Regulation of CDK4

Cyclin-dependent kinase (CDK)4 is a master integrator that couples mitogenic and antimitogenic extracellular signals with the cell cycle. It is also crucial for many oncogenic transformation processes. In this overview, we address various molecular features of CDK4 activation that are critical but remain poorly known or debated, including the regulation of its association with D-type cyclins, its subcellular location, its activating Thr172-phosphorylation and the roles of Cip/Kip CDK "inhibitors" in these processes. We have recently identified the T-loop phosphorylation of CDK4, but not of CDK6, as a determining target for cell cycle control by extracellular factors, indicating that CDK4-activating kinase(s) might have to be reconsidered.     

β-Catenin Inactivation Is a Pre-Requisite for Chick Retina Regeneration

In the present study we explored the role of β-catenin in mediating chick retina regeneration. The chick can regenerate its retina by activating stem/progenitor cells present in the ciliary margin (CM) of the eye or via transdifferentiation of the retinal pigmented epithelium (RPE). Both modes require fibroblast growth factor 2 (FGF2). We observed, by immunohistochemistry, dynamic changes of nuclear β-catenin in the CM and RPE after injury (retinectomy). β-catenin nuclear accumulation was transiently lost in cells of the CM in response to injury alone, while the loss of nuclear β-catenin was maintained as long as FGF2 was present. However, nuclear β-catenin positive cells remained in the RPE in response to injury and were BrdU-/p27+, suggesting that nuclear β-catenin prevents those cells from entering the cell cycle. If FGF2 is present, the RPE undergoes dedifferentiation and proliferation concomitant with loss of nuclear β-catenin. Moreover, retinectomy followed by disruption of active β-catenin by using a signaling inhibitor (XAV939) or over-expressing a dominant negative form of Lef-1 induces regeneration from both the CM and RPE in the absence of FGF2. Our results imply that β-catenin protects cells of the CM and RPE from entering the cell cycle in the developing eye, and specifically for the RPE during injury. Thus inactivation of β-catenin is a pre-requisite for chick retina regeneration.     

Targeted deletion of integrin-linked kinase reveals a role in T-cell chemotaxis and survival

Integrin-linked kinase (ILK) is a serine/threonine kinase that is important in cell-matrix interactions and cell signaling. To examine the role of ILK in leukocyte trafficking and survival, we generated T cell-specific ILK knockouts by breeding ILK(flox/flox) mice to transgenic mice expressing Cre recombinase under control of the Lck proximal promoter. Thymic T cells from Lck-Cre(+)/ILK(flox/flox) mice had a marked reduction (>95%) in ILK protein levels. Thymic cellularity was comparable in 3- to 4-week-old mice, but a threefold diminution of thymic T cells became evident by 6 to 8 weeks of age in the T cell-specific ILK knockout mice due to increased cell death of double-positive (DP) T cells. Analysis of peripheral T cells by quantitative PCR and by breeding Lck-Cre(+)/ILK(flox/flox) mice to a YFP-transgenic reporter strain demonstrated an approximate 20-fold enrichment of ILK-competent cells, suggesting these cells have a competitive advantage in trafficking to and/or survival in peripheral lymphatic organs. We explored mechanisms related to altered cell trafficking and survival that might explain the decreases in thymic cellularity and enrichment for ILK-competent cells in the spleen and lymph nodes. We observed a >50% reduction in chemotaxis of ILK-deficient T cells to the chemokines CXCL12 (stromal cell-derived factor [SDF]-1alpha) and CCL19 (macrophage inflammatory protein [MIP]-3beta), as well as enhanced apoptosis of ILK-deficient cells upon stress. Signaling studies in ILK-deficient T cells demonstrated diminished phosphorylation of Akt on the activating phosphorylation site, Ser 473, and a concordant decrease in Akt kinase activity following stimulation with the chemokine SDF-1. Rac1 activation was also markedly diminished in ILK-deficient T cells following chemokine stimulation. These data extend the role of ILK to immune-cell trafficking and survival via modulation of Akt- and Rac-dependent substrates, and have implications for cell recruitment in both homeostatic and pathological processes.     

Protein expression changes induced in murine peritoneal macrophages by Group B Streptococcus

Protein expression changes induced in thioglycolate‐elicited peritoneal murine macrophages (MΦ) by infection with type III Group B Streptococcus (GBS) are described. Proteins from control MΦ and MΦ incubated 2 h with live or heat‐inactivated GBS were separated by 2‐DE. Proteins whose expression was significantly different in infected MΦ, as compared with control cells, were identified by MS/MS analysis. Changes in the expression level of proteins involved in both positive and negative modulation of phagocytic functions, stress response and cell death were induced in MΦ by GBS infection. In particular, expression of enzymes playing a key role in production of reactive oxygen species was lowered in GBS‐infected MΦ. Significant alterations in the expression of some metabolic enzymes were also observed, most of the glycolytic and of the pentose‐cycle enzymes being down‐regulated in MΦ infected with live GBS. Finally, evidence was obtained that GBS infection affects the expression of enzymes or enzyme subunits involved in ATP synthesis and in adenine nucleotides interconversion processes.     

Enhancing effect of a protein from <Emphasis Type="Italic">Lonomia obliqua</Emphasis> hemolymph on recombinant protein production

Gene expression in animal cells allows large scale production of proteins used for either structure and function studies or therapeutic purposes. Maximizing recombinant protein production is necessary to optimize cell growth and protein expression. Some studies have demonstrated the presence of pharmacologically active substances in insect hemolymph. In this work, we have identified and purified a protein from Lonomia obliqua hemolymph able to increase the production of the rabies virus glycoprotein, expressed in Drosophila melanogaster S2 cells, by about 59%.     

Active form of Notch imposes T cell fate in human progenitor cells

The crucial role of Notch signaling in cell fate decisions in hematopoietic lineage and T lymphocyte development has been well established in mice. Overexpression of the intracellular domain of Notch mediates signal transduction of the protein. By retroviral transduction of this constitutively active truncated intracellular domain in human CD34+ umbilical cord blood progenitor cells, we were able to show that, in coculture with the stromal MS-5 cell line, depending on the cytokines added, the differentiation toward CD19+ B lymphocytes was blocked, the differentiation toward CD14+ monocytes was inhibited, and the differentiation toward CD56+ NK cells was favored. The number of CD7+cyCD3+ cells, a phenotype similar to T/NK progenitor cells, was also markedly increased. In fetal thymus organ culture, transduced CD34+ progenitor cells from umbilical cord blood cells or from thymus consistently generated more TCR-gammadelta T cells, whereas the other T cell subpopulations were largely unaffected. Interestingly, when injected in vivo in SCID-nonobese diabetic mice, the transduced cells generated ectopically human CD4+CD8+ TCR-alphabeta cells in the bone marrow, cells that are normally only present in the thymus, and lacked B cell differentiation potential. Our results show unequivocally that, in human, Notch signaling inhibits the monocyte and B cell fate, promotes the T cell fate, and alters the normal T cell differentiation pathway compatible with a pretumoral state.     

Expression and role of retinoic acid receptor alpha in lens regeneration

The role of retinoids in eye development has been well studied. Retinoids and their receptors regulate gene expression and morphogenesis of the eye. In this study, a highly specific antagonist of retinoic acid receptor (RAR)-alpha was used in an attempt to study its function in lens regeneration. It was found that this antagonist inhibited lens regeneration and lens fiber differentiation. It was also shown that RAR-alpha is expressed in the lens during the process of regeneration. These results indicate that different RAR might have unique as well as redundant effects and patterns of expression in the regenerating lens.