PODXL Knockout Cell Lysate

Podocalyxin (Podxl) is a CD34 orthologue and cell surface sialomucin reported to have roles in renal podocyte diaphragm slit development; vascular cell integrity; and the progression of blood, breast, and prostate cancers. Roles for Podxl during nonmalignant hematopoiesis, however, are largely undefined. We have developed a Vav-Cre Podxl knockout (KO) 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 granulocyte colony -stimulating factor (G-CSF) in myeloid progenitors and by thrombopoietin in human stem cells. In keeping with low-level Podxl expression at steady state, Vav-Cre deletion of Podxl did not markedly alter peripheral blood cell levels. A G-CSF challenge in Podxl-KO mice, in contrast, hyperelevated peripheral blood neutrophil and monocyte levels. Podxl-KO also substantially heightened neutrophil levels after 5-fluorouracil myeloablation. These loss-of-function phenotypes were selective, and Podxl-KO did not alter lymphocyte, basophil, or eosinophil levels. Within bone marrow (and after G-CSF challenge), Podxl deletion moderately decreased colony forming units granulocytes, eyrthrocytes, monocyte/macrophages, megakaryocytes and CD16/32P'CD11bP' progenitors but did not affect Gr-11' cell populations. Notably, Podxl-KO did significantly heighten peripheral blood neutrophil migration capacities. To interrogate Podxl's action mechanisms, a coimmunoprecipitation plus liquid chromatography-mass spectrometry approach was applied using hematopoietic progenitors from G-CSF-challenged mice. Rapla, a Ras-related small GTPase, was a predominant co-retrieved Podxl partner. In bone marrow human progenitor cells, PodxlKO led to heightened G-CSF activation of RaplaGTP, and RaplaGTP inhibition attenuated Podxl-KO neutrophil migration. Studies have revealed novel roles for Podxl as an important modulator of neutrophil and monocyte formation and of Rapla activation during stress hematopoiesis. Copyright (C) 2017 ISEH- International Society for Experimental Hematology. Published by Elsevier Inc.

Background: MicroRNAs (miRNAs) are small non-coding RNA molecules that mediate the biological cellular processes via regulation of target genes through translational repression or mRNA degradation. Among various miRNAs, miRNA-199a (miR-199a) has been known to be involved in cancer development and progression, protection of cardiomyocyte, and skeletal formation. Objective: Although miR-199a-5p was studied in various cell types, the role of miR-199a-5p and its target genes in skin keratinocyte have not been documented. In this study, we identified target genes of miR-199a-5p in skin keratinocyte. Methods: In order to identify the target of miR-199a-5p in keratinocyte, microarray analysis was performed. The relative expression of candidate target genes was investigated using quantitative RT-PCR and western blot analysis. To determine whether their expression was directly regulated by miR-199a-5p, luciferase reporter assay was performed. In order to investigate expression of target genes in cutaneous squamous cell carcinoma, immunohistochemistry was performed. Results: We identified new target genes, Bcam, Fzd6, and Wnt7a, as well as previously known targets, Ddr1 and Podxl. We found that their expressions were directly regulated by miR-199a-5p in the skin keratinocyte using in vitro study and observed that expression of miR-199a-5p was inversely correlated with those of BCAM, FZD6 and DDR1 in the cSCC. In addition, overexpression of miR-199a-5p resulted in inhibition of the migratory capability of the skin keratinocyte. Conclusion: These results suggested that miR-199a-5p plays a role in pathogenesis of cSCC via inhibition of invasiveness through regulation of BCAM, FZD6 and DDR1 expression. (C) 2015 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.