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Coordination of IL-7 Receptor and T-Cell Receptor Signaling by Cell-Division Cycle 42 in T-Cell Homeostasis

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Coordination of IL-7 Receptor and T-Cell Receptor Signaling by Cell-Division Cycle 42 in T-Cell Homeostasis Coordination of IL-7 receptor and T-cell receptor signaling by cell-division cycle 42 in T-cell homeostasis Fukun Guoa,1, David Hildemanb, Pulak Tripathib, Chinavenmeni S. Velub, H. Leighton Grimesb, and Yi Zhenga Divisions of aExperimental Hematology and Cancer Biology and bImmunobiology, Children’s Hospital Research Foundation, Cincinnati, OH 45229 Edited by N. Avrion Mitchison, University College London Medical School, London, United Kingdom, and approved September 15, 2010 (received for review July 15, 2010) T-cell homeostasis is essential for normal functioning of the im- of BCL2-associated agonist of cell death (Bad), and actin re- mune system. IL-7 receptor (IL-7R) and T-cell receptor (TCR) sig- organization (12–14). The Wiskott–Aldrich syndrome protein naling are pivotal for T-cell homeostatic regulation. The detailed (WASP) and its homologous molecule, N-WASP, were shown to mechanisms regulating T-cell homeostasis and how IL-7R and TCR couple Cdc42 GTPase to the actin-related protein 2/3 (Arp2/3) signaling are coordinated are largely unknown. Here we demon- complex in actin nucleation and polymerization (15–17). Parti- strate that T cell-specific deletion of cell-division cycle 42 (Cdc42) tioning-defective protein 6 (Par6) has been suggested to regulate GTPase causes a profound loss of mature T cells. Deletion of Cdc42 Cdc42-mediated cell polarity through interaction with glycogen β β leads to a markedly increased expression of growth factor indepen- synthase kinase 3 (GSK3 ) and adenomatous polyposis coli (APC) dence-1 (Gfi-1) and represses expression of IL-7Rα. In the absence of (18). The multidomain adaptor molecules IQ motif containing Cdc42, aberrant ERK1/2 MAP kinase activity results in enhanced, GTPase-activating protein 1 (IQGAP1) and IQGAP2 may be im- TCR-mediated T-cell proliferation. In vivo reconstitution of effec- portant for connecting Cdc42 signaling to actin and microtubule tor-binding–defective Cdc42 mutants and the effector p21 protein- cytoskeleton structures (19). Recent studies also revealed that activated kinase 1 (PAK1) into Cdc42-deficient T cells showed that IQGAP1 can interact directly with APC for cell polarization and migration (20). Structurally, Cdc42 recognizes its effectors through PAK1 is both necessary and sufficient for Cdc42-regulated T-cell the GTP-binding–sensitive switch I/II regions and additional distal homeostasis. Thus, T-cell homeostasis is maintained through a con- fi – – sites that have been pinpointed recently by site-directed mutagen- certed regulation of G -1 IL-7R controlled cytokine responsiveness esis studies (21–24). These studies have identified a panel of effec- and ERK-mediated TCR signaling strength by the Cdc42-PAK1 sig- tor-binding–defective mutants of Cdc42, among which Cdc42D38A naling axis. is defective in binding to PAK, Cdc42D63H is defective in binding to IQGAP, and Cdc42I173AL174A is defective in binding to WASP -cell homeostasis is critical for both protective immunity and and Par6. The availability of such specific effector-recognition–de- Tlimitation of autoimmunity (1). T cells mature in the thymus fective mutants allows a fine definition of the physiologic role of and then enter the periphery, where they are maintained as individual effectors among the Cdc42-regulated pathways. CELL BIOLOGY resting naive cells. The mechanisms by which T-cell homeostasis Previous studies , using the overexpression of dominant active or is maintained are not fully defined. On one hand, the availability negative Cdc42 mutants, suggest that Cdc42 is involved in T-cell of several soluble cytokine “survival factors” regulates the overall cytoskeletal polarization (25, 26). However, the role and mecha- size of the T-cell compartment (2). One of these factors, IL-7, is nism of signal transduction of Cdc42 in T-cell homeostatic regula- critical for T-cell survival and increases expression of the anti- tion remains unclear. In the present studies, we generated a mouse apoptotic molecule B-cell leukemia/lymphoma 2 (Bcl-2) (2, 3). strain with T cell-specificdeletionofCdc42 by cross-breeding the On the other hand, MHC/T-cell receptor (TCR) signals also lymphocyte-specific protein tyrosine kinase (Lck)-cause re- fl fl contribute to T-cell homeostasis (4). MHC/TCR interactions combination (Cre) transgenic mice with Cdc42 ox/ ox mice. By promote actin cytoskeletal rearrangement and immunological characterizing this mouse model, we demonstrate that Cdc42, synapse formation that are followed by activation of proximal through activation of its effector PAK1, positively regulates T-cell ζ TCR signaling molecules, including -chain (TCR)-associated homeostasis by coordinating growth factor independence-1 (Gfi-1)/ protein kinase 70 kDa (ZAP70) and linker for activation of T cells IL-7 receptor (IL-7R)-mediated survival signaling and ERK kinase- (LAT), and distal TCR signaling molecules, such as MAP kinases mediated TCR signaling. Our study proposes forward a regulatory (5). Low-level activation of the TCR signaling pathways has been mechanism for T-cell homeostasis that may have broad implications shown to be critical for maintaining T-cell homeostasis (6). in T-cell biology. However, how the TCR- and cytokine-mediated signals are in- tegrated in T cells to control their homeostasis remains unclear. Results Cell-division cycle 42 (Cdc42) of the Rho GTPase family is an Cdc42 fi intracellular signal transducer that cycles between an inactive De ciency Causes a Defect in T-Cell Homeostasis. To de- GDP-bound form and an active GTP-bound form under tight reg- termine the physiological function of Cdc42 in T cells, we gen- ulation (7, 8). Upon activation, Cdc42 can engage multiple ef- erated a mouse strain bearing a conditional deletion of Cdc42 in the T-cell lineage by cross-breeding Lck-Cre transgenic mice fector molecules directly to elicit regulatory functions in cell actin fl fl with previously described Cdc42 ox/ ox mice (27) (Fig. 1A). The cytoskeleton reorganization, adhesion, migration, proliferation, fi and survival (9, 10). To date, more than 10 putative Cdc42 effective removal of Cdc42 protein was con rmed by Western effectors have been identified, mostly by direct protein pulldown or yeast two-hybrid approaches (10, 11). These findings may in- dicate that Cdc42 utilizes an individual effector or a combination Author contributions: F.G., D.H., H.L.G., and Y.Z. designed research; F.G., D.H., P.T., and of effectors to mediate a defined cell function. Many of these C.S.V. performed research; F.G., D.H., H.L.G., and Y.Z. contributed new reagents/analytic effectors contain the Cdc42/Ras-related C3 botulinum toxin tools; F.G., D.H., H.L.G., and Y.Z. analyzed data; and F.G. and Y.Z. wrote the paper. substrate (Rac)-interactive binding (CRIB) motif or a Rac/Cdc42 The authors declare no conflict of interest. (p21) binding domain (PBD) domain. Among them, a class of This article is a PNAS Direct Submission. serine/threonine kinases, group A p21-activated kinases (PAK), 1To whom correspondence should be addressed. E-mail: [email protected]. are activated upon binding to Cdc42, resulting in activation of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. PI3K, JNK, and p38 pathways, inhibition of proapoptotic effects 1073/pnas.1010249107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1010249107 PNAS | October 26, 2010 | vol. 107 | no. 43 | 18505–18510 Downloaded by guest on September 28, 2021 flox/flox blot of thymocytes from Cdc42 ; Lck-Cre mice (Fig. 1A). + A B CD4 CD8 + fi + WT Ablation of Cdc42 led to a signi cant reduction in mature CD4 WT Cdc42-/- + Cdc42 -/- WT and CD8 T cells in a variety of tissues, including thymus, lymph Cdc42 -/- nodes, and spleen (Fig. 1B). Analysis of naive and effector/ − − memory T cells in Cdc42 / mice revealed a marked decrease in + low high + low naive T cells (CD4 /CD44 /CD62L and CD8 /CD44 / (%) Apoptosis CD62Lhigh) and a modest increase in effector/effector mem- + high low + high CD4 CD8 ory T cells (CD4 /CD44 /CD62L and CD8 /CD44 / + + CD62Llow) (Fig. 1C). These results demonstrate that Cdc42 (%) death cell T of Inhibition plays a critical role in maintaining T-cell homeostasis. IL-7 dose (ng/ml) IL-7 dose (ng/ml) WT Deletion of Cdc42 Results in a Survival Defect. Cdc42 -/- WT Survival regulation C WT D Cdc42 -/- is pivotal for T-cell homeostasis. Annexin V-staining analysis Cdc42 -/- revealed that Cdc42-deficient CD4+ and CD8+ T cells were more susceptible to apoptosis than their WT counterparts (Fig. 2A). + − − Consistent with these data, we found that Cdc42 / T cells were IL-7R T cells (%) cells T IL-7R largely unresponsive to IL-7–induced survival, unlike WT T cells CD4 CD8 IL-7R Gfi-1 + + Relative fold of expression of fold Relative (Fig. 2B). These results suggest that one way Cdc42 maintains expression of fold Relative T-cell homeostasis is by promoting T-cell survival. The impaired IL-7–mediated survival of mutant T cells was as- WT+IL-7 Cdc42 -/- +IL-7 E -/- fi α WT Cdc42 sociated with signi cantly reduced expression of IL-7R mRNA Isotype Isotype and protein (Fig. 2C). A major negative regulator of IL-7Rα ex- fi pression in T cells is G -1 (28, 29). Previously we observed a sig- Max of % − − Max of % nificant increase of Gfi-1 expression in Cdc42 / hematopoietic progenitor cells (27), leading us to investigate whether increased Gfi-1 expression might explain decreased IL-7Rα expression in p-Stat5 p-Stat5 − − − − + + Cdc42 / T cells. Significantly, we found that Cdc42 / T cells CD4 CD4 had a roughly threefold increase in Gfi-1 mRNA (Fig. 2D). Fig. 2. Cdc42-deficient T cells display impaired cell survival. (A) Cdc42 de- ficiency causes increased apoptosis. Freshly isolated splenocytes were stained with anti-CD4, anti-CD8, anti-CD62L and anti-CD44 antibodies followed by staining with Annexin V.
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