Hindawi Publishing Corporation BioMed Research International Volume 2015, Article ID 207347, 8 pages http://dx.doi.org/10.1155/2015/207347 Research Article The Expression and Distributions of ANP32A in the Developing Brain Shanshan Wang,1 Yunliang Wang,2 Qingshan Lu,3 Xinshan Liu,1 Fuyu Wang,4 Xiaodong Ma,4 Chunping Cui,5 Chenghe Shi,5 Jinfeng Li,2 and Dajin Zhang5 1 Weifang Medical University, Weifang 261042, China 2 Department of Neurology, The 148th Hospital, Zibo 255300, China 3 Zhengzhou University, Zhengzhou 450001, China 4 Department of Neurosurgery, PLA 301 Hospital, Beijing 100853, China 5 Center for Basic Medical Sciences, Navy General Hospital of Chinese PLA, Beijing 100048, China Correspondence should be addressed to Jinfeng Li; [email protected] and Dajin Zhang; [email protected] Received 9 July 2014; Revised 20 August 2014; Accepted 20 August 2014 Academic Editor: Hongwei Wang Copyright © 2015 Shanshan Wang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Acidic (leucine-rich) nuclear phosphoprotein 32 family, member A (ANP32A), has multiple functions involved in neuritogenesis, transcriptional regulation, and apoptosis. However, whether ANP32A has an effect on the mammalian developing brain is still in question. In this study, it was shown that brain was the organ that expressed the most abundant ANP32A by human multiple tissue expression (MTE) array. The distribution of ANP32A in the different adult brain areas was diverse dramatically, with high expression in cerebellum, temporal lobe, and cerebral cortex and with low expression in pons, medulla oblongata, and spinal cord. The expression of ANP32A was higher in the adult brain than in the fetal brain of not only humans but also mice in a time-dependent manner. ANP32A signals were dispersed accordantly in embryonic mouse brain. However, ANP32A was abundant in the granular layer of the cerebellum and the cerebral cortex when the mice were growing up, as well as in the Purkinje cells of the cerebellum. The variation of expression levels and distribution of ANP32A in the developing brain would imply that ANP32A may playan important role in mammalian brain development, especially in the differentiation and function of neurons in the cerebellum and the cerebral cortex. 1. Introduction LRRs shape a globular head domain. LRRs, belonging to a superfamilywithdiversebioactivity,maypotentiallyfunction ANP32A is a member of acidic nuclear phosphoprotein in mediating protein-protein interaction [8]. The extended 32 kDa (ANP32) family [1, 2]. The nomenclature of ANP32A and hydrophilic C-terminal domain is highly unusual in its familymembersisconfusingbecausethesameproteinhas amino acid composition, containing abundant aspartic and beengivenmorethanonenamebasedonthecontextof glutamic acid residues, and a putative nuclear localization isolation. The family members are comprised of ANP32A signal (NLS) [9]. According to the structure characteristic, it (also known as PP32, LANP, HPPCn, I1PP2A, MAPM, or is not surprising to find ANP32 proteins involved in a variety PHAPIa), ANP32B (PAL31, APRIL, or PHAPIb) [3], ANP32C of cellular processes in both nucleus and cytoplasm, including (PP32r1) [4], ANP32D (PP32r2) [5], and ANP32E (Cpd1, signaling, apoptosis, protein degradation, and morphogene- LANP-L, or PHAPIII) [6]. sis. All ANP32 proteins share two highly conserved regions: More studies have focused on ANP32A, the founding the N-terminal leucine-rich repeats (LRRs) sequence and the member of the ANP32 family. ANP32A (PP32) was orig- C-terminal acidic tail [7]. For ANP32A, the hydrophobic inally found as a tumor suppressor [10], associated with 2 BioMed Research International cancer cell survival and drug efficacy. Meanwhile its closely 2. Materials and Methods related homologue PP32r1 is oncogenic and is overexpressed in breast cancer and prostate cancer [11]. Family member 2.1. Animals. Adult C57 BL/6 mice were kept with free ANP32B was indicated as a potential prognostic marker of accesses to food and water. The day of insemination was human breast cancer [2]. In fact, ANP32A has also been designated as embryonic day 0 (E0). The day of birth was identified having a potential oncogenic and drug-resistant designated as postnatal day 0 (P0). Brains from different function in hepatocellular carcinoma [12], colorectal can- embryonic period (E12 and E16), early time points after birth cer [13], and pancreatic tumor [14]. The confused role in (P0, P5, and P12), pubescent male mice (approximately 5-6 tumorigenesis of ANP32A may be due to its functions and weeks old), and adult male mice (approximately 8–10 weeks − ∘ subcellular localizations being almost bewildering in variety. old) were frozen quickly and stored at 80 Cuntilrequired ANP32A is known to be a key component of the inhibitor for experiments. E12 and E16 brains were collected under of acetyltransferase (INHAT) complex in the nucleus, the dissection microscope and the mesenchymal tissues involved in regulating chromatin remodeling or transcription were removed with fine forceps as much as possible. For initiation [15]. ANP32A forms a multisubunit heterocomplex immunohistochemistry preparation, brains were fixed in × with HuR, regulating the nucleocytoplasmic shuttling of formalin (4% formaldehyde in 1 PBS, pH7.4) for 24 hours HuR, which is essential for RNA stability and transport [16]. and then embedded in paraffin. Serial sections (4 m) were In the cytoplasm, ANP32A (mapmodulin) is positioned to mounted onto silane-coated slides (Dako, Denmark). microtubule associated proteins (MAPs), involved in regu- lating microtubule function and microtubule-based vesicular 2.2. Human Multiple Tissue Expression (MTE) Array Analysis. trafficking17 [ ]. ANP32A may control enzymatic activities by The human MTE array (BD, America) was a positively + inhibition of protein phosphatase 2A (PP2A) or activation charged nylon membrane to which poly A RNAs from dif- of caspases [18]. ANP32A (LANP) and SET were observed ferent human tissues had been normalized and immobilized attheinnersurfaceoftheplasmamembraneoflympho- in separate dots, along with several controls. The MTE array cytesandsupposedlyplayaroleinsignaltransduction made it possible to determine the relative expression levels [19]. ANP32A (HPPCn) was the first factor that it could of a target mRNA in different tissues and developmental + transport to the extracellular space and act as an autocrine stages [26]. To this end, poly A RNA samples on each MTE factor to promote DNA synthesis and suppress apoptosis by array had been normalized to the mRNA expression levels of upregulating myeloid cell leukemia-1 [20]. These functions eight different “housekeeping” genes, which minimized the taken together suggest that ANP32A could therefore be an small tissue-specific variations in expression of any single important regulator of cellular homeostasis. housekeeping gene [27]. The human MTE array gave a ANP32A plays essential roles in a variety of neural relative convenient and convincing way to demonstrate the pathophysiology processes. The level of ANP32A (I1PP2A) is levels of ANP32A mRNA in 20 different areas of the human increasedinAlzheimer’sdisease(AD)andmaybeinvolvedin brain, as well as adult whole brain and fetal brain. regulatory mechanism of affecting Tau phosphorylation and A750bpANP32A probe was amplified from human impairing the microtubule network and neurite outgrowth ANP32A gene (Gene ID: 8125) in the pET-24a(+) plas- [21]. ANP32A (LANP) regulates neuronal differentiation by mid (kept by our laboratory) with primers 5 -CGG- epigenetic modulating expression of the neurofilament light GATCCATGGAGATGGGCAGACGGATT-3 (forward) chain, an important neuron-specific cytoskeletal gene [22]. and 5 -AACTGCAGGTCAT-CATCTTCTCCCTCATC-3 ANP32A can interact with the retinoblastoma protein Rb in (reverse). Probe used for the human MTE assay was radioac- both young and mature neurons and is implicated in the 32 tively labeled with -[ P]dCTP using the DNA labeling kit regulation of neuronal survival by CXCL12/CXCR4 [23]. The (Promega, America) as described by the operation manual. decreased levels of ANP32A, as a potent and selective PP2A ∘ Prehybridization of the MTE array was performed at 68 C inhibitor, may contribute to abnormal neuritic morphology for 2 hours in ExpressHyb hybridization (Clontech, America) in a dominantly inherited neurodegenerative disorder of the with sheared salmon testis DNA added to a final con- spinocerebellar ataxia type 1 [24]. centration of 0.1 mg/mL. The denatured radiolabeled probe The expression characteristic of ANP32A in the devel- was mixed directly into the prehybridization solution and oping brain, especially details on the expression and distri- ∘ hybridized overnight at 68 C. After hybridization, the array butions of ANP32A in the human brain, had rarely been ∘ was washed three times at 37 Cinsolution2× SSC with reported [25]. In this study, the distribution of ANP32A in ∘ 0.1% SDS for 10min each time, repeated at 65 Cinsolution different human brain areas, as well as ANP32A abundance 0.1× SSC with 0.1% SDS for 20 min each time. Array was in the human fetal and adult brain, was identified. For more ∘ exposed to X-ray film at −70 C for 48 hours. ANP32A details of the expression and localization of ANP32A in mRNA levels were determined by densitometric scanning of the developing