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• W2901883599 abstract "Article16 November 2018Open Access Transparent process Attenuation of PKCδ enhances metabolic activity and promotes expansion of blood progenitors Tata Nageswara Rao Corresponding Author Tata Nageswara Rao [email protected] orcid.org/0000-0002-9928-5944 Department of Stem Cell and Regenerative Biology, Harvard Stem Cell Institute, Harvard University, Cambridge, MA, USA Section on Islet Cell and Regenerative Biology, Joslin Diabetes Center, Boston, MA, USA Search for more papers by this author Manoj K Gupta Manoj K Gupta Section on Islet Cell and Regenerative Biology, Joslin Diabetes Center, Boston, MA, USA Search for more papers by this author Samir Softic Samir Softic Section on Integrative Physiology and Metabolism, Joslin Diabetes Center, Boston, MA, USA Division of Gastroenterology, Hepatology and Nutrition, Boston Children's Hospital, Boston, MA, USA Search for more papers by this author Leo D Wang Leo D Wang Department of Stem Cell and Regenerative Biology, Harvard Stem Cell Institute, Harvard University, Cambridge, MA, USA Section on Islet Cell and Regenerative Biology, Joslin Diabetes Center, Boston, MA, USA Division of Pediatric Hematology/Oncology/Stem Cell Transplantation, Dana-Farber/Boston Children's Center for Cancer and Blood Disorders, Boston, MA, USA Search for more papers by this author Young C Jang Young C Jang Department of Stem Cell and Regenerative Biology, Harvard Stem Cell Institute, Harvard University, Cambridge, MA, USA Section on Islet Cell and Regenerative Biology, Joslin Diabetes Center, Boston, MA, USA Search for more papers by this author Thomas Thomou Thomas Thomou Section on Integrative Physiology and Metabolism, Joslin Diabetes Center, Boston, MA, USA Search for more papers by this author Olivier Bezy Olivier Bezy orcid.org/0000-0003-4367-5043 Section on Integrative Physiology and Metabolism, Joslin Diabetes Center, Boston, MA, USA Search for more papers by this author Rohit N Kulkarni Rohit N Kulkarni Section on Islet Cell and Regenerative Biology, Joslin Diabetes Center, Boston, MA, USA Search for more papers by this author C Ronald Kahn C Ronald Kahn orcid.org/0000-0002-7583-9228 Section on Integrative Physiology and Metabolism, Joslin Diabetes Center, Boston, MA, USA Search for more papers by this author Amy J Wagers Corresponding Author Amy J Wagers [email protected] orcid.org/0000-0002-0066-0485 Department of Stem Cell and Regenerative Biology, Harvard Stem Cell Institute, Harvard University, Cambridge, MA, USA Section on Islet Cell and Regenerative Biology, Joslin Diabetes Center, Boston, MA, USA Search for more papers by this author Tata Nageswara Rao Corresponding Author Tata Nageswara Rao [email protected] orcid.org/0000-0002-9928-5944 Department of Stem Cell and Regenerative Biology, Harvard Stem Cell Institute, Harvard University, Cambridge, MA, USA Section on Islet Cell and Regenerative Biology, Joslin Diabetes Center, Boston, MA, USA Search for more papers by this author Manoj K Gupta Manoj K Gupta Section on Islet Cell and Regenerative Biology, Joslin Diabetes Center, Boston, MA, USA Search for more papers by this author Samir Softic Samir Softic Section on Integrative Physiology and Metabolism, Joslin Diabetes Center, Boston, MA, USA Division of Gastroenterology, Hepatology and Nutrition, Boston Children's Hospital, Boston, MA, USA Search for more papers by this author Leo D Wang Leo D Wang Department of Stem Cell and Regenerative Biology, Harvard Stem Cell Institute, Harvard University, Cambridge, MA, USA Section on Islet Cell and Regenerative Biology, Joslin Diabetes Center, Boston, MA, USA Division of Pediatric Hematology/Oncology/Stem Cell Transplantation, Dana-Farber/Boston Children's Center for Cancer and Blood Disorders, Boston, MA, USA Search for more papers by this author Young C Jang Young C Jang Department of Stem Cell and Regenerative Biology, Harvard Stem Cell Institute, Harvard University, Cambridge, MA, USA Section on Islet Cell and Regenerative Biology, Joslin Diabetes Center, Boston, MA, USA Search for more papers by this author Thomas Thomou Thomas Thomou Section on Integrative Physiology and Metabolism, Joslin Diabetes Center, Boston, MA, USA Search for more papers by this author Olivier Bezy Olivier Bezy orcid.org/0000-0003-4367-5043 Section on Integrative Physiology and Metabolism, Joslin Diabetes Center, Boston, MA, USA Search for more papers by this author Rohit N Kulkarni Rohit N Kulkarni Section on Islet Cell and Regenerative Biology, Joslin Diabetes Center, Boston, MA, USA Search for more papers by this author C Ronald Kahn C Ronald Kahn orcid.org/0000-0002-7583-9228 Section on Integrative Physiology and Metabolism, Joslin Diabetes Center, Boston, MA, USA Search for more papers by this author Amy J Wagers Corresponding Author Amy J Wagers [email protected] orcid.org/0000-0002-0066-0485 Department of Stem Cell and Regenerative Biology, Harvard Stem Cell Institute, Harvard University, Cambridge, MA, USA Section on Islet Cell and Regenerative Biology, Joslin Diabetes Center, Boston, MA, USA Search for more papers by this author Author Information Tata Nageswara Rao *,1,2,6, Manoj K Gupta2, Samir Softic3,4, Leo D Wang1,2,5,7, Young C Jang1,2,8, Thomas Thomou3, Olivier Bezy3,9, Rohit N Kulkarni2, C Ronald Kahn3 and Amy J Wagers *,1,2 1Department of Stem Cell and Regenerative Biology, Harvard Stem Cell Institute, Harvard University, Cambridge, MA, USA 2Section on Islet Cell and Regenerative Biology, Joslin Diabetes Center, Boston, MA, USA 3Section on Integrative Physiology and Metabolism, Joslin Diabetes Center, Boston, MA, USA 4Division of Gastroenterology, Hepatology and Nutrition, Boston Children's Hospital, Boston, MA, USA 5Division of Pediatric Hematology/Oncology/Stem Cell Transplantation, Dana-Farber/Boston Children's Center for Cancer and Blood Disorders, Boston, MA, USA 6Present address: Department of Biomedicine, Experimental Hematology, University Hospital Basel and University of Basel, Basel, Switzerland 7Present address: Departments of Immunooncology and Pediatrics, Beckman Research Institute, City of Hope, Duarte, CA, USA 8Present address: Georgia Institute of Technology, School of Biological Sciences, Parker H. Petit Institute for Bioengineering and Bioscience, Atlanta, GA, USA 9Present address: Internal Medicine Research Unit, Pfizer Inc., Cambridge, MA, USA *Corresponding author. Tel: +41 768030539; E-mail: [email protected] *Corresponding author. Tel: +1 6174960586; E-mail: [email protected] The EMBO Journal (2018)37:e100409https://doi.org/10.15252/embj.2018100409 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract A finely tuned balance of self-renewal, differentiation, proliferation, and survival governs the pool size and regenerative capacity of blood-forming hematopoietic stem and progenitor cells (HSPCs). Here, we report that protein kinase C delta (PKCδ) is a critical regulator of adult HSPC number and function that couples the proliferative and metabolic activities of HSPCs. PKCδ-deficient mice showed a pronounced increase in HSPC numbers, increased competence in reconstituting lethally irradiated recipients, enhanced long-term competitive advantage in serial transplantation studies, and an augmented HSPC recovery during stress. PKCδ-deficient HSPCs also showed accelerated proliferation and reduced apoptosis, but did not exhaust in serial transplant assays or induce leukemia. Using inducible knockout and transplantation models, we further found that PKCδ acts in a hematopoietic cell-intrinsic manner to restrict HSPC number and bone marrow regenerative function. Mechanistically, PKCδ regulates HSPC energy metabolism and coordinately governs multiple regulators within signaling pathways implicated in HSPC homeostasis. Together, these data identify PKCδ as a critical regulator of HSPC signaling and metabolism that acts to limit HSPC expansion in response to physiological and regenerative demands. Synopsis Hematopoietic stem and progenitor cells (HSPC) exert critical roles in the cell expansion required during homeostasis and regeneration of the blood system. Here, the protein kinase C delta (PKCδ) is shown to limit mouse HSPC output by controlling energy metabolism and hematopoietic differentiation. PKCδ restricts HSPC pool size by restraining cell cycle progression and regulating apoptosis. PKCδ deletion enhances recovery of HSPCs in settings of BM transplant and myelosuppression. PKCδ deletion enhances metabolic activity of HSPCs. PKCδ sets a threshold for HSPC activation and coordinately regulates multiple targets within progenitor cell signaling. Introduction Hematopoietic stem cells (HSCs) are a specialized subset of cells equipped with two cardinal features, self-renewal and multi-lineage differentiation potency, which are critical for their function in bone marrow transplantation (Orkin & Zon, 2008). The regulation of HSC self-renewal and differentiation in balance with proliferation and apoptosis governs the size and functional capacity of the stem cell pool, while defects in such regulation can result in leukemic transformation or depletion of normal hematopoietic activity (Yilmaz et al, 2006; Pietras et al, 2011; Wagers, 2012). Recent studies have revealed multiple important HSC regulators and suggest that an orchestrated interaction among cell-intrinsic signals (transcription factors, cell surface receptors, cell cycle regulators, and signal transducers) and extrinsic mediators (bone marrow niche components and soluble growth factors) controls HSC fate (Orkin & Zon, 2008; Wilson et al, 2009; Ehninger & Trumpp, 2011; Gazit et al, 2013). Furthermore, growing evidence suggests a link between the metabolic activity of HSCs and their capacity to preserve stem cell functions and hematopoietic differentiation potential (Yu et al, 2013; Burgess et al, 2014; Kohli & Passegue, 2014). Despite these advances, however, many of the molecular pathways and mechanisms that regulate the self-renewal, survival, expansion, and regenerative functions of blood-forming stem cells remain unknown. Improved understanding of the molecular machinery that determines HSC function will aid the development of innovative strategies to expand HSCs ex vivo and to prevent their involvement in hematopoietic cancers. Protein kinase δ (PKCδ) is a member of the novel subclass of PKC serine/threonine kinase isoforms and has been implicated in regulating key cellular processes including proliferation, apoptosis, differentiation, and metabolism via its role in diverse downstream signal transduction pathways (Basu & Pal, 2010; Bezy et al, 2011). Although the role of PKCδ in apoptosis appears to be stimulus- and context-dependent, in most cases, overexpression or activation of PKCδ induces apoptosis (Basu & Pal, 2010). PKCδ can be activated by diacyl glycerol (DAG) and phorbol esters (such as PMA) (Basu & Pal, 2010), which triggers a pro-apoptotic signaling cascade that may include proteolytic activation and translocation of PKCδ to the mitochondria (Limnander et al, 2011). PKCδ is widely expressed in the mouse and human hematopoietic systems (Limnander et al, 2011), and one of its best-studied functions in hematopoiesis is in B-cell signaling, where PKCδ deficiency enhances B-cell proliferation and leads to autoimmunity in mice and man (Miyamoto et al, 2002; Guo et al, 2004; Limnander et al, 2011). However, the role of PKCδ in regulating more primitive hematopoietic precursors, including blood-forming hematopoietic stem and progenitor cells (HSPCs), has not been interrogated. Given the established role of PKCδ in regulating cell survival, proliferation, and metabolism (Basu & Pal, 2010; Bezy et al, 2011), and the fact that perturbations in cell survival, proliferation, or metabolic signals can disrupt the functional integrity of HSPCs, we hypothesized that PKCδ may play a role in regulating HSPC homeostasis. Here, we test this hypothesis using in vivo and in vitro approaches and demonstrate that PKCδ restricts HSPC number and function in the steady-state and during hematopoietic stress conditions. PKCδ−/− mice showed increased numbers of HSPCs in the bone marrow and better competence in bone marrow transplantation assays. Mechanistically, these phenotypes could be attributed to accelerated cell cycle progression and reduced apoptosis in PKCδ-deficient HSPCs. Finally, we found that PKCδ-deficient HSPCs display enhanced mitochondrial activity and oxidative phosphorylation. Our results indicate a pivotal role for PKCδ in the regulation of HSPC proliferation and apoptosis and identify PKCδ as a critical rheostat controlling HSPC expansion. Thus, targeting of PKCδ may represent an attractive strategy to stimulate ex vivo expansion of HSPCs and enhance hematopoietic recovery following HSPC transplantation. Results PKCδ deficiency expands the primitive HSC pool in vivo To investigate the role of PKCδ in hematopoietic homeostasis, we first examined its expression during steady-state blood cell differentiation in the bone marrow of wild-type mice. Real-time PCR analysis of PKCδ mRNA in FACS-sorted Lineage negative (Lin−), Lin−Sca1+c-Kit+ (LSK) cells, LT-HSC, ST-HSC, MPP, myeloid progenitors (GMP, CMP, MEP), and common lymphoid progenitor (CLP) subsets revealed that PKCδ is expressed at variable levels by all HSPC populations, with the highest expression in CLP, LT-HSC, and MPPs. The lowest levels of PKCδ expression were observed in megakaryocyte-erythroid progenitors (MEP) (Fig 1A). This expression pattern suggests that PKCδ functions in primitive LT-HSCs, as well as in multiple other stages of hematopoiesis. Figure 1. PKCδ restricts HSPC pool size in the bone marrow A. Quantitative real-time PCR analysis of PKCδ mRNA levels in FACS-sorted Lin−, LT-HSC, ST-HSC, MPP, L−S−K+, GMP, CMP, MEP, and CLP subsets from C56BL/6 wild-type (6- to 9-week-old) mice bone marrow. Levels of PKCδ expression were normalized to an internal control gene (β-actin). Expression of PKCδ is shown relative to Lineage negative (Lin−) cells whose expression was arbitrarily set to 1 (n = 4 mice analyzed for each population). B, C. (B) FACS plots depict the percentage of live, Lin−Sca1+c-Kit+ (LSK) (left panel), and primitive CD34−Flt3−, CD34+Flt3−, and CD34+Flt3+ HSPCs among LSK cells (right panel). (C) Bar charts show the average frequency (left) and absolute number (right) ± SEM of the indicated populations, analyzed from 2 femurs and 2 tibias per mouse (n = 10 mice analyzed for each genotype). D. FACS plots show percentages of “SLAM code” based stem and progenitor cells in the LSK population. Bar graphs show the average frequency (left) and absolute numbers (right) ± SEM of LT-HSC per 2 femurs and 2 tibias. (n = 10 mice analyzed for each genotype). E. FACS plots illustrate gating and frequencies of myeloid progenitors (MP), granulocyte–monocyte progenitor (GMP), common myeloid progenitor (CMP), and megakaryocyte–erythroid progenitor (MEP) population. Bar graphs show the frequencies (left) and absolute numbers (right) of L−S−K+ MP and GMP, CMP, and MEP populations in the BM of WT (n = 12) and PKCδ KO (n = 12). F. FACS plots illustrate the gating and percentages of common lymphoid progenitors (CLP). Bar graph shows the absolute number of CLPs per 2 femurs and 2 tibias (n = 5 mice for each genotype). G. FACS plots illustrate the percentages of uncommitted lymphoid progenitors (Lin− IL-7Rα+Flt3+Ly-6D−) and B lymphoid progenitors (BLP). Bar graph represents the average frequencies of CLPs, BLP, and uncommitted lymphoid progenitors in total BM (n = 5 mice per genotype). H. Limiting dilution analysis (LDA) demonstrates an increased frequency of long-term repopulating HSCs in PKCδ KO BM (solid red lines) compared with WT BM (black solid lines). Engraftment data shown at 14 weeks post-BMT. Plots depict the percentages of recipient mice containing < 1% CD45.2+ blood nucleated cells. Dotted lines represent the 95% confidence interval of the same (P = 0.0005). Data information: All data are presented as mean ± SEM. *P < 0.05; **P < 0.01, and ***P < 0.001 by with one-way ANOVAs with Sidak's multiple comparisons test (A) or two-tailed Student's unpaired t-test (C-G) analysis for comparison of WT to PKCδ KO mice. Overall, test for differences in stem cell frequencies between WT to PKCδ KO mice was determined with likelihood ratio test of single-hit model (H). Download figure Download PowerPoint As an initial approach to probe the importance of PKCδ signaling in bone marrow (BM) HSPCs, we exposed FACS-purified wild-type HSPCs to Indolactam V (Indo-V), a non-specific PKC isoform activator, or Mallotoxin (MTX, also called Rottlerin), a PKC inhibitor that shows some selectivity for PKCδ when used at lower concentrations (3–5 μM) (Gschwendt et al, 1994). Interestingly, Indo-V accelerated acquisition of lineage markers and depletion of primitive LSK HSPCs from the culture, whereas MTX increased the relative proportion of Lin− cells and LSK cells (Fig EV1A–C). Consistent with enhanced maintenance of more primitive HSPCs in PKC-inhibited cultures, transplantation of MTX-treated HSPCs resulted in 2- to 3-fold higher contributions to mature hematopoietic lineages than transplantation of untreated control cells (Fig EV1D), although levels of engraftment were generally low in both experimental groups. These data suggest that the PKC pathway plays a role in regulating the number and/or function of hematopoietic reconstituting cells. To evaluate this possibility more specifically, we next determined the frequencies and absolute numbers of BM HSPCs in germline PKCδ knockout mice (Bezy et al, 2011), which allow for highly specific inactivation of PKCδ in vivo. PKCδ knockout mice displayed a slight but significant increase in BM cellularity (Appendix Fig S1A), and immunophenotypic analysis further revealed an increase in the frequency and absolute numbers (~2- to 3-fold increase) of LSK cells in the BM of PKCδ-deficient mice (Fig 1B, left plots). This expansion of LSK cells was unique to the PKCδ-deficient BM, as analysis of e14.5 fetal livers revealed equivalent frequencies of LSK cells in PKCδ−/−, PKC−/−, and PKCδ+/+ mice (Appendix Fig S1B). More comprehensive analysis of the adult LSK compartment using Flt3- and CD34-based immunophenotypic fractionation indicated a significant increase in the frequency and absolute numbers of LT-HSC (CD34−Flt3−LSK), ST-HSC (CD34−Flt3−LSK), and MPP (CD34+Flt3+LSK) (Fig 1B and C). Consistent with this, “SLAM code” based analysis (Kiel et al, 2005) of BM LSK cells confirmed an ~3-fold increase in the frequency and numbers of LT-HSC (CD150+CD48−LSK) in PKCδ-deficient mice compared with wild-type littermates (Fig 1D). Click here to expand this figure. Figure EV1. Pharmacological modulation of PKCδ activity preserves HSPC activity in vitro A, B. One hundred HSCs were sorted from WT mice and cultured in 96-well (U-bottom) plates in triplicate in the presence of mSCF (20 ng/ml) and mTPO (20 ng/ml) and with or without Mallotoxin (MTX, 5 μM) or Indolactam V (Indo-V, 10 μM) for indicated time. (A) FACS histograms and (B) bar graph show the percentage of Lin- cells (pre-gated on live cells). C. At the indicated time of culture, cells were analyzed for LSK phenotype. Representative FACS plots showing the percentage of cells retaining LSK phenotype. Data are representative of two independent experiments (n = 5 mice total per treatment group). D. Schematic of competitive reconstitution analysis of MTX-treated WT HSPCs. After 13 days in culture, cells were transplanted into recipient mice along with 1 × 106 competitive total BM cells (CD45.1+). Percentage of total donor-derived cells (CD45.2+), B cells (B220+), and myeloid cells (CD11b+ Gr1+) in the peripheral blood was analyzed at indicated time (n = 6 mice per condition). Data information: All data shown as mean ± SEM. *P < 0.05 and **P < 0.01 by repeated measures one-way ANOVA analysis with Bonferroni posttest. Download figure Download PowerPoint Intriguingly, despite an increased frequency of primitive HSPCs in the BM of PKCδ−/− mice, analysis of myeloid progenitors (Lin−Sca1−kit+, MyP) and further fractionation of these heterogeneous cell types into GMP (Lin−Sca1−kit+CD34+FcεRγIII/II+), CMP (Lin−Sca1−kit+CD34+FcεRγIII/Illo), and MEP (Lin−Sca1−kit+CD34+ FcεRγIII/II+) revealed that the frequencies of these subsets were generally unchanged, with a modest and selective decrease in frequency and absolute number of CMPs in the absence of PKCδ (Fig 1E). Consistent with these observations, PKCδ−/− BM cells showed a significantly increased frequency of in vitro colony-forming cells (CFU-C), measured at day 12 (Appendix Fig S1C). Furthermore, in vivo colony-forming unit-spleen (CFU-S) assays (Zhang et al, 2009), in which transplanted BM cells form hematopoietic colonies in recipient spleens, revealed equivalent numbers of colonies at day 8 after transplantation (CFU-S8) and significantly higher numbers of colonies at day 13 (CFU-S13) in recipients of KO BM (Appendix Fig S1D). The frequencies of mature circulating myeloid and erythroid cells were not detectably perturbed in PKCδ-deficient mice (Table EV1). In both mice and humans, PKCδ deficiency causes increased B-cell proliferation and autoimmunity (Miyamoto et al, 2002; Kuehn et al, 2013). To determine whether this B-cell hyperproliferative phenotype due to PKCδ deficiency may be evident at the progenitor stage, we measured the frequency and absolute numbers of common lymphoid progenitors (CLP, Lin−IL-7Rα+Flt3+) and B lineage-committed progenitors (BLPs, Lin−IL7-Rα+Flt3+Ly-6D+). Our analyses indicated similar frequencies and absolute numbers of CLPs and BLPs in the bone marrow of PKCδ+/+ and PKCδ−/− mice (Fig 1F and G). Finally, to assess whether the increase in phenotypic HSCs in PKCδ-deficient mice correlated with an increase in hematopoietic reconstituting cells, we performed limit dilution transplantation assays, in which graded numbers of PKCδ+/+ or PKCδ−/− BM cells (CD45.2+) were mixed with a fixed number (2.5 × 105) of recipient type BM cells (CD45.1+) and transplanted into lethally irradiated CD45.1 recipients. These studies revealed a significant increase in reconstituting cell frequency in PKCδ−/− as compared to PKCδ+/+ BM cells (1 in 11,042 versus 1 in 50,364, P = 0.0005, Fig 1H). Thus, deficiency of PKCδ results in a significant immunophenotypic expansion of the LT-HSC, ST-HSC, and MPP populations, an increase in primitive CFU-S13 activity, and an almost four-fold increase in the frequency of hematopoietic reconstituting cells as assayed by limit dilution engraftment studies. Collectively, these data suggest that functional PKCδ acts normally to limit expansion of the phenotypic and functional pool of HSPCs in adult bone marrow. Accelerated proliferation and reduced apoptosis of PKCδ-deficient HSPCs in vivo We next investigated the underlying mechanisms that may account for the increased HSPC pool size in the BM of adult PKCδ-deficient mice. Because PKCδ has been implicated in regulating cell cycle and apoptosis in various cell lines (Basu & Pal, 2010), and loss of PKCδ is associated with a dramatic increase in HSPCs in vivo (Fig 1), we hypothesized that increased HSPC numbers in PKCδ-deficient BM could reflect an altered proliferation rate or decreased spontaneous cell death in vivo. To test this hypothesis, we examined possible changes in the cell cycle status of HSPCs during steady-state hematopoiesis using intracellular staining with Ki67/Hoechst. These analyses revealed a significant increase (two-fold) in the percentage of cycling (S/G2/M, identified as Ki67hi Hoechsthi) HSPCs and early progenitors in PKCδ-knockout mice as compared to WT controls (Fig 2A). PKCδ-KO HSCs further exhibited a substantial decrease in the quiescent G0 phase (Ki67low Hoechstlow) and a concomitant increase in the fraction of cells in the active G1 phase (Ki67+Hoechstlow) of cell cycle. Next, we performed a short-term (20 hr) in vivo BrdU labeling assay to quantify the frequency of actively proliferating cells in HSPC subsets (Fig 2B). In line with our findings using combinatorial Ki67/Hoechst staining, BrdU incorporation revealed an approximately 2.5-fold higher rate of BrdU incorporation in LT-HSCs from KO mice compared to WT controls (~20% versus 7.5%, Fig 2C). A moderate increase in BrdU+ cells was also observed in PKCδ-deficient ST-HSCs and MPPs, but not among myeloid progenitor stages (Fig 2B and C). These data suggest that loss of PKCδ activates cell cycle progression of primitive HSPCs, which in turn leads to their expansion. Figure 2. Accelerated proliferation and reduced apoptosis in subsets of PKCδ-deficient HSPCs Representative FACS profiles of HSPC cell cycle analysis using combinatorial staining for Ki67 and Hoechst 33342. Bar charts depict the average percentage of cells in each phase of the cell cycle for each LSK subset from WT (n = 6) and PKCδ KO (n = 7) mice. Data compiled from two independent experiments. FACS profiles of indicated BM subsets from WT and PKCδ KO mice 20 hr after BrdU injection. Average percentages of cells in each phase of the cell cycle phases for each of the indicated HSPC subsets from WT and PKCδ KO mice. Data are pooled from two independent experiments (totaling n = 6–7 mice per genotype). Representative FACS plots and summary of FACS data analyzing the frequency of apoptotic HSPC cells using co-staining for Annexin V and 7-AAD (WT and PKCδ−/− (n = 7 per genotype). Data information: All data are presented as mean ± SEM, *P < 0.05 and **P < 0.001, with significance determined by two-tailed Student's unpaired t-test analysis. Download figure Download PowerPoint We next assessed the frequencies of apoptotic cells among HSPC subsets using Annexin V/7-AAD staining. Percentages of Annexin V+/7-AAD− (apoptotic) LT-HSCs were not substantially different between WT and PKCδ−/− mice; however, ST-HSCs, MPPs, and myeloid progenitors from PKCδ−/− mice exhibited a significantly lower rate (two-fold reduction) of spontaneous cell death as compared to their WT counterparts (Fig 2D). These data suggest that loss of PKCδ signaling in progenitor cells downstream of LT-HSCs exerts a protective role that reduces apoptotic rate. Collectively, these results indicate that the expansion of the primitive HSPC compartment apparent in PKCδ-deficient BM reflects alterations in both cell cycle progression and survival of HSPCs, with differential impact on distinct subsets of multi-potent and oligopotent progenitors. Loss of PKCδ enhances competitive repopulation capacity The accumulation of primitive stem and progenitor cells in PKCδ-deficient mice BM could result from loss of PKCδ activity within HSPCs themselves or from defects in microenvironmental cues arising due to loss of PKCδ in hematopoietic or non-hematopoietic lineages that could indirectly affect their numbers. To distinguish hematopoietic system intrinsic versus extrinsic effects of PKCδ deficiency on HSPC function, we performed competitive BM transplants, in which total BM cells from WT or PKCδ−/− mice were transplanted into lethally irradiated congenic WT mice (CD45.1+) along with equal numbers of recipient type (CD45.1+) whole BM cells (Fig 3A). Analysis of peripheral blood chimerism in primary recipients revealed a slight but significant increase in engraftment of CD45.2+ hematopoietic lineages, with particular enhancement of B-cell (B220+) (Limnander et al, 2011) and myeloid (Gr1+Mac-1+) reconstitution, in mice receiving BM cells from PKCδ−/− as compared to control mice (Fig 3B). Figure 3. Loss of PKCδ enhances competitive repopulation and self-renewal of HSPCs in vivo without exhaustion Schematic of competitive BM transplantation assay. Percent of total donor-derived, hematopoietic cells (CD45.2+), B cells (B220+), myeloid cells (CD11b+Gr1+), and T cells (CD3+) in the peripheral blood (PB) of recipient mice, as determined by FACS at the indicated time points. The statistical significance of differences was determined using two-way ANOVAs with Holm–Sidak's multiple comparisons tests (n = 8 recipients per genotype in each experiment). Percent donor-derived HSPCs in the BM of secondary (left) and tertiary (right) recipients of WT or PKCδ−/− marrow. Data are compiled from two independent experiments (n = 8 recipients per genotype). Data information: All data are presented as mean ± SD. *P < 0.05, **P < 0.001, with significance determined by repeated measures two-way ANOVA analysis with Sidak's multiple comparison tests for comparison of recipients of WT and PKCδ KO marrow at each time point and for each cell population. Download figure Download PowerPoint To test the long-term self-renewal and differentiation capacities of PKCδ−/− HSPCs, we next conducted serial transplantation studies, which impose overt proliferative stress on HSCs (Min et al, 2008; Pietras et al, 2014; Rao et al, 2015). Defects in regulating self-renewal capacity or aberrant cell cycle activity can lead to HSC exhaustion in this setting. Total BM cells were harvested from primary recipients 20 weeks after initial transplantation and infused into lethally irradiated secondary recipients. Hematopoietic reconstitution was monitored by staining of peripheral blood cells sampled over 16 weeks after transplant (Fig 3A and B). Then, at 16 weeks after secondary transplant, total BM cells were harvested again to assess reconstitutio" @default.
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• W2901883599 date "2018-11-16" @default.
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• W2901883599 title "Attenuation of PKC δ enhances metabolic activity and promotes expansion of blood progenitors" @default.
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