Fig. 1.

CamkII is activated in the prehypertrophic zone. (A) The growth plate is composed of four zones: resting (RZ), proliferative (PZ), prehypertrophic (PHZ) and hypertrophic (HZ). (B) Western blot analysis of total chick growth plate confirms the presence of CamkIIα and CamkIIβ, including phosphorylated forms (pCamkII). (C) Lysates of total chick growth plates pretreated with λ protein phosphatase show substantial reduction in pCamkII without significantly altering CamkIIα (β-actin loading control), demonstrating phospho-specificity. (D) Analysis of adjacent sections of the chick growth plate (n=6) by fluorescent in situ hybridization (FISH) with probes for Col2a1 (proliferative zone, green, left-hand column) and Ihh (prehypertrophic zone, red, left-hand column), or immunofluorescence (IF) with anti-pCamkII (green, right-hand column) revealed that nuclear pCamkII increases in chondrocytes at the PZ to PHZ transition (white dashed line). (E) Immunofluorescence of mouse metatarsals treated with calyculin A (n=6 per concentration) shows global activation of CamkII in chondrocytes (arrows), suggesting that negative regulators oppose CamkII activity. White dashed line indicates the border between the proliferative and the prehypertrophic zones. (F) Parallel western blot analysis (n=20 metatarsals; two experiments) shows increased phosphorylation of CamkII following calyculin A treatment that occurs without changes in total CamkII or PP2A levels. Scale bars: 500 μm in D; 100 μm in E.