We report here that different cell stresses regulate the stability of cyclin D1 protein. Exposition of Granta 519 cells to osmotic shock, oxidative stress, and arsenite induced the post-transcriptional down-regulation of cyclin D1. In the case of osmotic shock, this effect was completely reversed by the addition of p38 SAPK2 -specific inhibitors (SB203580 or SB220025), indicating that this effect is dependent on p38 SAPK2 activity. Moreover, the use of proteasome inhibitors prevented this down-regulation. Thus, osmotic shock induces proteasomal degradation of cyclin D1 protein by a p38 SAPK2 -dependent pathway. The effect of p38 SAPK2 on cyclin D1 stability might be mediated by direct phosphorylation at specific sites. We found that p38 SAPK2 phosphorylates cyclin D1 in vitro at Thr 286 and that this phosphorylation triggers the ubiquitination of cyclin D1. These results link for the first time a stress-induced MAP kinase pathway to cyclin D1 protein stability, and they will help to understand the molecular mechanisms by which stress transduction pathways regulate the cell cycle machinery and take control over cell proliferation.Mammalian cell cycle progression depends on the sequential activation of different members of a family of serine-threonine kinases named cyclin-dependent kinases (CDKs). 1 The activity of these kinases is positively regulated by cyclin binding, and phosphorylation by the CDK-activating kinase. The activity is also modulated negatively by phosphorylation at specific residues of the CDKs and by the binding of CDK inhibitors (1-3). Progression through G 1 phase is controlled first by CDK4 and CDK6, which bind combinatorially to cyclins D1, D2, and D3; and later on by CDK2, which associates with cyclin E (4). During G 1 these complexes are responsible for the phosphorylation of different members of the pocket proteins family, which includes the retinoblastoma protein, p107, and p130 (5-8). The hyperphosphorylation of the pocket proteins leads to the transactivation of genes that are necessary for the onset and progression of DNA replication (9 -11).Quiescent cells contain low levels of D-type cyclins. After growth factor stimulation, their synthesis is induced, and then cyclin D1-CDK4/6 complexes can be formed during G 1 (12, 13). Cyclin D1 expression, assembly of cyclin D-CDK complexes, and their nuclear translocation require the activation of Ras, Raf1, MAP kinase kinase 1/2, ERKs, and the transcription factor c-Ets-2 (14 -17). The maintenance of active cyclin D1-CDK4/6 complexes requires persistent mitogenic signaling, and mitogen withdrawal cancels cyclin D1 synthesis and cyclin D1-CDK4 complexes rapidly dissipate (18). Cyclin D1 turnover is regulated by degradation, mediated by phosphorylation of a specific threonine residue (Thr 286 ) located near the carboxyl terminus. This phosphorylation promotes its polyubiquitination and subsequent degradation by the 26 S proteasome (19). Recently, an alternative mechanism of cyclin D1 ubiquitination, independent of Thr 286 phosphorylation, has be...

Cytokine secretion by T lymphocytes plays a central role in mounting adaptive immune responses. However, little is known about how newly synthesized cytokines, once produced, are routed within T cells and about the mechanisms involved in regulating their secretions. In this study, we investigated the role of cytoskeleton remodeling at the immunological synapse (IS) in cytokine secretion. We show that a key regulator of cytoskeleton remodeling, the Rho GTPase Cdc42, controls IFN-γ secretion by primary human CD4+ T lymphocytes. Surprisingly, microtubule organizing center polarity at the IS, which does not depend on Cdc42, is not required for cytokine secretion by T lymphocytes, whereas microtubule polymerization is required. In contrast, actin remodeling at the IS, which depends on Cdc42, controls the formation of the polymerized actin ring at the IS, the dynamic concentration of IFN-γ–containing vesicles inside this ring, and the secretion of these vesicles. These results reveal a previously unidentified role of Cdc42-dependent actin remodeling in cytokine exocytosis at the IS.

Stroke induces inflammation that can aggravate brain damage. This work examines whether interleukin-10 (IL-10) deficiency exacerbates inflammation and worsens the outcome of permanent middle cerebral artery occlusion (pMCAO). Expression of IL-10 and IL-10 receptor (IL-10R) increased after ischemia. From day 4, reactive astrocytes showed strong IL-10R immunoreactivity. Interleukin-10 knockout (IL-10 KO) mice kept in conventional housing showed more mortality after pMCAO than the wild type (WT). This effect was associated with the presence of signs of colitis in the IL-10 KO mice, suggesting that ongoing systemic inflammation was a confounding factor. In a pathogen-free environment, IL-10 deficiency slightly increased infarct volume and neurologic deficits. Induction of proinflammatory molecules in the IL-10 KO brain was similar to that in the WT 6 hours after ischemia, but was higher at day 4, while differences decreased at day 7. Deficiency of IL-10 promoted the presence of more mature phagocytic cells in the ischemic tissue, and enhanced the expression of M2 markers and the T-cell inhibitory molecule CTLA-4. These findings agree with a role of IL-10 in attenuating local inflammatory reactions, but do not support an essential function of IL-10 in lesion resolution. Upregulation of alternative immunosuppressive molecules after brain ischemia can compensate, at least in part, the absence of IL-10.

The presented results point to a critical involvement of S1P and its signalling axis in the pathogenesis of hypertension. Specifically, SphK2 evolves as key player in immune cell trafficking and vascular dysfunction contributing to the development of overt hypertension.

␤-Catenin and plakoglobin are two related armadillo proteins necessary for the establishment of adhesion junctions and desmosomes. Moreover, ␤-catenin can also act as a transcriptional co-activator through its interaction with the members of Tcf/LEF-1 transcriptional factor family. We show here that Tcf-4 can be phosphorylated in vitro by protein kinase CK2 stoichiometrically in amino acids Ser-58 -Ser-59 -Ser-60. Phosphorylation of these residues does not modify the interaction of Tcf-4 with ␤-catenin but reduces its association to plakoglobin. The binding sites of Tcf-4 for these two proteins were compared; whereas ␤-catenin requires the N-terminal first 50 amino acids, plakoglobin interacts mainly with residues 51-80. Tcf-4-(51-80) binds plakoglobin in the region of armadillo repeats 1-6. Ternary complexes composed by ␤-catenin/Tcf-4/plakoglobin could be detected in vitro, demonstrating that simultaneous binding of the two armadillo proteins to Tcf-4 is possible. Experiments performed using a Tcf-4 mutant with decreased interaction to plakoglobin demonstrated that binding to this protein negatively affected the transcriptional activity of Tcf-4. These results indicate that Tcf-4 contains two different sites for binding of ␤-catenin and plakoglobin, and the interaction of the latter hinders the transcriptional activity of the complex.

Material

Brain proteins are detected in the cerebrospinal fluid (CSF) and blood of stroke patients and their concentration is related to the extent of brain damage. Antibodies against brain antigens develop after stroke, suggesting a humoral immune response to the brain injury. Furthermore, induced immune tolerance is beneficial in animal models of cerebral ischemia. The presence of circulating T cells sensitized against brain antigens, and antigen presenting cells (APCs) carrying brain antigens in draining lymphoid tissue of stroke patients support the notion that stroke might induce antigen-specific immune responses. After stroke, brain proteins that are normally hidden from the periphery, inflammatory mediators, and danger signals can exit the brain through several efflux routes. They can reach the blood after leaking out of the damaged blood-brain barrier (BBB) or following the drainage of interstitial fluid to the dural venous sinus, or reach the cervical lymph nodes through the nasal lymphatics following CSF drainage along the arachnoid sheaths of nerves across the nasal submucosa. The route and mode of access of brain antigens to lymphoid tissue could influence the type of response. Central and peripheral tolerance prevents autoimmunity, but the actual mechanisms of tolerance to brain antigens released into the periphery in the presence of inflammation, danger signals, and APCs, are not fully characterized. Stroke does not systematically trigger autoimmunity, but under certain circumstances, such as pronounced systemic inflammation or infection, autoreactive T cells could escape the tolerance controls. Further investigation is needed to elucidate whether antigen-specific immune events could underlie neurological complications impairing recovery from stroke.

Patients presenting with genetic deficiencies in IFNGR1, IFNGR2, IL-12B, and IL-12RB1 display increased susceptibility to mycobacterial infections. We analyzed in this group of patients the cross-talk between human CD4+ T lymphocytes and dendritic cells (DCs) that leads to maturation of DC into producers of bioactive IL-12 and to activation of T cells into IFN-γ producers. We found that this cross-talk is defective in all patients from this group. Unraveling the mechanisms underlying this deficiency, we showed that IL-12 signaling in T cells is required to induce expression of costimulatory molecules and secretion of IL-12 by DCs and that IFNGR expression is required on both DCs and CD4+ T cells to induce IL-12 secretion by DCs. These data suggest that CD4+ T cell-mediated activation of DCs plays a critical role in the defense against mycobacterial infections in humans.