Rho GTPase Cdc42 is essential for human T-cell development.
BACKGROUND
Rho GTPases are involved in the regulation of many cell functions, including some related to the actin cytoskeleton. Different Rho GTPases have been shown to be important for T-cell development in mice. However, their role in human T-cell development has not yet been explored.


METHODS
We examined the expression and activation of Rho GTPases along different stages of T-cell development in the human thymus. Early stage human thymocytes were transduced with constitutively active and dominant negative mutants of different Rho GTPases to explore their role in human T-cell development, as analyzed in fetal thymus organ cultures. The use of these mutants as well as Rho GTPase-specific inhibitors allowed us to explore the role of GTPases in thymocyte migration.


RESULTS
We found that the expression of several Rho GTPases is differently regulated during successive stages of T-cell development in man, suggesting a specific role in human thymopoiesis. In chimeric fetal thymus organ culture, T-cell development was not or only mildly affected by expression of dominant negative Rac1 and Rac2, but was severely impaired in the presence of dominant negative Cdc42, associated with enhanced apoptosis and reduced proliferation. Kinetic analysis revealed that Cdc42 is necessary in human T-cell development both before and after expression of the pre-T-cell receptor. Using inhibitors and retrovirally transferred mutants of the aforementioned Rho GTPases, we showed that only Rac1 is necessary for migration of different thymocyte subsets, including the early CD34(+) fraction, towards stromal cell-derived factor-1 alpha. Constitutively active mutants of Rac1, Rac2 and Cdc42 all impaired migration towards stromal cell-derived factor-1 alpha and T-cell development to different degrees.


CONCLUSIONS
This is the first report on Rho GTPases in human T-cell development, showing the essential role of Cdc42. Our data suggest that enhanced apoptotic death and reduced proliferation rather than disturbed migration explains the decreased thymopoiesis induced by dominant negative Cdc42.
Smits
Kaatje
K
Department of Clinical Chemistry, Microbiology and Immunology, Ghent University Hospital, Ghent, Belgium.
Iannucci
Veronica
V
Stove
Veronique
V
Van Hauwe
Peter
P
Naessens
Evelien
E
Meuwissen
Pieter J
PJ
Ariën
Kevin K
KK
Bentahir
Mostafa
M
Plum
Jean
J
Verhasselt
Bruno
B
D015153:Blotting, Western; D002465:Cell Movement / Q000502:physiology; D016764:Cell Polarity; D054377:Chemokine CXCL12 / Q000494:pharmacology; D002634:Chemotaxis, Leukocyte / Q000502:physiology; D002648:Child; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D015870:Gene Expression; D006801:Humans; D008213:Lymphocyte Activation / Q000502:physiology; D009924:Organ Culture Techniques; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000502:physiology*; D013950:Thymus Gland / Q000166:cytology / Q000196:embryology* / Q000201:enzymology; D020764:cdc42 GTP-Binding Protein / Q000502:physiology*; D020744:rac GTP-Binding Proteins / Q000235:genetics; D020830:rac1 GTP-Binding Protein / Q000235:genetics; D000097766:RAC2 GTP-Binding Protein
D054377:Chemokine CXCL12; D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D020764:cdc42 GTP-Binding Protein; D020744:rac GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein; D000097766:RAC2 GTP-Binding Protein; C107953:RAC1 protein, human