Abstract
SUMMARYMutations in the WAS gene cause Wiskott-Aldrich syndrome (WAS), which is characterized by eczema, immunodeficiency and microthrombocytopenia. Although the role of WASP in lymphocytes and myeloid cells is well characterized, its role on megakaryocyte (MK) development is poorly understood. In order to develop a human cellular model that mimics the megakaryocytic-derived defects observed in WAS patients we used K562 cells, a well-known model for study of megakaryocytic development. We knocked out the WAS gene in K562 cells using a zinc-finger nuclease (ZFN) pair targeting the WAS intron 1 and a homologous donor DNA that disrupted WASP expression. Knockout of WASP on K562 cells (K562WASKO cells) resulted in several megakaryocytic-related defects such as morphological alterations, lower expression of CD41ɑ, lower increments in F-actin polymerization upon stimulation, reduced CD43 expression and increased phosphatidylserine exposure. All these defects have been previously described either in WAS-knockout mice or in WAS patients, validating K562WASKO as a cell model for WAS. However, K562WASPKO cells showed also increased basal F-actin and adhesion, increased expression of CD61 and reduced expression of TGFβ and Factor VIII, defects that have never been described before for WAS-deficient cells. Interestingly, these phenotypic alterations correlate with different roles for WASP in megakaryocytic differentiation. All phenotypic alterations observed in K562WASKO cells were alleviated upon expression of WAS following lentiviral transduction, confirming the role of WASP in these phenotypes. In summary, in this work we have validated a human cellular model, K562WASPKO, that mimics the megakaryocytic-related defects found in WAS-knockout mice and have found evidences for a role of WASP as regulator of megakaryocytic differentiation. We propose the use of K562WASPKO cells as a tool to study the molecular mechanisms involved in the megakaryocytic-related defects observed in WAS patients and as a cellular model to study new therapeutic strategies.
Highlights
Wiskott-Aldrich syndrome (WAS) is an X-linked inherited disease that combines immunodeficiency and platelet dysfunction
To obtain a human cellular model that mimics the megakaryocytic-derived defects observed in patients with WAS, the authors knocked out the WAS gene in K562 cells, a human leukemia cell line that produces megakaryocytes on activation with phorbol 12myristate 13-acetate (PMA)
WASP-knockout K562 cells show some additional phenotypes not previously associated with WASP deficiency, such as increased basal levels of polymerized F-actin and enhanced adhesion in the absence of PMA activation
Summary
Wiskott-Aldrich syndrome (WAS) is an X-linked inherited disease that combines immunodeficiency and platelet dysfunction. The role of WASP in immune-related defects is well characterized. WASP binds the actin-related protein complex 2/3 (Arp2/3), allowing actin nucleation and the generation of new actin filaments (Gallego et al, 1997). Microthrombocytopenia (a decrease in the number and size of platelets) is an invariable characteristic of Wiskott-Aldrich Syndrome (WAS), a primary immunodeficiency caused by mutations in the WAS gene that cause the absence or inactivity of the WASP protein. The role of WASP in lymphocytes and myeloid cells is well characterized, its role in the development of megakaryocytes (the bone marrow cells that gives rise to platelets) is poorly understood, in part because WAS-knockout mice models exhibit only mild thrombocytopenia and have platelets of a normal size
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