title: Megakaryocytes regulate expression of Pyk2 isoforms and caspase-mediated cleavage of actin in osteoblasts.
abstract: The proliferation and differentiation of osteoblast (OB) precursors are essential for elaborating the bone-forming activity of mature OBs. However, the mechanisms regulating OB proliferation and function are largely unknown. We reported that OB proliferation is enhanced by megakaryocytes (MKs) via a process that is regulated in part by integrin signaling. The tyrosine kinase Pyk2 has been shown to regulate cell proliferation and survival in a variety of cells. Pyk2 is also activated by integrin signaling and regulates actin remodeling in bone-resorbing osteoclasts. In this study, we examined the role of Pyk2 and actin in the MK-mediated increase in OB proliferation. Calvarial OBs were cultured in the presence of MKs for various times, and Pyk2 signaling cascades in OBs were examined by Western blotting, subcellular fractionation, and microscopy. We found that MKs regulate the temporal expression of Pyk2 and its subcellular localization. We also found that MKs regulate the expression of two alternatively spliced isoforms of Pyk2 in OBs, which may regulate OB differentiation and proliferation. MKs also induced cytoskeletal reorganization in OBs, which was associated with the caspase-mediated cleavage of actin, an increase in focal adhesions, and the formation of apical membrane ruffles. Moreover, BrdU incorporation in MK-stimulated OBs was blocked by the actin-polymerizing agent, jasplakinolide. Collectively, our studies reveal that Pyk2 and actin play an important role in MK-regulated signaling cascades that control OB proliferation and may be important for therapeutic interventions aimed at increasing bone formation in metabolic diseases of the skeleton.
authors.affiliation: Department of Orthopaedic Surgery, Indiana University School of Dentistry, Indianapolis, Indiana 46202. Electronic address: mkacena@iupui.edu.
authors.forename: Melissa A
authors.initials: MA
authors.lastname: Kacena
authors.affiliation: Department of Oral Biology, Indiana University School of Dentistry, Indianapolis, Indiana 46202.
authors.forename: Pierre P
authors.initials: PP
authors.lastname: Eleniste
authors.affiliation: Department of Orthopaedic Surgery, Indiana University School of Dentistry, Indianapolis, Indiana 46202.
authors.forename: Ying-Hua
authors.initials: YH
authors.lastname: Cheng
authors.affiliation: Department of Oral Biology, Indiana University School of Dentistry, Indianapolis, Indiana 46202.
authors.forename: Su
authors.initials: S
authors.lastname: Huang
authors.affiliation: Department of Oral Biology, Indiana University School of Dentistry, Indianapolis, Indiana 46202.
authors.forename: Mahesh
authors.initials: M
authors.lastname: Shivanna
authors.affiliation: Department of Orthopaedic Surgery, Indiana University School of Dentistry, Indianapolis, Indiana 46202.
authors.forename: Tomas E
authors.initials: TE
authors.lastname: Meijome
authors.affiliation: Herman B. Wells Center for Pediatric Research, Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indiana University School of Dentistry, Indianapolis, Indiana 46202.
authors.forename: Lindsey D
authors.initials: LD
authors.lastname: Mayo
authors.affiliation: Department of Oral Biology, Indiana University School of Dentistry, Indianapolis, Indiana 46202. Electronic address: abruzzan@iupui.edu.
authors.forename: Angela
authors.initials: A
authors.lastname: Bruzzaniti
mesh_terms: D000199:Actins / Q000378:metabolism*; D017398:Alternative Splicing; D000818:Animals; D020169:Caspases / Q000378:metabolism*; D002454:Cell Differentiation / Q000502:physiology; D049109:Cell Proliferation; D002478:Cells, Cultured; D051418:Focal Adhesion Kinase 2 / Q000096:biosynthesis*; D022001:Focal Adhesions / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D007527:Isoenzymes / Q000096:biosynthesis; D008533:Megakaryocytes / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism*
chemical_list: D000199:Actins; D007527:Isoenzymes; D051418:Focal Adhesion Kinase 2; C496053:Ptk2b protein, mouse; D020169:Caspases