Patrick Lorès

Enhanced apoptosis in the thymus of transgenic mice expressing constitutively activated forms of human Rac2GTPase.

Rac proteins constitute a subgroup of the Rho family of small GTPases and include Rac1, which is expressed ubiquitously, and Rac2, a highly homologous protein only expressed in myelo-monocytic and lymphoid cell lineages. In fibroblasts, Rac1 plays a crucial role in control of actin cytoskeleton organisation, cell growth and Ras-induced transformation. In phagocytes, Rac1 and Rac2 regulate a specific enzymatic complex, NADPH oxidase. These multiple functions have been ascribed to Rac proteins only on the basis of cell culture and in vitro biochemical studies. To examine the role of Rac2 in vivo in a T cell lineage, we have expressed either wild-type or constitutively-activated forms of human Rac2 (Rac2V12 and Rac2L61) in transgenic mice under control of the thymus specific lck proximal promoter. We report here a striking atrophy of the thymus in mice expressing even low levels of either of the activated mutants of Rac2, while expression of Rac2wt has no effect. This phenotype is correlated with a marked decrease in the number of double positive (CD4+CD8+) and single positive (CD4+CD8− and CD8+CD4−) thymocytes. Cellular and molecular analyses demonstrate that this defect is due to an increase in apoptosis among thymocytes. As Rac2 is normally expressed in thymocytes and activated T cells, we propose that Rac2 dependent pathways could play an important role in control of growth and death of T cells.

Pre-B-cell development in the absence of λ5 in transgenic mice expressing a heavy-chain disease protein

Abstract Background: Heavy-chain diseases (HCDs) are human lymphoproliferative neoplasias that are characterized by the secretion of truncated immunoglobulin heavy chains devoid of light chains. We have previously proposed — by analogy to the process by which mutated growth factor receptors can be oncogenic — that because the genetic defects in HCDs result in the production of abnormal membrane-associated heavy chains lacking an antigen-binding domain, these abnormal B-cell antigen receptors might engage in ligand-independent signalling. Normal pre-B-cell development requires the presence of the pre-B-cell receptor, formed by the association of μ heavy chains with two polypeptides — so-called surrogate light chains, V pre-B and λ5 — that are homologous to the variable and constant portions of immunoglobulin light chains, respectively. To assess whether amino-terminal truncation of membrane-associated heavy chains results in their constitutive activation, we have examined the ability of a HCD-associated μ protein to promote pre-B-cell development in transgenic mice. Results When the μ HCD transgene is introduced into SCID mice, CD43 − pre-B cells develop normally. To determine whether this pre-B-cell development requires surrogate light chains, we backcrossed mice expressing full-length or truncated μ transgenes with λ5-deficient mice. Our results show that the truncated heavy chain, but not the normal chain, is able to promote pre-B-cell development in the absence of λ5. We also show that truncated μ chains spontaneously aggregate at the surface of bone marrow cells. Conclusion Expression of the truncated μ heavy chain overrides a tightly controlled step of pre-B-cell development, which strongly suggests that a constitutive signal is delivered by the truncated μ chain disease protein. The self-aggregation of μ chain disease proteins might account for this constitutive activation. We conclude that amino-terminal truncation of heavy chains could play a role in the genesis of HCD neoplasia if it occurs at an appropriate stage of B-cell differentiation, namely in a mature B cell.

Activated Rac1, but not the tumorigenic variant Rac1b, is ubiquitinated on Lys 147 through a JNK-regulated process.

Ubiquitination and proteasomal degradation have recently emerged as an additional level of regulation of activated forms of Rho GTPases. To characterize this novel regulatory pathway and to gain insight into its biological significance, we studied the ubiquitination of two constitutively activated forms of Rac1, i.e. the mutationally activated Rac1L61, and the tumorigenic splice variant Rac1b, which is defective for several downstream signaling pathways, including JNK activation. Whereas Rac1L61 undergoes polyubiquitination and subsequent proteasomal degradation in HEK293 cells, Rac1b is poorly ubiquitinated and appears to be much more resistant to proteasomal degradation than Rac1L61. Mutational analysis of all lysine residues in Rac1 revealed that the major target site for Rac1 ubiquitination is Lys147, a solvent‐accessible residue that has a similar conformation in Rac1b. Like Rac1L61, Rac1b was found to be largely associated with plasma membrane, a known prerequisite for Rac1 ubiquitination. Interestingly, Rac1b ubiquitination could be stimulated by coexpression of Rac1L61, suggesting positive regulation of Rac1 ubiquitination by Rac1 downstream signaling. Indeed, ubiquitination of Rac1L61 is critically dependent on JNK activation. In conclusion: (a) Rac1b appears to be more stable than Rac1L61 with regard to the ubiquitin–proteasome system, and this may be of importance for the expression and tumorigenic capacity of Rac1b; and (b) ubiquitination of activated Rac1 occurs through a JNK‐activated process, which may explain the defective ubiquitination of Rac1b. The JNK‐dependent activation of Rac1 ubiquitination would create a regulatory loop allowing the cell to counteract excessive activation of Rac1 GTPase.

Transgenic Expression of Fas Ligand on Thyroid Follicular Cells Prevents Autoimmune Thyroiditis

“Immune privilege” is defined as tissue resistance to aggression by specifically activated lymphocytes, and involves the interaction between Fas expressed on infiltrating cells and Fas ligand (FasL) constitutively expressed on the target tissue. To test whether ectopic expression of FasL on thyrocytes could prevent autoimmune aggression of the thyroid by activated lymphoid cells, three lines of transgenic mice expressing low, intermediate, and high levels of functional FasL on thyroid follicular cells were generated. Experimental autoimmune thyroiditis was induced by immunization with mouse thyroglobulin. In all of the experiments, the effects were dependent on the level of FasL expression. Low and intermediate expression had no or only weak preventive effects, respectively, whereas high FasL expression strongly inhibited lymphocytic infiltration of the thyroid. Anti-mouse thyroglobulin-proliferative and cytotoxic T cell responses, as well as autoantibody production, were diminished in transgenic mice expressing high levels of FasL relative to controls. Furthermore, in these latter mice Th1 responses to mouse thyroglobulin were profoundly down-regulated, uncovering a new potential role for FasL in peripheral tolerance to organ-specific Ags. In sum, the prevention of experimental autoimmune thyroiditis by FasL on thyrocytes is dependent on the level of FasL expression.

Transgenic Expression of CD95 Ligand on Thyroid Follicular Cells Confers Immune Privilege upon Thyroid Allografts

Constitutive Fas ligand (FasL) expression by specialized cells in the body participates in the immune privilege status of tissues containing these cells. This property has been used to prevent rejection of allogeneic grafts. Nevertheless, the mechanism responsible for such protection has not been fully elucidated. Unfortunately, grafting of FasL transgenic (TG) tissues has been unsuccessful. We have generated TG mice expressing FasL (soluble + membrane bound) on thyroid follicular cells (TFC), and used them to show that ectopic FasL expression prevents thyroid allograft rejection. FasL expression on TFC led to markedly decreased anti-allogeneic, cytotoxic, and helper T lymphocyte activities. The alloantibody response in TG thyroid recipients was either completely inhibited or switched toward a T2-Ab response. Surprisingly, the beneficial effect of FasL on TG thyroid grafts was abolished by host CD4+ T cell depletion. Host CD8+ T cell depletion improved nontransgenic (NTG), but not TG graft survival. Altogether, our results suggest that FasL-induced tolerance is concomitant with a move away from a T1 type response, and a CD4 T cell-mediated regulation of the allocytotoxic T cell response. These results were dependent upon the level of FasL expression on TFC, in that low expression of FasL led to a less marked effect compared with the effect observed with high expression of FasL. These results provide some insight into the role of FasL in regulating destructive alloimmune responses in the case of whole organ grafting, and they have important implications for the development of FasL-based immunotherapy in organ transplantation.

The testis anion transporter TAT1 (SLC26A8) physically and functionally interacts with the cystic fibrosis transmembrane conductance regulator channel: a potential role during sperm capacitation

The Slc26 gene family encodes several conserved anion transporters implicated in human genetic disorders, including Pendred syndrome, diastrophic dysplasia and congenital chloride diarrhea. We previously characterized the TAT1 (testis anion transporter 1; SLC26A8) protein specifically expressed in male germ cells and mature sperm and showed that in the mouse, deletion of Tat1 caused male sterility due to a lack of sperm motility, impaired sperm capacitation and structural defects of the flagella. Ca(2+), Cl(-) and HCO(3)(-) influxes trigger sperm capacitation events required for oocyte fertilization; these events include the intracellular rise of cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA)-dependent protein phosphorylation. The cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in mature sperm and has been shown to contribute to Cl(-) and HCO(3)(-) movements during capacitation. Furthermore, several members of the SLC26 family have been described to form complexes with CFTR, resulting in the reciprocal regulation of their activities. We show here that TAT1 and CFTR physically interact and that in Xenopus laevis oocytes and in CHO-K1 cells, TAT1 expression strongly stimulates CFTR activity. Consistent with this, we show that Tat1 inactivation in mouse sperm results in deregulation of the intracellular cAMP content, preventing the activation of PKA-dependent downstream phosphorylation cascades essential for sperm activation. These various results suggest that TAT1 and CFTR may form a molecular complex involved in the regulation of Cl(-) and HCO(3)(-) fluxes during sperm capacitation. In humans, mutations in CFTR and/or TAT1 may therefore be causes of asthenozoospermia and low fertilizing capacity of sperm.

Soluble Fcγ Receptors: Interaction with Ligands and Biological Consequences

Soluble Fcγ receptors are produced by cleavage of the membrane receptors or by alternative splicing. They are found in biologic fluids. After a brief description of the structure and mode of production of soluble FcγR, we address the question of ligands and function of the soluble FcγR by using recombinant molecules and transgenic animals. We show that soluble FcγR are not only IgG-binding factors which interfere with, and block, Fc-dependent immune reactions but also molecules that interact, in vitro, with non-Ig-ligands such as CR3 and CR4 and are trigger or regulate immune functions via these receptors.

The SWI/SNF protein BAF60b is ubiquitinated through a signalling process involving Rac GTPase and the RING finger protein Unkempt

The SWI/SNF chromatin remodelling complexes are important regulators of transcription; they consist of large multisubunit assemblies containing either Brm or Brg1 as the catalytic ATPase subunit and a variable subset of approximately 10 Brg/Brm‐associated factors (BAF). Among these factors, BAF60 proteins (BAF60a, BAF60b or BAF60c), which are found in most complexes, are thought to bridge interactions between transcription factors and SWI/SNF complexes. We report here on a Rac‐dependent process leading to BAF60b ubiquitination. Using two‐hybrid cloning procedures, we identified a mammalian RING finger protein homologous to drosophila Unkempt as a new partner of the activated form of RacGTPases and demonstrated that mammalian Unkempt specifically binds to BAF60b and promotes its ubiquitination in a Rac1‐dependent manner. Immunofluorescence studies demonstrated that Unkempt is primarily localized in the cytoplasmic compartment, but has the ability to shuttle between the nucleus and the cytoplasm, suggesting that the Rac‐ and Unkempt‐dependent process leading to BAF60b ubiquitination takes place in the nuclear compartment. Ubiquitinated forms of BAF60b were found to accumulate upon treatment with the proteasome inhibitor MG132, indicating that BAF60b ubiquitination is of the degradative type and could regulate the level of BAF60b in SWI/SNF complexes. Our observations support the new idea of a direct connection between Rac signalling and chromatin remodelling.

Expression of a V region-less B cell receptor confers a tolerance-like phenotype on transgenic B cells.

Neoplastic B cells from H chain disease patients express a truncated B cell receptor (BCR), comprising a membrane Ig that lacks part of its extracellular domain. It has been speculated that deletion of the Ag binding domain would confer a constitutive activity on the BCR, as it has been shown for oncogenic growth factor receptors. A V region-less BCR has constitutive activity, because in transgenic mice it causes inhibition of endogenous H chain gene rearrangements and relieves the requirement for surrogate L chain in pre-B cell development. However, it has been speculated that normal Ag receptors also display constitutive activity. Here we show that transgenic B cells expressing a membrane H chain disease protein on their surface are phenotypically and functionally similar to B cells developing in the presence of their cognate Ag and that cells with normal levels of mutant BCR are eliminated in spleen via a bcl-2 sensitive pathway while progressing toward the mature stage. In contrast, cells with lower levels of mutant receptors develop as mature B cells. These findings support the view that the truncated BCR has a constitutive activity that mimics ligand binding, in analogy to what has been shown for oncogenic growth factor receptors.

Deletion of MgcRacGAP in the male germ cells impairs spermatogenesis and causes male sterility in the mouse.

MgcRacGAP (RACGAP1) is a GTPase Activating Protein (GAP), highly produced in the mouse embryonic brain and in the human and mouse post-natal testis. MgcRacGAP negatively controls the activity of Rac and Cdc42, which are key molecular switches acting on the microtubule and actin cytoskeleton and controlling various cell processes such as proliferation, adhesion and motility. Previous studies demonstrated that MgcRacGAP plays a critical role in the cytokinesis of somatic cells; hence homozygous inactivation of the gene in the mouse and mutation in Caenorhabditis elegans led to embryonic lethality due to the inability of MgcRacGAP-null embryos to assemble the central spindle and to complete cytokinesis. In the testis, the germ cells do not complete cytokinesis and remain connected as a syncytium throughout the entire process of spermatogenesis. Interestingly, MgcRacGAP was shown to locate to the intercellular bridges, connecting these germ cells. In order to determine the function(s) of MgcRacGAP in the male germline, we generated a conditional knock-out mouse using Stra8 promoter driven Cre recombinase to induce the specific deletion of MgcRacGAP in the pre-meiotic germ cells. We found that the absence of MgcRacGAP induced a germline depletion and male sterility. Consistent with the role of MgcRacGAP in the establishment of the cytoplasm constriction during cytokinesis of the somatic cells, we observed that MgcRacGAP deletion in the germ cells prevented the formation of the intercellular bridges and induced a proliferation arrest. While we assume that inherited homozygous loss of function mutations in MgcRacGAP would be lethal in human, de novo mutations in the testis might account for some cases of non-obstructive oligo- and/or azoo-spermia syndromes, whose genetic causes are altogether still poorly defined.