Pharmacological activation of guanine nucleotide exchange factors for the small GTPase Rap1 recruits high-affinity beta1 integrins as coreceptors for parvovirus B19: improved ex vivo gene transfer to human erythroid progenitor cells.
Parvovirus B19 has potential as a gene therapy vector because of its restricted tropism for human erythroid progenitor cells in the bone marrow. B19 binds to the cell surface through P antigen and we identified activated beta(1) integrins as coreceptors for internalization. Because differentiation with phorbol ester induces beta(1) integrin coreceptor activity, but cell differentiation is not desirable in gene transfer to human progenitor cells and one of the downstream effectors of phorbol esters is the small GTPase Rap1, the role of Rap1 in the recruitment of beta(1) integrins on hematopoietic cells was examined. Expression of a constitutively active Rap1 (63E) was sufficient to recruit beta(1) integrin coreceptors in erythroleukemic K562 cells by inducing high-affinity integrin conformation. A crucial role of actin polymerization in Rap1-mediated beta(1) integrin recruitment was documented by complete inhibition of the 63E Rap1 effect with low-dose cytochalasin D and by the ability of a constitutively active mutant of the actin cytoskeleton regulator Rac1 to sensitize K562 cells to the pharmacological activation of endogenous Rap1, using the Rap1 exchange factor-specific 8-pCPT-2'-O-Me-cAMP [8-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate]. Interestingly, in primary human erythroid progenitor cells, 8-pCPT-2'-O-Me-cAMP was sufficient to significantly increase B19-mediated gene transfer, suggesting that these cells possess the cytoskeleton organization capacity required for efficient recruitment of beta(1) integrins by brief pharmacological stimulation of Rap1 GTP loading. Because 8-pCPT-2'-O-Me-cAMP has been implicated in enhanced homing of progenitor cells, these results identify a novel tool with which to optimize ex vivo B19-mediated gene transfer and potentially improve homing of transduced cells by Rap1-beta(1) integrin activation with 8-pCPT-2'-O-Me-cAMP.
Weigel-Van Aken
Kirsten A K
KA
Division of Cellular and Molecular Therapy, Department of Pediatrics, Powell Gene Therapy Center, University of Florida College of Medicine, Gainesville, FL 32610, USA. weigel@ufl.edu
D000818:Animals; D002460:Cell Line; D000242:Cyclic AMP / Q000031:analogs & derivatives* / Q000494:pharmacology; D015638:Cytochalasin D / Q000494:pharmacology; D015672:Erythroid Precursor Cells / Q000187:drug effects* / Q000378:metabolism; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000819:agonists* / Q000378:metabolism; D006801:Humans; D019012:Integrin beta1 / Q000378:metabolism*; D051379:Mice; D016732:Parvovirus B19, Human / Q000235:genetics / Q000378:metabolism*; D013873:Thionucleotides / Q000494:pharmacology*; D020827:rap1 GTP-Binding Proteins / Q000819:agonists* / Q000378:metabolism
D020662:Guanine Nucleotide Exchange Factors; D019012:Integrin beta1; D013873:Thionucleotides; D015638:Cytochalasin D; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; D000242:Cyclic AMP; D020827:rap1 GTP-Binding Proteins
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