PKC-epsilon deficiency alters progenitor cell populations in favor of megakaryopoiesis

Abstract

Background: It has long been postulated that Protein Kinase C (PKC) is an important regulator of megakaryopoiesis. Recent contributions to the literature have outlined the functions of several individual PKC isoforms with regard to megakaryocyte differentiation and platelet production. However, the exact role of PKCε remains elusive.

Objective: To delineate the role of PKCε in megakaryopoiesis.

Approach and results: We used a PKCε knockout mouse model to examine the effect of PKCε deficiency on platelet mass, megakaryocyte mass, and bone marrow progenitor cell distribution. We also investigated platelet recovery in PKCε null mice and TPO-mediated signaling in PKCε null megakaryocytes. PKCε null mice have higher platelet counts due to increased platelet production compared to WT littermate controls (p<0.05, n = 8). Furthermore, PKCε null mice have more bone marrow megakaryocyte progenitor cells than WT littermate control mice. Additionally, thrombopoietin-mediated signaling is perturbed in PKCε null mice as Akt and ERK1/2 phosphorylation are enhanced in PKCε null megakaryocytes stimulated with thrombopoietin. Finally, in response to immune-induced thrombocytopenia, PKCε null mice recovered faster and had higher rebound thrombocytosis than WT littermate control mice.

Conclusions: Enhanced platelet recovery could be due to an increase in megakaryocyte progenitor cells found in PKCε null mice as well as enhanced thrombopoietin-mediated signaling observed in PKCε deficient megakaryocytes. These data suggest that PKCε is a negative regulator of megakaryopoiesis.

Fig 1. Platelet mass is enhanced in PKCε deficient mice.

A) Western blot analysis of PKCε, PKCα, and PKCδ in PKCε^+/+ and PKCε^-/- mouse megakaryocytes (n = 3). Actin was used to assess loading. B) Platelet counts from PKCε^-/- and WT littermate control (PKCε^+/+) whole blood (n = 9). C) Reticulated “new” platelets expressed as a percent of total blood cells in PKCε^+/+ and PKCε^-/- mice (n = 6). D) Platelet clearance in PKCε^+/+ and PKCε^-/- mice (n = 3). E) Representative images of femur sections stained with H&E. White arrows denote megakaryocytes. Images were captures used a Nikon E1000 microscope at 200X magnification. F) Quantitation of megakaryocytes per field of view (FOV). * p < 0.05, n = 11.

Fig 2. PKCε^-/- mice have a reduced LSK population, but a heightened megakaryocyte progenitor cell population.

A) Schematic showing gates used to define each progenitor cell population. LK cells are defined as Lineage (Lin) negative cells that stain C-Kit+, Sca-1-. LSK cells stain Lin-, Sca-1+, C-Kit+. Megakaryocyte progenitors (MkP) are from the LK population and stain CD41+, CD150+. The LSK population is used to define multipotent progenitor cells (MPP), which stain CD105- and CD150-, as well as hematopoietic stem cells (HSC), which stain CD105+ and CD150+. B-F) Quantification of each progenitor cell population as defined in A in PKCε^+/+ and PKCε^-/- mouse bone marrow expressed as a percentage of total bone marrow cells. * p < 0.05 compared to PKCε^+/+, n = 4.

Fig 3. Bone marrow and splenic megakaryocyte number and DNA content are unaltered in PKCε^-/- mice.

A) Quantification of bone marrow megakaryocyte DNA content expressed as a percentage of total megakaryocytes (n = 7). B) Quantification of bone marrow megakaryocyte number expressed as percent nucleated bone marrow cells (n = 7). C) Quantification of splenic megakaryocyte DNA content expressed as a percentage of total megakaryocytes (n = 8). D) Quantification of splenic megakaryocyte number expressed as a percentage of nucleated spleen cells (n = 8).

Fig 4. Megakaryocyte number is enhanced in PKCε^-/- bone marrow cultured with exogenous TPO.

A) Megakaryocyte DNA content in PKCε^-/- and PKCε^+/+ bone marrow cultured in the presence of 50 ng/mL TPO. B) Megakaryocyte number is cultured bone marrow from PKCε^-/- and PKCε^+/+ mice supplemented with 50 ng/mL TPO. * p < 0.05 compared to PKCε^+/+, n = 7.

Fig 5. TPO-mediated Akt and ERK1/2 phosphorylation is enhanced in PKCε^-/- megakaryocytes.

A) Western blot showing Akt phosphorylation at Ser473 in lysates from megakaryocytes isolated from PKCε^+/+ or PKCε^-/- mice treated with 50 ng/mL TPO (+) or not (-). Lane loading was assessed using total Akt (n = 4). B) Quantification from A expressed as a ratio of phosphorylated Akt to total Akt and normalized to PKCε^+/+ without TPO treatment. C) Western blot showing ERK1/2 phosphorylation in PKCε^+/+ and PKCε^-/- megakaryocytes treated with or without TPO. Lane loading was assessed using total ERK (n = 7). D) Quantification of C expressed as a ratio of phosphorylated ERK1/2 to total ERK1/2 and normalized to PKCε^+/+ without TPO treatment. * p < 0.05 compared to PKCε^+/+ with TPO treatment.

Fig 6. Platelet recovery and rebound thrombocytosis is enhanced in PKCε^-/- mice following immune-induced thrombocytopenia.

A) PKCε^+/+ and PKCε^-/- Mice were injected (I.P.) with 50 μg/kg anti-mouse CD41 antibody at day 0. Blood was collected daily via submandibular puncture for 5 days and again on day 7 and platelets were enumerated. * p < 0.05 compared to corresponding PKCε^+/+ time point, n = 7. B) Representative images of proplatelet producing megakayrocytes. C) Quantitation of proplatelet production taken from several fields per experiment, expressed as a percentage of total megakaryocytes. *p < 0.05, n = 11.