PKCδ Deficiency Enhances Megakaryopoiesis and Recovery from Thrombocytopenia

Abstract

Objective

We previously determined that Protein Kinase C delta (PKCδ) regulates platelet function. However, the function of PKCδ in megakaryopoiesis is unknown.

Approach, Results

Using PKCδ^−/− and WT littermate mice we found that deficiency of PKCδ caused an increase in white blood cell and platelet counts, as well as bone marrow and splenic megakaryocytes (p < 0.05). Additionally, megakaryocyte number and DNA content were enhanced in PKCδ^−/− mouse bone marrow following culture with exogenous thrombopoietin (Tpo) compared to WT (p < 0.05). Importantly, Tpo-induced signaling was also altered with PKCδ deletion as both ERK1/2 and Akt308 phosphorylation were heightened in PKCδ^−/− megakaryocytes compared to WT. Finally, PKCδ^−/− mice recovered faster and had a heightened rebound thrombocytosis following thrombocytopenic challenge.

Conclusions

These data suggest that PKCδ is an important megakaryopoietic protein, which regulates signaling induced by Tpo, and represents a potential therapeutic target.

Introduction

Megakaryocytes, which arise chiefly in the bone marrow, are responsible for producing platelets. They accomplish this task by projecting proplatelets into bone marrow sinusoidal vessels ^1,2. However, the megakaryocyte must undergo a unique maturation process in order to develop the resources necessary to perform this function. The hallmark of this maturation process is a modified cell cycle known as endomitosis, in which the megakaryocyte replicates its DNA but does not divide ^3-5. Instead, through repeated rounds of endomitosis, a large polyploid cell capable of producing platelets is formed. The primary regulator of this process is the cytokine thrombopoietin (Tpo) ^6-8. Tpo engagement of its receptor, c-Mpl, leads to activation of a number of signaling pathways including Janus kinase 2, signal transducers and activators of transcription, phosphoinositide 3-kinase (PI3K), Akt, and mitogen activated protein kinase ^9-14. Inhibition of any of these signaling pathways will inhibit megakaryocyte differentiation. Recently, negative regulators of Tpo-mediated signaling have been identified including focal adhesion kinase, the Src-family kinase Lyn, and calcium- and integrin-binding protein 1 ^15-17. Together, a complex view of Tpo-mediated signaling is beginning to emerge. While the role of PKC's in phorbol ester-mediated endomitosis is defined, very little regarding the function of PKC's in primary megakaryopoiesis has been reported ^18-22.

PKC's are a family of serine/threonine protein kinases that have a wide range of functions. There are three classes of PKC's; conventional (α, βI, βII, γ) which require diacylglycerol and Ca2+ for activation, novel (δ, ε, η, θ) which require only diacylglycerol for activation, and atypical (ζ, ι/λ) which require phospholipids for their activation. We have previously reported that PKCδ is important for platelet function ^23,24. In platelets, PKCδ negatively regulates GPVI-mediated dense granule secretion, but positively regulates PAR4-mediated dense granule secretion ²³. In megakaryocytes PKCδ transcript and protein expression are enhanced compared to CD34+ progenitor cells ^25,26. However, the function of PKCδ in primary megakaryocyte differentiation has not yet been elucidated.

To define the role of PKCδ in megakaryopoiesis, we utilized a murine knockout model. We determined that PKCδ^−/− mice have more platelets and megakaryocytes than WT littermate control mice. Furthermore, culture of PKCδ^−/− bone marrow using exogenous Tpo produced a greater number of and more highly polyploid megakaryocytes than WT littermate bone marrow. This may be due to enhanced Tpo-mediated signaling, as PKCδ^−/− megakaryocytes have elevated extracellular signal-regulated kinase (ERK1/2) and Akt308 phosphorylation compared to WT littermate megakaryocytes after stimulation with Tpo. Importantly, in an in vivo experiment PKCδ^−/− mice recovered faster than WT littermate mice from thrombocytopenia. These data strongly suggest that PKCδ is an endogenous negative regulator of Tpo-mediated megakaryocyte differentiation.

METHODS

Methods available in online-only supplement.

RESULTS

PKCδ expression is elevated during megakaryocyte differentiation

Previous reports show that PKCδ mRNA expression and PKCδ protein expression is enhanced in human megakaryocytes compared to progenitor cells ^25,26. To determine whether or not PKCδ is also important for mouse megakaryopoiesis, we performed similar experiments using mouse progenitors and megakaryocytes. First, we isolated bone marrow progenitor cells from WT mice and incubated them in Iscove's Modified Dulbecco's Medium either containing 50 ng/mL recombinant mouse Tpo, or not. After 7 days of culture we analyzed the DNA content of megakaryocytes from both Tpo- and Tpo+ cultures. We were able to generate a sizeable quantity of mature megakaryocytes from Tpo+ cultures (Figure 1A). Therefore, we harvested progenitor cells (0 days) and cells after 3, 5, and 7 days of culture to analyze PKCδ protein expression throughout this time period. Interestingly, we found that PKCδ protein expression was elevated after 3 days of culture and continued to increase up to 7 days of culture (Figure 1B), suggesting that PKCδ expression increases during megakaryocyte differentiation. Additionally, we compared PKCδ expression in megakaryocytes purified using a discontinuous BSA gradient after culturing bone marrow, and found that PKCδ expression was much greater in megakaryocytes than bone marrow mononuclear cells (Figure 1C). These data suggest that PKCδ protein expression is enhanced during megakaryocyte differentiation and that PKCδ may play an important role in megakaryopoiesis.

Figure 1.

PKCδ protein expression is enhanced during megakaryocyte differentiation. A) CD34+ progenitor cells were cultured with or without 50ng/mL recombinant mouse Tpo for 7 days and DNA content was quantified via flow cytometry. B) CD34+ cells cultured in the presence of 50ng/mL exogenous Tpo for 0, 3, 5, or 7 days were lysed and resolved via SDS-PAGE, then probed for PKCδ. C) Mouse mononuclear bone marrow cells and megakaryocytes were lysed and resolved using SDS-PAGE, then probed for PKCδ. ERK1/2 was used to assess loading in both A and B.

PKCδ deletion enhances circulating platelet count

Using a hemavet blood analyzer and blood drawn via cardiac puncture, we determined that PKCδ^−/− mice have more circulating platelets than WT littermate mice (Table 1). Platelet volume was not altered, and there was a slight, but not significant decrease in plasma Tpo concentration with PKCδ deficiency (Table 1). Additionally, PKCδ^−/− mice also had enhanced lymphoproliferation (Table 1). These data suggest that deletion of PKCδ in mice causes increased circulating platelet counts.

Table 1.

Blood cell counts and plasma Tpo levels in PKCδ^−/− and WT littermate mice.

Parameter	WT	PKCδ−/−
Platelets (10−3/μL)	579 ± 49	1,100 ± 229*
Mean platelet volume (fL)	4.949 ± 0.058	5.359 ± 0.174
Plasma Tpo (pg/mL)	532.5 ± 78.97	377.5 ± 117.95
White blood cells (10−3/μL)	4.77 ± 0.19	8.19 ± 0.87*
Neutrophils (10−3/μL)	0.55 ± 0.08	0.64 ± 0.03
Lymphocytes (10−3/μL)	4.03 ± 0.19	7.16 ± 0.91*
Monocytes (10−3/μL)	0.18 ± 0.02	0.39 ± 0.05*

^*p < 0.05, n = 8.

PKCδ^−/− mice have enhanced bone marrow megakaryocyte proliferation and platelet production

Increased platelet count suggests that megakaryopoiesis may be altered in PKCδ deficient mice. To determine that PKCδ deficiency did not result in changes of other PKC isoform protein expression, we performed western blot analysis of PKCδ^−/− and WT littermate control megakaryocyte lysate. We found that all PKC isoforms tested (α, β, ε, θ) had equal expression in PKCδ^−/− compared to WT control (data not shown). Therefore, we analyzed bone marrow megakaryocyte number and DNA content in PKCδ^−/− and WT littermate mice using flow cytometry. Interestingly, we did not observe any differences in DNA content between PKCδ^−/− and WT littermate megakaryocytes isolated directly from bone marrow (Figure 2A). However, we did find that PKCδ^−/− mice contain more bone marrow megakaryocytes than WT littermate mice (Figure 2B). This is in agreement with data presented in Table 1, which shows that PKCδ^−/− mice produce more platelets than WT littermate mice. Increased circulating platelet number suggests that platelet production may be altered in PKCδ^−/− mice. Therefore, we quantified the number of “new” platelets using thiazole orange to identify reticulated platelets. We observed that the increase in platelet number with PKCδ deletion was indeed due to increased platelet production, as PKCδ^−/− mice had significantly more reticulated platelets per blood cell than WT littermate mice (Figure 2C). Furthermore, we determined platelet clearance in PKCδ^−/− mice and found that it was unchanged compared to WT littermate control mice, suggesting that enhanced circulating platelet counts observed in PKCδ^−/− mice is due to enhanced platelet production (Figure 2D).

Figure 2.

Bone marrow megakaryocyte number and platelet production is enhanced with PKCδ deletion. A) Megakaryocyte DNA content from WT littermates and PKCδ^−/− mice was quantified using flow cytometry. B) Bone marrow megakaryocyte number from WT littermates and PKCδ^−/− mice was quantified via flow cytometry and expressed per nucleated bone marrow cell. C) Anticoagulated blood from WT littermates and PKCδ^−/− mice was stained with thiazole orange. The total number of thiazole orange positive platelets were quantified and expressed per 50,000 total blood cells. D) Platelet clearance was assessed via flow cytometry. The number of biotin positive platelets was quantified and expressed as percent original. *p < 0.05, n = 6.

Splenic megakaryopoiesis is enhanced in PKCδ-deficient mice

Platelet production is altered in PKCδ^−/− mice, so we investigated megakaryocyte production in the spleen, which is another site of hematopoiesis in mice. We found that PKCδ^−/− mice had much larger spleens than WT littermate mice (Figure 3A-B) in agreement with a previous report ²⁷. This could be due to enhanced lymphocyte production as previously reported. However, megakaryopoiesis in PKCδ^−/− mouse spleens has not yet been described. Therefore, we sectioned PKCδ^−/− and WT littermate mouse spleens, and stained with Hematoxylin & Eosin. We determined that PKCδ^−/− mouse spleens contained more megakaryocytes than WT littermate spleens, which is in agreement with our data collected in bone marrow (Figure 3C-D).

Figure 3.

Splenic megakaryopoiesis is enhanced with PKCδ deficiency. A) Representative spleens from WT littermates and PKCδ^−/− mice. B) Spleen weight was quantified and expressed per gram body weight (n = 8). C) Representative spleen sections from WT littermates and PKCδ^−/− mice, stained with Hematoxylin & Eosin. White arrows indicate megakaryocytes. Images were taken using a Nikon Eclipse E600 microscope with a Nikon DMX1200 camera. D) Megakaryocytes were quantified from each section and expressed per high power field (n = 5). *p < 0.05.

Deletion of PKCδ enhances megakaryocyte DNA content and proliferation after bone marrow culture

In order to further characterize the role of PKCδ in megakaryopoiesis, we cultured bone marrow from PKCδ^−/− and WT littermate mice in the presence of 50 ng/mL exogenous Tpo. Using flow cytometry, we observed an increase in the proportion of highly polyploid megakaryocytes present in PKCδ^−/− bone marrow cultures compared to WT littermate cultures (Figure 4A). To quantify this difference we compared the proportion of mature (16N+) megakaryocytes to that of immature (2N-8N) megakaryocytes in both WT littermate and PKCδ^−/− mouse bone marrow. We found that the majority of megakaryocytes in WT littermate cultures had DNA content equal to or less than 8N. However, the majority of megakaryocytes in PKCδ^−/− cultures had DNA content greater than 8N (Figure 4B), suggesting that PKCδ deletion enhances megakaryocyte differentiation. Additionally, we quantified the number of megakaryocytes compared to nucleated cells in each culture and found a higher proportion of megakaryocytes in the PKCδ^−/− bone marrow cultures compared to cultures from WT littermate bone marrow (Figure 4C).

Figure 4.

Megakaryocyte DNA content and number is enhanced with PKCδ deletion following culture of bone marrow cells. A) Representative histograms of megakaryocyte DNA content from WT littermates and PKCδ^−/− mouse bone marrow cultures. B) Quantification of megakaryocyte DNA content expressed as immature (2N-8N) megakaryocytes and mature (16N+) megakaryocytes. C) Quantification of megakaryocyte number from WT littermates and PKCδ^−/− bone marrow cultures, expressed per nucleated cell. *p < 0.05, n = 6.

PKCδ regulates apoptosis in a variety of cells. One reason for the enhanced number of megakaryocytes observed in PKCδ^−/− mouse bone marrow and spleen could be resistance to apoptosis due to PKCδ deficiency. Therefore, we assessed apoptosis in WT and PKCδ^−/− megakaryocytes via flow cytometry. We did not observe any differences between WT and PKCδ^−/− cells (data not shown) suggesting that PKCδ does not regulate megakaryocyte apoptosis and that the reason for enhanced megakaryopoiesis in PKCδ^−/− mice must be something else.

PKCδ is a negative regulator of Tpo-induced signaling

Because we noted that megakaryopoiesis was heightened with PKCδ deficiency we aimed to determine whether or not PKCδ regulates Tpo-induced signaling. Following 5 days culture, megakaryocytes from WT littermate and PKCδ^−/− bone marrow were purified, and treated with 50 ng/mL Tpo to induce signaling. SDS-PAGE of megakaryocyte lysates revealed that both ERK and Akt308 phosphorylation was significantly enhanced in PKCδ^−/− mouse megakaryocytes (Figure 5A-B). ERK and Akt are known regulators of Tpo-mediated signaling ^9,10. To verify that the observed increase in ERK and Akt phosphorylation is not attributed to altered cell surface expression of the TPO receptor, we analyzed surface expression of c-Mpl on PKCδ^−/− and WT littermate control megakaryocytes via flow cytometry. We determined that PKCδ deficiency does not alter surface expression of c-Mpl (data not shown). Therefore, these data suggest that PKCδ is an integral negative regulator of Tpo-induced signaling.

Figure 5.

Megakaryocytes were purified and treated with 50ng/mL TPO for 10 min, then lysed and separated by SDS-PAGE. Membranes were probed for phosphorylated ERK (A), phosphorylated Akt T308 (B), and total proteins to verify loading. Band intensity was quantified and normalized to WT –TPO for phosphor-ERK and WT +TPO for phosphor-Akt. *p < 0.05, n = 4.

Proplatelet production is unaltered with PKCδ^−/− deficiency

To determine whether or not PKCδ influences proplatelet production we plated purified bone marrow megakaryocytes on immobilized fibrinogen and quantified megakaryocytes that were producing proplatelets. We observed no differences in the frequency of proplatelet producing megakaryocytes (Figure 6A-B). Additionally, proplatelet-producing megakaryocytes from PKCδ^−/− bone marrow appeared to be structurally similar to proplatelet-producing megakaryocytes from littermate control bone marrow. These data suggest that PKCδ does not influence proplatelet production.

Figure 6.

PKCδ^−/− mice recover from thrombocytopenia faster that WT littermate control mice although proplatelet production is unaltered. A) Megakaryocytes from PKCδ^−/− and littermate WT control bone marrow were plated on 100mg/mL immobilized fibrinogen overnight to observe proplatelets. B) The frequency of proplatelet-producing megakaryocytes was determined. C) PKCδ^−/− and WT littermate control mice were injected with anti-mouse CD41 antibody and platelet counts were monitored every 24 hours for 5 days. *p < 0.05, n = 6

Recovery from immune-mediated thrombocytopenia is enhanced in PKCδ^−/− mice

To elucidate whether or not the alterations in megakaryopoiesis we observed with PKCδ deficiency in vitro were relevant in vivo, we created an immune-mediated thrombocytopenia and monitored recovery. Platelet counts in both WT littermate and PKCδ^−/− mice were greatly reduced 24 hours after injection of a mouse anti-CD41 antibody (Figure 6C). However, the PKCδ^−/− mice recovered platelet counts at a higher rate than WT littermates mice such that PKCδ^−/− mice were fully recovered after 3 days, while WT littermates took nearly 4 days. Additionally, rebound thrombocytosis was greatly enhanced in PKCδ^−/− mice compared to WT littermates. These data suggest that PKCδ regulates megakaryopoiesis and platelet production in vivo and establishes PKCδ as an important therapeutic target for thrombocytopenia.

DISCUSSION

In this report we demonstrate for the first time that PKCδ is an important regulator of Tpo-mediated megakaryopoiesis. We demonstrated that PKCδ protein expression is enhanced during megakaryocyte differentiation, suggesting that it may be a regulatory protein. Deletion of PKCδ in mice caused an increase in platelet production possibly due to enhanced megakaryocyte production in the bone marrow and spleen. Furthermore, culture of mouse bone marrow produced heightened megakaryocyte differentiation and proliferation in PKCδ^−/− tissue compared to WT littermates. We also revealed that PKCδ could be a regulator of Tpo-mediated signaling as phosphorylated ERK1/2 and Akt308 was enhanced with PKCδ deficiency. Finally, recovery from immune mediated thrombocytopenia was enhanced in PKCδ^−/− mice compared to WT littermates. These data strongly suggest that PKCδ is an important regulator of bone marrow megakaryopoiesis.

PKCδ protein expression is enhanced during megakaryocyte differentiation, and PKCδ protein expression is greater in megakaryocytes than mononuclear bone marrow cells. This data is in agreement with previous reports regarding both mRNA expression and protein expression in megakaryocytes compared to CD34+ progenitor cells ^25,26. A possible explanation for increased PKCδ protein expression in megakaryocytes could be to insure adequate PKCδ expression in platelets, since platelets have limited ability to undergo protein synthesis. Furthermore, we have previously reported that PKCδ is an important regulator of platelet function ^23,24. However, the data presented in this report suggest that PKCδ protein expression is also tied to megakaryocyte differentiation. Therefore we theorize that PKCδ regulates both megakaryopoiesis and platelet function after it is transported to the platelet from the megakaryocyte.

Deletion of PKCδ in mice causes enhanced platelet production. Likewise, PKCδ^−/− mice have more circulating platelets than WT littermates. Increased platelet count can arise from either increased platelet production or inhibited platelet destruction. In this report we show that platelet production is greatly increased with PKCδ deletion while platelet clearance remains unaltered, suggesting that the reason for enhanced platelet counts is due to heightened platelet production. Additionally, we report here that megakaryocyte number is enhanced in the bone marrow of PKCδ^−/− mice, further implying that enhanced platelet production is responsible for the increase in platelet counts observed in PKCδ^−/− mice.

PKCδ may be a negative regulator of Tpo-dependent signaling as pERK1/2 and pAkt308 are elevated in PKCδ^−/− megakaryocytes following treatment with exogenous Tpo. Furthermore, bone marrow megakaryocyte number is enhanced in PKCδ^−/− mice, and megakaryocyte DNA content and number are enhanced following culture of PKCδ^−/− bone marrow. We have previously reported that PKCδ is a negative regulator of GPVI-mediated signaling via interactions with the Src-family kinase Lyn, and SH2 domain-containing inositol phosphatase-1 (SHIP-1) ²⁴. Interestingly, Lyn, which is activated by FAK downstream of c-Mpl, is also a negative regulator of Tpo-mediated signaling ^16,17. In Lyn^−/− megakaryocytes Tpo-mediated ERK and Akt phosphorylation is enhanced, while SHIP-1 tyrosine phosphorylation is depressed. Therefore, it is possible that PKCδ is activated by Lyn downstream of c-Mpl, and is responsible for phosphorylating and activating SHIP-1, which is known to regulate PI3K ²⁸. Additionally, knockdown of SHIP-1 in erythroleukemia cells caused enhanced PI3K and ERK1/2 phosphorylation compared to control ²⁹. Together, these data suggest that PKCδ regulates Tpo-dependent megakaryocyte differentiation perhaps through interactions with both Lyn and SHIP-1. It would be most interesting to determine if PKCδ interacts with these two proteins following stimulation of megakaryocytes with Tpo. In addition to enhanced megakaryopoiesis and platelet production, PKCδ^−/− mice have enhanced lymphoproliferation, which is in agreement with a previous report ²⁷. Interestingly, two PKCδ deficient patients have recently been described ^30,31. The patients have enhanced lymphoproliferation, lymphadenopathy, and splenomegaly similar to the PKCδ^−/− mouse. Unfortunately no data regarding platelets or megakaryocytes has been reported. However, the similarities between the mouse and human data suggest that alterations in megakaryopoiesis in humans with PKCδ deficiency is likely. Any such data would further cement PKCδ as a potential therapeutic target.

We demonstrated that PKCδ is an important mediator of megakaryocyte differentiation and subsequent platelet production. PKCδ^−/− mice have more platelets and megakaryocytes than WT littermate mice, and they recover faster following immune thrombocytopenia. PKCδ may regulate megakaryopoiesis by inhibiting Tpo-mediated signaling as both ERK and Akt308 phosphorylation were enhanced with PKCδ deficiency. Therefore, PKCδ may represent an important therapeutic target for thrombocytopenia.

Significance.

Thrombocytopenia or low platelet count is a condition that can be caused by reduced production of platelets, increased destruction of platelets, or the use of certain therapeutics. Current treatment of thrombocytopenia includes platelet transfusion. However, there are significant risks associated with this approach, and the donor pool cannot currently support demand. Thrombopoietin therapy is also used and can be effective. However, another therapeutic that could synergize with thrombopoietin therapy could be most useful. In this report we evaluate the role of PKCδ in megakaryopoiesis. We demonstrate that PKCδ deficiency augments Tpo-mediated signaling, megakaryopoiesis and thrombopoiesis. Importantly, we note that PKCδ deficiency enhances recovery from thrombocytopenia in mice. These data suggest that PKCδ could be a therapeutic target, and since specific inhibitors of PKCδ are currently in production testing this hypothesis may soon be possible.

Acronyms and Abbreviations

PKC

Protein Kinase C

Tpo

Thrombopoietin

WT

Wild-type littermate control