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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.

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-1β, 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.

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.

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% O 2 , 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.

It is known that growth hormone (GH) is expressed in immune cells, where it exerts immunomodulatory effects. However, the mechanisms of expression and release of GH in the immune system remain unclear. We analyzed the effect of growth hormone-releasing hormone (GHRH), thyrotropin-releasing hormone (TRH), ghrelin (GHRL), and somatostatin (SST) upon GH mRNA expression, intracellular and released GH, Ser133-phosphorylation of CREB (pCREB S133 ), intracellular Ca 2+ levels, as well as B-cell activating factor (BAFF) mRNA expression in bursal B-lymphocytes (BBLs) cell cultures since several GH secretagogues, as well as their corresponding receptors (-R), are expressed in B-lymphocytes of several species. The expression of TRH/TRH-R, ghrelin/GHS-R1a, and SST/SST-Rs (Subtypes 1 to 5) was observed in BBLs by RT-PCR and immunocytochemistry (ICC), whereas GHRH/GHRH-R were absent in these cells. We found that TRH treatment significantly increased local GH mRNA expression and CREB phosphorylation. Conversely, SST decreased GH mRNA expression. Additionally, when added together, SST prevented TRH-induced GH mRNA expression, but no changes were observed in pCREB S133 levels. Furthermore, TRH stimulated GH release to the culture media, while SST increased the intracellular content of this hormone. Interestingly, SST inhibited TRH-induced GH release in a dose-dependent manner. The coaddition of TRH and SST decreased the intracellular content of GH. After 10 min. of incubation with either TRH or SST, the intracellular calcium levels significantly decreased, but they were increased at 60 min. However, the combined treatment with both peptides maintained the Ca 2+ levels reduced up to 60-min. of incubation. On the other hand, BAFF cytokine mRNA expression was significantly increased by TRH administration. Altogether, our results suggest that TRH and SST are implicated in the regulation of GH expression and release in BBL cultures, which also involve changes in pCREB S133 and intracellular Ca 2+ concentration. It is likely that TRH, SST, and GH exert autocrine/paracrine immunomodulatory actions and participate in the maturation of chicken BBLs.

In addition to its role as an endocrine messenger, growth hormone (GH) also acts as a neurotrophic factor in the central nervous system (CNS), whose effects are involved in neuroprotection, axonal growth, and synaptogenic modulation. An increasing amount of clinical evidence shows a beneficial effect of GH treatment in patients with brain trauma, stroke, spinal cord injury, impaired cognitive function, and neurodegenerative processes. In response to injury, Müller cells transdifferentiate into neural progenitors and proliferate, which constitutes an early regenerative process in the chicken retina. In this work, we studied the long-term protective effect of GH after causing severe excitotoxic damage in the retina. Thus, an acute neural injury was induced via the intravitreal injection of kainic acid (KA, 20 µg), which was followed by chronic administration of GH (10 injections [300 ng] over 21 days). Damage provoked a severe disruption of several retinal layers. However, in KA-damaged retinas treated with GH, we observed a significant restoration of the inner plexiform layer (IPL, 2.4-fold) and inner nuclear layer (INL, 1.5-fold) thickness and a general improvement of the retinal structure. In addition, we also observed an increase in the expression of several genes involved in important regenerative pathways, including: synaptogenic markers (DLG1, NRXN1, GAP43); glutamate receptor subunits (NR1 and GRIK4); pro-survival factors (BDNF, Bcl-2 and TNF-R2); and Notch signaling proteins (Notch1 and Hes5). Interestingly, Müller cell transdifferentiation markers (Sox2 and FGF2) were upregulated by this long-term chronic GH treatment. These results are consistent with a significant increase in the number of BrdU-positive cells observed in the KA-damaged retina, which was induced by GH administration. Our data suggest that GH is able to facilitate the early proliferative response of the injured retina and enhance the regeneration of neurite interconnections.

This brief review of the neurological effects of growth hormone (GH) and gonadotropin-releasing hormone (GnRH) in the brain, particularly in the cerebral cortex, hypothalamus, hippocampus, cerebellum, spinal cord, neural retina, and brain tumors, summarizes recent information about their therapeutic potential as treatments for different neuropathologies and neurodegenerative processes. The effect of GH and GnRH (by independent administration) has been associated with beneficial impacts in patients with brain trauma and spinal cord injuries. Both GH and GnRH have demonstrated potent neurotrophic, neuroprotective, and neuroregenerative action. Positive behavioral and cognitive effects are also associated with GH and GnRH administration. Increasing evidence suggests the possibility of a multifactorial therapy that includes both GH and GnRH., Competing Interests: The authors declare no conflicts of interest.