Elsevier

General and Comparative Endocrinology

Volume 224, 1 December 2015, Pages 148-159
General and Comparative Endocrinology

Direct antiapoptotic effects of growth hormone are mediated by PI3K/Akt pathway in the chicken bursa of Fabricius

https://doi.org/10.1016/j.ygcen.2015.07.010Get rights and content

Highlights

  • GH has a direct anti-apoptotic effect on chicken cultured bursal B lymphocytes.

  • Direct anti-apoptotic effects of GH are mediated by PI3K/Akt pathway.

  • Bcl-2 expression is stimulated by GH in cultured B lymphocytes.

  • IGF-I production in cultured B lymphocytes is GH-independent.

Abstract

Growth hormone (GH) is expressed in several extra-pituitary tissues, including the primary and secondary lymphoid organs of the immune system. In birds, GH mRNA and protein expression show a specific developmental distribution pattern in the bursa of Fabricius (BF), particularly in epithelial and B cells. Changes in the bursal concentration and distribution of locally produced GH during ontogeny suggest it is involved in B cell differentiation and maturation, as well as in a functional survival role in this organ, which may be mediated by paracrine/autocrine mechanisms. Here, we analyzed the anti-apoptotic effect of GH in BF and the intracellular signaling pathways involved in this activity. Also, we studied if this effect was exerted directly by GH or mediated indirectly by IGF-I. Bursal cell cultures showed an important loss of their viability after 4 h of incubation and a significant increase in apoptosis. However, treatment with 10 nM GH or 40 nM IGF-I significantly increased B cell viability (16.7 ± 0.67% and 13.4 ± 1.12%, respectively) when compared with the untreated controls. In addition, the presence of apoptotic bodies (TUNEL) dramatically decreased (5.5-fold) after GH and IGF-I treatments, whereas co-incubation with anti-GH or anti-IGF-I, respectively, blocked their anti-apoptotic effect. Likewise, both GH and IGF-I significantly inhibited caspase-3 activity (by 40 ± 2.0%) in these cultures. However, the use of anti-IGF-I could not reverse the GH anti-apoptotic effects, thus indicating that these were exerted directly. The addition of 100 nM wortmannin (a PI3K/Akt inhibitor) blocked the GH protective effects. Also, GH stimulated (3-fold) the phosphorylation of Akt in bursal cells, and adding wortmannin or an anti-GH antibody inhibited this effect. Furthermore, GH was capable to stimulate (7-fold) the expression of Bcl-2. Taken together, these results indicate that the direct anti-apoptotic activity of GH observed in the chicken bursal B cell cultures might be mediated through the PI3K/Akt pathway.

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.

References (75)

  • M. Luna et al.

    Heterogeneity of growth hormone immunoreactivity in lymphoid tissues and changes during ontogeny in domestic fowl

    Gen. Comp. Endocrinol.

    (2005)
  • M. Luna et al.

    Immune growth hormone (GH): localization of GH and GH mRNA in the bursa of Fabricius

    Dev. Comp. Immunol.

    (2008)
  • M. Luna et al.

    Expression and function of chicken bursal growth hormone (GH)

    Gen. Comp. Endocrinol.

    (2013)
  • A.J. Rodríguez-Méndez et al.

    Growth hormone expression in stromal and non-stromal cells in the bursa of Fabricius during bursal development and involution: Causal relationships?

    Gen. Comp. Endocrinol.

    (2010)
  • E.J. Sanders et al.

    Growth hormone expression and neuroprotective activity in a quail neural retina cell line

    Gen. Comp. Endocrinol.

    (2010)
  • E.J. Sanders et al.

    Growth hormone promotes the survival of retinal cells in vivo

    Gen. Comp. Endocrinol.

    (2011)
  • E.J. Sanders et al.

    Retinal growth hormone is an anti-apoptotic factor in embryonic retinal ganglion cell differentiation

    Exp. Eye Res.

    (2005)
  • E.J. Sanders et al.

    Retinal ganglion cell survival in development: Mechanisms of retinal growth hormone action

    Exp. Eye Res.

    (2006)
  • E.J. Sanders et al.

    Growth hormone-mediated survival of embryonic retinal ganglion cells: signaling mechanisms

    Gen. Comp. Endocrinol.

    (2008)
  • A. Scheepens et al.

    Growth hormone as a neuronal rescue factor during recovery from CNS injury

    Neuroscience

    (2001)
  • H.B. Segard et al.

    Autocrine growth hormone production prevents apoptosis and inhibits differentiation in C2C12 myoblasts

    Cell. Signal.

    (2003)
  • N. Shved et al.

    Growth hormone (GH) treatment acts on the endocrine and autocrine/paracrine GH/IGF-axis and on TNF-alpha expression in bony fish pituitary and immune organs

    Fish Sellfish Immunol.

    (2011)
  • K. Sumita et al.

    Effects of growth hormone on the differentiation of mouse B-lymphoid precursors

    J. Pharmacol. Sci

    (2005)
  • D.A. Weigent

    High molecular weight isoforms of growth hormone in cells of the immune system

    Cell. Immunol.

    (2011)
  • D.A. Weigent

    Lymphocyte GH-axis hormones in immunity

    Cell. Immunol.

    (2013)
  • C. Arámburo et al.

    Desarrollo de un radioinmunoensayo homólogo y específico para la determinación de la hormona de crecimiento de pollo (cGH)

    Vet. Mex.

    (1989)
  • R.E. Arnold et al.

    The inhibition of apoptosis in EL4 lymphoma cells overexpressing growth hormone

    NeuroImmunoModulation

    (2004)
  • S. Arkins et al.

    Murine macrophages express abundant insulin-like growth factor-I class I Ea and Eb transcripts

    Endocrinology

    (1993)
  • S. Arkins et al.

    The colony-stimulating factors induce expression of insulin-like growth factor-I messenger ribonucleic acid during hematopoiesis

    Endocrinology

    (1995)
  • S. Arkins et al.

    Interferon-gamma inhibits macrophage insulin-like growth factor-I synthesis at the transcriptional level

    Mol. Endocrinol.

    (1995)
  • J.B. Baxter et al.

    Characterization of immunoreactive insulin-like growth factor-I from leukocytes and its regulation by growth hormone

    Endocrinology

    (1991)
  • D. Bikle et al.

    The skeletal structure of insulin-like growth factor I-deficient mice

    J. Bone Miner. Res.

    (2001)
  • J. Castillo et al.

    IGF-I and insulin receptor signal transduction in trout muscle cells

    Am. J. Physiol. Reg. Integr. Comp. Physiol.

    (2006)
  • H. Chung et al.

    IGF-I inhibition of apoptosis is associated with decreased expression of prostate apoptosis response-4

    J. Endocrinol.

    (2007)
  • R. Clark

    The somatogenic hormones and insulin like growth factor-I: stimulators of lymphopoiesis and immune function

    Endocr. Rev.

    (1997)
  • B.L. Conway-Campbell et al.

    Nuclear targeting of the growth hormone receptor results in dysregulation of cell proliferation and tumorigenesis

    Proc. Natl. Acad. Sci. U.S.A.

    (2007)
  • J.A. Costoya et al.

    Activation of growth hormone receptor delivers an antiapoptotic signal: evidence for a role of Akt in this pathway

    Endocrinology

    (1999)
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