Date of Award

8-17-2009

Document Type

Open Access Thesis

Degree Name

Medical Doctor (MD)

First Advisor

Melissa Kacena, Ph.d

Abstract

MEGAKARYOCYTE-BONE CELL INTERACTIONS: THE ROLE OF GAP JUNCTIONS, MATURATION, AND LONGEVITY. Wendy A. Ciovacco, Carolyn G. Goldberg, Amanda F. Taylor, Justin M. Lemieux, Henry J. Donahue, Ying-Hua Cheng, Mark C. Horowitz, and Melissa A. Kacena. Department of Orthopaedic Surgery, Indiana University School of Medicine, Indianapolis, IN. Research shows that megakaryocytes (MKs) can enhance bone volume by increasing osteoblast (OB) proliferation and inhibiting osteoclast (OC) formation. This cumulative work first explores the role of gap junction intercellular communication (GJIC) in MK-OB interactions, and secondly examines the effect of MK maturation state and MK number on skeletal homeostasis. In both studies, cell lineages were cultured as described below. In the gap junction (GJ) study, we used real-time PCR to test for MK expression of connexin 43 (Cx43), the predominant GJ protein found in bone cells. A dual-label parachute assay and FACS analysis assessed GJIC between MKs and OBs. Proliferation and differentiation assays of OBs cultured with and without MKs were performed. Here we demonstrate that: 1) MKs express Cx43, 2) MKs can functionally communicate with OBs via GJIC, 3) the addition of two distinct GJ uncouplers inhibits this communication, 4) inhibiting MK-mediated GJIC further enhances the ability of MKs to stimulate OB proliferation and, 5) blocking GJIC does not result in MK-induced reduction of OB differentiation. In the second study, increasing numbers of MKs were co-cultured with bone cells to see if increased MK number correlated with increased OB proliferation and decreased OC formation. In addition, MKs were separated using flow cytometry into 3 subpopulations based on maturation and effects on OB proliferation and OC formation were assessed. Finally, longevity studies on wild-type and mutant MKs were also conducted. In the second study we show that: 1) increased MK number corresponds with increased OB proliferation and decreased OC formation, 2) MK maturation stage does not alter the effect of MKs on bone cell lineages beyond the megakaryoblast stage, and, 3) GATA-1 deficient MKs survive longer than wild-type controls. Thus we demonstrate a novel interaction between two cell lineages only recently shown to be functionally connected, and make steps towards understanding how MKs exert their osteogenic effects.

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This is an Open Access Thesis.

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