Kiichiro Tomoda

If it were possible to reprogram differentiated human somatic cells into a pluripotent state, patient-specific and disease-specific stem cells could be developed. Previous work generated induced pluripotent stem (iPS) cells capable of germline transmission from murine somatic cells by transducing 4 transcription factors: Oct3/4, Sox2, Klf4, and c-Myc. The investigators now report generating iPS cells from adult human dermal fibroblasts using the same 4 factors. The first step was to optimize retroviral transduction in human fibroblasts as well as conditions of culture. Amphotrophic retrovirus was utilized. Colonies resembling human embryonic stem (hES) cell colonies were observed about 25 days after sampling cells from the facial dermis of a 36-year-old Caucasian female. In general, the human iPS cells expressed hES cell-specific surface antigens rather than stage-specific embryonic antigen. The cells expressed many undifferentiated ES cell-marker genes and they exhibited high telomerase activity. The cells proliferated exponentially for at least 4 months. It proved possible for iPS cells to differentiate into 3 germ layers in vitro. The cells resembled hES cells with regard to morphology, proliferation, gene expression, and epigenetic status of pluripotent cell-specific genes. Among the cell types formed from directed differentiation of human iPS cells were dopaminergic neurons and cardiac myocytes. Injection of human iPS cells subcutaneously into immunodeficient mice was followed after 9 weeks by teratomas containing gut-like epithelial tissues, striated muscle, cartilage, neural tissues, and keratin-containing epidermal tissues. The human iPS clones were not a result of cross-contamination. Although human iPS cells are not identical to hES cells, they should find applications in regenerative medicine once safety issues are overcome.

The fifth component of the COP9 signalosome complex, Jab1/CSN5, directly binds to and induces specific down-regulation of the cyclin-dependent kinase inhibitor p27 (p27(Kip1)). Nuclear-cytoplasmic translocation plays an important role because leptomycin B (LMB), a chemical inhibitor of CRM1-dependent nuclear export, prevents p27 degradation mediated by Jab1/CSN5. Here we show that Jab1/CSN5 functions as an adaptor between p27 and CRM1 to induce nuclear export and subsequent degradation. Jab1/CSN5, but not p27, contains a typical leucine-rich nuclear export signal (NES) sequence conserved among different species, through which CRM1 bound to Jab1/CSN5 in an LMB-sensitive manner. Alteration of conserved leucine residues to alanine within Jab1/CSN5-NES abolished the interaction with CRM1 in vitro and impaired LMB-sensitive nuclear export and the ability to induce p27 breakdown in cultured cells. A Jab1/CSN5 truncation mutant lacking NES reversed p27 down-regulation induced by the full-length Jab1/CSN5, indicating that this mutant functions as a dominant negative (DN-Jab1). Introduction of DN-Jab1 into proliferating fibroblasts increased the level of p27 protein, thereby inducing growth arrest of the cells. Random mutagenesis analysis revealed that specific aspartic acid, leucine, and asparagine residues contained in the Jab1/CSN5-binding domain of p27 were required for interaction with Jab1/CSN5 and for down-regulation of p27. Glycerol gradient and cell fractionation experiments showed that at least two different forms of Jab1/CSN5-containing complexes existed within the cell. One is the conventional 450-kDa COP9 signalosome (CSN) complex located in the nucleus, and the other is much smaller (around 100-kDa), containing only a subset of CSN components (CSN4-8 but not CSN1-3), and mainly located in the cytoplasm. Treatment of cells with LMB greatly reduced the level of the smaller complex, suggesting that it originated from the CSN complex by nuclear export. Besides Jab1/CSN5, CSN3, -6, -7, and -8 were capable of inducing p27 down-regulation, when ectopically expressed. These results indicate that cytoplasmic shuttling regulated by Jab1/CSN5 and other CSN components may be a new pathway to control the intracellular abundance of the key cell cycle regulator.

Jab1 interacts with a variety of signaling molecules and regulates their stability in mammalian cells. As the fifth component of the COP9 signalosome (CSN) complex, Jab1 (CSN5) plays a central role in the deneddylation of the cullin subunit of the Skp1-Cullin-F box protein ubiquitin ligase complex. In addition, a CSN-independent function of Jab1 is suggested but is less well characterized. To elucidate the function of Jab1, we targeted the Jab1 locus by homologous recombination in mouse embryonic stem cells. Jab1-null embryos died soon after implantation. Jab1-/- embryonic cells, which lacked other CSN components, expressed higher levels of p27, p53, and cyclin E, resulting in impaired proliferation and accelerated apoptosis. Jab1 heterozygous mice were healthy and fertile but smaller than their wild-type littermates. Jab1+/- mouse embryonic fibroblast cells, in which the amount of Jab1-containing small subcomplex, but not that of CSN, was selectively reduced, proliferated poorly, showed an inefficient down-regulation of p27 during G1, and was delayed in the progression from G0 to S phase by 3 h compared with the wild-type cells. Most interestingly, in Jab1+/- mouse embryonic fibroblasts, the levels of cyclin E and deneddylated Cul1 were unchanged, and p53 was not induced. Thus, Jab1 controls cell cycle progression and cell survival by regulating multiple cell cycle signaling pathways.