Hyaluronan Increases RANKL Expression in Bone Marrow Stromal Cells Through CD44

Abstract

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.Introduction: 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. Materials and 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 ␣I 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 CT, 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.

Importance of CD44 in the proliferation and mineralization of periodontal ligament cells

“…The results of this study are in accordance with previous studies using different strategies to block CD44 signaling (6,17,18). Tanimoto et al (17) demonstrated that cell proliferation is inhibited by the treatment of anti-CD44 neutralizing antibody in cultured PDL fibroblasts.…”

“…Tanimoto et al (17) demonstrated that cell proliferation is inhibited by the treatment of anti-CD44 neutralizing antibody in cultured PDL fibroblasts. The study of Cao et al, reveals that the treatment with anti-CD44 antibody inhibits alkaline phosphatase activity and calcification nodule formation from bone marrow stromal cells stimulated with hyaluronic acid (hyaluronan; HA) (18). CD44 is well accepted as a marker for MSCs that are characterized by their great capacity for self-renewal while maintaining their multipotency (5,19).…”

Specific concentration of hyaluronan amide derivative induces osteogenic mineralization of human mesenchymal stromal cells: Evidence of RUNX2 and COL1A1 genes modulation

“…CD44 (Kaviani et al, 2019), an important receptor of HA regulating the process of proliferation and mineralization (Yeh et al, 2014). Mineralization effect of HA on dental pulp cells occur via CD44 (Cao et al, 2005;Chen et al, 2013;Chen et al, 2016) and it is known that the knockdown of CD44 receptor block mineralization and COL15A1 (Yeh et al, 2014), these data suggest that also HAD could act through the same signaling mechanism.…”

Trabecular Bone Deterioration in col9a1+/− Mice Associated With Enlarged Osteoclasts Adhered to Collagen IX–Deficient Bone

“…Recently, extracellular matrix molecules have been implicated in influencing the expression of RANKL. Cao et al (34) reported that the effect of hyaluronan on RANKL expression was mediated through CD44, a cell surface receptor for hyaluronic acid as well as OPN and collagens. Our histomorphometric data on osteoblast numbers indicated that there were no significant differences between col9a1 +/− and wildtype mice ( Fig.…”