Direct antiapoptotic effects of growth hormone are mediated by PI3K/Akt pathway in the chicken bursa of Fabricius
Introduction
It is known that growth hormone (GH) has an important role on several processes like cell cycle progression, differentiation, energy metabolism and survival (Jeay et al., 2001). GH may exert these effects acting either directly through its specific cell receptor or mediated indirectly by the insulin growth factor I (IGF-I) (Fryburg, 1994). The GH receptor (GHR) belongs to the cytokine type I family and its activation triggers several signal pathways such as JAK/STAT, MAPK, PI3K/Akt pathway, which are involved in important processes such as cell cycle regulation, glucose uptake, oxidative stress and apoptosis, as has been shown in several cell types such as Chinese hamster ovary cells, rat and chicken neuron and chondrocytes cells, among others (Brooks et al., 2008, Conway-Campbell et al., 2007, Costoya et al., 1999, Frago et al., 2002, Lanning and Carter-Su, 2006, Zermeño et al., 2006).
It has been described that GH shows anti-apoptotic effects in diverse cell types such as rat and human B cells, rat and mice T cells, human pancreatic cells, and chicken cerebellar cells (Alba-Betancourt et al., 2013, Clark, 1997, Dorshkind and Horseman, 2000, Jeay et al., 2002, Jensen et al., 2005, Lempereur et al., 2003). It is also known that in models such as cultured chicken cerebellar cells, chicken embryonic retinal ganglion (RGCs) cells and murine pro-B Ba/F3 cells, GH activates PI3K/Akt pathway and inhibits apoptosis through this signaling pathway (Alba-Betancourt et al., 2013, Jeay et al., 2001, Sanders et al., 2008).
It has been shown that both GH and GHR are expressed in chicken immune tissues (bursa of Fabricius, thymus and spleen) during development. The bursa of Fabricius (BF) is a primary lymphoid organ found only in birds and is located in the terminal portion of the gastrointestinal tract; it serves as a place for antigen processing and maturation of B cells. In the BF both GH mRNA and GH expression were documented and its content showed a parallel pattern in relation to growth and involution of this tissue (Luna et al., 2005, Luna et al., 2008, Luna et al., 2013, Render et al., 1995). GH immunoreactivity (GH-IR) and GH mRNA expression were detected in stromal and non-stromal cells, mainly in B lymphocytes, but also in macrophage-like cells, reticulo-endothelial cells and dendritic cells, suggesting a functional role for local GH upon B cells during BF differentiation (Luna et al., 2008, Luna et al., 2013; Rodríguez-Méndez et al., 2010).
Although GH was related to apoptosis inhibition within the BF (Rodríguez-Méndez et al., 2010); it is not yet clear if it exerts this effect directly or indirectly through IGF-I mediation, and the signaling pathway involved needs to be elucidated. Therefore in this work we investigated the mechanisms that mediate the anti-apoptotic effect of chicken GH in bursal primary B cell cultures.
Our data show that GH is able to inhibit apoptosis in the BF in a direct manner and this effect is mediated through the PI3K/Akt pathway by increasing phosphorylation of Akt. Also, GH stimulates the expression of the anti-apoptotic protein Bcl-2. Moreover, blocking this signal pathway with wortmannin disrupts the anti-apoptotic effect of GH.
Section snippets
Animals
Male White Leghorn chickens were kept on a 12L:12D photoperiod and had access to commercial food and water ad libitum in the avian facilities at the Institute of Neurobiology. The birds were killed by decapitation, following the protocol approved by the Institute’s Bioethics Committee.
Primary B-cell cultures of BF
Primary B-cell cultures were prepared after the BFs were aseptically removed from 4 weeks-old male chicks, as described elsewhere (Rodríguez-Méndez et al., 2010). In brief, the bursae were minced in RPMI 1640 and
BF primary cell culture characterization
Three different antibodies were used to characterize the cell subpopulations present in the primary cultures of BF: α-Bu-1a as a marker of bursal B cells (Igyártó et al., 2008); α-chicken IgM to identify immature B cells; and α-chicken/turkey IgG to identify mature B cells. As shown in Fig. 1, most of the cultured cells (95.4 ± 0.9%) corresponded to B cells, since they presented Bu-1a immunoreactivity (Bu 1a-IR), out of which 69.07 ± 7.2% were mature IgG-expressing B lymphocytes, whereas 24.8 ± 7.2%
Discussion
Earlier work has provided evidence that GH plays a significant role in the immune system (Clark, 1997, Gelato, 1993, Luna et al., 2013, Postel-Vinay et al., 1997, Sumita et al., 2005). It has been shown that GH deficiency provokes a deleterious effect on immune function, which can be corrected by addition of exogenous GH (Khansari and Gustad, 1991). It is also known that GH stimulates growth of immune organs (Villanua et al., 1992), and modulates several developmental functions, including
Acknowledgments
The participation of Gerardo Courtois (Lab assistant) is acknowledged. This work was supported by grants from CONACYT (178335) and PAPIIT-UNAM (IN206813 and IN206115). JLLA, CAB, CGMM were enrolled in the Biomedical Sciences PhD Program whereas CR was a student of the MSc Program in Neurobiology, at UNAM and received fellowships from CONACYT (200220, 184939, 185024, 225281, respectively). CGMM currently holds a postdoctoral fellowship (206148) from CONACYT.
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