In this report we provide evidence for the involvement of p27 in controlling chondrocyte differentiation and endochondral bone development in mice. We have generated and analyzed the phenotype of p107−/− p27D51/D51
mice. The combination of these deficiencies resulted in phenotypes similar to those previously reported for p107−/− p130−/−
mice and recapitulated here, including neonatal lethality (Tables and ), aberrant or ectopic endochondral ossification with increased chondrocyte proliferation in cartilaginous regions of the limbs and sternum (Table ; Fig. and ) (13
), and chondrocyte maturation ex vivo (Fig. and ).
Endochondral ossification begins when mesenchymal cells are induced to enter a chondrocytic lineage. Many of the signaling molecules and the transcription factors involved in chondrocyte maturation have been identified (28
). A fetal cartilage mold is established and chondrocytes begin to produce PTHrP, which stimulates proliferation and further differentiation into postmitotic hypertrophic chondrocytes. Transforming growth factor β and Ihh both positively regulate chondrocytic PTHrP expression. On the other hand, fibroblast growth factor (FGF) signaling induces differentiation and the withdrawal of chondrocytes from the cell cycle. Ultimately, this process is controlled by a variety of transcription factors. Sox5 and -6 are essential for the induction of the chondrocytic lineage from mesenchymal cells (55
). Sox9 is involved in the expansion of chondrocytic cells (5
). Runx2/Cbfa1 is a determinant of the chondro-osteoprogenitor lineage in mesenchymal cells but must be downregulated for perichondrial progenitors to enter the chondrocyte lineage (24
). Runx2 is, however, required for hypertrophic chondrocyte maturation. Runx2 activation of vascular endothelial growth factor in hypertrophic chondrocytes is essential for progression of endochondral bone formation (69
). Runx2 mediates PTH/PTHrP signaling in the hypertrophic zone (23
) and FGF signaling in the perichondrium (24
). Thus, it is not surprising that skeletal malformations arise when the chondrocytic differentiation program is deregulated by either chronic activation or insufficient activation of Runx 2 (61
Skeletal manifestations can arise as a consequence of changes in chondrocyte proliferation. Skeletal defects in p107−/− p130−/−
), and E2F1 transgenic mice (51
) are associated with increased chondrocyte proliferation, without disruption of the normal morphological patterning of the growing cartilage column. We documented similar defects in bone development in p107−/− p27D51/D51
mice. The exaggeration of the p107 skeletal phenotype by either p130 or p27 insufficiency suggests that these proteins might share a function controlling proliferation and enforcing a p107-dependent cell cycle block.
How the cell cycle program is coordinated with chondrocyte maturation is becoming increasingly clear. Transforming growth factor β and PTHrP stimulate chondrocyte proliferation by increasing cyclin D1 (2
) and decreasing p57Kip2
). Consistent with this, cyclin D1-deficient mice have reduced growth plate proliferation (54
), and the proliferation of chondrocytes continues in p57−/−
mice, leading to defects in endochondral ossification (71
). However, the most compelling piece of evidence supporting a link between PTHrP signaling and p57 accumulation is the observation that skeletal abnormalities associated with PTHrP deficiency are not seen in a p57-deficient background (39
). Interestingly, these skeletal abnormalities are also reversed in a p107−/− p130−/−
), suggesting that p57 and p107/p130 may act in a common pathway during PTHrP-regulated endochondral ossification.
Conversely, FGF induces chondrocyte growth arrest and maturation by inducing p107 and p130 (33
). p107- and p130-deficient mice have defects in endochondral ossification associated with increased chondrocyte proliferation (13
). It has been suggested that p57 might act by inhibiting CDK activity, allowing p107 and p130 to accumulate and support chondrocyte differentiation (36
). However, dephosphorylation of p107 is rapid, occurring within 1 hour of FGF treatment, and is not dependent on gene transcription or protein translation (30
). Consequently, it is more likely that changes in the pocket proteins precede cell cycle exit induced by p57.
The regulation of p27 levels in relation to p57 might account for the differences in the penetrance of the skeletal phenotype that arise from compound mutations in the p27 and p107 loci (17
). In osteoblasts, the level of p27 is correlated with Runx2 and inversely related to proliferation. p57 levels are increased as chondrocytes exit the cell cycle with decreased Runx2, whereas p27 levels only increase in conjunction with the reactivation of Runx2 in hypertrophic chondrocytes. There are a number of reports that show that p27 accumulates in hypertrophic chondrocytes (1
) However, as there is little change in the spatial or temporal regulation of chondrocyte proliferation in p27-deficient mice, it was suggested that its contribution might be minimal (18
). Our crossing the p27 deficiency into the sensitized background of p107-deficient mice clearly unmasks a significant contributory function for p27. In the chondrogenic lineage, differences in the temporal regulation of these two CDK inhibitors may cause either protein to become genetically indispensable for normal cartilage.
Thus, there are four important cell cycle regulators implicated in chondrocyte maturation: p57, p27, p107, and p130. To understand the relationships between these proteins, we find it helpful to think of the impact of the cell cycle on chondrocytic maturation in two stages (14
). First, because the loss of either p57 or p107 alone had the greatest impact on chondrocyte maturation in both mice and primary cultures, we think these are at the top of the heirarchy. Additionally, we suspect that p107 is the major input through which signaling affects maturation, because p107 accumulates before p130 and before cell cycle exit occurs in FGF-treated RCS cells (14
). In the absence of p107, compensatory upregulation of p130 might substitute for the initial decision to exit the cycle, as suggested previously (13
), but it is also plausible that a parallel pathway controlled by increasing p57 as cells enter the G1
phase would operate a bit more inefficiently to induce the initial growth arrest. Indeed, it is clear that p107 single mutant mice do have a subtle phenotype, albeit subclinical in regards to viability. So what do p27 and p130 do? Because any role for p27 and p130 is only unmasked in p107-deficient cells, p27 accumulates during the hypertrophic transition in vivo and in vitro, and p130 accumulates in RCS cells once they exit the cell cycle, we suspect that these proteins normally accumulate only when the cells have exited the cell cycle. Thus, they might serve to buffer the cell against continual mitogenic signaling which could otherwise induce promiscuous proliferation. Thus, p130 and p27 may simply reinforce the cell cycle exit decision made by p107 and p57. Ultimately, collaboration between the molecules that induce cell cycle exit and those that maintain quiescence might be required to permit the elaboration of appropriate transcriptional program leading to the hypertrophic fate.