The Role of Rho/Rac Pathways in Proplatelet Formation of Megakaryocytes.

We reported that Rho and/or Rac signal pathways were involved in proplatelet formation (PPF) of megakaryocytes in the last ASH Meeting. Recently Nagata et al. reported that 3β-hydroxysteroid dehydrogenase (3β-HSD) was a target of p45 NF-E2 and that estradiole, synthesized by 3β-HSD in megakaryocytes, stimulated PPF (GENES & DEVELOPMENT 2003). In this study we focused on the signal pathways, activated by estradiole, and the predominant pathway in the process of PPF using murine megakaryocytes. Murine megakaryocytes were isolated from bone marrow cells by Percoll® density centrifugation and BSA gradient method. The purity of the isolated megakaryocytes was more than 40% in all experiments.

Isolated megakaryocytes (300/well) were cultured in serum free condition with 17β-estradiol (E2) or/and Y27632 (Rho kinase inhibitor) or PP1(Src family kinase inhibitor) or LY294002 (PI3K inhibitor). Y27632 inhibits Rho pathway and the 2 latters inhibits Rac activation. Y27632 alone stimulated PPF of murne megakaryocytes (0 μM:100%, 1μM:116.2±0.2%,10μM:129.2±4.25% (p<0.01),100μM: 159.8±13.3% (p<0.01))(n=4). E2 alone stimulated PPF of murne megakaryocytes in the dose-dependent manner (0μM:100%,1μM:108.1±4.2%,10μM:118.7±9.5%(p<0.05),100μM: 123.7±3.7% (p<0.01))(n=4). While, PP1 or LY294002 alone inhibited PPF of murine megakaryocytes (PP1; 0μM: 100%, 2μM: 82.1±7.3%(p<0.05), 20μM: 36.2±4.5%(p<0.01), LY294002; 0μM: 100%, 1μM: 94.8±2.3%, 10μM:66.5±4.7%(p<0.01),100μM:43.9±4.2%(p<0.01))(n=4). Based on these results, Rho pathway inhibition or activated Rac stimulated PPF. The addition of E2 to Y27632 resulted in no inhibition/stimulation of PPF (E2/Y27632; 0 nM/10 μM: 100%, 2.3 nM/10 μM: 113±3.5%, 23 nM/10 μM 116.9±9.0%, 230 nM/10 μM: 115.8±4.6 %, 0 nM/100 μM:100%, 2.3 nM/100 μM: 97.2±6.9%, 23 nM/100 μM: 93.1±5.1%, 230 nM/100 μM: 99.4±5.7%)(n=4).The addition of E2 to PP1 or LY294002 resulted in the dose-dependent manner reduction of PPF (E2/PP1; 230 nM/0 μM: 100%, 230 nM/2 μM: 61.9±8.5%, 230 nM/20 μM: 51.0±4.1%, E2/LY294002; 23 nM/0μM: 100%, 23 nM/10μM: 66.8±2.0%, 23 nM/100μM: 68.4±3.3%)(n=4). The degree in reduction with E2/PP1 or E2/LY294002 was similar to that with PP1 or LY294002 alone.These results strongly suggested that that PPF of megakaryocytes, stimulated by E2, might not be involved in Rho but Rac pathway.

Next, we investigated the predominance between Rho and Rac pathways in the process of PPF.The addition of PP1 or LY294002 to Y27632 resulted in no inhibition of PPF (PP1/Y27632; 0μM/100 μM:100%, 2μM /100 μM:105.6±6.0%, 20 μM/100 μM: 107.5±4.7, LY294002/Y27632; 0μM/100 μM:100%, 1μM/100 mμM:92.5±13.8%, 10μM /100 μM:92.6±6.6%, 100μM/100 μM: 97.8±8.0%, ( n=4)).

These results indicated that Rho pathway inhibition stimulated PPF of megakaryocytes, even if activated Rac decreased. Therfore, Rho pathway is more predominant than Rac pathway in PPF of megakaryocytes.