Elsevier

Neuroscience Research

Volume 74, Issues 3–4, December 2012, Pages 269-276
Neuroscience Research

Pleiotrophin promotes microglia proliferation and secretion of neurotrophic factors by activating extracellular signal-regulated kinase 1/2 pathway

https://doi.org/10.1016/j.neures.2012.09.001Get rights and content

Abstract

Pleiotrophin (PTN) is an effective neuroprotective factor and its expression is strikingly increased in microglia after ischemia/reperfusion injury. However, whether PTN could provide neurotrophic support to neurons by regulating microglia function is not clear. In this study, we demonstrated that the expression of PTN was induced in microglia after oxygen-glucose deprivation/reperfusion. PTN promoted the proliferation of microglia by enhancing the G1 to S phase transition. PTN also stimulated the secretion of brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF) and nerve growth factor (NGF) in microglia, but did not upregulate the expression of proinflammatory factors such as TNF-α, IL-1β and iNOS. Mechanistically, we found that PTN increased the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 in microglia in both concentration-dependent and time-dependent manners. In addition, ERK1/2 inhibitor U0126 abolished the proliferation and G1 to S phase transition of microglia stimulated by PTN, and inhibited the production of BDNF, CNTF and NGF induced by PTN. In conclusion, our results demonstrated that PTN-ERK1/2 pathway plays important role in regulating microglia growth and secretion of neurotrophic factors. These findings provide new insight into the neuroprotective role of PTN and suggest that PTN is a new target for therapeutic intervention of stroke.

Highlights

PTN was induced in microglia after oxygen-glucose deprivation/reperfusion. ► PTN promoted the proliferation of microglia by enhancing G1 to S phase transition. ► PTN stimulated the secretion of neurotrophic factors in microglia. ► PTN increased the phosphorylation of ERK 1/2 in microglia. ► ERK1/2 inhibitor U0126 abolished the stimulatory effects of PTN on microglia.

Introduction

Pleiotrophin (PTN), also called Heparin affin regulatory peptide (HARP) or heparin-binding growth-associated molecule (HB-GAM), is an 18-kDa secreted growth factor which binds three cell surface receptors, syndecan-3, anaplastic lymphoma kinase (ALK), and protein tyrosine phosphatase receptor (RPTPβ/ζ) (Papadimitriou et al., 2004, Weng and Liu, 2010). PTN is an effective neuroprotective factor by promoting axonal growth and regeneration and supporting the survival of neurons (Herradon and Ezquerra, 2009, Hida et al., 2003, Poulsen et al., 2000). It has been shown that the expression of PTN is strikingly increased in microglia after ischemia/reperfusion injury (Yeh et al., 1998). However, whether PTN could potentially provide neurotrophic support to neurons by regulating microglia function is not clear.

CD11b is one of the most important microglia surface markers during microglia activation (Roy et al., 2008). The activated microglia proliferate, migrate, and secret growth factors and neurotrophic factors such as BDNF, CNTF, FGF-2, IGF-1, GDNF and NGF to promote tissue repairing and neuronal rescuing (Batchelor et al., 2002, Garden and Moller, 2006, Lalancette-Hebert et al., 2007, Lambert et al., 2004, Nakajima and Kohsaka, 2004). Meanwhile, overactivated microglia secret cytotoxins such as TNF-α, IL-1β and NO to initiate inflammatory processes, which are harmful for the recovery of stroke (Kaushal and Schlichter, 2008, Mabuchi et al., 2000). Cerebral ischemia/reperfusion is known to lead to the upregulation of CD11b, indicating the activation of microglia in this process (Batchelor et al., 2002). In the present study, we used an in vitro model of cerebral ischemia/reperfusion to demonstrate that PTN promotes microglial proliferation and the production of growth factors and neurotrophins and further elucidate the underlying signaling pathways.

Section snippets

Cell culture

Pregnant Sprague-Dawley rats (20–21 days) were purchased from Medical Laboratory Animal Center (Guangdong, China) and were maintained on a 12-hour light/dark cycle with free access to food and water. Neonatal rats, age 1–2 days, were used for cell culture. All experiments were conducted according to the National Institutes of Health (NIH) guidelines on laboratory animal use and care (Publication no. 80-23), and were approved by the Institutional Animal Care and Use Committee of Sun Yat-sen

PTN promotes microglia proliferation

The purity of the microglia culture was determined by immunocytochemical staining of Mac-1. The results showed that more than 98% of the purified microglia cell cultures were stained positively by Mac-1 antibody, indicating the purity of the cultured microglia (Fig. 1).

To clarify whether OGD/reperfusion affects PTN expression in microglia, we measured the protein level of PTN at different time points (0, 15 min, 30 min, 1 h and 2 h) after OGD/reperfusion. It showed that PTN protein level was

Discussion

In the present study, for the first time, we demonstrated the key role of PTN in the regulation of microglia. PTN promotes the proliferation of microglia by enhancing G1 to S phase transition, and stimulates the secretion of BDNF, CNTF, and NGF by microglia. Furthermore, we established that these effects of PTN on microglia are mediated by the activation of ERK1/2 pathway.

Microglia is known to show enhanced proliferation in pathological conditions such as cerebral ischemia (Yenari et al., 2010

Competing interests

The authors declare that they have no competing interests.

Acknowledgements

This study was funded by Science and Technology Foundation of Guangdong Province of China (No. 2009B080701072, No. 2009B060700041 and No. 9451008901002160) and National Natural Science Foundation of China (No. 81171101). We thank Prof. Guanghui Jin of Xiamen University for intellectual and technical assistance.

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