Fig. 7.

Proliferative potential of growth plate chondrocytes is regulated by CamkII activity. pCamkII and Pthrp might exert opposing effects on hypertrophy through the common target Runx2. (A) To test this possibility, DF-1 fibroblast cultures were transiently transfected with RCAS(A)-HA-daCamkII (red) and RCAS(A)-Runx2 (green). In two experiments, an average of 58% of co-transfected cells (40/76 and 31/46 in experiments 1 and 2, respectively) showed strong, predominantly nuclear localization of Runx2 (white arrows). By contrast, in cells that displayed only Runx2 expression, only 8% (5/59 and 2/25 in experiments 1 and 2, respectively) showed predominantly nuclear Runx2 (open arrows show a cell with both nuclear and cytosolic signal). (B) Luciferase reporter experiments (three experiments in triplicate per condition) show that co-transfection with pCAG-HA-daCamkII upregulates pcDNA3.1-Runx2-induced transcription from the Ihh promoter. ^**Differences between compared data sets (brackets) are statistically significant, P<0.001. Data are expressed as mean ± s.d. (C) A model for regulation of proliferation and hypertrophy by CamkII. Left: a summary of the genetic pathway that regulates chondrocyte maturation and CamkII activity in the growth plate. The green triangle represents a hypothetical gradient of CamkII activation across the PZ. Red indicates the novel interactions described in this paper. The `X' represents hypothetical intermediates regulated by the Fzd pathway that act upstream to repress CamkII activation. Right: we propose that a gradient of Pthrp signaling (purple triangle) generates a gradient of phosphatase activity (yellow outline on chondrocytes) that results in an inverse gradient of CamkII activity (green in chondrocytes). CamkII activity reaches a threshold at the PZ/PHZ boundary necessary for high-level nuclear enrichment (green circles) of pCamkII. Thus, antagonistic interactions between opposing gradients of Pthrp and Ihh (orange triangle) signaling define the distal end of the PZ (green dashed line) and the inhibitory action of Pthrp on CamkII activity defines the proximal boundary of the PZ (red dashed line). This distance, which is dependent on the relative strengths of Pthrp and Ihh signals, determines the maximum number of proliferative chondrocytes that can be generated from a single mother row cell because each daughter cell occupies ∼7 μm along the longitudinal axis (Hunziker, 1994). Scale bar: 25 μm in A. PZ, proliferative zone; PHZ, prehypertrophic zone.