Calcium- and integrin-binding protein 1 regulates megakaryocyte ploidy, adhesion, and migration

Abstract

Megakaryocytes are large, polyploid cells that produce platelets. We have previously reported that calcium-and integrin-binding protein 1 (CIB1) regulates endomitosis in Dami cells. To further characterize the role of CIB1 in megakaryopoiesis, we used a Cib1 ؊/؊ mouse model. Cib1 ؊/؊ mice have more platelets and BM megakaryocytes than wild-type (WT) controls (P < .05). Furthermore, subsequent analysis of megakaryocyte-CFU production revealed an increase with Cib1 deletion compared with WT (P < .05). In addition, BM from Cib1 ؊/؊ mice, cultured with thrombopoietin (TPO) for 24 hours, produced more highly polyploid megakaryocytes than WT BM (P < .05). Subsequent analysis of TPO signaling revealed enhanced Akt and ERK1/2 phosphorylation, whereas FAK Y925 phosphorylation was reduced in Cib1 ؊/؊ megakaryocytes treated with TPO. Conversely, platelet recovery in Cib1 ؊/؊ mice after platelet depletion was attenuated compared with WT (P < .05). This could be the result of impaired adhesion and migration, as adhesion to fibrinogen and fibronectin and migration toward an SDF-1␣ gradient were reduced in Cib1 ؊/؊ megakaryocytes compared with WT (P < .05). In addition, Cib1 ؊/؊ megakaryocytes formed fewer proplatelets compared with WT (P < .05), when plated on fibrinogen. These data suggest that CIB1 plays a dual role in megakaryopoiesis, initially by negatively regulating TPO signaling and later by augmenting proplatelet production. IntroductionMature megakaryocytes produce platelets by extending proplatelet projections into sinusoidal vessels in BM. 1 Before this process, the megakaryocyte must differentiate from progenitor cells by undergoing multiple rounds of endomitosis. Megakaryocyte endomitosis occurs primarily through cell signaling initiated by the cytokine thrombopoietin (TPO), which is produced constitutively in the liver. 2 TPO binding to its receptor, c-Mpl, results in the activation of Janus kinase 2 (Jak2). Jak2 phosphorylates tyrosine residues on c-Mpl, which enables the subsequent activation of other signaling pathways, such as PI3K/Akt and MAPK. [3][4][5][6][7][8] In addition, focal adhesion kinase (FAK) is activated and acts as a negative regulator of TPO-induced signaling by activating Lyn, an Src-family kinase. 9 Specifically, Western blot analysis revealed that impaired TPO-induced FAK phosphorylation is concomitant with heightened Akt and ERK1/2 phosphorylation. 9 Once mature, megakaryocytes must migrate from an "osteoblastic niche" to a "vascular niche" where they interact with BM endothelial cells. 10 Integrins play a key role in cell migration, and the most abundantly expressed integrin on the megakaryocyte is ␣ IIb ␤ 3 , which is the major receptor for the highly expressed BM extracellular matrix (ECM) protein fibronectin (Fn). 11,12 The exact mechanism underlying megakaryocyte migration toward the vasculature is unknown, but stromal cell-derived factor-1␣ (SDF-1␣) appears to provide the homing signal. 13 Calcium-and integrin-binding protein 1 (CIB1) was first identified as a binding partner of th...

PKCθ is dispensable for megakaryopoiesis

“…injection of 50 mg/kg anti-mouse CD41 antibody, and monitored recovery. After twenty-four hours we observed greater than 85% platelet reduction in both PKCy À/À and WT littermate control mice, which is consistent with previous reports [16,17]. However, recovery up to 7 days was unaltered with PKCy deficiency suggesting that PKCy does not contribute to in vivo megakaryopoiesis or platelet production ( Figure 2).…”

Sympathetic stimulation facilitates thrombopoiesis by promoting megakaryocyte adhesion, migration, and proplatelet formation

“…4 Previous studies were primarily focused on the early and middle stages of thrombopoiesis, including the commitment to MK lineage and the MK proliferation. 20,32 Here, we demonstrate that the sympathetic transmitters NE and EPI stimulate platelet production by promoting MK adhesion, migration, PPF and platelet release, in addition to the expansion of CD34 1 cells. These findings are consistent with our recent study that showed that human growth hormone treatment resulted in a quick increase in platelet production by regulating the late stage of thrombopoiesis, including the promotion of PPF and platelet release.…”

“…20,32,36 Furthermore, ERK1/2 activation downregulates the RhoA activity in mature MKs upon treatment with NE or EPI. These effects led to the rearrangement of the actin and microtublin cytoskeletons, which was a known key determinant for PPF, platelet formation, and platelet release.…”

Pak2 restrains endomitosis during megakaryopoiesis and alters cytoskeleton organization

“…35 DNA content in CD41 1 megakaryocytes was determined by labeling red blood celllysed bone marrow with anti-CD41-efluor450, followed by fixation with 0.5% formalin. Cells were permeabilized with 70% ice-cold methanol and washed and incubated with 10 mg RNase A, followed by brief incubation with 1 mg/mL propidium iodide.…”