A soluble transforming growth factor-beta (TGF-beta ) type I receptor mimics TGF-beta responses.

Transforming growth factor-beta (TGF-beta) signaling requires a ligand-dependent interaction of TGF-beta receptors Tau beta R-I and Tau beta R-II. It has been previously demonstrated that a soluble TGF-beta type II receptor could be used as a TGF-beta antagonist. Here we have generated and investigated the biochemical and signaling properties of a soluble TGF-beta type I receptor (Tau beta RIs-Fc). As reported for the wild-type receptor, the soluble Tau beta R-I does not bind TGF-beta 1 on its own. Surprisingly, in the absence of TGF-beta1, the Tau beta RIs-Fc mimicked TGF-beta 1-induced transcriptional and growth responses in mink lung epithelial cells (Mv1Lu). Signaling induced by the soluble TGF-beta type I receptor is mediated via the obligatory presence of both TGF-beta type I and type II receptors at the cell surface since no signal was observed in Mv1Lu-derivated mutants for TGF-beta receptors R-1B and DR-26. The comparison between the structures of TGF-betas and a three-dimensional model of the extracellular domain of Tau beta RI has shown that five residues of the supposed binding site of TGF-beta 1 (Lys(31), His(34), Glu(5), Tyr(91), and Lys(94)) were found with equivalent biochemical properties and similar spatial positions.

A transforming growth factor-beta antagonist unmasks the neuroprotective role of this endogenous cytokine in excitotoxic and ischemic brain injury.

Various studies describe increased concentrations of transforming growth factor-beta (TGF-beta) in brain tissue after acute brain injury. However, the role of endogenously produced TGF-beta after brain damage to the CNS remains to be clearly established. Here, the authors examine the influence of TGF-beta produced after an episode of cerebral ischemia by injecting a soluble TGF-beta type II receptor fused with the Fc region of a human immunoglobulin (TbetaRIIs-Fc). First, this molecular construct was characterized as a selective antagonist of TGF-beta. Then, the authors tested its ability to reverse the effect of TGF-beta1 on excitotoxic cell death in murine cortical cell cultures. The addition of 1 microg/mL of TbetaRIIs-Fc to the exposure medium antagonized the neuroprotective activity of TGF-beta1 in N-methyl-D-aspartate (NMDA)-induced excitotoxic cell death. These results are consistent with the hypothesis that TGF-beta1 exerts a negative modulatory action on NMDA receptor-mediated excitotoxicity. To determine the role of TGF-beta1 produced in response to brain damage, the authors used a model of an excitotoxic lesion induced by the intrastriatal injection of 75 nmol of NMDA in the presence of 1.5 microg of TbetaRIIs-Fc. The intrastriatal injection of NMDA was demonstrated to induce an early upregulation of the expression of TGF-beta1 mRNA. Furthermore, when added to the excitotoxin, TbetaRIIs-Fc increased (by 2.2-fold, P < 0.05) the lesion size. These observations were strengthened by the fact that an intracortical injection of TbetaRIIs-Fc in rats subjected to a 30-minute reversible cerebral focal ischemia aggravated the volume of infarction. In the group injected with the TGF-beta1 antagonist, a 3.5-fold increase was measured in the infarction size (43.3 +/- 9.5 versus 152.8 +/- 46.3 mm3; P < 0.05). In conclusion, by antagonizing the influence of TGF-beta in brain tissue subjected to excitotoxic or ischemic lesion, the authors markedly exacerbated the resulting extent of necrosis. These results suggest that, in response to such insults, brain tissue responds by the synthesis of a neuroprotective cytokine, TGF-beta1, which is involved in the limitation of the extent of the injury. The pharmacologic potentiation of this endogenous defensive mechanism might represent an alternative and novel strategy for the therapy of hypoxic-ischemic cerebral injury.

Analysis of in vivo immunosuppressive and in vitro interaction with constitutive heat shock protein 70 activity of LF08-0299 (Tresperimus) and analogues.

LF08-0299 (Tresperimus) is a new immunosuppressive analogue of Gusperimus (15-deoxyspergualin or DSG). Despite the fact that its mechanism of action remains unknown, DSG has previously been demonstrated to bind specifically to Hsc70 protein, a constitutive or cognate member of heat shock protein 70 family. Herein we further explore whether immobilised LF08-0299 will selectively retain the heat shock protein Hsc70. We analysed the correlation between biological activity in vivo in the prevention of murine graft-vs-host disease (GVHD) and the ability in vitro in dissociating Hsc70 from LF08-0299 resin for LF08-0299 and structural analogues. The Hsc70 protein bound to the LF08-0299 immobilised specifically via the spermidine primary amino group and could be successfully eluted from the column by various analogues of LF08-0299. All immunosuppressants tested were able to competitively bind Hsc70 although some biological inactive compounds could as well. Our data suggests that LF08-0299 and its active analogue effects were not mediated directly through the interaction of molecules with Hsc70. The mechanism of action probably occurred by more than one step, the first being the binding of Hsc70.

Chimeric extracellular domain type II transforming growth factor (TGF)-beta receptor fused to the Fc region of human immunoglobulin as a TGF-beta antagonist.

Transforming growth factor-beta (TGF-beta) type-I and type-II receptors form a ligand-dependent heteromeric signalling complexes, in which transforming growth factor-beta receptor type II (TbetaRII) trends to act as the primary receptor. In the present study, we used a chimeric soluble type-II receptor fused with the Fc regions of human immunoglobulin (TbetaRIIs-Fc) in order to obtain a putative TGF-beta antagonist. Biochemical studies revealed that TbetaRIIs-Fc shared the same properties as the wild-type receptor. The TbetaRIIs-Fc receptor displayed an affinity of 1370 +/- 363 pM which was similar to those of the wild-type TbetaRII when expressed alone in Cos-1 cells (1122 +/- 413 pM). Furthermore, the chimeric receptor showed the same selectivity for TGF-beta isoforms as the native receptor. Although both TGF-beta1 and TGF-beta3 were able to bind TbetaBRIIs-Fc, TGF-beta2 could not compete with the binding of TGF-beta1 to TbetaRIIs-Fc. It was noted that this type of fused Fc receptor could be used in FlashPlate screening for potent agonism and antagonism of TGFbeta. Moreover, biological activities of the chimeric receptor showed it to be a potent TGF-beta1-antiproliferative and TGF-beta1-extracellular matrix transcriptional inhibitor on responses in Mv1Lu cells. To conclude, our results clearly show that the TbetaRIIs-Fc chimeric receptor could be used as a potent TGF-beta antagonist. These data raised the possibility that this TbetaRIIs-Fc construct might act successfully as an antagonist of both TGF-beta1 and TGF-beta3 in vivo.

A monoclonal antibody, raised against mammalian centrosomes and screened by recognition of plant microtubule organizing centers, identifies a pericentriolar component in different cell types.

We have used monoclonal antibodies raised against isolated native calf thymus centrosomes to probe the structure and composition of the pericentriolar material. To distinguish prospective antibodies as specific to conserved elements of this material, we screened clones by their identification of microtubule organizing centers (MTOCs) in different animal and plant cells. Among the clonal antibodies that reacted with MTOCs in both plant and mammalian cells, we describe one (mAb 6C6) that was found to immunostain centrosomes in a variety of bovine and human cells. In cycling cells this signal persisted through the entire cell cycle. Microscopy showed that the mAb 6C6 antigen was a component of the pericentriolar material and this was confirmed by biochemical analysis of centrosomes. Using immunoblot analysis of protein fractions derived from purified components of centrosomes, we have characterized the mAb 6C6 antigen as a 180 kDa polypeptide. We conclude that we have identified a protein component permanently associated with the pericentriolar material. Surprisingly, monoclonal antibody 6C6 also stained other mitotic organelles in mammalian cells, in a cell-cycle-dependent manner. During prometaphase and metaphase the antibody stained both centrosomes and kinetochores. At the onset of anaphase the kinetochore-specific staining dissociated from chromosomes and was subsequently redistributed onto a newly characterized organelle, the telophase disc while the centrosomal stain remained intact. It is not known if the 180 kDa centrosomal protein itself redistributes during mitosis, or if the pattern observed represents other antigens with shared epitopes. The pericentriolar material is thought to be composed of conserved elements, which appeared very early during the evolution of eukaryotes. Our results strongly suggest that mAb 6C6 identifies one of these elements.

The intercentriolar linkage is critical for the ability of heterologous centrosomes to induce parthenogenesis in Xenopus.

Centrosomes isolated from various sources, including human cells, have the capacity to induce parthenogenetic development when injected into unfertilized amphibian eggs. We recently isolated calf thymus centrosomes and showed that they differ structurally and functionally from previously isolated centrosomes of KE37 cells, in that the two centrioles in calf thymocytes are linearly associated by their proximal ends through a mass of electron dense material and nucleate few microtubules from their distal ends (Komesli, S., F. Tournier, M. Paintrand, R. Margolis, D. Job, and M. Bornens. 1989. J. Cell Biol. 109:2869-2878). We report here that these centrosomes are also unable to induce egg cleavage and examine the various possibilities which could account for this lack of competence. The results show that: (a) the kinetics of microtubule assembly on calf thymus centrosomes in Xenopus extracts are comparable to those of KE37 centrosomes; (b) centrosomes isolated from thymus of calves raised under controlled conditions (without anabolic agents) also lack competence; (c) centrosomes isolated from bovine cells of other tissues are competent; (d) centrosomes isolated from thymus of three other species (rat, mouse, and human) are competent. Since the lack of activity of calf thymus centrosomes apparently was not linked to species or tissue differences, we compared the ultrastructure of the centrosomes in the various centrosome preparations. The results show a strict correlation between the linear arrangement of centrioles and the lack of activity of the centrosomes. They suggest that the centrosome cycle can be blocked when the centrioles are prevented from separating into a nonlinear configuration, a step which might be critical for the initiation of procentriole budding. They also indicate that the centrosome may be involved in the G0-G1 transition.

Mass isolation of calf thymus centrosomes: identification of a specific configuration.

Centrosomes from calf thymocytes were isolated using a simple preparative procedure that provides large yields of free organelles. A comparative study with centrosomes isolated from human cultured lymphoblasts has led to the discovery of important differences in the structure of the two isolates and in their capacity to nucleate microtubules from purified tubulin. The possibility that the centrosomal structure depends upon the growth state of cells is discussed.