Novel Roles for Podocalyxin in Regulating Stress Myelopoiesis, Rap1a and Neutrophil Migration

• Published in: Experimental hematology 2017-06, Vol.50, p.77-83.e6
• Main Authors: Li, Pan, Karaczyn, Aldona A, McGlauflin, Rose, Favreau, Amanda, Jachimowicz, Edward, Vary, Calvin, Xu, Kailin, Wojchowski, Don M, Sathyanarayana, Pradeep
• Format: Article
• Language: English
• Subjects: Advanced Basic Science; Animals; Gene Expression Profiling; Gene Expression Regulation - drug effects; Gene Order; Genetic Loci; Granulocyte Colony-Stimulating Factor - pharmacology; Hematology, Oncology and Palliative Medicine; Hematopoietic Stem Cells - cytology; Hematopoietic Stem Cells - drug effects; Hematopoietic Stem Cells - metabolism; Humans; Mice; Mice, Knockout; Myelopoiesis - genetics; Neutrophils - physiology; rap1 GTP-Binding Proteins; Sialoglycoproteins - genetics; Sialoglycoproteins - metabolism; Stress, Physiological - genetics
• ISSN: 0301-472X; 1873-2399
• DOI: 10.1016/j.exphem.2017.04.001

Abstract Podocalyxin (Podxl) is a CD34 orthologue and cell surface sialomucin with reported roles in renal podocyte diaphragm slit development, vascular cell integrity, and the progression of blood, breast, and prostate cancers. Roles for Podxl during non-malignant hematopoiesis, however, are largely undefined. Presently we have developed a Vav-Cre Podxl knockout mouse model, and report on novel roles for Podxl in governing stress myelopoiesis. At steady-state, Podxl expression among hematopoietic progenitor cells was low-level but was induced by GCSF (granulocyte colony stimulating factor) in myeloid progenitors, and by TPO (thrombopoietin) in HSCs. In keeping with low level Podxl expression at steady-state, Vav-Cre deletion of Podxl did not markedly alter peripheral blood cell levels. G-CSF challenge in Podxl-KO mice, in contrast, hyper-elevated peripheral blood neutrophil and monocyte levels. Podxl-KO also substantially heightened neutrophil levels following 5-fluorouracil myeloablation. These LOF phenotypes were selective, and Podxl-KO did not alter lymphocyte, basophil or eosinophil levels. Within bone marrow (and following G-CSF challenge), Podxl deletion moderately decreased CFU-GEMM and CD16/32pos CD11bpos progenitors but did not affect Gr-1pos cell populations. Notably, Podxl-KO did significantly heighten peripheral blood neutrophil migration capacities. To interrogate Podxl’s action mechanisms, a co-immunoprecipitation plus LC-MS/MS (liquid chromatography – mass spectrometry) approach was applied using hematopoietic progenitors from G-CSF-challenged mice. Rap1a, a Ras-related small GTPase, was a predominant co-retrieved Podxl partner. In bone marrow HPC’s, Podxl-KO led to heightened GCSF activation of Rap1aGTP , and Rap1aGTP inhibition attenuated Podxl-KO neutrophil migration. Studies reveal novel roles for Podxl as an important modulator of neutrophil and monocyte formation, and of Rap1a activation, during stress hematopoiesis.