Apoptosis-inducing factor 1 mediates Vibrio splendidus-induced coelomocyte apoptosis via importin ß dependent nuclear translocation in Apostichopus japonicus.

As is well known, apoptosis is an important form of immune response and immune regulation, particularly playing a crucial role in combating microbial infections. Apoptosis-inducing factor 1 (AIF-1) is essential for apoptosis to induce chromatin condensation and DNA fragmentation via a caspase-independent pathway. The nuclear translocation of AIF-1 is a key step in apoptosis but the molecular mechanism is still unclear. In this study, the homologous gene of AIF-1, named AjAIF-1, was cloned and identified in Apostichopus japonicus. The mRNA expression of AjAIF-1 was significantly increased by 46.63-fold after Vibrio splendidus challenge. Silencing of AjAIF-1 was found to significantly inhibit coelomocyte apoptosis because the apoptosis rate of coelomocyte decreased by 0.62-fold lower compared with the control group. AjAIF-1 was able to promote coelomocyte apoptosis through nuclear translocation under the V. splendidus challenge. Moreover, AjAIF-1 and Ajimportin ß were mainly co-localized around the nucleus in vivo and silencing Ajimportin ß significantly inhibited the nuclear translocation of AjAIF-1 and suppressed coelomocyte apoptosis by 0.64-fold compared with control. In summary, nuclear translocation of AjAIF-1 will likely mediate coelomocyte apoptosis through an importin ß-dependent pathway in sea cucumber.

WW domains form a folded type of nuclear localization signal to guide YAP1 nuclear import.

The nuclear translocation of YAP1 is significantly implicated in the proliferation, stemness, and metastasis of cancer cells. Although the molecular basis underlying YAP1 subcellular distribution has been extensively explored, it remains to be elucidated how the nuclear localization signal guides YAP1 to pass through the nuclear pore complex. Here, we define a globular type of nuclear localization signal composed of folded WW domains, named as WW-NLS. It directs YAP1 nuclear import through the heterodimeric nuclear transport receptors KPNA-KPNB1, bypassing the canonical nuclear localization signal that has been well documented in KPNA/KPNB1-mediated nuclear import. Strikingly, competitive interference with the function of the WW-NLS significantly attenuates YAP1 nuclear translocation and damages stemness gene activation and sphere formation in malignant breast cancer cells. Our findings elucidate a novel globular type of nuclear localization signal to facilitate nuclear entry of WW-containing proteins including YAP1.

Bioinformatics analysis proposes a possible role for long noncoding RNA MIR17HG in retinoblastoma.

BACKGROUND: Retinoblastoma (RB) is the most common prevalent intraocular malignancy among infants and children, particularly in underdeveloped countries. With advancements in genomics and transcriptomics, noncoding RNAs have been increasingly utilized to investigate the molecular pathology of diverse diseases. AIMS: This study aims to establish the competing endogenous RNAs network associated with RB, analyse the function of mRNAs and lncRNAs, and finds the relevant regulatory network. METHODS AND RESULTS: This study establishes a network of competing endogenous RNAs by Spearman correlation analysis and prediction based on RB patients and healthy children. Enrichment analyzes based on Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes are conducted to analyze the potential biological functions of lncRNA and mRNA networks. Weighted gene co-expression network analysis (WGCNA) is employed to identify gene cluster modules exhibiting the strongest correlation with RB. The results indicate a significant correlation between the lncRNA MIR17HG (R = .73, p = .02) and the RB phenotype. ceRNA networks reveal downstream miRNAs (hsa-mir-425-5p and hsa-mir455-5p) and mRNAs (MDM2, IPO11, and ITGA1) associated with MIR17Hg. As an inhibitor of the p53 signaling pathway, MDM2 can suppress the development of RB. CONCLUSION: In conclusion, lncRNAs play a role in RB, and the MIR17HG/hsa-mir-425-5p/MDM2 pathway may contribute to RB development by inhibiting the p53 signaling pathway.

Stabilization of KPNB1 by deubiquitinase USP7 promotes glioblastoma progression through the YBX1-NLGN3 axis.

BACKGROUND: Glioblastoma (GBM) is the most common malignant tumor of the central nervous system. It is an aggressive tumor characterized by rapid proliferation, diffuse tumor morphology, and poor prognosis. Unfortunately, current treatments, such as surgery, radiotherapy, and chemotherapy, are unable to achieve good outcomes. Therefore, there is an urgent need to explore new treatment targets. A detailed mechanistic exploration of the role of the nuclear pore transporter KPNB1 in GBM is lacking. This study demonstrated that KPNB1 regulated GBM progression through a transcription factor YBX1 to promote the expression of post-protrusion membrane protein NLGN3. This regulation was mediated by the deubiquitinating enzyme USP7. METHODS: A tissue microarray was used to measure the expression of KPNB1 and USP7 in glioma tissues. The effects of KPNB1 knockdown on the tumorigenic properties of glioma cells were characterized by colony formation assays, Transwell migration assay, EdU proliferation assays, CCK-8 viability assays, and apoptosis analysis using flow cytometry. Transcriptome sequencing identified NLGN3 as a downstream molecule that is regulated by KPNB1. Mass spectrometry and immunoprecipitation were performed to analyze the potential interaction between KPNB1 and YBX1. Moreover, the nuclear translocation of YBX1 was determined with nuclear-cytoplasmic fractionation and immunofluorescence staining, and chromatin immunoprecipitation assays were conducted to study DNA binding with YBX1. Ubiquitination assays were performed to determine the effects of USP7 on KPNB1 stability. The intracranial orthotopic tumor model was used to detect the efficacy in vivo. RESULTS: In this study, we found that the nuclear receptor KPNB1 was highly expressed in GBM and could mediate the nuclear translocation of macromolecules to promote GBM progression. Knockdown of KPNB1 inhibited the progression of GBM, both in vitro and in vivo. In addition, we found that KPNB1 could regulate the downstream expression of Neuroligin-3 (NLGN3) by mediating the nuclear import of transcription factor YBX1, which could bind to the NLGN3 promoter. NLGN3 was necessary and sufficient to promote glioma cell growth. Furthermore, we found that deubiquitinase USP7 played a critical role in stabilizing KPNB1 through deubiquitination. Knockdown of USP7 expression or inhibition of its activity could effectively impair GBM progression. In vivo experiments also demonstrated the promoting effects of USP7, KPNB1, and NLGN3 on GBM progression. Overall, our results suggested that KPNB1 stability was enhanced by USP7-mediated deubiquitination, and the overexpression of KPNB1 could promote GBM progression via the nuclear translocation of YBX1 and the subsequent increase in NLGN3 expression. CONCLUSION: This study identified a novel and targetable USP7/KPNB1/YBX1/NLGN3 signaling axis in GBM cells.

Yeast 26S proteasome nuclear import is coupled to nucleus-specific degradation of the karyopherin adaptor protein Sts1.

In eukaryotes, the ubiquitin-proteasome system is an essential pathway for protein degradation and cellular homeostasis. 26S proteasomes concentrate in the nucleus of budding yeast Saccharomyces cerevisiae due to the essential import adaptor protein Sts1 and the karyopherin-α protein Srp1. Here, we show that Sts1 facilitates proteasome nuclear import by recruiting proteasomes to the karyopherin-α/ß heterodimer. Following nuclear transport, the karyopherin proteins are likely separated from Sts1 through interaction with RanGTP in the nucleus. RanGTP-induced release of Sts1 from the karyopherin proteins initiates Sts1 proteasomal degradation in vitro. Sts1 undergoes karyopherin-mediated nuclear import in the absence of proteasome interaction, but Sts1 degradation in vivo is only observed when proteasomes successfully localize to the nucleus. Sts1 appears to function as a proteasome import factor during exponential growth only, as it is not found in proteasome storage granules (PSGs) during prolonged glucose starvation, nor does it appear to contribute to the rapid nuclear reimport of proteasomes following glucose refeeding and PSG dissipation. We propose that Sts1 acts as a single-turnover proteasome nuclear import factor by recruiting karyopherins for transport and undergoing subsequent RanGTP-initiated ubiquitin-independent proteasomal degradation in the nucleus.

Proteomic and yeast 2-hybrid screens to identify PTEN binding partners.

PTEN is a phosphoinositide lipid phosphatase and an important tumour suppressor protein. PTEN function is reduced or lost in around a third of all human cancers through diverse mechanisms, from gene deletion to changes in the function of proteins which regulate PTEN through direct protein binding. Here we present data from SILAC (Stable Isotope Labelling by Amino acids in Cell culture) proteomic screens to identify proteins which bind to PTEN. These experiments using untransformed epithelial cells and glioma cells identified several novel candidate proteins in addition to many previously identified PTEN binding partners and many proteins which are recognised as common false positives using these methods. From subsequent co-expression pull-down experiments we provide further evidence supporting the physical interaction of PTEN with MMP1, Myosin 18A and SHROOM3. We also performed yeast two-hybrid screens which identify the previously recognised PTEN binding partner MSP58 in addition to the nuclear import export receptor TNPO3. These experiments identify several novel candidate binding partners of PTEN and provide further data addressing the set of proteins that interact with this important tumour suppressor.

Human RTEL1 Interacts with KPNB1 (Importin ß) and NUP153 and Connects Nuclear Import to Nuclear Envelope Stability in S-Phase.

Regulator of TElomere Length Helicase 1 (RTEL1) is a helicase required for telomere maintenance and genome replication and repair. RTEL1 has been previously shown to participate in the nuclear export of small nuclear RNAs. Here we show that RTEL1 deficiency leads to a nuclear envelope destabilization exclusively in cells entering S-phase and in direct connection to origin firing. We discovered that inhibiting protein import also leads to similar, albeit non-cell cycle-related, nuclear envelope disruptions. Remarkably, overexpression of wild-type RTEL1, or of its C-terminal part lacking the helicase domain, protects cells against nuclear envelope anomalies mediated by protein import inhibition. We identified distinct domains in the C-terminus of RTEL1 essential for the interaction with KPNB1 (importin ß) and NUP153, respectively, and we demonstrated that, on its own, the latter domain can promote the dynamic nuclear internalization of peptides that freely diffuse through the nuclear pore. Consistent with putative functions exerted in protein import, RTEL1 can be visualized on both sides of the nuclear pore using high-resolution microscopy. In all, our work points to an unanticipated, helicase-independent, role of RTEL1 in connecting both nucleocytoplasmic trafficking and nuclear envelope integrity to genome replication initiation in S-phase.

Structural convergence endows nuclear transport receptor Kap114p with a transcriptional repressor function toward TATA-binding protein.

The transcription factor TATA-box binding protein (TBP) modulates gene expression in nuclei. This process requires the involvement of nuclear transport receptors, collectively termed karyopherin-ß (Kap-ß) in yeast, and various regulatory factors. In previous studies we showed that Kap114p, a Kap-ß that mediates nuclear import of yeast TBP (yTBP), modulates yTBP-dependent transcription. However, how Kap114p associates with yTBP to exert its multifaceted functions has remained elusive. Here, we employ single-particle cryo-electron microscopy to determine the structure of Kap114p in complex with the core domain of yTBP (yTBPC). Remarkably, Kap114p wraps around the yTBPC N-terminal lobe, revealing a structure resembling transcriptional regulators in complex with TBP, suggesting convergent evolution of the two protein groups for a common function. We further demonstrate that Kap114p sequesters yTBP away from promoters, preventing a collapse of yTBP dynamics required for yeast responses to environmental stress. Hence, we demonstrate that nuclear transport receptors represent critical elements of the transcriptional regulatory network.

Defective recognition of a nonclassical nuclear localization signal in neurodevelopmental disorders.

In this issue of Structure, Gonzalez et al. present the cryo-EM structure of Karyopherin-ß2 bound to the proline-tyrosine nuclear localization signal (PY-NLS) of heterogeneous nuclear ribonucleoprotein H2 (HNRNPH2). The structure advances our understanding of not only the diversity of PY-NLSs but also the pathogenic mechanisms arising from HNRNPH2 variants.

Structure of the Hepatitis B virus capsid quasi-6-fold with a trapped C-terminal domain reveals capsid movements associated with domain exit.

Many viruses undergo transient conformational change to surveil their environments for receptors and host factors. In Hepatitis B virus (HBV) infection, after the virus enters the cell, it is transported to the nucleus by interaction of the HBV capsid with an importin α/ß complex. The interaction between virus and importins is mediated by nuclear localization signals on the capsid protein's C-terminal domain (CTD). However, CTDs are located inside the capsid. In this study, we asked where does a CTD exit the capsid, are all quasi-equivalent CTDs created equal, and does the capsid structure deform to facilitate CTD egress from the capsid? Here, we used Impß as a tool to trap transiently exposed CTDs and examined this complex by cryo-electron microscopy. We examined an asymmetric reconstruction of a T = 4 icosahedral capsid and a focused reconstruction of a quasi-6-fold vertex (3.8 and 4.0 Å resolution, respectively). Both approaches showed that a subset of CTDs extended through a pore in the center of the quasi-6-fold complex. CTD egress was accompanied by enlargement of the pore and subtle changes in quaternary and tertiary structure of the quasi-6-fold. When compared to molecular dynamics simulations, structural changes were within the normal range of capsid flexibility. Although pore diameter was enlarged in the Impß-bound reconstruction, simulations indicate that CTD egress does not exclusively depend on enlarged pores. In summary, we find that HBV surveillance of its environment by transient exposure of its CTD requires only modest conformational change of the capsid.

KPNB1-mediated nuclear import in cancer.

Dysregulation of nucleocytoplasmic shuttling impairs cellular homeostasis and promotes cancer development. KPNB1 is a member of karyopherin ß family, mediating the transportation of proteins from the cytoplasm to the nucleus. In a variety of cancers, the expression of KPNB1 is upregulated to facilitate tumor growth and progression. Both downregulation of KPNB1 level and inhibition of KPNB1 activity prevent the entry of cancer-related transcription factors into the nucleus, subsequently suppressing the proliferation and metastasis of cancer cells. Currently, five KPNB1 inhibitors have been reported and exhibited good efficacy against cancer. This paper provides an overview of the role and mechanism of KPNB1 in different cancers and KPNB1-targeted anticancer compounds which hold promise for the future.

Regulation of FLC nuclear import by coordinated action of the NUP62-subcomplex and importin ß SAD2.

Flowering locus C (FLC) is a central transcriptional repressor that controls flowering time. However, how FLC is imported into the nucleus is unknown. Here, we report that Arabidopsis nucleoporins 62 (NUP62), NUP58, and NUP54 composed NUP62-subcomplex modulates FLC nuclear import during floral transition in an importin α-independent manner, via direct interaction. NUP62 recruits FLC to the cytoplasmic filaments and imports it into the nucleus through the NUP62-subcomplex composed central channel. Importin ß supersensitive to ABA and drought 2 (SAD2), a carrier protein, is critical for FLC nuclear import and flower transition, which facilitates FLC import into the nucleus mainly through the NUP62-subcomplex. Proteomics, RNA-seq, and cell biological analyses indicate that the NUP62-subcomplex mainly mediates the nuclear import of cargos with unconventional nuclear localization sequences (NLSs), such as FLC. Our findings illustrate the mechanisms of the NUP62-subcomplex and SAD2 on FLC nuclear import process and floral transition, and provide insights into the role of NUP62-subcomplex and SAD2 in protein nucleocytoplasmic transport in plants.

Toxoplasma Gondii Importin α Shows Weak Auto-Inhibition.

Importin α is a nuclear transporter that binds to nuclear localization signals (NLSs), consisting of 7-20 positively charged amino acids found within cargo proteins. In addition to cargo binding, intramolecular interactions also occur within the importin α protein due to binding between the importin ß-binding (IBB) domain and the NLS-binding sites, a phenomenon called auto-inhibition. The interactions causing auto-inhibition are driven by a stretch of basic residues, similar to an NLS, in the IBB domain. Consistent with this, importin α proteins that do not have some of these basic residues lack auto-inhibition; a naturally occurring example of such a protein is found in the apicomplexan parasite Plasmodium falciparum. In this report, we show that importin α from another apicomplexan parasite, Toxoplasma gondii, harbors basic residues (KKR) in the IBB domain and exhibits auto-inhibition. This protein has a long, unstructured hinge motif (between the IBB domain and the NLS-binding sites) that does not contribute to auto-inhibition. However, the IBB domain may have a higher propensity to form an α-helical structure, positioning the wild-type KKR motif in an orientation that results in weaker interactions with the NLS-binding site than a KRR mutant. We conclude that the importin α protein from T. gondii shows auto-inhibition, exhibiting a different phenotype from that of P. falciparum importin α. However, our data indicate that T. gondii importin α may have a low strength of auto-inhibition. We hypothesize that low levels of auto-inhibition may confer an advantage to these important human pathogens.

A new Karyopherin-ß2 binding PY-NLS epitope of HNRNPH2 linked to neurodevelopmental disorders.

The HNRNPH2 proline-tyrosine nuclear localization signal (PY-NLS) is mutated in HNRNPH2-related X-linked neurodevelopmental disorder, causing the normally nuclear HNRNPH2 to accumulate in the cytoplasm. We solved the cryoelectron microscopy (cryo-EM) structure of Karyopherin-ß2/Transportin-1 bound to the HNRNPH2 PY-NLS to understand importin-NLS recognition and disruption in disease. HNRNPH2 206RPGPY210 is a typical R-X2-4-P-Y motif comprising PY-NLS epitopes 2 and 3, followed by an additional Karyopherin-ß2-binding epitope, we term epitope 4, at residues 211DRP213; no density is present for PY-NLS epitope 1. Disease variant mutations at epitopes 2-4 impair Karyopherin-ß2 binding and cause aberrant cytoplasmic accumulation in cells, emphasizing the role of nuclear import defect in disease. Sequence/structure analysis suggests that strong PY-NLS epitopes 4 are rare and thus far limited to close paralogs of HNRNPH2, HNRNPH1, and HNRNPF. Epitope 4-binidng hotspot Karyopherin-ß2 W373 corresponds to close paralog Karyopherin-ß2b/Transportin-2 W370, a pathological variant site in neurodevelopmental abnormalities, suggesting that Karyopherin-ß2b/Transportin-2-HNRNPH2/H1/F interactions may be compromised in the abnormalities.

Long noncoding RNA ARTA controls ABA response through MYB7 nuclear trafficking in Arabidopsis.

In eukaryotes, transcription factors are a crucial element in the regulation of gene expression, and nuclear translocation is the key to the function of transcription factors. Here, we show that the long intergenic noncoding RNA ARTA interacts with an importin ß-like protein, SAD2, through a long noncoding RNA-binding region embedded in the carboxyl terminal, and then it blocks the import of the transcription factor MYB7 into the nucleus. Abscisic acid (ABA)-induced ARTA expression can positively regulate ABI5 expression by fine-tuning MYB7 nuclear trafficking. Therefore, the mutation of arta represses ABI5 expression, resulting in desensitization to ABA, thereby reducing Arabidopsis drought tolerance. Our results demonstrate that lncRNA can hijack a nuclear trafficking receptor to modulate the nuclear import of a transcription factor during plant responses to environmental stimuli.

MicroRNA-130a-3p impedes the progression of papillary thyroid carcinoma through downregulation of KPNB1 by targeting PSME3.

BACKGROUND: Papillary thyroid carcinoma (PTC) is the main type of thyroid cancer (THCA). Despite the good prognosis, some PTC patients may deteriorate into more aggressive disease, leading to poor survival. Our study aimed to explore the role of microRNA (miR)-130a-3p in regulating PTC. METHODS: After transfection with miR-130a-3p-mimic, OE-PSME3, or miR-130a-3p-mimic + OE-KPNB1 in PTC cells (TPC-1), CCK-8, Transwell, scratch, and flow cytometry experiments were performed to analyze TPC-1 cell proliferation, invasion, migration, and apoptosis. Western blotting was used to detect proliferation or invasion-related protein markers (PCNA, E-cadherin, and N-cadherin). The RNA22 database, dual-luciferase reporter assay, and RNA pull-down assay were applied for the prediction and verification of the binding site between miR-130a-3p and PSME3. Pan-cancer software identified a positive correlation between PSME3 and KPNB1 in THCA. Co-immunoprecipitation was utilized to verify the interaction of PSME3 with KPNB1. Nude mice were transplanted with TPC-1 cells overexpressing miR-130a-3p. The tumors were isolated for detection of the expression of miR-130a-3p, PSME3, KPNB1, Ki-67, and CD31. RESULTS: miR-130a-3p was lowly expressed in PTC cell lines. Upregulation of miR-130a-3p repressed the expression of PSME3 and KPNB1 and reduced the malignancy of TPC-1 cells in vitro, shown by inhibited cell proliferation, invasion, migration, and the expression of PCNA and N-cadherin. Also, overexpressed miR-130a-3p inhibited the growth of xenograft tumors in nude mice. miR-130a-3p bound to PSME3 which interacted with KPNB1. CONCLUSION: miR-130a-3p impedes the progression of PTC by downregulating PSME3/KPNB1.

IRF5-TNOP3 polymorphisms are associated with elite control of HIV infection: A retrospective study.

IRF5-TNPO3 polymorphisms have previously been related to immune response, and TNPO3 plays a role in human immunodeficiency virus (HIV)-1 infection after nuclear import. Therefore, we analyzed the genetic association between IRF5-TNPO3 polymorphisms and the HIV elite control in long-term nonprogressors (LTNPs). We performed a retrospective cohort study on 183 LTNPs, who were antiretroviral therapy-naïve with CD4+ ≥ 500 cells/mm3 , viral load ≤10 000 copies/mL, and asymptomatic over 10 years after HIV seroconversion. The primary outcome variable was HIV elite control (undetectable viral load in at least 90% of the measurements for at least 1 year). Seven IRF5-TNPO3 polymorphisms were genotyped using Agena Bioscience's MassARRAY platform. We found a significant association between specific IRF5-TNPO3 genotypes and HIV elite control: rs2004640 TT (aOR = 2.05; p = 0.041), rs10954213 AA (aOR = 1.95; p = 0.035), rs2280714 TT (aOR = 2.02; p = 0.031), and rs10279821 CC (aOR = 2.12; p = 0.017). We also found a significant association between IRF5-TNPO3 haplotype TATC composed of the favorable significant polymorphisms (rs2004640, rs10954213, rs2280714, and rs10279821) and the HIV elite control (aOR = 1.59; p = 0.048). IRF5-TNPO3 rs2004640, rs10954213, rs2280714, and rs10279821 polymorphisms were related to HIV elite control in LTNPs. Our data provide new knowledge about the impact of IRF5-TNPO3 polymorphisms on HIV pathogenesis to understand the phenomenon of natural HIV control.

Progress in the study of parvovirus entry pathway.

A group of DNA viruses called parvoviruses that have significant effects on cancer therapy and genetic engineering applications. After passing through the cell membrane to reach the cytosol, it moves along the microtubule toward the nuclear membrane. The nuclear localization signal (NLS) is recognized by importin-beta (impß) and other proteins from the complex outside the nuclear membrane and binds to enter the nucleus via the nuclear pore complex (NPC). There are two main pathways for viruses to enter the nucleus. The classical pathway is through the interaction of imp α and impß with NLS via NPC. The other is the NPC mediated by the combination of impß and it. While the capsid is introduced into the nucleus through classical nuclear transduction, there is also a transient nuclear membrane dissolution leading to passive transport into the nucleus, which has been proposed in recent years. This article mainly discusses several nuclear entry pathways and related proteins, providing a reference for subsequent research on viral entry pathways.

Augmin is a Ran-regulated spindle assembly factor.

Mitotic spindles are composed of microtubules (MTs) that must nucleate at the right place and time. Ran regulates this process by directly controlling the release of spindle assembly factors (SAFs) from nucleocytoplasmic shuttle proteins importin-αß and subsequently forms a biochemical gradient of SAFs localized around chromosomes. The majority of spindle MTs are generated by branching MT nucleation, which has been shown to require an eight-subunit protein complex known as augmin. In Xenopus laevis, Ran can control branching through a canonical SAF, TPX2, which is nonessential in Drosophila melanogaster embryos and HeLa cells. Thus, how Ran regulates branching MT nucleation when TPX2 is not required remains unknown. Here, we use in vitro pulldowns and total internal reflection fluorescence microscopy to show that augmin is a Ran-regulated SAF. We demonstrate that augmin directly interacts with both importin-α and importin-ß through two nuclear localization sequences on the Haus8 subunit, which overlap with the MT-binding site. Moreover, we show that Ran controls localization of augmin to MTs in both Xenopus egg extract and in vitro. Our results demonstrate that RanGTP directly regulates augmin, which establishes a new way by which Ran controls branching MT nucleation and spindle assembly both in the absence and presence of TPX2.

Activin and BMP Signalling in Human Testicular Cancer Cell Lines, and a Role for the Nucleocytoplasmic Transport Protein Importin-5 in Their Crosstalk.

Testicular germ cell tumours (TGCTs) are the most common malignancy in young men. Originating from foetal testicular germ cells that fail to differentiate correctly, TGCTs appear after puberty as germ cell neoplasia in situ cells that transform through unknown mechanisms into distinct seminoma and non-seminoma tumour types. A balance between activin and BMP signalling may influence TGCT emergence and progression, and we investigated this using human cell line models of seminoma (TCam-2) and non-seminoma (NT2/D1). Activin A- and BMP4-regulated transcripts measured at 6 h post-treatment by RNA-sequencing revealed fewer altered transcripts in TCam-2 cells but a greater responsiveness to activin A, while BMP4 altered more transcripts in NT2/D1 cells. Activin significantly elevated transcripts linked to pluripotency, cancer, TGF-ß, Notch, p53, and Hippo signalling in both lines, whereas BMP4 altered TGF-ß, pluripotency, Hippo and Wnt signalling components. Dose-dependent antagonism of BMP4 signalling by activin A in TCam-2 cells demonstrated signalling crosstalk between these two TGF-ß superfamily arms. Levels of the nuclear transport protein, IPO5, implicated in BMP4 and WNT signalling, are highly regulated in the foetal mouse germline. IPO5 knockdown in TCam-2 cells using siRNA blunted BMP4-induced transcript changes, indicating that IPO5 levels could determine TGF-ß signalling pathway outcomes in TGCTs.