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.

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 (pCREBS133), intracellular Ca2+ 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 pCREBS133 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 Ca2+ 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 pCREBS133 and intracellular Ca2+ concentration. It is likely that TRH, SST, and GH exert autocrine/paracrine immunomodulatory actions and participate in the maturation of chicken BBLs.

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Thyroid hormones (THs) regulate tissue remodeling processes during early- and post-embryonic stages in vertebrates. The Mexican axolotl (Ambystoma mexicanum) is a neotenic species that has lost the ability to undergo metamorphosis; however, it can be artificially induced by exogenous administration of thyroxine (T4) and 3,3′,5-triiodo-L-thyronine (T3). Another TH derivative with demonstrative biological effects in fish and mammals is 3,5-diiodo-L-thyronine (3,5-T2). Because the effects of this bioactive TH remains unexplored in other vertebrates, we hypothesized that it could be biologically active in amphibians and, therefore, could induce metamorphosis in axolotl. We performed a 3,5-T2 treatment by immersion and observed that the secondary gills were retracted, similar to the onset stage phenotype; however, tissue regeneration was observed after treatment withdrawal. In contrast, T4 and T3 immersion equimolar treatments as well as a four-fold increase in 3,5-T2 concentration triggered complete metamorphosis. To identify the possible molecular mechanisms that could explain the contrasting reversible or irreversible effects of 3,5-T2 and T3 upon gill retraction, we performed a transcriptomic analysis of differential expression genes in the gills of control, 3,5-T2–treated, and T3-treated axolotls. We found that both THs modify gene expression patterns. T3 regulates 10 times more genes than 3,5-T2, suggesting that the latter has a lower affinity for TH receptors (TRs) or that these hormones could act through different TR isoforms. However, both TH treatments regulated different gene sets known to participate in tissue development and cell cycle processes. In conclusion, 3,5-T2 is a bioactive iodothyronine that promoted partial gill retraction but induced full metamorphosis in higher concentrations. Differential effects on gill retraction after 3,5,-T2 or T3 treatment could be explained by the activation of different clusters of genes related with apoptosis, regeneration, and proliferation; in addition, these effects could be initially mediated by TRs that are expressed in gills. This study showed, for the first time, the 3,5,-T2 bioactivity in a neotenic amphibian.