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.
Emergency,demand-adapted or reactive granulopoiesis is a physiological process to restore neutrophil levels in response to chemotherapy, ionizing radiation, allergic responses, autoimmune diseases or infections that cause neutropenia (low levels of neutrophils). After an infection has been cleared, this process needs to be restrained to enable the blood system to restore normal conditions, as excessive neutrophils has detrimental effects, such as the development of inflammatory diseases. Successful completion of the proposed studies will lead to better understanding of the key cellular and molecular pathways that are involved in regulation of demand-adapted and emergency granulopoiesis and will eventually lead to new therapeutic targets and approaches for treating neutropenia associated with chemotherapy or ionizing radiation and neutropenia arising from infectious diseases.
