title: Pyk2 and Megakaryocytes Regulate Osteoblast Differentiation and Migration Via Distinct and Overlapping Mechanisms.
abstract: Osteoblast differentiation and migration are necessary for bone formation during bone remodeling. Mice lacking the proline-rich tyrosine kinase Pyk2 (Pyk2-KO) have increased bone mass, in part due to increased osteoblast proliferation. Megakaryocytes (MKs), the platelet-producing cells, also promote osteoblast proliferation in vitro and bone-formation in vivo via a pathway that involves Pyk2. In the current study, we examined the mechanism of action of Pyk2, and the role of MKs, on osteoblast differentiation and migration. We found that Pyk2-KO osteoblasts express elevated alkaline phosphatase (ALP), type I collagen and osteocalcin mRNA levels as well as increased ALP activity, and mineralization, confirming that Pyk2 negatively regulates osteoblast function. Since Pyk2 Y402 phosphorylation is important for its catalytic activity and for its protein-scaffolding functions, we expressed the phosphorylation-mutant (Pyk2(Y402F) ) and kinase-mutant (Pyk2(K457A) ) in Pyk2-KO osteoblasts. Both Pyk2(Y402F) and Pyk2(K457A) reduced ALP activity, whereas only kinase-inactive Pyk2(K457A) inhibited Pyk2-KO osteoblast migration. Consistent with a role for Pyk2 on ALP activity, co-culture of MKs with osteoblasts led to a decrease in the level of phosphorylated Pyk2 (pY402) as well as a decrease in ALP activity. Although, Pyk2-KO osteoblasts exhibited increased migration compared to wild-type osteoblasts, Pyk2 expression was not required necessary for the ability of MKs to stimulate osteoblast migration. Together, these data suggest that osteoblast differentiation and migration are inversely regulated by MKs via distinct Pyk2-dependent and independent signaling pathways. Novel drugs that distinguish between the kinase-dependent or protein-scaffolding functions of Pyk2 may provide therapeutic specificity for the control of bone-related diseases.
authors.affiliation: Department of Biomedical and Applied Sciences, Indiana University School of Dentistry, Indianapolis, Indiana.
authors.forename: Pierre P
authors.initials: PP
authors.lastname: Eleniste
authors.affiliation: Department of Biomedical and Applied Sciences, Indiana University School of Dentistry, Indianapolis, Indiana.
authors.forename: Vruti
authors.initials: V
authors.lastname: Patel
authors.affiliation: Department of Biomedical and Applied Sciences, Indiana University School of Dentistry, Indianapolis, Indiana.
authors.forename: Sumana
authors.initials: S
authors.lastname: Posritong
authors.affiliation: Department of Biomedical and Applied Sciences, Indiana University School of Dentistry, Indianapolis, Indiana.
authors.forename: Odette
authors.initials: O
authors.lastname: Zero
authors.affiliation: Department of Biomedical and Applied Sciences, Indiana University School of Dentistry, Indianapolis, Indiana.
authors.forename: Heather
authors.initials: H
authors.lastname: Largura
authors.affiliation: Department of Orthopaedic Surgery, Indiana University School of Medicine, Indianapolis, Indiana.
authors.forename: Ying-Hua
authors.initials: YH
authors.lastname: Cheng
authors.affiliation: Department of Orthopaedic Surgery, Indiana University School of Medicine, Indianapolis, Indiana.
authors.forename: Evan R
authors.initials: ER
authors.lastname: Himes
authors.affiliation: Department of Orthopaedic Surgery, Indiana University School of Medicine, Indianapolis, Indiana.
authors.forename: Matthew
authors.initials: M
authors.lastname: Hamilton
authors.affiliation: Department of Orthopaedic Surgery, Indiana University School of Medicine, Indianapolis, Indiana.
authors.forename: Jenna T B
authors.initials: JTB
authors.lastname: Ekwealor
authors.affiliation: Department of Orthopaedic Surgery, Indiana University School of Medicine, Indianapolis, Indiana.
authors.forename: Melissa A
authors.initials: MA
authors.lastname: Kacena
authors.affiliation: Department of Biomedical and Applied Sciences, Indiana University School of Dentistry, Indianapolis, Indiana.
authors.forename: Angela
authors.initials: A
authors.lastname: Bruzzaniti
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D002465:Cell Movement; D002478:Cells, Cultured; D018920:Coculture Techniques; D051418:Focal Adhesion Kinase 2 / Q000235:genetics* / Q000378:metabolism*; D005786:Gene Expression Regulation; D055786:Gene Knockout Techniques; D008533:Megakaryocytes / Q000166:cytology* / Q000378:metabolism; D051379:Mice; D010006:Osteoblasts / Q000166:cytology* / Q000378:metabolism; D010766:Phosphorylation; D015398:Signal Transduction
chemical_list: D051418:Focal Adhesion Kinase 2; C496053:Ptk2b protein, mouse
keywords: ALKALINE PHOSPHATASE; BONE FORMATION; MINERALIZATION; PHOSPHORYLATION; TYROSINE KINASE