Supplementary MaterialsSupplementary Information 41467_2019_11490_MOESM1_ESM. Similarly, DJ001 administration accelerates hematologic recovery in mice?treated with 5-fluorouracil chemotherapy. DJ001 displays high specificity for PTP and antagonizes PTP via unique IWR-1-endo non-competitive, allosteric binding. Mechanistically, DJ001 suppresses radiation-induced HSC apoptosis via activation of the RhoGTPase, RAC1, and induction of BCL-XL. Furthermore, treatment of irradiated human HSCs with DJ001 promotes the regeneration of human HSCs capable of multilineage in vivo repopulation. These studies demonstrate the therapeutic potential of selective, small-molecule PTP inhibitors for human Rabbit polyclonal to YIPF5.The YIP1 family consists of a group of small membrane proteins that bind Rab GTPases andfunction in membrane trafficking and vesicle biogenesis. YIPF5 (YIP1 family member 5), alsoknown as FinGER5, SB140, SMAP5 (smooth muscle cell-associated protein 5) or YIP1A(YPT-interacting protein 1 A), is a 257 amino acid multi-pass membrane protein of the endoplasmicreticulum, golgi apparatus and cytoplasmic vesicle. Belonging to the YIP1 family and existing asthree alternatively spliced isoforms, YIPF5 is ubiquitously expressed but found at high levels incoronary smooth muscles, kidney, small intestine, liver and skeletal muscle. YIPF5 is involved inretrograde transport from the Golgi apparatus to the endoplasmic reticulum, and interacts withYIF1A, SEC23, Sec24 and possibly Rab 1A. YIPF5 is induced by TGF1 and is encoded by a genelocated on human chromosome 5 hematopoietic regeneration. isomer) (represented as ball and stick in yellow color) to the PTP allosteric binding site located between domain name 1 (green) and domain name 2 (blue) of PTP. e At left, substrate titration reveals DJ001 as a non-competitive inhibitor that inhibits substrate catalysis (mice displayed increased recovery of BM CFCs at day?+?10 compared with irradiated mice (Fig.?2b). These results suggested that deletion of or PTP inhibition comparably promoted hematopoietic progenitor cell regeneration following irradiation. Open in a separate windows Fig. 2 PTP inhibition promotes hematopoietic regeneration. a Mean numbers IWR-1-endo of CFCs from BM KSL cells following 300?cGy irradiation and culture for 3 days in TSF media??DJ001 (and mice at IWR-1-endo day?+?10 following 600?cGy TBI (mice caused no switch in RAC1-GTP levels, suggesting that DJ001-mediated activation of RAC1 occurred specifically via PTP (Fig.?3c). DJ009 also did not induce RAC1 activation in BM cells from mice, suggesting comparable selectivity of DJ009 for PTP (Supplementary Fig.?7c). Treatment of BM KSL cells with DJ001 increased phosphorylation of p21-activated kinase 1 (PAK1), a substrate of RAC1, and concomitant treatment with the RAC inhibitor, EHT186428, abrogated DJ001-mediated phosphorylation of PAK1 (Fig.?3d). Open in a separate window Fig. 3 DJ001 promotes HSC regeneration via RAC1 activation and induction of BCL-XL. a %p250GAP phospho-tyrosine (pTyr) in BM lin? cells cultured??DJ001 (media, and mice treated??DJ001 (or ((and expression was RAC pathway dependent (Fig.?3h). In order to confirm that DJ001-mediated effects on irradiated HSPCs were dependent on RAC1 and BCL-XL, we transduced BM KSL cells separately with lentiviral short hairpin RNAs (shRNAs) targeting or and measured hematopoietic progenitor cell recovery following 300?cGy irradiation. Silencing of either or blocked DJ001-mediated recovery of hematopoietic progenitor cells from irradiated BM KSL cells (Fig.?3i). Taken together, these results suggested that DJ001-mediated HSPC recovery after irradiation was dependent on RAC1 and BCL-XL. Table 1 Primers utilized for mouse gene detection in irradiated BM KSL cells (Fig.?4b and Table?1). However, DJ001 treatment significantly increased the expression of and in irradiated KSL cells. Transduction of BM KSL cells with and and in KSL cells was RAC1 dependent (Fig.?4c). Furthermore, treatment of irradiated BM KSL cells with SU9516, a specific CDK2 inhibitor, or the RAC inhibitor, EHT1864, suppressed DJ001-mediated induction of cell cycle progression in BM KSL cells following irradiation (Fig.?4d). These results suggested that DJ001-mediated HSC cell cycle progression following irradiation was dependent on RAC pathway activation and CDK2. Open in a separate windows Fig. 4 DJ001 promotes HSC proliferation via induction of CDK2. a At left, representative cell cycle analysis of BM KSL cells at 36?h following 300?cGy and culture with media??1?g/mL DJ001. At right, imply percentages of KSL cells in G0 (Ki67?7AAD?), G1 (Ki67+7AAD?) and G2/S/M phase (Ki67+7AAD+) are shown (and media treatment. c Fold changes (2?Ct) of and expression in BM lin? cells in response to DJ001 at 48?h after 300?cGy, with and without and and gene expression in CD34+CD38? cells at 12?h after 300?cGy in media??DJ001 (and media treatment. Two-way ANOVA with Sidaks multiple comparison test. e Schematic representation of NSG mice transplantation assay using the progeny of human BM CD34+ cells irradiated with 300?cGy and treated with or without DJ001??36?h. f Representative circulation cytometric analysis of human CD45+ cells, human CD34+ cells, human CD19+ B cells, human CD33+ myeloid cells, and human CD3+ T cells engraftment in the BM of NSG mice at 12 weeks post transplantation. g Percent engraftment of human hematopoietic cell subsets in the BM of NSG mice at 12 weeks post transplantation (in the hematopoietic compartment of mice has been shown to cause decreased HSPC engraftment, homing, and niche localization30,31,36, whereas expression of a dominant-negative increased HSPC apoptosis36,59. RAC1 has.