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. 2012 Jan 19;119(3):838-46.
doi: 10.1182/blood-2011-04-346098. Epub 2011 Nov 29.

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

John C Kostyak  1 , Meghna U Naik, Ulhas P Naik
Affiliations

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

John C Kostyak et al. Blood. .

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-1α 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.

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Figures

Figure 1
Figure 1
Platelet production and platelet clearance are unchanged in Cib1−/− mice. (A) Quantification of in vivo biotinylated platelets 24, 48, 72, and 168 hours after thiazole orange injection expressed as a percentage of baseline (1 hour after injection; n = 5). (B) Quantification of the percentage of reticulated platelets from WT and Cib1−/− mouse blood (n = 5).
Figure 2
Figure 2
Spleens of Cib1−/− mice contain fewer megakaryocytes than WT mice. (A) Fluorescent microscopic images of mouse spleen sections stained with anti-CD41 to label the megakaryocytes and Draq-5 to label nuclei. White arrows indicate megakaryocytes. Images were captured using a Zeiss 5-live confocal microscope (original magnification ×100). (B) Quantification of panel A expressed as megakaryocytes per field. *P < .05 vs WT (n = 7).
Figure 3
Figure 3
CFU-MK production is heightened with Cib1 deletion. (A) Representative histograms of DNA content of CD41+ BM cells derived from WT and Cib1−/− mice. (B) Quantification of the ploidy distribution of WT and Cib1−/− BM megakaryocytes expressed as a percentage of total megakaryocytes (n = 6). (C) Representative light-microscopic images of CFU-MK stained with acetylcholinesterase after 7 days in culture. Arrows indicate CFU-MK colonies. (D) Quantification of the number of CFU-MK in each well after 12 days of incubation. Colonies were composed of at least 3 cells. *P < .05 vs WT (n = 4).
Figure 4
Figure 4
Cib1 deletion increases megakaryocyte ploidy in response to exogenous TPO. (A) Representative histograms of CD41+ BM cells from WT and Cib1−/− mice after addition of 50 ng/mL TPO for 24 hours. (B) Quantification of megakaryocyte ploidy from panel A. *P < .05 vs WT (n = 7). (C) Representative histograms of CD41+ BM cells from WT and Cib1−/− mice after 50 ng/mL TPO treatment for 5 days. (D) Quantification of megakaryocyte ploidy from panel C. *P < .05 vs WT (n = 6).
Figure 5
Figure 5
TPO-induced signaling is enhanced in Cib1−/− megakaryocytes. (A) Western blots of lysates from untreated or TPO-treated WT and Cib1−/− BM-derived megakaryocytes purified using a discontinuous BSA gradient. Membranes were initially probed for phosphorylated AktT308 and phosphorylated ERK1/2, then stripped and reprobed for total Akt or total ERK1/2, respectively. (B) Quantification of band intensity from panel A normalized to untreated WT sample. *P < .05 vs WT + TPO (n = 5). (C) Western blots of lysates from WT and Cib1−/− megakaryocytes treated with or without TPO. Membranes were probed for phosphorylated FAKY925, then stripped and reprobed for total FAK. (D) Quantification of band intensity from panel B, normalized to untreated WT sample. *P < .05 vs WT + TPO (n = 5).
Figure 6
Figure 6
Platelet recovery is attenuated in Cib1−/− mice after immune-induced thrombocytopenia. (A) Time course of platelet recovery from WT and Cib1−/− mice, every 24 hours for 7 days after injection of an anti–mouse CD41 antibody. *P < .05 vs WT (n = 8). (B) Time course of platelet recovery after experimentally induced thrombocytopenia expressed as a percentage of original platelet count. (C) Quantification of DNA content from WT and Cib1−/− megakaryocytes 72 hours after injection of a mouse anti-CD41 antibody.
Figure 7
Figure 7
Cib1 deletion causes inhibition of megakaryocyte migration and attachment to ECM proteins. (A) Percentage of WT and Cib1−/− megakaryocytes attached to BSA, Fg, or Fn. *P < .05 vs WT (n = 6). (B) Diff-Quik stained membranes from transwell migration assays using WT and Cib1−/− megakaryocytes. Arrows point to megakaryocytes. Images were captured using a Nikon Eclipse TS100 microscope: top (original magnification ×40) and bottom (original magnification ×200). (C) Quantification of megakaryocytes per well after transwell migration assays. *P < .05 vs WT (n = 7). (D) Phase-contrast images of BM-derived megakaryocytes purified using discontinuous BSA gradient from WT and Cib1−/− mice attached for 5 hours to immobilized Fg. The arrow indicates a proplatelet-producing megakaryocyte. Images were captured using a Zeiss Axiovert microscope (original magnification ×200). (E) Quantification of the percentage of proplatelet-producing megakaryocytes derived from WT and Cib1−/− mice. *P < .05 vs WT (n = 6).
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