Stress inoculation modeled in mice.

Stress inoculation entails intermittent exposure to mildly stressful situations that present opportunities to learn, practice and improve coping in the context of exposure psychotherapies and resiliency training. Here we investigate behavioral and hormonal aspects of stress inoculation modeled in mice. Mice randomized to stress inoculation or a control treatment condition were assessed for corticosterone stress hormone responses and behavior during open-field, object-exploration and tail-suspension tests. Stress inoculation training sessions that acutely increased plasma levels of corticosterone diminished subsequent immobility as a measure of behavioral despair on tail-suspension tests. Stress inoculation also decreased subsequent freezing in the open field despite comparable levels of thigmotaxis in mice from both treatment conditions. Stress inoculation subsequently decreased novel-object exploration latencies and reduced corticosterone responses to repeated restraint. These results demonstrate that stress inoculation acutely stimulates glucocorticoid signaling and then enhances subsequent indications of active coping behavior in mice. Unlike mouse models that screen for the absence of vulnerability to stress or presence of traits that occur in resilient individuals, stress inoculation training reflects an experience-dependent learning-like process that resembles interventions designed to build resilience in humans. Mouse models of stress inoculation may provide novel insights for new preventive strategies or therapeutic treatments of human psychiatric disorders that are triggered and exacerbated by stressful life events.

A rho exchange factor mediates thrombin and Galpha(12)-induced cytoskeletal responses.

Thrombin induces astrocytoma cell rounding through a Rho-dependent pathway (Majumdar, M., Seasholtz, T. M., Goldstein, D., de Lanerolle, P., and Brown, J. H. (1998) J. Biol. Chem. 273, 10099-10106). The involvement of the G(12) family of G proteins and the role of specific Rho exchange factors in transducing signals from the thrombin receptor to Rho-dependent cytoskeletal responses was examined. Microinjection of cDNAs for activated Galpha(12) or Galpha(13) induced cell rounding, and antibodies to Galpha(12) or Galpha(13) blocked the response to thrombin. In contrast, activation or inhibition of Galpha(q) function had relatively little effect. The cytoskeletal response to Galpha(12) was inhibited by microinjection of C3 exoenzyme, indicating Rho dependence. Two Rho-specific guanine nucleotide exchange factors (GEFs), oncogenic lbc and p115, increased the percentage of rounded cells 4-5-fold, and this was inhibited by C3. Mutant GEFs lacking the Dbl homology (DH) domain required for exchange factor activity failed to induce cell rounding. However, the DH mutants of lbc and p115 were efficacious inhibitors of rounding induced by thrombin or Galpha(12). The effects of lbc were dependent on an intact pleckstrin homology domain, which may be required for appropriate targeting of the Rho-GEF. These findings identify the Galpha(12) protein family as transducers of thrombin signaling to the cytoskeleton and provide the first evidence that a Rho-GEF transduces signals between G protein-coupled receptors and Rho-mediated cytoskeletal responses.

Inhibition of DNA synthesis by RB: effects on G1/S transition and S-phase progression.

The retinoblastoma tumor suppressor protein, RB, is a negative regulator of cell proliferation. Growth inhibitory activity of RB is attenuated by phosphorylation. Mutation of a combination of phosphorylation sites leads to a constitutively active RB. In Rat-1 cells, the phosphorylation-site-mutated (PSM)-RB, but not wild-type RB, can inhibit S-phase entry. In PSM-RB-arrested G1 cells, normal levels of cyclin E and cyclin E-associated kinase activity were detected, but the expression of cyclin A was inhibited. The ectopic expression of cyclin E restored cyclin A expression and drove the PSM-RB expressing cells into S phase. Interestingly, Rat-1 cells coexpressing cyclin E and PSM-RB could not complete DNA replication. Microinjection of cells that have passed through the G1 restriction point with plasmids expressing PSM-RB also led to the inhibition of DNA synthesis. The S-phase inhibitory activity of PSM-RB could be attenuated by the coinjection of SV40 T-antigen, adenovirus E1A, or a high level of E2F-1 expression plasmids. However, the S-phase inhibitory activity of PSM-RB could not be overcome by the coinjection of cyclin E or cyclin A expression plasmids. These results reveal a novel role for RB in the inhibition of S-phase progression that is distinct from the inhibition of the G1/S transition, and suggest that continued phosphorylation of RB beyond G1/S is required for the completion of DNA replication.

Thyrotropin-induced mitogenesis is Ras dependent but appears to bypass the Raf-dependent cytoplasmic kinase cascade.

Cellular growth control requires the coordination and integration of multiple signaling pathways which are likely to be activated concomitantly. Mitogenic signaling initiated by thyrotropin (TSH) in thyroid cells seems to require two distinct signaling pathways, a cyclic AMP (cAMP)-dependent signaling pathway and a Ras-dependent pathway. This is a paradox, since activated cAMP-dependent protein kinase disrupts Ras-dependent signaling induced by growth factors such as epidermal growth factor and platelet-derived growth factor. This inhibition may occur by preventing Raf-1 protein kinase from binding to Ras, an event thought to be necessary for the activation of Raf-1 and the subsequent activation of the mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinases (MEKs) and MAP kinase (MAPK)/ERKs. Here we report that serum-stimulated hyperphosphorylation of Raf-1 was inhibited by TSH treatment of Wistar rat thyroid cells, indicating that in this cell line, as in other cell types, increases in intracellular cAMP levels inhibit activation of downstream kinases targeted by Ras. Ras-stimulated expression of genes containing AP-1 promoter elements was similarly inhibited by TSH. On the other hand, stimulation of thyroid cells with TSH resulted in stimulation of DNA synthesis which was Ras dependent but both Raf-1 and MEK independent. We also show that Ras-stimulated DNA synthesis required the use of this kinase cascade in untreated quiescent cells but not in TSH-treated cells. These data suggest that in TSH-treated thyroid cells, Ras might be able to signal through effectors other than the well-studied cytoplasmic kinase cascade.

Microinjection of an nm23 specific antibody inhibits cell division in rat embryo fibroblasts.

Expression of the nm23/NDP-K gene correlates with reduced metastasis in some tumors and with increased proliferation in both nontransformed and transformed cells in culture. Decreased nm23/NDP-K expression results in mitotic arrest in neuroblasts of developing Drosophila. In order to better understand the biological role(s) of nm23 in non-transformed cells, an nm23-specific antibody was introduced into rat embryo fibroblasts and effects on DNA synthesis and cell cycle progression were analyzed. Microinjection of the nm23 antibody inhibited cell division with no apparent effect on DNA synthesis. Control experiments revealed that the survival of cells injected with the nm23 antibody was similar to that of control antibody injected cells in the absence of cell division. These results suggest that in mammalian fibroblasts, as in Drosophila, nm23 expression may be necessary for progression through the cell cycle.

Activation of c-myb by carboxy-terminal truncation: relationship to transformation of murine haemopoietic cells in vitro.

Murine retroviruses which encode c-myb proteins that have either complete or truncated carboxy (C) termini were used to infect haemopoietic cells from murine fetal liver. Using an agar colony assay, we could show that infection with the virus encoding the truncated protein resulted in the persistence of colony-forming cells well beyond the short period for which such cells are present in uninfected cultures. The resultant colonies failed to give rise to cell lines; however, clonal cell lines occasionally emerged from the original infected liquid cultures. The virus which encoded a myb protein with a complete C-terminus was virtually inactive in the colony assay; surprisingly, however, this virus could enhance proliferation in liquid cultures and has led to the generation of at least one cell line. In addition to demonstrating 'activation' of c-myb by C-terminal truncation, our results imply that an unaltered c-myb protein can also contribute to cellular transformation and that a second event is required to establish myb-transformed cells as a permanent cell line.