JNK Activity Is Essential for Atf4 Expression and Late-Stage Osteoblast Differentiation

Abstract

Osteoblasts differentiate from mesodermal progenitors and play a pivotal role in bone formation and mineralization. Several transcription factors including runt-related transcription factor 2 (RUNX2), Osterix (OSX), and activating transcription factor4 (ATF4) are known to be crucial for the process, whereas the upstream signal transduction controlling the osteoblast differentiation sequence is largely unknown. Here, we explored the role of c-jun N-terminal kinase (JNK) in osteoblast differentiation using in vitro differentiation models of primary osteoblasts and MC3T3-E1 cells with ascorbic acid/b-glycerophosphate treatment. Terminal osteoblast differentiation, represented by matrix mineralization, was significantly inhibited by the inactivation of JNK with its specific inhibitor and exogenous overexpression of MKP-M (MAP kinase phosphatase isolated from macrophages), which preferentially inactivates JNK. Conversely, enhanced mineral deposition was observed by inducible overexpression of p54 JNK2 , whereas it was not observed by the overexpression of p46 JNK1 or p46 JNK2 , indicating a distinct enhancing role of p54 JNK2 in osteoblast differentiation. Inactivation of JNK significantly inhibited late-stage molecular events of osteoblast differentiation, including gene expression of osteocalcin (Ocn) and bone sialoprotein (Bsp). In contrast, earlier differentiation events including alkaline phosphatase (ALP) activation and osteopontin (Opn) expression were not inhibited by JNK inactivation. Although the expression levels of two transcription factor genes, Runx2 and Osx, were not significantly affected by JNK inactivation, induction of Atf4 mRNA during osteoblast differentiation was significantly inhibited. Taken together, these data indicate that JNK activity is specifically required for the late-stage differentiation events of osteoblasts.

c-Jun N-terminal kinase 1 negatively regulates osteoblastic differentiation induced by BMP2 via phosphorylation of Runx2 at Ser104

“…(43) Moreover, blocking JNK activity has previously been found to upregulate BMP2-induced ALP activity and osteopontin (OPN) expression. (13,39) Taken together, these data support our conclusion that JNK1 functions as a negative regulator in BMP2-induced osteoblastic differentiation.…”

“…Recently, Matsuguchi et al reported that JNK2 activation by osteogenic supplements is essential for ATF4 expression and late-stage differentiation in pre-osteoblast cells. (39) Liu et al reported that activation of JNK1, but not JNK2, increases cellular responsiveness to BMP2 by increasing the phosphorylation of Smad1 and association of Smad1 with BMP type I receptor. (27) However, the activity of Smad1 regulated by phosphorylation depends on the phosphorylation region.…”

“…Moreover, inhibition of BMP2-mediated JNK1 activation by specific inhibitor, DN-JNK1 and shRNA specific for JNK1, further enhanced BMP2-induced ALP activity, OCN expression, and cell mineralization. Furthermore, the former two studies (27,39) failed to look into the underlying mechanism(s) of how JNK1 mediates regulation of osteoblastic differentiation through posttranslational modification of Runx2, an essential factor for osteoblastic differentiation.…”

Activation of c-Jun NH2-terminal kinase 1 increases cellular responsiveness to BMP-2 and decreases binding of inhibitory Smad6 to the type 1 BMP receptor

“…(22) JNK has been reported to be required for late-stage differentiation of preosteoblasts and BMP-2-induced differentiation of preosteoblasts and pleuripotent cells. (18,20,23,24) JNK is considered a critical regulator of activating transcription factor 4, osteocalcin, and bone sialoprotein expression in spontaneously differentiating osteoblasts. (21,23) These studies suggest that JNK plays an important role in the induction of osteogenesis by BMP-2.…”

“…(18,20,23,24) JNK is considered a critical regulator of activating transcription factor 4, osteocalcin, and bone sialoprotein expression in spontaneously differentiating osteoblasts. (21,23) These studies suggest that JNK plays an important role in the induction of osteogenesis by BMP-2. However, the exact mechanism of this interaction is still unclear.…”