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In brief: January 2003

Nuclear export

Nuclear export of microRNA precursors.
Lund, E. et al.
Science 20 November 2003 (doi:10.1126/science.1090599)

Nuclear and cytoplasmic RNase-III-like endonucleases are involved in the biogenesis of microRNAs (miRNAs) in mammalian cells. Pre-miRNAs are generated in the nucleus by Drosha, but need to be processed further in the cytoplasm by Dicer. In this report, Kund et al. show that exportin-5 (Exp5), which binds minihelix-containing RNAs, stimulates pre-miRNA export in a RanGTP-dependent way. Efficient nuclear export requires pre-miRNAs to be correctly processed, and the binding is direct, as adaptor proteins did not enhance the Exp5–pre-miRNA association.

Cytoskeleton

Regulation of cell polarity and protrusion formation by targeting RhoA for degradation.
Wang, H.-R. et al.
Science 302, 1775–1779 (2003) | Article | PubMed |

Until now, ubiquitin ligases have not been reported to regulate cell shape, motility or polarity. But the authors of this paper show that Smurf1, a HECT-domain E3 ubiquitin ligase, regulates protrusive activity and polarity in fibroblasts. Small interfering RNA against Smurf1 decreases cell motility and restores a non-transformed morphology to tumour cells. Protein kinase C gamma , a component of the PAR6–PKC gamma complex, recruits Smurf1 to filopodia and lamellipodia, where it locally mediates Rho degradation by ubiquitylation. This probably inhibits inappropriate stress-fibre formation during remodelling of protrusions.

Autoimmunity

HSP70 promotes antigen-presenting cell function and converts T-cell tolerance to autoimmunity in vivo.
Millar, D. G. et al.
Nature Med. 16 November 2003 (doi:10.1038/nm962)

Here, Millar et al. show that administration of HSP70 increases the stimulatory capacity of antigen-presenting cells(APCs) in vivo. Mice engineered to express the lymphocytic choriomeningitis virus glycoprotein (LCMV-GP) in pancreatic beta -cells and in which CD8⁺ T cells are specific for LCMV-GP are tolerant when administered the immunodominant LCMV-GP-derived epitope gp33. HSP70 converts tolerance to autoimmunity when co-administered with gp33, rapidly inducing diabetes. HSP70 did not increase the expression of MHC class II molecules or CD80/CD86 by APCs, but its ability to induce diabetes was dependent on CD4⁺ T-cell help and CD40 expression by APCs. The authors, therefore, suggest that HSP70 partially activates APCs, but subsequent CD40 ligation is required for full activation and the onset of diabetes.

Gene regulation

A conserved signal-responsive sequence mediates activation-induced alternative splicing of CD45.
Rothrock, C. et al.
Mol. Cell 12, 1317–1324 (2003) | PubMed |

Alterative splicing generates the distinct CD45 isoforms expressed by naive and activated T cells. In this paper, Rothrock et al. identify a consensus sequence — the activation-responsive sequence (ARS) — that is required for the exclusion of exons 4, 5 and 6 from CD45 after stimulation with phorbol 12-myristate 13-acetate (PMA). The ARS within exon 4 was mapped and when compared with exons 5 and 6 for regions of sequence homology, a conserved imperfect repeat sequence was observed. ARS-homology regions were found and shown to mediate PMA-induced alternative splicing in other PMA-regulated genes, including cytotoxic T lymphocyte antigen 4 (CTLA4), Agrin (AGRN) and FYN. These studies indicate that a common pathway might regulate activation-induced alternative splicing of a large number of diverse genes.

HIV

Use of a small molecule CCR5 inhibitor in macaques to treat simian immunodeficiency virus infection or prevent simian–human immunodeficiency virus infection.
Veazey, R. S. et al.
J. Exp. Med. 198, 1551–1562 (2003) | Article | PubMed |

Absence of the chemokine receptor CCR5 protects against HIV infection, and therapies for HIV based on CCR5-specific antibodies are being developed. In this study, the effects of a CCR5-specific receptor agonist CMPD167 on established simian immunodeficiency virus (SIV) infection were investigated. CMPD167 was shown to have antiviral activity against SIVmac251 and SIVB670 replication in vivo, resulting in marked decreases in plasma viraemia in six rhesus macaques, which was maintained in three animals. Vaginal administration of gel-formulated CMPD167 reduced the extent of viraemia after infection with SHIV-162P4, but was unable to prevent vaginal transmission.

T-cell development

Critical roles for transcription factor GATA-3 in thymocyte development.
Pai, S. -Y.
Immunity 19, 863–875 (2003) | PubMed |

The zinc finger transcription factor GATA3 is expressed throughout thymocyte development, but its role in thymocyte differentiation is unknown. In this study, the authors generated mice in which GATA3 was deleted in the T-cell lineage at early and late stages of development. Absence of GATA3 expression in double-negative (DN) thymocytes resulted in reduced numbers of mature T cells and a partial arrest in development at the DN3 to DN4 transition, indicating a role for GATA3 at the beta -selection checkpoint. Deletion of GATA3 later, after beta -selection, showed that this transcription factor is also required at later stages of development in the generation of single-positive CD4⁺ thymocytes.

Transplantation

Pro-inflammatory functions of vascular endothelial growth factor in alloimmunity.
Reinders, M. E. J. et al.
J. Clin. Invest. 112, 1655–1665 (2003) | Article | PubMed |

In both experimental models and human transplantation, the expression of vascular endothelial growth factor (VEGF) is associated with allograft rejection, but the mechanism for this is unknown. VEGF is a potent angiogenic factor, but it is also associated with pro-inflammatory responses. These authors show that in a humanized SCID mouse model, the infiltration of human T cells into skin allografts is enhanced by VEGF-induced endothelial expression of monocyte chemoattractant protein 1 and interleukin-8. In synergy with interferon- gamma (IFN- gamma ), VEGF also enhanced endothelial expression of the T-cell chemoattractant IFN-inducible protein 10 (IP-10). In line with this, a VEGF-specific antibody inhibited T-cell infiltration and acute rejection of heart transplants in mice. Therefore, VEGF-induced leukocyte recruitment is an important component of graft rejection, and VEGF might be a therapeutic target after transplantation.

Therapeutics

Suppression of breast cancer by chemical modulation of vulnerable zinc fingers in estrogen receptor.
Wang, L. H. et al.
Nature Med. 14 Dec 2003 (doi:10.1038/nm969)

Breast cancer therapies that involve blocking ligand binding to the oestrogen receptor (ER) are limited, as the drugs used can activate the ER in some cancer cells. Wang et al. found that electrophilic agents that disrupt zinc fingers in the DNA-binding domain of the ER downregulate expression from oestrogen-responsive elements and inhibit the proliferation of breast tumour cells in a mouse xenograft model, indicating that these agents could be used as alternative treatments for ER-expressing breast cancers.

Tumour progression

Role of thymosin- beta ₄ in tumor metastasis and angiogenesis.
Cha, H. -J. , Jeong, M. -J. & Kleinman, H. K.
J. Natl Cancer Inst. 95, 1674–1680 (2003) | Article | PubMed |

Thymosin- beta ₄ expression is associated with increased metastatic potential in tumour cells, but the mechanisms underlying this are unclear. Cha et al. overexpressed thymosin- beta ₄ in tumour cells and saw increases in the numbers of blood vessels associated with tumours, and increased cell migration and expression of vascular endothelial growth factor (VEGF). So, thymosin- beta ₄ seems to promote tumour progression by stimulating angiogenesis — either directly or by increasing VEGF expression — and by promoting cell migration, which might be due to its actin-binding function.

Clinical trials

Farnesyltransferase inhibitor R115777 in myelodysplastic syndrome: clinical and biologic activities in the phase 1 setting.
Kurzrock, R. et al.
Blood 102, 4527–4534 (2003) | Article | PubMed |

Farnesylation is essential for the activity of several proteins, including the RAS oncoprotein. The farnesyltransferase inhibitor R115777 inhibits tumour growth in vitro and in animal models. Kurzrock et al. carried out Phase I clinical trials of R115777 in patients with myelodysplastic syndrome, a group of pro-leukaemic disorders in which RAS mutations are seen in $sim$ 25% of cases. Responses to R115777 were seen in 30% of patients, although only one-third of these had RAS mutations.

Tumour suppressors

Arf tumor suppressor promoter monitors latent oncogenic signals in vivo.
Zindy, F. et al.
Proc. Natl Acad. Sci. USA 100, 15930–15935 (2003) | Article | PubMed |

Activation of the tumour suppressor Arf is difficult to detect in vivo, probably because cells that express Arf die or undergo growth arrest. Zindy et al. made transgenic mice in which the Arf coding region was replaced by a green fluorescent protein (GFP) complementary DNA. They monitored GFP fluorescence to confirm that the Arf promoter is activated in response to a range of oncogenic signals.

Cell biology of the neuron

AMPA receptor tetramerization is mediated by Q/R editing.
Greger, I. H. et al.
Neuron 40, 763–774 (2003) | PubMed |

The channel properties and trafficking of AMPA ( alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors depend largely on their subunit composition. RNA editing of the GluR2 subunit at the 'Q/R site' controls trafficking of AMPA receptors from the endoplasmic reticulum to the synapse, and this study shows that RNA editing at this site also regulates tetramerization of AMPA receptor subunits. Edited GluR2 subunits are retained in the endoplasmic reticulum (ER) and do not readily tetramerize, whereas most unedited subunits are exported from the ER to the synapse and assemble into receptor tetramers.

Anticancer Drugs

A small-molecule antagonist of CXCR4 inhibits intracranial growth of primary brain tumors.
Rubin, J. B. et al.
Proc. Natl Acad. Sci. USA 100, 13513–13518 (2003). | Article | PubMed |

Malignant brain tumours are a major cause of cancer mortality in both adults and children. Rubin et al. show that antagonism of the chemokine receptor CXCR4 by the small molecule AMD 3100 inhibits the growth of brain tumours in vivo in mice. This finding could rapidly lead to clinical trials, as AMD 3100 is already known to be well tolerated in humans from studies of this agent as a potential anti-HIV treatment.