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A Hematopoietic Growth Factor, Thrombopoietin, Has a Proapoptotic Role in the Brain

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A Hematopoietic Growth Factor, Thrombopoietin, Has a Proapoptotic Role in the Brain A hematopoietic growth factor, thrombopoietin, has a proapoptotic role in the brain Hannelore Ehrenreich*†, Martin Hasselblatt*, Friederike Knerlich*, Nico von Ahsen‡, Sonja Jacob*, Swetlana Sperling*, Helge Woldt*, Katalin Vehmeyer§, Klaus-Armin Nave*, and Anna-Leena Sire´ n* *Max Planck Institute of Experimental Medicine and ‡Departments of Clinical Chemistry and §Hematology and Oncology, Georg-August University, 37075 Goettingen, Germany Edited by Anthony Cerami, The Kenneth S. Warren Institute, Kitchawan, NY, and approved December 2, 2004 (received for review August 15, 2004) Central nervous and hematopoietic systems share developmental Ͼ95% neurons). Neuronal cell number and viability was assessed features. We report that thrombopoietin (TPO), a stimulator of by trypan blue dye exclusion method. Spontaneous cell death rate platelet formation, acts in the brain as a counterpart of erythro- in neuronal cultures at the time of experiments (5 days plus 15 h) poietin (EPO), a hematopoietic growth factor with neuroprotective was 17 Ϯ 9% (mean Ϯ SD, n ϭ 40). Neuronal survival on properties. TPO is most prominent in postnatal brain, whereas EPO experimental conditions is expressed as percent of spontaneous is abundant in embryonic brain and decreases postnatally. Upon death rate in each particular experiment. Effects of growth factors hypoxia, EPO and its receptor are rapidly reexpressed, whereas on extracellular signal-regulated kinase (ERK)1͞2 phosphorylation neuronal TPO and its receptor are down-regulated. Unexpectedly, were tested on day 5 by incubation at 37°C for 10 min. For TPO is strongly proapoptotic in the brain, causing death of newly measuring long-term effects on neuronal growth, TPO (10 pM) was generated neurons through the Ras-extracellular signal-regulated added to the culture medium at the time of plating and supple- ͞ kinase 1 2 pathway. This effect is not only inhibited by EPO but mented again on day 3. also by neurotrophins. We suggest that the proapoptotic function Primary astrocyte cultures were prepared from the cortices of of TPO helps to select for neurons that have acquired target- 1-day-old Wistar-Imamichi rats as described in refs. 25 and 26, derived neurotrophic support. yielding 98% positive staining for glial fibrillary acidic protein at 2–3 weeks, i.e., the time of experiments. astrocytes ͉ erythropoietin ͉ neurons ͉ differentiation ͉ development Hypoxic conditions were induced by purging an incubator with a mixture of 95% N2͞5% CO2 (neurons) or 90% N2͞10% CO2 n the hematopoietic system, survival, proliferation, and dif- (astrocytes) as described in refs. 25 and 26 and maintained for Iferentiation of cells are regulated by a plethora of growth 15 h. Control experiments were simultaneously performed on factors (1–4). The effect of erythropoietin (EPO) on the gen- the same cell batch under normoxic conditions. For drug treat- eration of red blood cells is well known. The hematopoietic ments, see Supporting Materials and Methods. growth factor thrombopoietin (TPO) stimulates megakaryopoi- esis and thrombocyte formation (1–3, 5, 6). During hematopoi- Experiments in Vivo. For induction of hypoxic͞ischemic brain esis, EPO and TPO can interact in a synergistic and an antag- onistic fashion (1–3, 7). injury, a standard method for immature rats (P14 Wistar- EPO and TPO exhibit significant homology in their receptor- Imamichi) was used (27), combining common carotid artery binding domain (20% identity and 25% similarity). Likewise, ligation with hypoxia for 1 h (‘‘moderate’’) or 2 h (‘‘severe’’) in they bind to receptors, erythropoietin receptor (EPOR) and a standardized airtight chamber, flushed with 8% oxygen. ͞ thrombopoietin receptor (TPOR), respectively, that belong to RhTPO (R & D Systems) (1 nmol kg), rhEPO (Janssen-Cilag, the same cytokine receptor superfamily (1–3, 8–10). Previous Neuss, Germany) (1.4 nmol͞kg), or vehicle were injected i.p. studies reported a neurotrophin-like motif in the N-terminal immediately before, 24 h after, and 48 h after hypoxia exposure. receptor binding region of the TPO molecule, with conflicting Brains were removed 72 h after hypoxia. Hematoxylin-eosin data about the presence of TPO in the brain (5, 6, 11–13). stained sections were scored for structural damage in cortex and For EPO, it is well established that the gene is expressed in the hippocampus ipsilateral to the ligation (two to three coronal embryonic CNS. EPO has a marked effect as a survival factor for sections per brain; 20 250ϫ fields per section) from 0–3 (0, Յ1 neurons and their progenitors (14, 15), presumably to overcome apoptotic cell per field; 1, 1–3 apoptotic or dark shrunken phases of physiological hypoxia (16, 17). The widespread but neurons per field; 2, 4–10 apoptotic, dark shrunken, or eosino- rather ‘‘unspecific’’ neuroprotective potential of EPO is regained philic neurons per field; 3, Ͼ10 apoptotic or eosinophilic, in the adult CNS upon distress or injury. This finding has been necrotic cells per field, cortical infarcts). confirmed in rodent models of cerebral ischemia (18–23), brain trauma (18), and neurodegenerative disease (18), as well as in a Expression of TPO and EPO During Brain Development. Forebrain and clinical study with stroke patients (24). hindbrain from C57B6 mice fetuses [embryonic days (E)11, 13, Here we show that TPO plays a previously unrecognized 15, and 18], newborn [postnatal day (P) 0], 14-day-old (P14), and proapoptotic role in the brain. adult mice were used for mRNA or protein extraction. For densitometric analysis of TPO and EPO protein, Western blots Materials and Methods were analyzed by National Institutes of Health image densitom- All experiments were approved by and conducted in accordance etry with ␣-tubulin III as an internal standard. with the regulations of the local Animal Care and Use Com- mittee. For detailed information on all methods see Supporting Materials and Methods, which is published as supporting infor- This paper was submitted directly (Track II) to the PNAS office. mation on the PNAS web site. Abbreviations: En, embryonic day n; EPO, erythropoietin; EPOR, EPO receptor; ERK, extra- cellular signal-regulated kinase; ISOL, in situ oligo ligation; PI3K, phosphatidylinositol Cell Culture. Primary hippocampal neuronal cultures were prepared 3-kinase; Pn, postnatal day n; TPO, thrombopoietin; TPOR, TPO receptor. from newborn Wistar-Imamichi rats, cultured under serum-free †To whom correspondence should be addressed. E-mail: [email protected]. conditions (25, 26), and used for experiments after five days (purity: © 2005 by The National Academy of Sciences of the USA 862–867 ͉ PNAS ͉ January 18, 2005 ͉ vol. 102 ͉ no. 3 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0406008102 Downloaded by guest on September 28, 2021 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 Ͻ 0.001) and an inverse relationship (similar with hindbrain, data not shown). E11–P0, n ϭ 4; P14 and adult, n ϭ 3; *, P Ͻ 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 ϭ 5; *, P Ͻ 0.05; **, P Ͻ 0.01 compared with normoxia. (a–c) Elongation factor was used as reference gene. Expression Analysis by Quantitative Real-Time RT-PCR. First-strand expression of EPOR (increased) and TPOR (decreased) in cDNA was generated from total RNA by random priming cultured neurons. In astrocytes, however, both TPOR and (GIBCO͞Pharmacia, Freiburg, Germany). Detailed informa- EPOR mRNA were augmented by hypoxia (Fig. 1c). tion on rat and mouse primer pairs is available in Supporting Materials and Methods. PCR reactions were carried out on a Antagonistic TPO͞TPOR and EPO͞EPOR Protein Expression in Neurons LightCycler real-time PCR machine (Roche Molecular Bio- and Astrocytes During Brain Development and After Hypoxia. At the chemicals). RACE of rat TPOR͞Mpl mRNA was performed to protein level, TPO and TPOR expression was weak in the fetal allow for the design of rat-specific primers in the 3Ј UTR that are brain and prominent in the juvenile hippocampus, with no not sensitive toward differentially spliced isoforms (28). obvious difference (in whole-tissue lysates) 6 h and 24 h after moderate hypoxia͞ischemia (data not shown). Quantification of Western Blotting. Protein extracts transferred to nitrocellulose protein levels between E11 and adult stages indicated again a membranes were incubated with rabbit͞goat anti-TPO (Sigma), regulatory dissociation of the two growth factors: whereas TPO EPO, TPOR͞Mpl, or EPOR antibodies (Santa Cruz Biotechnol- peaked in the adult brain, EPO decreased to a nearly undetect- ogy), mouse anti-phospho-p44͞42-ERK (Thr-202͞Tyr-204) (New able level (Fig. 2a). For both TPO and TPOR, cultured neurons England Biolabs) or rabbit pan-ERK polyclonal antibody (New revealed weak but distinct specific labeling of cell bodies and England Biolabs). Immunoreactive bands were visualized by using processes (Fig. 2a, and Fig. 5 a–d, which is published as sup- secondary antibodies coupled to horseradish peroxidase by en- porting information on the PNAS web site). In agreement with hanced chemoluminescence (Amersham Pharmacia). the mRNA data, immunostaining was reduced in neurons after 15 h of hypoxia, whereas that of EPO and EPOR was enhanced. Immunohistochemistry and in Situ Oligo Ligation (ISOL). For infor- In hypoxic astrocytes, staining of TPO was weaker and staining mation on this subject, see Supporting Materials and Methods. of TPOR was unchanged as compared with normoxia (Fig. 2b). Taken together, neuronal EPO and TPO systems are regulated Statistical Analysis. Data, expressed as mean Ϯ SEM in figures NEUROSCIENCE and text, were compared by ANOVA with post hoc planned comparisons or Duncan test, Kruskal-Wallis ANOVA with Mann–Whitney U test, or the Fisher exact probability test.
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