Inactivation of the Integrin-Linked Kinase (ILK) in osteoblasts increases mineralization

In osteoblasts, Integrin-Linked Kinase (ILK)-dependent phosphorylation of the cJUN transcriptional coactivator, alpha NAC, induces the nuclear accumulation of the coactivator and potentiates cJUN-dependent transcription. Mutation of the ILK phosphoacceptor site within the alpha NAC protein leads to cytoplasmic retention of the coactivator and cell-autonomous increases in osteoblastic activity. In order to gain further insight into the ILK-alpha NAC signaling cascade, we inactivated ILK using RNA knockdown in osteoblastic cells and engineered mice with specific ablation of ILK in osteoblasts. ILK knockdown in MC3T3-E1 osteoblast-like cells reduced phosphorylation of its downstream target glycogen synthase kinase 3 beta (GSK3 beta), which led to cytoplasmic retention of alpha NAC and increased mineralization with augmented expression of the osteoblastic differentiation markers, pro-alpha 1 (I) collagen (col1A1), Bone Sialoprotein (Bsp) and Osteocalcin (Ocn). Cultured ILK-deficient primary osteoblasts also showed increased cytoplasmic aNAC levels, and augmented mineralization with higher Runx2, Coital and Bsp expression. Histomorphometric analysis of bones from mutant mice with ILK-deficient osteoblasts (Col1-Cre;Ilk(-/fI)) revealed transient changes, with increased bone volume in newborn animals that was corrected by two weeks of age. Our data suggest that the ILK-alpha NAC cascade acts to reduce the pace of osteoblast maturation. We propose that in vivo, functional redundancy is able to compensate for the loss of ILK activity, leading to the absence of an obvious phenotype when osteoblast-specific Ilk-deficient mice reach puberty. (C) 2013 Elsevier B.V. All rights reserved.