The role of gap junctions in megakaryocyte-mediated osteoblast proliferation and differentiation.
Gap junctions (GJs) are membrane-spanning channels that facilitate intercellular communication by allowing small signaling molecules (e.g. calcium ions, inositol phosphates, and cyclic nucleotides) to pass from cell to cell. Over the past two decades, many studies have described a role for GJ intercellular communication (GJIC) in the proliferation and differentiation of many cells, including bone cells. Recently, we reported that megakaryocytes (MKs) enhance osteoblast (OB) proliferation by a juxtacrine signaling mechanism. Here we determine whether this response is facilitated by GJIC. First we demonstrate that MKs express connexin 43 (Cx43), the predominant GJ protein expressed by bone cells, including OBs. Next, we provide data showing that MKs can communicate with OBs via GJIC, and that the addition of two distinct GJ uncouplers, 18alpha-glycyrrhetinic acid (alphaGA) or oleamide, inhibits this communication. We then demonstrate that inhibiting MK-mediated GJIC further enhances the ability of MKs to stimulate OB proliferation. Finally, we show that while culturing MKs with OBs reduces gene expression of several differentiation markers/matrix proteins (type I collagen, osteocalcin, and alkaline phosphatase), reduces alkaline phosphatase enzymatic activity, and decreases mineralization in OBs, blocking GJIC does not result in MK-induced reductions in OB gene expression, enzymatic levels, or mineralized nodule formation. Overall, these data provide evidence that GJIC between MKs and OBs is functional, and that inhibiting GJIC in MK-OB cultures enhances OB proliferation without apparently altering differentiation when compared to similarly treated OB cultures. Thus, these observations regarding MK-OB GJIC inhibition may provide insight regarding potential novel targets for anabolic bone formation.
Department of Orthopaedics and Rehabilitation, Yale University School of Medicine, New Haven, CT, USA.
Wendy A
WA
Ciovacco
Carolyn G
CG
Goldberg
Amanda F
AF
Taylor
Justin M
JM
Lemieux
Mark C
MC
Horowitz
Henry J
HJ
Donahue
Melissa A
MA
Kacena
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D002118:Calcium / Q000378:metabolism; D002450:Cell Communication / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D018031:Connexin 43 / Q000378:metabolism; D005110:Extracellular Space / Q000187:drug effects / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D017629:Gap Junctions / Q000187:drug effects / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D006034:Glycyrrhetinic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D008533:Megakaryocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D009829:Oleic Acids / Q000494:pharmacology; D010006:Osteoblasts / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D011247:Pregnancy; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014316:Tritium
D018031:Connexin 43; D009829:Oleic Acids; D014316:Tritium; C119129:18alpha-glycyrrhetinic acid; C029407:oleylamide; D000469:Alkaline Phosphatase; D006034:Glycyrrhetinic Acid; D002118:Calcium