In the present studies, we demonstrate for the first time that cell cycle proteins regulate the key transcription factor Runx2 by inducing its phosphorylation and degradation through the ubiquitin-proteasome pathway. The serine-472 residue plays a critical role in cyclin D1-induced Runx2 phosphorylation and subsequent ubiquitination and degradation. Furthermore, cyclin D1-Cdk4 controls Runx2 transcriptional activity and physiological function in osteogenic progenitor C3H10T1/2 cells.
Although regulation by cell cycle proteins has not been characterized for Runx2 in bone and cartilage, regulation of differentiation-associated transcription factors by cell cycle proteins has been well established in other cell types, such as myoblasts. MyoD is a key transcription factor controlling the myoblast differentiation. Its phosphorylation and degradation is controlled and regulated by cyclin B-Cdk1/Cdk2. Cyclin B induces phosphorylation of serine-200 of MyoD protein and triggers MyoD ubiquitination and rapid degradation (32
An inverse relationship between proliferation and differentiation has been reported in many developmental processes, such as myogenesis and neural development. Previous findings suggest that this inverse relationship is regulated at different levels. For example, cyclin-Cdk complexes phosphorylate MyoD and induce its degradation (10
). Forced expression of MyoD induces p21 expression in myoblasts (11
). In addition, hypophosphorylated Rb acts as a co-repressor for E2F-induced gene expression of cell cycle proteins, and it also serves as a co-activator for Runx2-induced osteoblast differentiation (34
). In the present studies, we report that cyclin D1-Cdk4 directly phosphorylate Runx2 and induce Runx2 ubiquitination and proteasome degradation, suggesting that coordinated relationship between proliferation and differentiation also exists during skeletal development (). The existence of these mutual inhibitions provides the handles for regulation of cellular functions by different signaling pathways.
Previously, we have shown that the ubiquitin-protein isopeptide ligase (E3) Smurf1 induces Runx2 degradation (35
) and that Smad6 enhances Smurf1-induced Runx2 degradation (27
). In the present studies, we have analyzed the effect of Smurf1 on the degradation of WT- and SA-Runx2 and found that Smurf1 induced WT as well as SA mutant Runx2 in a similar efficiency, suggesting that Smurf1 is not involved in the cyclin D1-induced, phosphorylation-dependent degradation of Runx2. Further investigation is required to identify the novel E3 ligase that is involved in the cyclin D1-Cdk4-induced Runx2 ubiquitination and degradation.
Proper development of bone tissues requires precise spatial and temporal control of cell proliferation and differentiation. Although Runx2 is expressed during early skeletal development (embryonic days 11.5-14.5), the formation of hypertrophic chondrocytes and mature osteoblasts does not occur in most bones until embryonic days 14.5-15.5 (15
). These observations suggest that Runx2 function must be suppressed during early bone development to allow chondrocyte and osteoblast pools to proliferate and expand. One possible mechanism for the suppression of Runx2 function during early bone development is through cyclin D1-Cdk4-induced degradation. More detailed in vivo
studies need to be conducted to further investigate this hypothesis.
In summary, our findings demonstrate that the cyclin D1-Cdk4 complex phosphorylates Runx2 and induces Runx2 degradation in an ubiquitin-proteasome-dependent manner. This study reveals a tightly regulated mechanism between bone cell growth and differentiation.