The goal of this research is to define the role of Podocalyxin (PODXL) in the regulation of
granulopoiesis. PODXL is a cell surface transmembrane protein belonging to the CD34 family of proteins. Our
preliminary data show that Podocalyxin is expressed in myeloid progenitors and is specifically induced by
granulocyte colony stimulating factor (G-CSF), the principal cytokine regulating granulopoiesis. To decipher the
role of Podocalyxin in G-CSF-induced granulopoiesis, we have generated a Podxl-flox/flox mouse model.
Vav1-Cre driver mice were used to selectively delete Podxl in hematopoietic cells, including myeloid lineages.
G-CSF challenge in Podxl–conditional knockout (cKO) mice resulted in significantly elevated levels of
peripheral blood (PB) neutrophils. G-CSF-induced granulopoiesis, also known as demand-adapted or reactive
granulopoiesis, is one of the principal pathways to meet neutrophil requirements under conditions such as post
chemotherapy or ionizing radiation that cause neutropenia. Further, G-CSF-induced granulopoiesis is critical to
resolve inflammation during infections, often referred to as emergency granulopoiesis. Elevated levels of G-
CSF-induced neutrophils in the absence of PODXL imply a negative regulatory role for PODXL in reactive
granulopoiesis. Podxl-cKO mice challenged with G-CSF exhibited significantly elevated levels of granulocyte-
monocyte progenitors (GMP) and immature neutrophils in bone marrow (BM). Interestingly, we found that high
levels of immature neutrophils were concomitant with a significant reduction of mature neutrophils, most likely
due to accelerated release to PB. Notably, Podxl- deficient PB neutrophils showed significantly heightened
migration abilities. To interrogate Podxl's mechanisms of action, a co-immunoprecipitation plus mass
spectrometry (LC-MS/MS) approach was applied using myeloid progenitors from G-CSF-challenged mice.
Rap1a, a Ras-related small GTPase, was a predominant co-retrieved Podxl partner. In bone marrow
hematopoietic progenitor cells, Podxl-cKO led to heightened G-CSF activation of Rap1aGTP, and Rap1aGTP
inhibition attenuated Podxl-cKO neutrophil migration. Importantly, G-CSF-induced Podxl deficient neutrophils
exhibited increased phagocytotic activity. Thus, we hypothesize that PODXL plays a negative regulatory role in
G-CSF-induced reactive granulopoiesis where Podxl exerts proliferation and differentiation effects at GMPs
and immature neutrophils, and Podxl-Rap1 is a signaling axis that regulates neutrophil trafficking via outside-in
and inside-out signaling. Thus the specific aims are: Aim 1: To characterize the functional role of Podocalyxin
during G-CSF-induced reactive granulopoiesis. Aim 2: To define the role of the Podocalyxin-Rap1a axis in
neutrophil trafficking during reactive granulopoiesis. The proposed studies will significantly advance the field in
understanding the critical regulatory mechanism that restrains emergency granulopoiesis. Further, successful
completion of the proposed studies may lead to novel drug targets to treat neutropenia resulting from infectious
diseases, chemotherapy, ionizing radiation and autoimmune disorders.