Hyaluronan increases RANKL expression in bone marrow stromal cells through CD44.
HA activates CD44 to stimulate RANKL expression in bone marrow stromal cells. HA stimulation of RANKL is blocked by anti-CD44 antibody and is absent in cells from CD44(-/-) mice. CD44(-/-) mice exhibit thicker cortical bone and a smaller medullary cavity, but indices of bone resorption are not affected.


BACKGROUND
Hyaluronan (HA), the major nonprotein glycosaminoglycan component of the extracellular matrix in mammalian bone marrow, functions in part through its receptor, CD44, to stimulate a series of intracellular signaling events that lead to cell migration, adhesion, and activation. To determine whether HA activation of CD44 influences RANKL and osteoprotegerin (OPG) expression and whether CD44 is functionally important in bone metabolism, we studied whole bone and bone marrow stromal cells (BMSCs) from wildtype and CD44(-/-) mice.


METHODS
BMSCs from wildtype and CD44(-/-) mice at 7 weeks of age were cultured and treated with either HA or anti-CD44 antibody. The levels of mRNA of RANKL, OPG, CD44, alkaline phosphatase (ALP), osteocalcin (OC), and alphaI collagen (COLL) were determined by quantitative real-time RT-PCR. Levels of RANKL and CD44 protein were measured by immunoblotting, and expression of CD44 in whole bone was determined by immunohistochemical staining. Double immunofluorescence staining and confocal microscopy were used to study colocalization of Cbfa1, CD44, and HA. Tibias were imaged using muCT, and cancellous and cortical parameters were measured. Osteoblast and osteoclast surface in the distal femoral metaphysis and osteoclast on the endocortical surface at the tibio-fibular junction were measured using quantitative histomorphometry. Differences were analyzed using ANOVA and the Newman-Keuls test.


RESULTS
Addition of HA dose-dependently increased RANKL mRNA (3.6-fold) and protein (3-fold) levels in BMSCs. Stimulation of RANKL by HA could be blocked with anti-CD44 antibody. Treatment of cells with HA or anti-CD44 antibody had no significant effect on OPG mRNA levels. Both CD44 and HA localized on the plasma membrane in cells expressing Cbfa1. HA localization on the cell membrane disappeared when cells were preincubated with anti-CD44 antibody. Compared with control mice, cortical bone of CD44(-/-) was thicker, and medullary area was smaller at both 7 and 17 weeks, but at 7 weeks, indices of bone resorption were normal. At 17 weeks of age, tibial mass of CD44(-/-) mice was higher than control mice. CD44(-/-) animals expressed less RANKL in whole bone (-30%) and in BMSCs (-50%). Cells from CD44(-/-) animals failed to respond to either HA or CD44 antibody treatment.


CONCLUSIONS
HA can increase RANKL expression in BMSCs through CD44.
Cao
Jay J
JJ
Division of Endocrinology, Veterans Affairs Medical Center, San Francisco, California 94121, USA.
Singleton
Patrick A
PA
Majumdar
Sharmila
S
Boudignon
Benjamin
B
Burghardt
Andrew
A
Kurimoto
Pam
P
Wronski
Thomas J
TJ
Bourguignon
Lilly Y W
LY
Halloran
Bernard P
BP
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D001854:Bone Marrow Cells / Q000737:chemistry / Q000166:cytology* / Q000378:metabolism; D001842:Bone and Bones / Q000378:metabolism; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D050659:Core Binding Factor Alpha 1 Subunit; D015870:Gene Expression / Q000187:drug effects; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism; D018960:Hyaluronan Receptors / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D006820:Hyaluronic Acid / Q000032:analysis / Q000494:pharmacology / Q000502:physiology*; D008297:Male; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D009154:Mutation / Q000235:genetics; D009363:Neoplasm Proteins / Q000032:analysis / Q000378:metabolism; D053244:Osteoprotegerin; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism; D018124:Receptors, Tumor Necrosis Factor; D017154:Stromal Cells / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D014157:Transcription Factors / Q000032:analysis / Q000378:metabolism; D015854:Up-Regulation
D000906:Antibodies; D002352:Carrier Proteins; D050659:Core Binding Factor Alpha 1 Subunit; D006023:Glycoproteins; D018960:Hyaluronan Receptors; D008562:Membrane Glycoproteins; D009363:Neoplasm Proteins; D053244:Osteoprotegerin; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D018160:Receptors, Cytoplasmic and Nuclear; D018124:Receptors, Tumor Necrosis Factor; C505687:Tnfrsf11a protein, mouse; C506031:Tnfrsf11b protein, mouse; C505689:Tnfsf11 protein, mouse; D014157:Transcription Factors; D006820:Hyaluronic Acid