Regulation of Rb family proteins by Cdk6/Ccnd1 in growth plates

The growth plate lacks vascular tissues and is solely composed of chondrocytes. The growth plate is composed of three layers, i.e., the resting, proliferating and hyper-trophic chondrocyte layers. The chon-drocytes in the resting and proliferating layers proliferate and then they uniformly maturate toward the diaphysis to become hypertrophic chondrocytes, which lose the ability to proliferate. As the growth plate lacks vasculature, apoptotic chondrocytes are not phagocytosed, and the fragmented DNA detected by TUNEL remains until they reach the bone marrow. Therefore, the growth plate is an appropriate tissue to use for evaluation of cell proliferation, differentiation, and apoptosis. We examined the effects of Cdk6 and Ccnd1 (cyclin D1) in chondrocyte proliferation, differentiation, and apop-tosis. 1 Transgenic mice overexpressing either Cdk6 or Ccnd1 in chondrocytes show no increase in the number of BrdU-positive cells, and the chondrocytes normally maturate, although CCND1 is a well-established human oncogene. In contrast, transgenic mice overexpressing both Cdk6 and Ccnd1 show dwarfism. In Cdk6/Ccnd1 double transgenic mice, chondrocyte maturation to hypertrophic chondrocytes is inhibited, and the number of BrdU-positive cells is increased, but the proliferation of chondrocytes is not enhanced, and the frequency of apop-totic chondrocytes is greatly increased. The apoptosis is rescued by p53 deletion, but chondrocyte proliferation is still not enhanced in p53 –/– Cdk6/Ccnd1 double transgenic mice. Cdk6 and Ccnd1 regulate the activity of E2fs by phosphorylating Rb family proteins, Rb, p107, and p130, and trigger progression through the G 0 /G 1 and G 1 /S transitions of the cell cycle. The effects of targeted deletions of Rb family genes in chondrocytes were reported by two groups. 2-4 p107-knockout mice show most apparent inhibition of chondrocyte differentiation among the knockout mice of Rb family genes. Double knockout mice of p107/p130 or p107/Rb show more severe inhibition of chondrocyte differentiation and chondrocyte proliferation is enhanced without an increase in chondrocyte apop-tosis. Therefore, all of the Rb family proteins are involved in chondrocyte proliferation and differentiation, and p107 plays a major role among them. When we generated Cdk6/Ccnd1 double transgenic mice, we expected that overexpression of both Cdk6 and Ccnd1 would result in the inactivation of all Rb family proteins by their phosphoryla-tion. However, the phenotype of Cdk6/ Ccnd1 double transgenic mice was different from those of p107/p130 or p107/ Rb double knockout mice. BrdU-positive cells are increased, and chondrocyte differentiation is inhibited in p107/p130 and p107/Rb double knockout mice and Cdk6/ Ccnd1 double …