Expression and activity of Runx2 mediated by hyaluronan during chondrocyte differentiation

Abstract

During endochondral ossification, the production of hyaluronan (HA) is strictly and selectively regulated by chondrocytes, with a temporal peak at the hypertrophic stage. This study was conducted to clarify the effects of HA on expression and activity of runt-related gene 2 (Runx2), a potent transcription factor for chondrocyte differentiation in hypertrophic chondrocytes. Immature chondrocytes from an ATDC5 cell line were cultured and differentiated in DMEM/Ham's F12 with pre-defined supplements. Using real-time PCR, the gene expressions of type II collagen, MMP-13, HAS2, and Runx2 in cultured chondrocytes were analysed from days 0 to 18 of cell differentiation. The activity and expression of Runx2 in hypertrophic chondrocytes were analysed after the treatment with HA oligosaccharide (HAoligo) using AML-3/Runx2 binding, real-time PCR and Western blot analysis. The effects of pre-incubation of anti-CD44 antibody on Runx2 expression were also examined. Expression of type X collagen and Runx2 mRNAs reached a maximum at the terminal differentiation of chondrocytes. The activity and expression of Runx2 was significantly inhibited in hypertrophic chondrocytes treated with HAoligo compared to the untreated controls. High molecular weight-HA did not affect the expression or activity of Runx2. The expression of Runx2 mRNA was significantly decreased in hypertrophic chondrocytes treated with anti-CD44 antibody. These results suggest that HAoligo may affect the terminal differentiation of chondrocytes during the endochondral ossification by inhibiting the expression and activity of Runx2.

Conditional inactivation ofHas2reveals a crucial role for hyaluronan in skeletal growth, patterning, chondrocyte maturation and joint formation in the developing limb

“…Alternatively, we suggest that the interaction of pericellular HA with CD44 in hypertrophying chondrocytes might modulate or augment some of the signaling networks that regulate the progression of chondrocyte maturation. Significantly, in the chondrocytic cell line ATDC5, expression of Has2 and Runx2, a crucial transcriptional regulator of chondrocyte maturation, is concomitantly upregulated at the onset of hypertrophic maturation (Tanne et al, 2008). Moreover, disruption of HA-CD44 interactions by addition of HA oligosaccharides or anti-CD44 antibodies inhibits Runx2 expression, as well as the expression of hypertrophic markers including type X collagen (Tanne et al, 2008).…”

“…Significantly, in the chondrocytic cell line ATDC5, expression of Has2 and Runx2, a crucial transcriptional regulator of chondrocyte maturation, is concomitantly upregulated at the onset of hypertrophic maturation (Tanne et al, 2008). Moreover, disruption of HA-CD44 interactions by addition of HA oligosaccharides or anti-CD44 antibodies inhibits Runx2 expression, as well as the expression of hypertrophic markers including type X collagen (Tanne et al, 2008). These results further indicate that HA-CD44 interactions might indeed play an important role in regulating chondrocyte maturation.…”

Integrin αvβ3 and CD44 pathways in metastatic prostate cancer cells support osteoclastogenesis via a Runx2/Smad 5/receptor activator of NF-κB ligand signaling axis

“…Altered levels of CD44 have been seen in many epithelial neoplasms and expression of CD44 has been shown to carry prognostic implications [35,36]. RUNX2 expression is regulated by CD44 signaling [37]. A neutralizing antibody to CD44s significantly decreased the expression of Runx2 mRNA in hypertrophic chondrocytes [37].…”

Inhibition of Notch1 signaling by Runx2 during osteoblast differentiation

“…(25,(28)(29)(30) Recently, multiple biologic functions of Runx2 have been demonstrated in various cell types, including chondrocytes, endothelial cells, osteosarcoma cells, T-cell lymphomas, and breast cancer cells. (31)(32)(33)(34)(35) One of the central players in the osteoblast differentiation process is the Runx2 transcription factor, which coordinates multiple signals involved in osteoblast differentiation. Up to the present, several regulators such as Stat1, Twist, Hey1, and Notch1 have been shown to form inhibitory complexes with Runx2 and to inhibit osteoblast differentiation.…”