Ankyrin exposure induced by activated protein kinase C plays a potential role in erythrophagocytosis

https://doi.org/10.1016/j.bbagen.2015.10.017Get rights and content

Highlights

  • PMA induces ankyrin exposure on the erythrocyte surface and erythrophagocytosis.

  • Calcium internalization is required for ankyrin exposure.

  • Phosphorylation of membrane proteins is required for ankyrin exposure.

  • Calcium internalization and protein phosphorylation induce ankyrin exposure.

Abstract

Background

In physiological and pathological conditions activated protein kinace C (PKC) has been observed in the erythrocytes. Externalization of ankyrin followed by Arg–Gly–Asp (RGD)/integrin recognition also triggers erythrophagocytosis. In the present study, to test whether activated PKC is associated with ankyrin exposure in erythrophagocytosis.

Methods

Phorbol 12-myristate-13-acetate (PMA)-induced PKC activation and ankyrin phosphorylation were tested, and under different treatment conditions the subpopulation of erythrocytes with ankyrin exposure and the levels of intracellular calcium were analyzed by flow cytometry.

Results

Results showed that treatment of erythrocytes with PMA in a calcium-containing buffer led to ankyrin exposure. In the absence of extracellular calcium, no ankyrin exposure was observed. PKC inhibition with calphostin C, a blocker of the PMA binding site, completely prevented the calcium entry, protein phosphorylation and ankyrin exposure. PKC inhibition with chelerythrine chloride, an inhibitor of the active site, diminished the level of ankyrin-exposing cells and ankyrin phosphorylation; however it even led to a higher percentage of cells with increased levels of calcium than with PMA treatment alone. Although PKC was activated and ankyrin phosphorylation occurred, no ankyrin exposure was observed in the absence of extracellular calcium.

Conclusion

Analyses of results suggested that PMA induces calcium influx into the erythrocytes, leading to the activation of calcium-dependent enzymes and the phosphorylation of membrane proteins, ultimately inducing ankyrin exposure and erythrophagocytosis. This study may provide insights into the molecular mechanisms of removing aged or diseased erythrocytes.

Introduction

Although erythrocytes cannot synthesize proteins, erythrocyte aging and removal influence important processes in the body [1]. Every day, approximately 2 × 1011 human erythrocytes are being destroyed and replenished throughout the 100–120 d lifespan of these cells [2]. Thus, macrophages play an important role in mediating the sequestration of aged or diseased erythrocytes. In addition, the mechanisms for macrophage erythrophagocytosis must be very robust. The presence of phosphatidylserine (PS) at the outer leaflet of erythrocytes is documented as a signal for the clearing of circulating erythrocytes [3], [4]. Macrophages recognize, engulf, and degrade erythrocytes by using specific receptors for PS [5], [6]. In erythrocytes, RGD-containing ankyrin exposed on the cell surface can also trigger αvβ3 integrin-mediated erythrophagocytosis [7].

Proteins that contain the RGD attachment site interact with integrin receptors, which constitute a major recognition system for cell adhesion [8], [9]. The integrin–mediated cell adhesion influences and regulates cell migration, growth, differentiation, and apoptosis. Macrophages equipped with αvβ3 and other receptors phagocytose apoptotic cells (e.g., leukocytes), and the RGD peptide induces cell death of chondrocytes and synovial cells [10], [11], [12]. In erythrocytes ankyrin exposure followed by RGD/integrin recognition plays a potential role in the erythrocytes adheres to macrophages preceding phagocytosis. The RGD motif of ankyrin in erythrocytes can be recognized by the αvβ3 integrin receptor. This motif can also be exposed on the erythrocyte surface when cells are incubated with calcium and sheared at physiological levels [7], [8], [9].

A number of studies have documented that skeletal proteins can be phosphorylated by a number of different kinases and are dephosphorylated by various phosphatases [13], [14]. PKC is a family of serine/threonine-specific protein kinases consisting of at least 10 members [15]. Several subtypes of PKC are expressed in erythrocytes, and PKCα is the only phorbol ester-responding subtype reported to be present in erythrocytes [16]. PMA is a commonly used PKC-specific activator that induces membrane protein phosphorylation (e.g., band 4.1, 4.9, and adducin), and stimulates erythrocyte calcium entry and PS exposure [16], [17], [18]. Specifically, removal of extracellular glucose induces activation of PKC and further promotes phosphorylation of channel proteins, which in turn activates the cation channels. The following entry of cation results in activation of the calpain, and final eryptosis [17], [19]. In vitro studies have shown that cyclic AMP-dependent protein kinase phosphorylates ankyrin in whole erythrocytes or ghost membrane [13], [14]. Phosphorylation reduces the affinity of ankyrin for both the internal fragment of band 3 and spectrin. This process also weakens the interactions between the membrane skeleton and the rest of the membrane. Ankyrin phosphorylation by PKC also modulates membrane protein interactions [20], [21], [22]. Thus, although the cell surface exposure of RGD-containing ankyrin triggers αvβ3 integrin-mediated erythrophagocytosis and activated PKC induces programmed cell death, whether or not PKC activation is associated with ankyrin exposure remains unclear.

Based on these observations, we hypothesized that PKC activation using PMA could lead to ankyrin exposure. To test this hypothesis, we assessed the relationship between PMA-induced activation of PKC and ankyrin-exposing cells. We report here that activated PKC stimulated the influx of calcium into a subpopulation of erythrocytes and induced protein phosphorylation, ultimately leading to ankyrin exposure. We conclude that PKC activation is associated with ankyrin exposure, and this may provide an underlying molecular mechanism for removal aged or diseased erythrocytes.

Section snippets

Materials

PMA, 4α-phorbol 12, 13-didecanoate, calcium ionophore A23187, calphostin C, okadaic acid and chelerythrine chloride were obtained from Sigma. Integrin αvβ3 was obtained from R&D Systems. Anti-ankyrin antibody, goat anti-mouse IgG-HRP and polyclonal antibody against spectrin were purchased from Santa Cruz. Anti-PKCα antibody and goat anti-rabbit IgG-HRP were purchased from Abcam. Anti-phosphoserine antibody was from Chem Pharmaceuticals Inc. in Canada. Human monocytic cell line THP-1 (TIB-202)

PMA induced ankyrin exposure

Flow cytometry analysis was performed to test whether or not PMA induces ankyrin exposure on erythrocyte surfaces. PMA-induced erythrocytes were probed with the immune staining of ankyrin. Ankyrin exposure was clearly observed in the erythrocytes incubated with PMA at 3 or 6 μM in the presence of extracellular calcium (Fig. 1A). However, no ankyrin exposure was observed in the absence of extracellular calcium (right part in Fig. 2B). The subpopulation of erythrocytes with ankyrin exposure

Discussion

Biochemical and functional studies have documented that aging or damage induced by oxidative stress, osmotic shock, energy depletion, or hematological diseases results in erythrophagocytosis [32], [33]. Activated PKC is present in the erythrocytes of individuals with essential hypertension, senescent erythrocytes, sickle erythrocytes, and chronic myelogenous leukemia [34], [35], [36], which involves in regulating erythrocyte programmed death [17]. Previous studies elucidate the death mechanisms

Conflict of interest

The authors declare no conflict of interest.

Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (NSFC 11072275 and 31271229) and Chinese National 863 High Tech Research and development program (No. 2011AA02A103). F.T. designed experiments, analyzed the data, and wrote the paper; R.W., Y. R. and Y.X. performed research and analyzed the data; X.D., Y. Z. and D. C. designed experiments, analyzed the data.

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