrine actions. In the pituitary, GH expression is regulated by several hypothalamic neuropeptides including GHRH, PACAP, TRH and SST. GH, in turn, regulates IGF1 synthesis in several target tissues, adding complexity to the system since GH effects can be exerted either directly or mediated by IGF1. In reptiles, little is known about the SA components and their functional interactions. The aim of this work was to characterize the mRNAs of the principal SA components in the green iguana and to develop the tools that allow the study of the structural and functional evolution of this system in reptiles. By employing RT-PCR and RACE, the cDNAs encoding for GHRH, PACAP, TRH, SST and IGF1 were amplified and sequenced. Results showed that these cDNAs coded for the corresponding protein precursors of 154, 170, 243, 113, and 131 amino acids, respectively. Of these, GHRH, PACAP, SST and IGF1 precursors exhibited a high structural conservation with respect to its counterparts in other vertebrates. On the other hand, iguana's TRH precursor showed 7 functional copies of mature TRH (pyr-QHP-NH2), as compared to 4 and 6 copies of TRH in avian and mammalian proTRH sequences, respectively. It was found that in addition to its primary production site (brain for GHRH, PACAP, TRH and SST, and liver for IGF1), they were also expressed in other peripheral tissues, i.e. testes and ovaries expressed all the studied mRNAs, whereas TRH and IGF1 mRNAs were observed ubiquitously in all tissues considered. These results show that the main SA components in reptiles of the Squamata Order maintain a good structural conservation among vertebrate phylogeny, and suggest important physiological interactions (endocrine, autocrine and/or paracrine) between them due to their wide peripheral tissue expression.

Autocrine/paracrine proliferative effect of ovarian GH and IGF-I in chicken granulosa cell cultures.

It is known that growth hormone (GH) and its receptor (GHR) are expressed in granulosa cells (GC) and thecal cells during the follicular development in the hen ovary, which suggests GH is involved in autocrine/paracrine actions in the female reproductive system. In this work, we show that the knockdown of local ovarian GH with a specific cGH siRNA in GC cultures significantly decreased both cGH mRNA expression and GH secretion to the media, and also reduced their proliferative rate. Thus, we analyzed the effect of ovarian GH and recombinant chicken GH (rcGH) on the proliferation of pre-hierarchical GCs in primary cultures. Incubation of GCs with either rcGH or conditioned media, containing predominantly a 15-kDa GH isoform, showed that both significantly increased proliferation as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, proliferating cell nuclear antigen (PCNA) quantification and ((3)H)-thymidine incorporation ((3)H-T) assays in a dose response fashion. Both, locally produced GH and rcGH also induced the phosphorylation of Erk1/2 in GC cultures. Furthermore, GH increased IGF-I synthesis and its release into the GC culture incubation media. These results suggest that GH may act through local IGF-I to induce GC proliferation, since IGF-I immunoneutralization completely abolished the GH-induced proliferative effect. These data suggest that GH and IGF-I may play a role as autocrine/paracrine regulators during the follicular development in the hen ovary at the pre-hierarchical stage.

Neuroprotection by GH against excitotoxic-induced cell death in retinal ganglion cells.

Retinal growth hormone (GH) has been shown to promote cell survival in retinal ganglion cells (RGCs) during developmental waves of apoptosis during chicken embryonic development. The possibility that it might also against excitotoxicity-induced cell death was therefore examined in the present study, which utilized quail-derived QNR/D cells as an in vitro RGC model. QNR/D cell death was induced by glutamate in the presence of BSO (buthionine sulfoxamide) (an enhancer of oxidative stress), but this was significantly reduced (P<0.01) in the presence of exogenous recombinant chicken GH (rcGH). Similarly, QNR/D cells that had been prior transfected with a GH plasmid to overexpress secreted and non-secreted GH. This treatment reduced the number of TUNEL-labeled cells and blocked their release of lactate dehydrogenase (LDH). In a further experiment with dissected neuroretinal explants from ED (embryonic day) 10 embryos, rcGH treatment of the explants also reduced (P<0.01) the number of glutamate-BSO-induced apoptotic cells and blocked the explant release of LDH. This neuroprotective action was likely mediated by increased STAT5 phosphorylation and increased bcl-2 production, as induced by exogenous rcGH treatment and the media from GH-overexpressing QNR/D cells. As rcGH treatment and GH-overexpression cells also increased the content of IGF-1 and IGF-1 mRNA this neuroprotective action of GH is likely to be mediated, at least partially, through an IGF-1 mechanism. This possibility is supported by the fact that the siRNA knockdown of GH or IGF-1 significantly reduced QNR/D cell viability, as did the immunoneutralization of IGF-1. GH is therefore neuroprotective against excitotoxicity-induced RGC cell death by anti-apoptotic actions involving IGF-1 stimulation.

Growth hormone reverses excitotoxic damage induced by kainic acid in the green iguana neuroretina.

It is known that growth hormone (GH) is expressed in extrapituitary tissues, including the nervous system and ocular tissues, where it is involved in autocrine/paracrine actions related to cell survival and anti-apoptosis in several vertebrates. Little is known, however, in reptiles, so we analyzed the expression and distribution of GH in the eye of green iguana and its potential neuroprotective role in retinas that were damaged by the intraocular administration of kainic acid (KA). It was found, by Western blotting, that GH-immunoreactivity (GH-IR) was expressed as two isoforms (15 and 26kDa, under reducing conditions) in cornea, vitreous, retina, crystalline, iris and sclera, in varying proportions. Also, two bands for the growth hormone receptor (GHR)-IR were observed (70 and 44kDa, respectively) in the same tissues. By immunofluorescence, GH-IR was found in neurons present in several layers of the neuroretina (inner nuclear [INL], outer nuclear [ONL] and ganglion cell [GCL] layers) as determined by its co-existence with NeuN, but not in glial cells. In addition, GH and GHR co-expression was found in the same cells, suggesting paracrine/autocrine interactions. KA administration induced retinal excitotoxic damage, as determined by a significant reduction of the cell density and an increase in the appearance of apoptotic cells in the INL and GCL. In response to KA injury, both endogenous GH and Insulin-like Growth Factor I (IGF-I) expression were increased by 70±1.8% and 33.3±16%, respectively. The addition of exogenous GH significantly prevented the retinal damage produced by the loss of cytoarchitecture and cell density in the GCL (from 4.9±0.79 in the control, to 1.45±0.2 with KA, to 6.35±0.49cell/mm(2) with KA+GH) and in the INL (19.12±1.6, 10.05±1.9, 21.0±0.8cell/mm(2), respectively) generated by the long-term effect of 1mM KA intraocular administration. The co-incubation with a specific anti-GH antibody, however, blocked the protective effect of GH in GCL (1.4±0.23cell/mm(2)) and INL (11.35±1.06), respectively. Furthermore, added GH induced an increase of 90±14% in the retinal IGF-I concentration and the anti-GH antibody also blocked this effect. These results indicate that GH and GHR are expressed in the iguana eye and may be able to exert, either directly of mediated by IGF-I, a protective mechanism in neuroretinas that suffered damage by the administration of kainic acid.

Differential responses of the somatotropic and thyroid axes to environmental temperature changes in the green iguana.

Growth hormone (GH), together with thyroid hormones (TH), regulates growth and development, and has critical effects on vertebrate metabolism. In ectotherms, these physiological processes are strongly influenced by environmental temperature. In reptiles, however, little is known about the direct influences of this factor on the somatotropic and thyroid axes. Therefore, the aim of this study was to describe the effects of both acute (48h) and chronic (2weeks) exposure to sub-optimal temperatures (25 and 18°C) upon somatotropic and thyroid axis function of the green iguana, in comparison to the control temperature (30-35°C). We found a significant increase in GH release (2.0-fold at 25°C and 1.9-fold at 18°C) and GH mRNA expression (up to 3.7-fold), mainly under chronic exposure conditions. The serum concentration of insulin-like growth factor-I (IGF-I) was significantly greater after chronic exposure (18.5±2.3 at 25°C; 15.92±3.4 at 18°C; vs. 9.3±1.21ng/ml at 35°C), while hepatic IGF-I mRNA expression increased up to 6.8-fold. Somatotropic axis may be regulated, under acute conditions, by thyrotropin-releasing hormone (TRH) that significantly increased its hypothalamic concentration (1.45 times) and mRNA expression (0.9-fold above control), respectively; and somatostatin (mRNA expression increased 1.0-1.2 times above control); and under chronic treatment, by pituitary adenylate cyclase-activating peptide (PACAP mRNA expression was increased from 0.4 to 0.6 times). Also, it was shown that, under control conditions, injection of TRH stimulated a significant increase in circulating GH. On the other hand, while there was a significant rise in the hypothalamic content of TRH and its mRNA expression, this hormone did not appear to influence the thyroid axis activity, which showed a severe diminution in all conditions of cold exposure, as indicated by the decreases in thyrotropin (TSH) mRNA expression (up to one-eight of the control), serum T4 (from 11.6±1.09 to 5.3±0.58ng/ml, after 2weeks at 18°C) and T3 (from 0.87±0.09 to 0.05±0.01ng/ml, under chronic conditions at 25°C), and Type-2 deiodinase (D2) activity (from 992.5±224 to 213.6±26.4fmolI(125)T4/mgh). The reduction in thyroid activity correlates with the down-regulation of metabolism as suggested by the decrease in the serum glucose and free fatty acid levels. These changes apparently were independent of a possible stress response, at least under acute exposure to both temperatures and in chronic treatment to 25°C, since serum corticosterone had no significant changes in these conditions, while at chronic 18°C exposure, a slight increase (0.38 times above control) was found. Thus, these data suggest that the reptilian somatotropic and thyroid axes have differential responses to cold exposure, and that GH and TRH may play important roles associated to adaptation mechanisms that support temperature acclimation in the green iguana.

Growth hormone in the eye: A comparative update.

Comparative studies have previously established that the eye is an extrapituitary site of growth hormone (GH) production and action in fish, amphibia, birds and mammals. In this review more recent literature and original data in this field are considered.

Characterization of pituitary growth hormone and its receptor in the green iguana (Iguana iguana).

Pituitary growth hormone (GH) has been studied in most vertebrate groups; however, only a few studies have been carried out in reptiles. Little is known about pituitary hormones in the order Squamata, to which the green iguana (gi) belongs. In this work, we characterized the hypophysis of Iguana iguana morphologically. The somatotrophs (round cells of 7.6-10 µm containing 250- to 300-nm secretory granules where the giGH is stored) were found, by immunohistochemistry and in situ hybridization, exclusively in the caudal lobe of the pars distalis, whereas the lactotrophs were distributed only in the rostral lobe. A pituitary giGH-like protein was obtained by immuno-affinity chromatography employing a heterologous antibody against chicken GH. giGH showed molecular heterogeneity (22, 44, and 88 kDa by SDS-PAGE/Western blot under non-reducing conditions and at least four charge variants (pIs 6.2, 6.5, 6.9, 7.4) by isoelectric focusing. The pituitary giGH cDNA (1016 bp), amplified by PCR and RACE, encodes a pre-hormone of 218 aa, of which 190 aa correspond to the mature protein and 28 aa to the signal peptide. The giGH receptor cDNA was also partially sequenced. Phylogenetic analyses of the amino acid sequences of giGH and giGHR homologs in vertebrates suggest a parallel evolution and functional relationship between the GH and its receptor.