Importins and exportins in cellular differentiation

The importin/exportin transport system provides the machinery involved in nucleocytoplasmic transport. Alterations of the levels of importins and exportins may play crucial roles in development, differentiation and transformation. Employing human leukaemia HL-60 cells, we and others have revealed the differentiation-associated changes in the protein and gene expression of these factors. The recent finding that a switch to the importin-α subtype triggers neural differentiation of embryonic stem cells underscores the importance of nucleocytoplasmic transport factors in cellular events. This review focuses on current research into the roles of importins and exportins in cell differentiation.


Introduction
In eukaryotic cells, the nucleus is separated from the cytoplasm by a double-layered membrane, the nuclear envelope. Macromolecules such as RNA transcripts generated in the nucleus are exported to the ribosomes in the cytoplasm and proteins synthesized in the cytoplasm, such as histones, DNA and RNA polymerases and transcription factors, are imported into the nucleus. The importin/ exportin transport system provides the machinery involved in nucleocytoplasmic transport of cargo molecules larger than ~40 kD [1][2][3][4][5][6][7][8][9][10][11][12]. In this system, proteins that shuttle between the cytoplasm and the nucleus have generally a nuclear localization signal (NLS) sequence or a nuclear export signal (NES) sequence.
Importin-␣ recognizes to the classical NLS (cNLS) within a protein cargo and forms a ternary complex with importin-␤1 to enter into the nucleus (Fig. 1). In another system, the cargo molecule with the NLS directly binds to importin-␤ and is transported into the nucleus. Exportin recognizes the NES in the cargo protein and the complex is exported from the nucleus by binding with the guanosine triphosphate (GTP)-bound form of the guanine nucleotide-binding protein Ran (RanGTP) (Fig. 1).
Alterations in the expression of the components of the nuclear transport machinery would determine transport efficiency and plays crucial roles in development, differentiation and transformation. This review focuses on current research into the roles of importins and exportins in cellular differentiation.

Importins and exportins
The National Center for Biotechnology Information (NCBI) database shows that there are at least 18 importin and 6 exportin genes in human beings and 15 importin and 6 exportin genes in mice.

• Introduction • Importins and exportins • NLS and NES • Cargo molecules • Importins and exportins in cellular differentiation -HL-60 cell differentiation -Importin expression -Exportin expression -Monocyte differentiation -Terminal erythroid differentiation -Neural differentiation -Cardiac differentiation -Keratinocyte differentiation -Germ cell maturation -Muscle cell differentiation • Concluding remarks
Exportin-1 is frequently referred to as CRM1. Comparisons of these nucleocytoplasmic transport factors in different species have been hampered by the multiple names assigned. In this review, we use the terms for the human genes.
In the importin-␣-mediated nuclear import system, cytosolic importin-␤1 forms a complex with importin-␣, which binds to the cNLS contained in a cargo protein. After entering the nucleus through the nuclear pore complex (NPC), the ternary complex dissociates. The energy required for this dissociation is provided by GTP from RanGTP. Importin-␣ is recycled back to the cytoplasm in a complex with an importin-␣ reexporter, cellular apoptosis susceptibility gene (CAS), in the presence of RanGTP [1,9]. In some cases, importin-␤1 recognizes the cNLS within a cargo in the cytoplasm without the importin-␣ adapter for entrance into the nucleus. Importin-␤1 in the nucleus is recycled to the cytoplasm in a complex with RanGTP.
The nuclear export of proteins is mediated by exportins which bind to NES-containing cargo and RanGTP in the nucleus. The signal recognized by exportin-1 may be termed the classical NES. Dissociation of the ternary complex in the cytoplasm is promoted by Ran GTPase-activating protein to ensure the export of the cargo. Exportin-1 is known to be recycled into the nucleus by binding to an NPC component, Nup358 [6]. Various types of intra-and inter-molecular masking of these transport signals regulate the efficiency of nucleocytoplasmic transport. Phosphorylation, changes in calcium concentrations and conformational changes for self-inhibition are representative events for such masking [5,11]. Exportin-7 XPO7 IFN-␣1 mRNA [77] p50RhoGAP [78] Exportin-t XPOT Mature tRNAs [79] tRNA-attached ribozymes [80]  The down-regulation of the gene expression of proteins related to nucleocytoplasmic transport may explain the differentiationassociated suppression of the growth of HL-60 cells [27,28]. Another example of the involvement of importins in cell growth is the finding that RNAi-based down-regulation of the gene expression  [35]. Its precise role, however, is not clear at present.

The expression of importin-␣ subtypes is strictly regulated during the neural differentiation of mouse ES cells [20]. The level of importin-␣1 protein is high in undifferentiated ES cells, whereas the levels of importins-␣3 and -␣5 are low and undetectable, respectively (Table 2). The RNAi-based knockdown of importin-␣1, the overexpression of importin-␣5 or a combination thereof leads to neural differentiation. The transcription factors Oct3/4, SOX2 and Brn2 which play important roles in neural differentiation contain a single cNLS (Oct3/4 and Brn2) or two cNLSs (SOX2), and importin-␣1 is involved in the nuclear transport of Oct3/4, which has a critical role in the maintenance of an undifferentiated ES-cell state. A decrease in importin-␣1/Oct3/4 concomitant with the up-regulation of importin-␣5, which is involved in the nuclear transport of SOX and Brn2 appears to lead to neural differentiation. Thus, the coordinated regulation of importin subtypes and their transcription factors appears to have a key role in cell-fate determination.
Surprisingly, transgenic Imp-␣5 -/mice do not exhibit any obvious morphological or behavioural abnormalities [36]. Since the expression of importin-␣4 is markedly increased in the brains of these knockout mice, a compensative mechanism may cover the lack of an importin subtype in mammals. Supporting this notion, an in vitro transport assay has shown that both importin-␣5 and -␣4 can import Brn2, although with differences in efficiency [37]. Table 2), suggesting that the difference may be related to cell fate. The up-regulated gene expression of transportin-2 and Ran-binding protein 6 is noticeable and may be related to the nuclear entry of cardiac transcription factors such as Mef2C, Nkx2.5 and Gata4.

Germ cell maturation
Proteomic profiling of differentially expressed proteins in germinal vesicles and metaphase II arrested mouse oocytes has identified 12 proteins including importin-␣1 that migrated differently on electrophoresis in a two-dimensional gel [40]. Thus, post-translational modification appears to take place during the maturation of oocytes.
In spermatogenesis, the mRNA expression of individual importin-␣ isoforms is differentially regulated [41]. Importin-␣5 is expressed in differentiated spermatogonia through to the round spermatids in the adult mouse testis, suggesting its importance in mitotic and meiotic germ cells. The expression of importin-␣1 is very limited, as its mRNA is present in spermatocytes but absent once the spermatids begins to elongate. Importin-␣4 is expressed specifically in the mitotic germ cell populations, and importin-␣3 in pachytene spermatocytes. Thus, mammalian spermatogenesis appears to be a model useful for further examination of the roles and regulation of nucleocytoplasmic transport factors in cellular differentiation and development, and information derived therefrom may have relevance to reproductive medicine.

Muscle cell differentiation
The mouse muscle myoblast cell line C2C12 provides an excellent model for studying myogenesis in vitro and cell differentiation.
The RNA-binding protein HuR is critically involved in the formation of muscle fibres through its association with MyoD and myogenin mRNAs and is transported into the nucleus through a transportin-2-mediated pathway [45]. Transportin-2 is expressed in undifferentiated and differentiated C2C12 cells and transportin 1 appears to be expressed weakly only in mature myotubes. The involvement of transportin-2 in muscle cell differentiation has been demonstrated by an experiment in which RNAi-mediated depletion of transportin-2 expression lead to the expression of the myogenic transcription factors MyoD and myogenin. The disruption of the association between HuR and transportin-2 appears to be an important event leading to muscle cell differentiation.

Concluding remarks
Significant progress has been achieved in our understanding of the structure and function of nucleocytoplasmic transport factors including importins and exportins. Yet, information on which specific transport factors are expressed in which tissues and cells is still limited. Such information will be crucial to investigations aiming at human therapeutic applications. In addition, further studies should be done to see whether the results obtained from in vitro culture models will hold true in vivo as well.
Many trials are in progress as exemplified by the coupling of NLS peptides to DNA for gene therapy [11]