PKCθ is dispensable for megakaryopoiesis

• Blood platelets
• bone marrow
• megakaryocytes
• protein kinase C
• thrombocytopenia

Megakaryocytes are large polyploid cells that produce platelets. Megakaryocytes undergo an abbreviated cell cycle (known as endomitosis) that does not proceed to cytokinesis [1]. After repeated rounds of endomitosis, the fully mature megakaryocyte begins the process of platelet production by extending long microtubule-based extensions called proplatelets [2]. Megakaryocyte differentiation and maturation is driven primarily by the cytokine thrombopoietin (TPO) [3].

Protein kinase C (PKC) is a group of serine threonine kinases that have a wide range of functions in a variety of cell types. There are three classes of PKC’s, (conventional, novel, and atypical), which are grouped by their cofactor requirements. It is well known that PKCs play an important role in the differentiation of megakaryocytes from cell lines [4–8]. However, the function of specific PKC isoforms, with the exception of ɛ and α, in primary megakaryopoiesis is currently unknown [9–11]. A previous report suggests that PKCθ mRNA expression is enhanced during megakaryocyte differentiation from progenitor cells, more so than any other PKC isoform [12]. We, and others, have previously reported that PKCθ regulates platelet functional responses [13–15]. Therefore, we sought to determine the role of PKCθ in megakaryocyte differentiation using a knockout mouse model.

We began by examining blood cell counts in PKCθ^−/− mice. After a quick analysis we were able to determine that platelet count (659 ± 31 vs. 645 ± 31 10⁻³/μL), mean platelet volume (4.06 ± 0.05 vs. 4.05 ± 0.07 fL), and white blood cell count (4.29 ± 0.60 vs. 4.18 ± 0.32 10⁻³/μL) were all unaltered in PKCθ^−/− mice compared to WT littermates, respectively. Similarly, using flow cytometry we found that bone marrow megakaryocyte number and DNA content were not different between PKCθ^−/− and WT mice, respectively (Figure 1A and B). This suggests that basal megakaryopoiesis and platelet production are not influenced by PKCθ. Therefore, we evaluated the effect of exogenous TPO on megakaryocyte differentiation from PKCθ^−/− bone marrow cells.

Figure 1. Bone marrow megakaryopoiesis is not altered with PKCθ deficiency. Megakaryocytes from PKCθ^−/− and WT littermate control mouse bone marrow were labeled with CD41 and propidium iodide. Neither DNA (A) nor megakaryocyte number (B) were different.

Bone marrow from PKCθ^−/− and WT littermate control mice was cultured in exogenous TPO for 5 days. Megakaryocytes were labeled with anti-CD 41 antibody and propidium iodide was used to quantify DNA. However, DNA content was unchanged with PKCθ deficiency compared to littermate controls. This suggests that PKCθ is not involved in TPO-mediated signaling in megakaryocytes.

While it does not appear that PKCθ is important for basal megakaryopoiesis, it is possible that PKCθ may have a function during thrombocytopenic insult. Therefore, we induced an immune thrombocytopenia using one I.P. injection of 50 μg/kg anti-mouse CD41 antibody, and monitored recovery. After twenty-four hours we observed greater than 85% platelet reduction in both PKCθ^−/− and WT littermate control mice, which is consistent with previous reports [16, 17]. However, recovery up to 7 days was unaltered with PKCθ deficiency suggesting that PKCθ does not contribute to in vivo megakaryopoiesis or platelet production (Figure 2).

Figure 2. PKCθ does not influence recovery from immune thrombocytopenia. PKCθ^−/− and WT littermate control mice were injected with mouse anti-CD41 antibody to reduce platelet counts. Platelet recovery was monitored every 24 hours for 5 days.

Data from this study suggests that PKCθ is not necessary for megakaryopoiesis and platelet production. Although PKCθ mRNA expression increases during megakaryocyte differentiation, it does not appear that PKCθ protein expression is important [12]. It is therefore possible that the increase in PKCθ mRNA expression in the megakaryocyte is simply intended to provide the platelet with sufficient PKCθ.

Addendum

J. C. Kostyak designed experiments, collected and analyzed data, and wrote the paper. S. P. Kunapuli provided overall direction, designed experiments, and wrote the paper.

Declaration of interest

The authors have no conflicts of interest to disclose. This work was supported by National Institutes of Health grants HL93231 and HL118593 (S. P. K.). JCK is supported by a training grant in Thrombosis and Hemostasis from the NIH T32 HL07777 (S. P. K.).