TNF-alpha-induced apoptosis is prevented by erythropoietin treatment on SH-SY5Y cells

Ahmad

Laboratory Investigation

2010

The source of circulating erythropoietin (EPO), the mediators and the mechanisms involved in the upregulation of EPO gene expression during acute-phase reaction are still poorly understood. Acute-phase reaction was induced by either intramuscular turpentine oil (TO) or intraperitoneal lipopolysaccharide (LPS) administration into wild-type and interleukin (IL)-6 knockout (KO) mice. Animals were killed at different time points and blood, liver and muscle tissue were collected. Serum levels of EPO were measured by enzyme-linked immunoadsorbent assay; liver and injured muscle samples were processed for RNA isolation and for protein analysis. EPO, hypoxia-inducible factors 1a and 2a (HIF-1a and HIF-2a) mRNA were analyzed by RT-PCR and the protein levels were analyzed by western blot and electrophoretic mobility shift assay. HIF-1a and HIF-2a localization was performed through immunofluorescence staining. EPO, HIF-1 and HIF-2 gene and protein expression levels were also analyzed in isolated mouse hepatocytes after stimulation with IL-6. In the wild-type animals, EPO serum levels increased dramatically at 12 h after the insults together with the hepatic gene expression. In TO-treated animals, the EPO gene expression reached an 8.2-fold increase at 12 h, and in LPS-treated mice a similar induction was recorded at 6 h (about 4.5-fold increase). In the IL-6KO strain, the upregulation after the inflammatory stimuli was much lower (only 2.0-fold increase). A progressive upregulation of HIF-1a and HIF-2a was detectable until 6 h after the insults, but only HIF-1a upregulation was reduced in IL-6KO mice. In isolated hepatocytes, stimulation with a single dose of IL-6 induced a nuclear accumulation of HIF-1a, in parallel with an increase of EPO mRNA. No effect on HIF-2a expression was found. IL-6 appears to be the main regulator of EPO gene expression and a major contributor for HIF-1a induction in hepatocytes and Kupffer cells during acute-phase response. The increase of HIF-2a, predominantly expressed in endothelial cells and fibroblast-like cells, seems not to be affected by the lack of IL-6.

supporting

confidence: 90%

Cellular and molecular mechanisms regulating the hepatic erythropoietin expression during acute-phase response: a role for IL-6

Ahmad

Laboratory Investigation

2010

J of Cellular Biochemistry

“…In previous works, we demonstrated the role of Epo in preventing apoptosis induced by STP [Pregi et al, 2006] or TNF-a [Pregi et al, 2009] in undifferentiated SH-SY5Y cells. Different cell responses upon the differentiation agent have been described [Tieu et al, 1999].…”

Section: Effect Of Proapoptotic Agents Upon Undifferentiated and Atramentioning

confidence: 84%

“…However, its biological role has been expanded by the finding of specific receptors in non-hematopoietic tissues, such as endothelial [Anagnostou et al, 1994] and neuronal [Masuda et al, 1993] tissues. Evidence has shown that Epo exhibits neuroprotective effects in vitro against proapoptotic agents such as glutamate, hypoxia, tumor necrosis factor-alpha (TNF-a), and glucose/serum deprivation [Marti et al, 2000;Buemi et al, 2002;Celik et al, 2002;Pregi et al, 2009]. In vivo studies in adult and neonatal animal models have revealed a neuroprotective action of exogenous Epo administration [Sola et al, 2005;Noguchi et al, 2007].…”

Section: Abstract: Apoptosis; Differentiation; Erythropoietin; All-tmentioning

confidence: 99%

“…2). The experimental conditions in undifferentiated cell cultures were established on the basis of our own previous results [Pregi et al, 2006;Pregi et al, 2009] as well as on dose-response and time-response curves. Cultures were either exposed to 100 nM STP or 25 ng/ml TNF-a for 12 h, or incubated under an atmosphere of 5-7% O 2 for 16 h. A significant decrease in cell viability, parallel to an increase in the apoptotic cell number, suggested that undifferentiated SH-SY5Y cells were highly sensitive to the proapoptotic procedures applied.…”

Section: Effect Of Proapoptotic Agents Upon Undifferentiated and Atramentioning

confidence: 99%

See 1 more Smart Citation

Differential antiapoptotic effect of erythropoietin on undifferentiated and retinoic acid‐differentiated SH‐SY5Y cells

Wenker

Chamorro

Vota

et al. 2010

Erythropoietin (Epo) is known to have a significant role in tissues outside the hematopoietic system. In this work, we investigated the function of Epo in cells of neuronal origin subjected to differentiation. Treatment of SH-SY5Y cells with all-trans-retinoic acid (atRA) generated differentiated neuron-like cells, observed by increased expression of neuronal markers and morphological changes. Exposure of undifferentiated cells to proapoptotic stimuli such as staurosporine, TNF-alpha, or hypoxia, significantly increased programmed cell death, which was prevented by previous treatment with Epo. In contrast, atRA-differentiated cultures showed cell resistance to apoptosis. No additional effect of Epo was detected in previously differentiated cells. The inhibition of the PI3K/Akt pathway by Ly294002 abrogated the protective effects induced by either Epo or atRA. The effect of atRA was mediated by an increased expression of Bcl-2 whereas the Epo treatment upregulated not only Bcl-2 but also Bcl-xL. This upregulation by Epo was not detected in atRA-differentiated cells, thus confirming the lack of the protective effect of Epo. As expected, assays with AG490, an inhibitor of Jak2, blocked the Epo action only in undifferentiated cells. This reduced neuroprotective function of Epo on SH-SY5Y differentiated cells could be explained at least in part by downregulation of the Epo receptor expression, which was observed in atRA-differentiated cells. This study shows differential cellular protection induced by Epo at two stages of SH-SY5Y differentiation. The results allow us to suggest that this differential cell behavior can be ascribed to the interaction between atRA and the signaling pathways mediated by Epo.

“…EPO has the same characteristics as other neurotrophic factors, because EPO is neuroprotective in neural cells exposed to cytokines, such as tumor necrosis factor-␣ (Pregi et al, 2009), or toxins such as the A␤ amyloid peptide (Ma et al, 2009). The EPOR that mediates neuroprotection in brain is the same classical EPOR expressed in peripheral tissues (Um et al, 2007).…”

Section: Discussionmentioning

confidence: 99%

Drug Targeting of Erythropoietin Across the Primate Blood-Brain Barrier with an IgG Molecular Trojan Horse

Boado¹,

Hui²,

Lu³

et al. 2010

J Pharmacol Exp Ther

107

Erythropoietin (EPO) is a neurotrophic factor that could be developed as a new drug for brain disorders. However, EPO does not cross the blood-brain barrier (BBB). In the present study, human EPO was re-engineered by fusion to the carboxyl terminus of the heavy chain of a chimeric monoclonal antibody (MAb) to the human insulin receptor (HIR). The HIRMAb acts as a molecular Trojan horse to ferry the EPO into the brain via receptor-mediated transport on the endogenous BBB insulin receptor. The HIRMAb-EPO fusion protein was immunoreactive with antibodies to both human IgG and EPO. The HIRMAb-EPO fusion protein bound with high affinity to the extracellular domain of both the HIR (ED 50 ϭ 0.21 Ϯ 0.05 nM) and the EPO receptor (ED 50 ϭ 0.30 Ϯ 0.01 nM) and activated thymidine incorporation into human TF-1 cells with an ED 50 of 0.1 nM.Differentially radiolabeled EPO and the HIRMAb-EPO fusion protein were injected intravenously into adult rhesus monkeys. Whereas EPO did not cross the primate BBB, the HIRMAb-EPO fusion protein was rapidly transported into brain, at levels that produce pharmacologic elevations in brain EPO at small systemic doses. The HIRMAb fusion protein selectively targeted the brain relative to peripheral organs. In conclusion, a novel IgG-EPO fusion protein has been engineered, expressed, and shown to be bifunctional with retention of high-affinity binding to both the insulin and EPO receptors. The IgG-EPO fusion protein represents a new class of EPO neurotherapeutics that has been specifically re-engineered to penetrate the human BBB.