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Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells
Published on: September 1, 2019
Rap1a null mice have altered myeloid cell functions suggesting distinct roles for the closely related Rap1a and 1b
Yu Li1, Jingliang Yan, Pradip De
1Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis 46202, USA.
Abstract:
The Ras-related GTPases Rap1a and 1b have been implicated in multiple biological events including cell adhesion, free radical production, and cancer. To gain a better understanding of Rap1 function in mammalian physiology, we deleted the Rap1a gene. Although loss of Rap1a expression did not initially affect mouse size or viability, upon backcross into C57BL/6J mice some Rap1a-/- embryos died in utero. T cell, B cell, or myeloid cell development was not disrupted in Rap1a-/- mice. However, macrophages from Rap1a null mice exhibited increased haptotaxis on fibronectin and vitronectin matrices that correlated with decreased adhesion. Chemotaxis of lymphoid and myeloid cells in response to CXCL12 or CCL21 was significantly reduced. In contrast, an increase in FcR-mediated phagocytosis was observed. Because Rap1a was previously copurified with the human neutrophil NADPH oxidase, we addressed whether GTPase loss affected superoxide production. Neutrophils from Rap1a-/- mice had reduced fMLP-stimulated superoxide production as well as a weaker initial response to phorbol ester. These results suggest that, despite 95% amino acid sequence identity, similar intracellular distribution, and broad tissue distribution, Rap1a and 1b are not functionally redundant but rather differentially regulate certain cellular events.
Insights
Deleting the Rap1a gene impacts cell adhesion and function, revealing Rap1a and Rap1b GTPases have distinct roles in mammals. This study highlights Rap1a
Area of Science:
- Molecular Biology
- Cell Biology
- Immunology
Background:
- Ras-related GTPases, including Rap1a and Rap1b, are vital for cellular processes like adhesion and cancer.
- Understanding Rap1a's specific physiological role is crucial due to its involvement in diverse biological events.
Purpose of the Study:
- To investigate the in vivo function of Rap1a by generating and analyzing Rap1a-deficient mice.
- To determine the specific cellular and physiological consequences of Rap1a gene deletion.
Main Methods:
- Gene deletion of Rap1a in mice.
- Analysis of embryonic viability and hematopoietic cell development.
- Assessment of macrophage and leukocyte functions, including haptotaxis, adhesion, chemotaxis, and phagocytosis.
- Evaluation of neutrophil superoxide production.
Main Results:
- Rap1a deficiency led to in utero embryonic lethality in some genetic backgrounds.
- Macrophages showed increased haptotaxis and decreased adhesion, while leukocyte chemotaxis was reduced.
- FcR-mediated phagocytosis was enhanced.
- Neutrophils exhibited diminished superoxide production in response to stimuli.
Conclusions:
- Rap1a plays a critical, non-redundant role in regulating cell adhesion, migration, and phagocytosis.
- Despite high sequence homology, Rap1a and Rap1b GTPases have distinct functions in mammalian physiology.
- Rap1a is essential for normal neutrophil function, including superoxide production.
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