Philippine Journal of Science 149 (1): 21-26, March 2020 ISSN 0031 - 7683 Date Received: 30 Sep 2019

Importin-β Expression Modulation in Embryonic Stem Cells and Embryonic Fibroblasts of Mouse

Percival P. Sangel*

Institute of Animal Science, College of Agriculture and Food Science University of the Philippines Los Baños, College 4031 Laguna, Philippines

Importin-β proteins are transport proteins important in the shuttling of cargo proteins by binding to either nuclear localization signal (NLS) or (NES). This study investigated the in vitro expression modulation of selected importin-βs. Specifically, this study characterized the culture behavior of mouse embryonic stem cells after knockdown of selected importin-β proteins like Cse1L, IPO7, KPNB1, RanBP16, RanBP17, or XPO4. Also, this study assessed the effects of overexpressing RanBP17 or IPO7 during cellular reprogramming of mouse embryonic fibroblasts (MEFs). Results showed that Cse1L and KPNB1 are essential for the viability of mouse embryonic stem cells since knockdown of either one of these proteins resulted in the death of mouse embryonic stem cells. Meanwhile, the growth characteristics of RanBP17, XPO4, IPO7, or RanBP16 knockdown mouse embryonic stem cells were comparable with the control. Aside from round colonies, the appearance of flat cells and spreading growth characteristics in some colonies were observed, which indicated early signs of differentiation. On the other hand, the number of colonies with overexpressed Oct4, Sox2, Klf4, cMYC (OSKM) + RanBP17, or OSKM+ IPO7 was comparable to OSKM+Flag or OSKM (controls). This suggests that RanBP17 or IPO7 has limited application in the generation of induced pluripotent stem cells.

Keywords: cellular reprogramming, importin-β proteins, knockdown

INTRODUCTION reports available for importin-α contributing to a better understanding of its roles as a transporter. The previous Many intricate trafficking pathways involve the works of Sekimoto et al. (1997) lead to the identification transport of various substances in a living cell. In the of the different subtypes of importin-α. This opened case of the eukaryotic cell, its biomembrane creates a research platform to identify and analyze respective compartmentalization, giving rise to the different NLS in different cargo proteins and elucidate the specific organelles and affecting the transport of all cargo mechanisms involved in their nuclear transport. The molecules. Central to these transport processes is the study of Yasuhara et al. (2007) showed that functional nucleocytoplasmic transport system. This regulates the switching of the importin-α subtype may lead to neural shuttle of the different cargo proteins in and out of the lineage differentiation of embryonic stem cells. Their nucleus as they pass through the complex study also demonstrated significance in the regulation (NPC) of the nuclear membrane. The major key players of the activity of importin-α subtypes, which influence mediating most cargo transport via this system mostly the transport of transcription factors as protein are small proteins collectively called (alpha cargoes. Moreover, the same report indicated the role of and beta). Between the two importins, there are more importin-αs as indicators of cell lineage determination. *Corresponding Author: [email protected]

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The importin-β family also plays an important role in RNA Extraction nucleocytoplasmic transport system. This family consists of For mouse embryonic stem cells, the total RNA was soluble proteins that facilitate majority of macromolecular extracted using TRIZOL (Invitrogen), after which the nuclear transport. In mice, there are more than 20 proteins extracted RNA was DNase-treated (Zymo Research) identified under this family. Specifically, these importin-βs and then reverse-transcribed using the Transcriptor First are associated with nuclear transport of proteins and Strand cDNA Synthesis Kit (Roche). All procedures via the NPC. Importin-βs may function either as importins followed the manufacturer’s recommendations. or exportins. This is through recognition of either the NLS or NES, respectively, during the nuclear transport of cargo Reverse Transcription PCR and Quantitative PCR proteins. Although, some of them may have dual functions For cDNA synthesis, reverse transcription was done with (Ström and Weis 2001, Chook and Blobel 2001, Yuh and polymerase chain reaction (PCR) conditions set at 25 °C Suel 2011). This transport mechanism is regulated by for 10 min, 50 °C for 60 min, and 85 °C for 5 min. And (RanGTP), which is a member of Ras superfamily then, qPCR analysis was done using a 384-well plate in (Maherali et al. 2007). ABI PRISM 7900HT system (Applied Biosystems) with Like importin-α, importin-βs are also involved in FastStart™ Universal SYBR Green Master [Rox] (Roche). different cellular events like pluripotency or cell fate The qPCR conditions were set at a holding temperature determination as seen in the studies using embryonic of 95 °C for 30 s, and 40 cycles of 95 °C for 15 s, 60 °C stem cells (Sangel et al. 2014). Thus, importin-βs as for 30 s, and a standard dissociation stage. transporters are biologically important (Yuh and Suel For all the target , standard curves were generated. 2011), although there is also report available involving Extracted RNA from mouse embryonic stem cells at 0.8, importin-β protein in non-transport function vitally needed 4, 20, and 100 ng were used for serial dilutions. And then, for cellular viability (Mosammaparast and Pemberton 20 ng from the extracted total of siRNA-treated cells was 2004). This study demonstrated the effects of expression used as template. The Pfaffl method was used to determine modulation of some importin-β proteins in different cell the relative target mRNA expression levels and all values types. Specifically, this study characterized the culture were normalized with respective mRNA levels of GAPDH. behavior of mouse embryonic stem cells after knockdown of selected importin-β proteins. Also, this study assessed the potential use of selected importin-β proteins in cellular Treatment of siRNA Oligonucleotide reprogramming using MEFs. In all of mouse embryonic stem cell transfections, 2 × 105 cells were seeded on the surface of Corning™ polystyrene plates (Sigma) coated with 0.1% gelatin with DMEM supplemented with LIF, 10% FBS, 0.1 mM MATERIALS AND METHODS β-mercaptoethanol (Sigma Chemical), 10 mM of MEM nonessential amino acid (GIBCO), and 100 mM of MEM sodium pyruvate (GIBCO). After cell plating, transfection Mouse Embryonic Stem Cells Culture and was carried out using two different siRNA constructs for Maintenance each target genes (i.e., Cse1L, IPO7, KPNB1, RanBP16, Mouse embryonic stem cells (EB3) (Niwa et al. 1998, RanBP17, and XPO4) with LipofectamineTM RNAiMAX 2002) were used for the knockdown of selected importin-β (Invitrogen). Transfected cells were then incubated at proteins. Mouse embryonic stem cells were grown on 37 °C, in 5% CO2. The medium was changed after 48-h plate surface coated with 0.1% gelatin with Dulbecco’s incubation using 2 mL of the fresh medium not containing modified Eagles as culture medium (DMEM) (Sigma) LIF. This is followed by another transfection following the enriched by addition of leukemia inhibitory factor (LIF), same procedure. Transfected cells were again incubated 10% fetal bovine serum (FBS), 0.1 mM β-mercaptoethanol for another 48 h before they were microscopically (Sigma Chemical), 10 mM of MEM nonessential amino examined, trypsinized, and collected. acid (GIBCO), and 100 mM of MEM sodium pyruvate (GIBCO). The cells were maintained at 37 °C in 5% CO . 2 Plasmids Used in Cellular Reprogramming This study made use of pMXs retroviral vectors expressing MEF Culture and Maintenance transcriptional factors like Oct4, Sox2, Klf4, and c-Myc MEFs were used in cellular reprogramming. MEFs (Addgene) for cellular reprogramming. Importin-βs from C57BL/6NCrSlc (SLC) embryos (12.5–13.5 d – which are highly expressed in mESCs, specifically postcoitum) stored in cryotubes and maintained at –80 °C RanBP17 and IPO7 – were also used. Each of the were thawed and cultured in DMEM supplemented with candidate Importin-β genes was amplified through PCR. 10% FBS. The cells were maintained at 37 °C in 5% CO2. Table 1 shows the respective primer sequences used in

22 Philippine Journal of Science Sangel: Importin-β Proteins Expression Vol. 149 No. 1, March 2020 Modulation in Mouse Embryonic Cells the amplification of target importin-β genes. And then, a re-plated on the surface of mitomycin C-treated feeder cells pMXs-RanBP17 or pMXs-IPO7 construct was made by grown on gelatin-coated 100-mm dish. After another 24 h, inserting the coding sequence of either RanBP17 or IPO7 replacement of culture medium was done using the mouse at BamHI and SalI sites located inside the multi-cloning embryonic stem cells medium containing 20% knockout site of pMXs-Flag plasmid vector (Addgene). After that, serum replacement (Invitrogen). The culture medium was the constructed plasmids were cloned and sequenced. regularly changed every other day until colony formation was visible under microscopic examination. And then, Cellular Reprograming alkaline phosphatase staining was done to count the number The method described by Okada et al. (2010) was used in of colonies of mouse induced pluripotent stem cells using the generation of mouse induced pluripotent stem cells. leukocyte alkaline phosphatase kit (Sigma) following the In this study, pMXs retroviruses expressing transcription manufacturer’s protocol. factors like Oct4, Sox2, Klf4, and c-Myc (Addgene) with either RanBP17 or IPO7 were used to infect Plat-E cells Nucleotide Sequences through transfection using respective retroviral vectors Table 1 summarizes the oligonucleotide sequences used (pMXs) with Fugene HD transfection reagent (Roche). A in the study. fresh medium was provided 24 h post-transfection. The supernatant from infected Plat-E cells was collected and Statistical Analysis filtered through a 0.45-µm filter after 48 h. And then, the Analysis of data on mRNA expression levels was carried collected supernatant carrying specific viruses were used to out using the Student’s t-test, while assessment of infect MEFs. At 24 h after infection, 1 × 104 MEF cells were significant differences among reprogrammed cell groups

Table 1. Oligonucleotide sequences used in the study. Type Sequence Purpose GAPDH-F AGGTCGGTGTGAACGGATTTG Quantitative PCR GAPDH-R TGTAGACCATGTAGTTGAGGTCA Quantitative PCR XPO4-F GGCAGCATCGAGTCACTG Quantitative PCR XPO4-R CCCGAACATACTTTTGGAGGT Quantitative PCR IPO7-F TCAGAACAACTGGATTTACCTGTG Quantitative PCR IPO7-R CGATCAGGCCAATATTGTGTTA Quantitative PCR RanBP16-F TCCTCGAGAGAGGAAGTTCG Quantitative PCR RanBP16-R GCTTGGTAAGGCATGTAGCTG Quantitative PCR RanBP17-F GGGACTCACCACACTTGACA Quantitative PCR RanBP17-R GACCATGTAGTCTAAACTTGTACAGCA Quantitative PCR siRanBP17-1 GAAACUACAUCCUGAAUUA siRNA siRanBP17-3 CGAGAAGUAUUUCAGUGAA siRNA siXPO4-1 UGACAAGCAUUUCCAUAAA siRNA siXPO4-3 AGACUUACCUCCUGGUAGA siRNA siIPO7-1 UGAUGCCUCUUCUACAUAA siRNA siIPO7-3 AUAGGGAUGUACCUAAUGA siRNA siRanBP16-1 GCAAGAUGAUAACAAUGUA siRNA siRanBP16-3 CCAGCAAGAUGAUAACAAU siRNA siCse1L-1 UCACAUACUUCCUGAUUUA siRNA siCse1L-3 GCAAUAUGCUUGUCUAUAA siRNA siKPNB-1 CCAGCAAAUUUUACGCCAA siRNA siKPNB-3 GGAGGAGCCUAGUAACAAU siRNA IPO7-F(BamHI) CATGGGATCCACCATGGACCCCAACACCATCAT Cellular reprogramming IPO7-R (SalI) GATCGTCGACTCAATTCATCCCCGGTGCTG Cellular reprogramming RanBP17F (BamHI) CATGGGATCCACCATGGCGCTGCACTTTCAGAG Cellular reprogramming RanBP17R (SalI) GATCGTCGACTCAGCTCATCATGTCCAGGCT Cellular reprogramming

23 Philippine Journal of Science Sangel: Importin-β Proteins Expression Vol. 149 No. 1, March 2020 Modulation in Mouse Embryonic Cells was done through one-way ANOVA using SAS v.9.1. All p values ≤ 0.05 were considered to be significant (*, p < 0.05).

RESULTS AND DISCUSSION

Effect of Knockdown of Importin-βs on Mouse Embryonic Stem Cells A previous study has demonstrated the differential expression patterns of importin-β genes in mouse embryonic stem cells, MEFs, mouse neuroectodermal cells, and mouse mesoendodermal cells (Sangel et al. 2014). Importin-βs that are expressed abundantly in mouse embryonic stem cells – namely RanBP17, XPO4, IPO7, and Cse1L – were knocked down to assess whether they will have an effect on the capacity of mouse embryonic stem cells to maintain their pluripotency or viability as manifested by their microscopic characteristics in culture Figure 1. Phase-contrast images of siRNA treated mouse embryonic medium. RanBP16 and KPNB1 (Importinβ1) – despite stem cells (EB3) after 96-h incubation. The arrowheads being moderately expressed in mouse embryonic stem (yellow) in some images show cells undergoing early cells (Sangel et al. 2014) – were also considered because stages of differentiation. Bar scale (red line) measured RanBP16 compared to the other importin-β proteins at 100 μm. has a high sequence identity with RanBP17 (Chook and Blobel 2001), which is the highest expressed Importin-β as Cse1L attenuation in HeLa cells has been reported to protein in mouse embryonic stem cells, while KPNB1 is perturb cell cycle with observed delayed transition from also targeted since this protein is a well-known importer G2 to G1 and increased levels of cyclin B (Ogryzko et through importin-α protein association (Sekimoto et al. 1997). Meanwhile, KPNB1 – as mentioned earlier – is al. 1997). In addition, it was also speculated that the involved in most import activities of importin-α subtypes. withdrawal of the LIF after 48 h is needed to enhance the In a previous study, it was also shown that KPNB1 down- effects of transfection on mouse embryonic stem cells. regulation in HeLa cells caused a strong decline in cell LIF is a cytokine used to maintain the pluripotent state numbers (Quensel et al. 2004). In the present study, of mouse embryonic stem cells in the absence of feeder the death observed in KPNB1 knocked down mouse cells (Niwa et al. 1998). Moreover, the 48-h time window embryonic stem cells can be attributed to disrupted following this second transfection was critical because it Importin α/β pathway in the nuclear transport of cargoes is within the “temporal window” (Jackson et al. 2010) essential for cell survival and proliferation. where embryonic stem cells after their removal from pluripotency-promoting factors are still nonresponsive In contrast, the knockdown of the other target genes like to any differentiation-inducing agents. Thus, at 48 h, re- RanBP17, XPO4, IPO7, and RanBP16 resulted in viable transfection of selected target genes was performed on cells. Moreover, there were also observed microscopic mouse embryonic stem cells, after which the medium was characteristics suggestive of cells undergoing the initial changed to enriched DMEM without LIF (-LIF), and the stages of differentiation like spreading and emergence of transfected cells were maintained for another 48 h before flat cells in some colonies of transfected mouse embryonic microscopic examination and collection. stem cells, although majority of the observed colonies were seen to be morphologically comparable to the Results showed that, after 96 h of incubation, the low control siRNA treated mouse embryonic stem cells. The expression of Cse1L or KPNB1 through siRNA treatment study of Sangel et al. (2014) reported that low expression is fatal to mouse embryonic stem cells (Figure 1). These of RanBP17, XPO4, IPO7, or RanBP16 predisposes findings indicate that Cse1L and KPNB1 are essential for mouse embryonic stem cells to cellular differentiation as mouse embryonic stem cells survival. Moreover, the low indicated by low levels of Nanog expression. Thomson et expression of either one of them may lead to the death al. (2011) have shown the need for Nanog downregulation of the mouse embryonic stem cells. In the case of Cse1L for embryonic stem cells to undergo differentiation and knocked down mouse embryonic stem cells, this may be lineage selection. due to possible mitotic arrest and subsequent cell death

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Meanwhile – for the mouse embryonic stem cells (EB3) that were still viable after transfection, following trypsinization and total RNA extraction – successful knocked down of selected genes using 2 different siRNAs variants were confirmed through qPCR analysis, with a non-targeting siRNA used as a control (Figure 2).

Figure 3. Reprogramming efficiency of MEFs 14 d post-infection. (A) Representative images of plates after alkaline phosphatase staining. (B) Mouse-induced pluripotent stem cell colony count after alkaline phosphatase staining. Error bars represent SD from three independent Figure 2. Knockdown efficiency of selected importin-β genes. experiments. Analysis of knockdown efficiency determined by qPCR analysis for RanBP17, XPO4, IPO7, and RanBP16 in reprogramming efficiency of MEFs. This is the case even siRNA-treated mouse embryonic stem cells (EB3) with though these two importin-β proteins were found to be two siRNA variants for each at 96 h incubation. All samples were normalized against GAPDH levels, abundantly expressed in mouse embryonic stem cells and and expression level of each gene is shown as a fold were also shown to be involved in the maintenance of their change relative to the expression of the same gene in pluripotency (Sangel et al. 2014). This also suggests that non-targeting siRNA treated mouse embryonic stem cells highly expressed importin-βs in mouse embryonic stem (EB3) used as control. Significance was assessed and cells are may not be directly involved in the induction compared with the control using Student’s t-test (*, p < of cellular reprogramming of MEFs to mouse induced 0.05). Error bars represent SEM from three independent experiments. pluripotent stem cells. Taken together, these findings indicate that Cse1L and Overexpression of Importin-β During Cellular KPNB1 are important proteins for the survival of mouse Reprogramming embryonic stem cells. Knockdown of RanBP17, XPO4, The investigation was carried out to assess the possible IPO7, or RanBP16 in mouse embryonic stem cells leads to application of importin-βs in the generation of induced a microscopic characteristic growth pattern similar to the pluripotent stem cells (cellular reprogramming). In this control and suggestive of cells undergoing differentiation. study, RanBP17 or IPO7 was overexpressed in MEFs Lastly, RanBP17 and IPO7 have little application in using respective pMXs retrovirus and simultaneously cellular reprogramming. infected with a combination of pMXs retroviruses for mouse Oct4, Sox2, Klf4, and cMyc. The two importin-β genes were considered since they were abundantly expressed in mouse embryonic stem cells and it was also ACKNOWLEDGEMENT speculated that overexpressing either one of them may The author would like to extend his gratitude to improve the generation of mouse induced pluripotent stem Dr. Masahiro Oka and Dr. Yoshihiro Yoneda of the cells from MEFs. As shown in Figure 3, overexpression Biomolecular Dynamic Group, Graduate School of of RanBP17or IPO7 together with Oct4, Sox2, Klf4, and Frontier Bioscience, Osaka University, Japan for all the cMyc in the MEFs demonstrated lower colony counts of support provided in the conduct of this study. Special resulting reprogrammed cells after alkaline phosphatase acknowledgment is also given to Dr. Hitoshi Niwa (Riken, staining. Nevertheless, the reduction in the number of Japan) for the EB3 cells. colonies observed in OSKM+RanBP17 or OSKM+IPO7 was shown comparable to OSKM or OSKM+Flag (empty vector), which were used as controls. These results suggest that overexpression of RanBP17 or IPO7 does not improve

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STATEMENT OF CONFLICT OF SANGEL P, OKA M, YONEDA Y. 2014. The role of INTEREST Importin-βs in the maintenance and lineage commit- ment of mouse embryonic stem cells. FEBS Open Bio The author has no conflict of interest to declare. 4(1): 112–120. SEKIMOTO T, IMAMOTO N, NAKAJIMA K, HIRANO T, YONEDA Y. 1997. Extracellular signal-dependent REFERENCES nuclear import of Stat1 is mediated by nuclear pore- targeting complex formation with NPI-1, but not Rch1. CHOOK YM, BLOBEL G. 2001. and Embo J 23: 7067–7077. nuclear import. Current Opinion in Structural Biology 11: 703–715. STRÖM A-C, WEIS K. 2001. Importin-beta-like nuclear transport receptors. Genome Biol 2: reviews3008.1. JACKSON SA, SCHIESSER J, STANLEY EG, ELE- FANTY AG. 2010. Differentiating embryonic stem THOMSON M, LIU SJ, ZOU LN, SMITH Z, MEISSNER cells pass through ‘temporal window’ that mark respon- A, RAMANATHAN S. 2011. Pluripotency factors in siveness to exogenous and paracrine mesoendoderm embryonic stem cells regulate differentiation into germ inducing signals. PLoS ONE 5: e10706. layers. Cell 145: 875–889. MAHERALI N, SRIDHARAN R, XIE W, UTIKAL YASUHARA N, SHIBAZAKI N, TANAKA S, NAGAI J, EMINLI S, ARNOLD K, STADTFELD M, YA- M, KAMIKAWA Y, OE S, ASALLY M, KAMACHI CHECHKO R, TCHIEU J, JAENISCH R, PLATH K, Y, KONDOH H, YONEDA Y. 2007. Triggering neural HOCHEDLINGE K. 2007. Directly reprogrammed fi- differentiation of ES cells by subtype switching of broblasts show global epigenetic remodeling and wide- importin-alpha. Nat Cell Biol 9: 72–79. spread tissue contribution. Cell Stem Cell 1: 55–70. YUH MC AND SUEL KE. 2011. Nuclear import by MOSAMMAPARAST M, PEMBERTON LF. 2004. Karyo- karyopherin-βs: recognition and inhibition. Biochem- pherins: from nuclear-transport mediators to nuclear- ica et Biophysica Acta 1813: 1593–1606. function regulators. Trends Cell Biol 14: 547–556. NIWA H, BURDON T, CHAMBERS I, SMITH A. 1998. Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3. Genes and Dev 12: 2048–2060. NIWA H, MASUI S, CHAMBERS I, SMITH AG, MIYAZAKI J. 2002. Phenotypic complementation establishes requirements for specific POU domain and generic transactivation function of Oct-3/4 in embryonic stem cells. Mol Cell Biol 22: 1526–1536. OKADA M, OKA M, YONEDA Y. 2010. Effective culture conditions for the induction of pluripotent stem cells. Biochem Biophys Acta 1800: 956–963. OGRYZKO VV, BRINKMANN E, HOWARD BH, PAS- TAN I, BRINKMANN U. 1997. Antisense inhibition of CAS, the human homologue of the yeast segregation gene CSE, interferes with mitosis in HeLa cells. Biochemistry 36(31): 9493–9500. QUENSEL C, FRIEDRICH B, SOMMER T, HART- MANN E, KOHLER M. 2004. In Vivo Analysis of Importin α Proteins Reveals Cellular Proliferation Inhibition and Substrate Specificity. Mol Cell Biol 24(23): 10246–10255.

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