Research Article: Reduced PKC α Activity Induces Senescent Phenotype in Erythrocytes

Date Published: September 4, 2012

Publisher: Hindawi Publishing Corporation

Author(s): Rukmini B. Govekar, Poonam D. Kawle, Suresh H. Advani, Surekha M. Zingde.


The molecular mechanism mediating expression of senescent cell antigen-aggregated or cleaved band 3 and externalized phosphatidylserine (PS) on the surface of aged erythrocytes and their premature expression in certain anemias is not completely elucidated. The erythrocytes with these surface modifications undergo macrophage-mediated phagocytosis. In this study, the role of protein kinase C (PKC) isoforms in the expression of these surface modifications was investigated. Inhibition of PKC α by 30 μM rottlerin (R30) and 2.3 nM Gö 6976 caused expression of both the senescent cell marker-externalized PS measured by FACS analysis and aggregated band 3 detected by western blotting. In contrast to this observation, but in keeping with literature, PKC activation by phorbol-12-myristate-13-acetate (PMA) also led to the expression of senescence markers. We explain this antithesis by demonstrating that PMA-treated cells show reduction in the activity of PKC α, thereby simulating inhibition. The reduction in PKC α activity may be attributed to the known downregulation of PMA-activated PKC α, caused by its membrane translocation and proteolysis. We demonstrate membrane translocation of PKC α in PMA-treated cells to substantiate this inference. Thus loss of PKC α activity either by inhibition or downregulation can cause surface modifications which can trigger erythrophagocytosis.

Partial Text

Human erythrocytes have a definite lifespan of 120 ± 4 days in circulation and thereafter are marked for phagocytosis by cell surface modifications, such as aggregation or cleavage of protein band 3 and exposure of PS [1–3]. Time-compressed expression of these markers leads to premature eryptosis in anemias [4, 5]. Molecular events which mediate expression of these surface markers of senescence have been partly delineated in erythrocytes mainly under oxidative conditions [6, 7]. They appear to recapitulate the cytoplasmic events in apoptosis of nucleated cells such as translocation of Fas into rafts, formation of a Fas-associated complex, and activation of caspases 8 and 3 [8]. Activation of caspase 3 in turn is associated with cleavage of band 3 [9], which generates senescent cell antigen in erythrocytes [10], as well as causes impairment of aminophospholipid flippase activity and PS externalization [11]. This similarity of molecular events in eryptosis and apoptosis prompted us to explore the role of PKC isoforms, which have distinct tissue-specific roles in both cell survival and apoptosis of nucleated cells [12], in eryptosis.

We have earlier demonstrated that PKC α is the only DAG-dependent and thus PMA-activated PKC isoform expressed in erythrocytes, while PKC ζ, ι, and μ are atypical isoforms which are non-responsive to PMA [14]. Thus the significant increase (P = 0.021; Wilcoxon Signed Ranks Test) in cells expressing externalized PS upon the activation of PKC with PMA (Figure 1(a)) can be attributed to PKC α. This is in keeping with the literature reports on PKC-induced PS externalization [18, 19] as well as its attribution to PKC α [20]. In cells treated with 4αPDD, a biologically inactive structural analogue of PMA, the percentage of cells expressing PS remained unchanged. The effect of PMA was less obvious on aggregation of band 3 (Figure 1(b)).

Thus, PKC isoforms, which have distinct tissue-specific roles in both cell survival and apoptosis of nucleated cells, can also mediate eryptosis. While literature reports the role of activation of PKC/PKC α in the expression of externalized PS, we demonstrate that loss of PKC α activity due to inhibition or activation-linked downregulation can cause expression of not only externalized PS but also aggregated band 3. The study has thus unravelled a molecular event causative of the expression of two cell surface modifications, which can trigger erythrophagocytosis.




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