Activity and phosphorylation of myosin light chain (MLC) kinase, which is downstream of RhoA (Figure 4, A and B). RhoA-dependent signaling has been linked to the production of proplatelets (17, 18). As a result, we treated human megakaryocytes with Y27632, a selective inhibitor on the Rho-associated protein kinase p160ROCK, or with C3 transferase, a direct RhoA inhibitor. Y27632 and C3 transferase rescued proplatelet formation in bortezomib-treated cells (Figure 4C and Supplemental Figure 7). This response was probably on account of inhibition of downstream RhoA effectors, because Y27632 decreased phosphorylation of MLC kinase in the presence of bortezomib (Figure 4B). In agreement with the rescue of proplatelet formation observed in bortezomib-treated human megakaryocytes, mouse megakaryocytes treated with bortezomib plus Y27632 or with bortezomib plus fasudil, a a lot more clinically relevant p160ROCK inhibitor, formed proplatelets (Figure 4D). These final results in mouse megakaryocytes were similar to a current report by Murai et al. (19). Genetic deletion with the proteasome benefits in extreme thrombocytopenia and death. To additional dissect the function in the proteasome in thrombopoiesis, we focused on protease (prosome, macropain) 26S subunit, ATPase 1 (Psmc1; gene ID 19179) in mouse megakaryocytes and platelets. Psmc1 is an essential subunit in the 19S regula-tory particle that is important for ubiquitin-mediated protein degradation by the 26S proteasome complex (202). It is conserved at the protein level in human and mouse megakaryocytes (Supplemental Figure eight). mRNA for Psmc1 was also expressed in both species, although human megakaryocytes had reduce levels on the transcript compared with mouse megakaryocytes (Supplemental Table 1).FIPI web Psmc1fl/fl mice have been crossed with platelet aspect four Cre recombinase (Pf4-Cre) mice to disrupt proteasome activity in megakaryocytes and platelets.Ciglitazone custom synthesis Psmc1fl/fl Pf4-Cre mice had drastically lowered protein for PSMC1 in megakaryocytes, but not other tissues (Supplemental Figure 9).PMID:23398362 Ubiquinated proteins also accumulated in megakaryocytes from Psmc1fl/fl Pf4-Cre mice (Supplemental Figure ten). Despite a marked reduction in PSMC1 protein, the amount of megakaryocytes from Psmc1fl/fl Pf4-Cre mice in bone marrow or spleen was not decreased compared with Psmc1fl/wt mice (Supplemental Figure 11). As opposed to their littermate controls, nonetheless, Psmc1fl/fl Pf4-Cre mice had extreme thrombocytopenia at postnatal day 1 (P1), plus the majority of Psmc1fl/fl Pf4-Cre mice died just before weaning (Figure five, A and B). The reduction in platelet counts was a lot more serious than in c-Mpl knockout pups in the same age (Supplemental Figure 12). In addition to reduced numbers of platelets, Psmc1fl/fl Pf4-Cre mice had reduced hematocrits than Psmc1fl/wt mice, and bleeding was seen within the abdomen and limbs (Figure five, C and D). Pathological indicators of hemorrhage were also present within the bladder and testes of all animals and sometimes observed in the brain, lymph nodes, and intestines (Figure 5E and data not shown). Ultrastructure examination of megakaryocytes from Psmc1fl/fl Pf4-Cre mice revealed less cytoplasm compared with megakaryocytes from Psmc1fl/wt mice (Figure 6A). Additionally, Psmc1fl/fl Pf4Cre megakaryocytes lacked demarcation membranes, which have been readily visible in Psmc1fl/wt megakaryocytes (Figure 6A). Comparable to mouse megakaryocytes treated with bortezomib (Figure 2A), megakaryocytes from Psmc1fl/fl Pf4-Cre mice failed to create proplatelets (Figure 6B). Inhibit.