c-Jun N-Terminal Kinases (JNKs) Are Critical Mediators of Osteoblast Activity In Vivo

Abstract

The c-Jun N-terminal kinases (JNKs) are ancient and evolutionarily conserved regulators of proliferation, differentiation, and cell death responses. Currently, in vitro studies offer conflicting data about whether the JNK pathway augments or represses osteoblast differentiation, and the contribution of the JNK pathway to regulation of bone mass in vivo remains unclear. Here we show that Jnk1^-/- mice display severe osteopenia due to impaired bone formation, whereas Jnk2^-/- mice display a mild osteopenia only evident in long bones. In order to both confirm that these effects were osteoblast intrinsic and assess whether redundancy with JNK1 masks a potential contribution of JNK2, mice with a conditional deletion of both JNK1 and JNK2 floxed conditional alleles in osteoblasts (Jnk1-2^osx ) were bred. These mice displayed a similar degree of osteopenia to Jnk1^-/- mice due to decreased bone formation. In vitro, Jnk1^-/- osteoblasts display a selective defect in the late stages of osteoblast differentiation with impaired mineralization activity. Downstream of JNK1, phosphorylation of JUN is impaired in Jnk1^-/- osteoblasts. Transcriptome analysis showed that JNK1 is required for upregulation of several osteoblast-derived proangiogenic factors such as IGF2 and VEGFa. Accordingly, JNK1 deletion results in a significant reduction skeletal vasculature in mice. Taken together, this study establishes that JNK1 is a key mediator of osteoblast function in vivo and in vitro. © 2017 American Society for Bone and Mineral Research.

Keywords: ANGIOGENESIS; BONE FORMATION; JNK; JUN; MAPK; OSTEOBLASTS.

Figure 1. Jnk1 ^-/- and Jnk1/2^osx mice have low bone mass due to decreased bone formation​

(A) Expression analysis of Jnk1, 2, and 3 (n=4). (B-G) Micro computed tomography analysis (micro-CT) analysis of the trabecular bone in the femur (B and E) and skull (D) and histological (C, upper and F) and histomorphometric (C, lower and G) analysis of the L₃ vertebrae in male Jnk1^-/- and Jnk2^-/- mice at 6-weeks of age (male, n≥6). (H-K) Skeletal analysis of Jnk1^osx, Jnk2^osx, and Jnk1/2^osx mice. Micro-CT analysis of the trabecular bone in the femur (H and I) and histological (J, upper) and histomorphometric (J, lower and K) analysis of the L₃ vertebrae of male Jnk1^osx, Jnk2^osx, and Jnk1/2^osx mice in 6-week-old mice (male, n≥6). Bone volume/tissue volume (BV/TV, mineral apposition rate (MAR, μm day^-1), bone formation rate/bone surface (BFR/BS, μm³ μm^-2 day^-1), osteoclast number/bone perimeter (No. Oc./B. Pm) and osteoblast surface/bone surface (Ob.S/BS). Scale bars, 500μm. Error bars, mean ± standard deviation (s.d.) *P < 0.05.

Figure 2. JNK1 is essential for osteoblast function

(A) Gene expression analysis of Alp, Sp7, Ibsp and Bglap in Jnk1^-/- and Jnk2^-/- COBs after 7 days of osteoblast differentiation conditions (n=4). (B) ALP activity (left) and representative ALP staining (right) in Jnk1^-/- and Jnk2^-/- osteoblasts at day 7 of osteogenic induction (n=3). (C and D) Representative non-soluble ECM pellets and Von Kossa staining in differentiated Jnk1^-/- and Jnk2^-/- COBs at day 14 of osteogenic induction. (E and F) Immunoblotting of phospho-JUN in Jnk1^-/- COBs at day 7 of osteogenic induction. (G) and immunohistochemistry for phospho-JUN in Jnk1^-/- femurs (bone collar and cortex) in 6-week-old mice. (I) Gene expression analysis of Fosl1, Fosl2, Vegfa and Igf2 in Jnk1^-/- COBs at day 7 of osteogenic induction (n=4). (H) CateGOrizer summarizes biological process functions from Gene Ontology (GO) based on DEGs between WT and Jnk1^-/- COBs. The fraction of the piechart presents the proportion of genes involving with given function. (J) GO enrichment analysis of DEGs between WT and Jnk1^-/- COBs indicated ten significantly overrepresented functions using DAVID. Each bar is colored and labeled according to p-value of enrichment analysis. (K) Longitudinal sections of femurs from WT and Jnk1^-/- mice; OSX and Jnk1/2^osx mice at 6-weeks of age were subjected to immunofluorescence staining for endomucin (red) and DAPI (blue). Scale bars, 300μm. Error bars, mean ± s.d. *P < 0.05.