A hematopoietic growth factor, thrombopoietin, has a proapoptotic role in the brain

Abstract

Central nervous and hematopoietic systems share developmental features. We report that thrombopoietin (TPO), a stimulator of platelet formation, acts in the brain as a counterpart of erythropoietin (EPO), a hematopoietic growth factor with neuroprotective properties. TPO is most prominent in postnatal brain, whereas EPO is abundant in embryonic brain and decreases postnatally. Upon hypoxia, EPO and its receptor are rapidly reexpressed, whereas neuronal TPO and its receptor are down-regulated. Unexpectedly, TPO is strongly proapoptotic in the brain, causing death of newly generated neurons through the Ras-extracellular signal-regulated kinase 1/2 pathway. This effect is not only inhibited by EPO but also by neurotrophins. We suggest that the proapoptotic function of TPO helps to select for neurons that have acquired target-derived neurotrophic support.

Figures

Fig. 1.

Antagonistic gene expression of brain EPO and TPO systems. (a) RT-PCR illustrating presence of mRNA of TPO, EPO, and their receptors in fetal and adult rat tissues. HC, hippocampus; CX, cortex. (b) Quantitative PCR of the developing mouse forebrain demonstrates for TPO and EPO mRNA significant changes over time (P < 0.001) and an inverse relationship (similar with hindbrain, data not shown). E11-P0, n = 4; P14 and adult, n = 3; *, P < 0.01 compared with E11. (c) Quantitative PCR demonstrating an inverse response of TPO, EPO, TPOR, and EPOR mRNA to hypoxia (15 h) in primary hippocampal neurons and cortical astrocytes. n = 5; *, P < 0.05; **, P < 0.01 compared with normoxia. (a-c) Elongation factor was used as reference gene.

Fig. 2.

Antagonistic TPO/TPOR and EPO/EPOR protein expression in brain tissue and cultured neurons and astrocytes. (a) Densitometric analysis of 70-kDa (TPO) and 38-kDa (EPO) bands corresponding to known sizes of TPO (3) and EPO (22) in Western blots of developing mouse forebrain demonstrate significant changes over time (P < 0.01) and an inverse relationship of TPO and EPO (hindbrain similar, data not shown). E11-P0, n = 4; P14 and adult: n = 3-4; *, P < 0.05 compared with E11. (b) Confocal images illustrating presence of TPO, EPO, and their receptors in neurons and astrocytes in culture (green fluorescence). Red fluorescence shows nuclear counterstaining with propidium iodide. N, normoxia; H, hypoxia. (Scale bar, 50 μm.)

Fig. 3.

TPO and EPO exert opposite actions on neuronal survival. (a) Dose-response curves of TPO (filled circles) and EPO (open circles) effects on cell death rate in primary hippocampal neurons under normoxic conditions. n = 4; *, P < 0.05 compared with control. (b) EPO (100 pM) abolishes the effect of TPO on neuronal death. Black circles, TPO alone; gray circles, TPO+EPO; n = 4. Experiments independent from a. *, P < 0.05 compared with control, #, P < 0.05 compared with TPO. (c) Granulocyte colony-stimulating factor (GCSF), nerve growth factor (NGF), neurotrophin-3 (NT-3), and brain-derived neurotrophic factor (BDNF) (each at 1 nM) abolish the death-promoting effect of TPO (100 pM). n = 6-17; *, P < 0.001 compared with control; #, P < 0.001 compared with TPO alone. (d) Antagonism of TPO-induced (100 pM) neuronal death with a Janus kinase (JAK) 2-transphosphorylation inhibitor (AG490), an inhibitor of ERK1/2 (PD98059) or a caspase inhibitor (Ac-VAD-CHO) but not a PI3K inhibitor (LY294002). The latter abolishes the protection against TPO-induced cell death by EPO (100 pM). n = 4-5; *, P < 0.05 compared with control; #, P < 0.05 compared with TPO. (e) TPO (100 pM) and EPO (100 pM) induce ERK1/2 phosphorylation in hippocampal neurons (representative of five separate experiments). (f) Proposed EPOR-TPOR signaling cross talk. TPO dimerizes TPOR and transphosphorylates receptor-associated JAK-2. Activation of Ras-mitogen-activated protein kinase pathway leads to caspase activation and apoptosis. EPO opposes TPO effects through activation of PI3K-Akt/protein kinase B pathway. (g-i) Primary hippocampal cultures on day 4. Representative confocal images of nuclear staining with propidium iodide (Left; red fluorescence). Arrows mark apoptotic cells. Identical fields in Right demonstrate fluorescent labeling (green) with markers for neural precursors (nestin), early postmitotic neurons (β-tubulin III), or mature neurons (MAP2). (Scale bar, 25 μm.)

Fig. 4.

TPO increases tissue damage upon cerebral hypoxia/ischemia in juvenile rats. (a) Representative low-magnification photomicrograph depicting cortical (CX) and hippocampal (HC) areas of a placebo-treated (Left) and a TPO-treated (Center) rat 72 h after right carotid artery ligation and exposure to hypoxia (1 h, 8% O2). Underneath corresponding high (250×) magnification images. Damage scores (cortex plus hippocampus) of placebo-versus TPO-treated rats are shown in Right (n = 10). (b-d) Representative cortical sections and quantification of labeled cells in hypoxic/ischemic hemisphere (cortex plus hippocampus) of placebo-versus TPO-treated rats. (b) Cleaved caspase-3 (n = 8-9). (c) ISOL-positive apoptotic cells (n = 9). (d) TPOR immunoreactivity (n = 9). (a-d) *, P < 0.05, **, P < 0.01.