title: Megakaryocytes modulate osteoblast synthesis of type-l collagen, osteoprotegerin, and RANKL.
abstract: We have previously reported evidence that megakaryocytes may play a role in bone remodeling, possibly by interactions with cells at the bone surface. To investigate the direct effects of megakaryocytes on osteoblasts, maturing megakaryocytes (CD61 positive cells) were isolated and added to cultures of human osteoblasts. Osteoblasts alone and osteoblasts treated with CD61-negative (non-megakaryocytic) cells were used as control cultures. After 48 h in culture, megakaryocytes were removed and osteoblasts immunolocalized for type-1 collagen, osteoprotegerin (OPG), and RANKL expression. Similar cultures were used for RNA extraction with mRNA for Col 1A1, OPG, and RANKL in osteoblasts measured quantitatively by RT-PCR. Osteoblasts cultured alone showed high levels of expression of collagen with 74% (+/-7) of cells staining positively. When cultured with megakaryocytes, the number of positively staining cells remained similar but the intensity of expression was increased 1.54-fold (P < 0.02). OPG was expressed by 32% (+/-6.3) of osteoblasts increasing to 51% (+/-5.5) when cultured in the presence of megakaryocytes (P < 0.01) with a 1.63-fold increase in intensity of expression (P < 0.01). In contrast, osteoblasts cultured with megakaryocytes showed suppression of RANKL expression; 35.6% (+/-5.8) of osteoblasts cultured alone stained positively decreasing to 24.3% (+/-5.3) with a 1.6-fold diminished intensity of expression (P < 0.02). Osteoblasts co-cultured with CD61-negative cells showed no differences in collagen, OPG, or RANKL expression levels compared to osteoblasts cultured alone. mRNA data supported these findings with a 3.1-fold increase in Col 1A1 expression in megakaryocyte-treated cultures compared to controls (P < 0.02). Low-level OPG mRNA expression increased 8.14-fold in osteoblasts cultured in the presence of megakaryocytes (P < 0.01), while RANKL expression was suppressed 3.3-fold (P < 0.02). These results demonstrate that in vitro, megakaryocytes have direct effects on osteoblastic production of factors affecting both bone formation and resorption. These data provide further evidence that megakaryocytes may play an important role in bone remodeling.
authors.affiliation: Cambridge University School of Clinical Medicine, Addenbrooke's Hospital, Box 157, Cambridge CB2 2QQ, UK. sb201@cam.ac.uk
authors.forename: S
authors.initials: S
authors.lastname: Bord
authors.forename: E
authors.initials: E
authors.lastname: Frith
authors.forename: D C
authors.initials: DC
authors.lastname: Ireland
authors.forename: M A
authors.initials: MA
authors.lastname: Scott
authors.forename: J I O
authors.initials: JI
authors.lastname: Craig
authors.forename: J E
authors.initials: JE
authors.lastname: Compston
mesh_terms: D018952:Antigens, CD34 / Q000276:immunology; D001483:Base Sequence; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics; D024042:Collagen Type I / Q000096:biosynthesis* / Q000235:genetics; D017931:DNA Primers; D015870:Gene Expression; D006023:Glycoproteins / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D039661:Integrin beta3 / Q000276:immunology; D008533:Megakaryocytes / Q000502:physiology*; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000235:genetics; D010006:Osteoblasts / Q000276:immunology / Q000378:metabolism*; D053244:Osteoprotegerin; D053245:RANK Ligand; D012333:RNA, Messenger / Q000235:genetics; D053246:Receptor Activator of Nuclear Factor-kappa B; D018160:Receptors, Cytoplasmic and Nuclear / Q000096:biosynthesis* / Q000235:genetics; D018124:Receptors, Tumor Necrosis Factor / Q000096:biosynthesis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D018952:Antigens, CD34; D002352:Carrier Proteins; D024042:Collagen Type I; D017931:DNA Primers; D006023:Glycoproteins; D039661:Integrin beta3; D008562:Membrane Glycoproteins; D053244:Osteoprotegerin; D053245:RANK Ligand; D012333:RNA, Messenger; D053246:Receptor Activator of Nuclear Factor-kappa B; D018160:Receptors, Cytoplasmic and Nuclear; D018124:Receptors, Tumor Necrosis Factor; C505686:TNFRSF11A protein, human; C506030:TNFRSF11B protein, human; C505688:TNFSF11 protein, human