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

Abstract

Bone morphogenetic protein 2 (BMP-2) plays a critical role in the differentiation of precursor cells and has been approved for clinical application to induce new bone formation. To date, unexpectedly high doses of recombinant BMP-2 have been required to induce bone healing in humans. Thus, enhancing cellular responsiveness to BMP-2 potentially has critically important clinical implications. BMP responsiveness may be modulated in part by cross-talk with other signaling pathways, including mitogen-activated protein kinases (MAPKs). c-Jun NH 2 -terminal kinase (JNK) is a MAPK that has been reported to be required for late-stage differentiation of preosteoblasts and BMP-2-induced differentiation of preosteoblasts and pleuripotent cells. In this study we determined that MC3T3-E1-clone 24 cells (MC-24) can be induced by BMP-2 to differentiate into mineralizing osteoblast cultures. Using this inducible system, we employed both JNK loss-of-function and gain-of-function reagents to make three key observations: (1) JNK is required for phosphorylation of Smad1 by BMP-2 and subsequent activation of Smad1 signaling and osteoblast differentiation, (2) JNK1, but not JNK2, is required for BMP-2-induced formation of mineralized nodules, and (3) JNK1 activation decreases binding of inhibitory Smad6 to the type I BMP receptor (BMPR-I) and reciprocally increases binding of Smad1, both observations that would increase responsiveness to BMP-2. Understanding this and other pathways that lead to increased cellular responsiveness to BMPs could greatly aid more cost-effective and safe clinical delivery of these important molecules. ß

Activation of JNKs is essential for BMP9-induced osteogenic differentiation of mesenchymal stem cells

“…JNKs can be activated by several stimuli, such as cytokines, ultraviolet irradiation, and heat shock. The physiological functions of JNKs in osteogenic differentiation and bone formation have been investigated reviously (11,13,14). However, the obtained results are controversial, with some studies suggesting a stimulatory role of JNKs in osteogenic differentiation and bone formation (11,13), and others proposing that JNKs is inhibitory (14).…”

“…The physiological functions of JNKs in osteogenic differentiation and bone formation have been investigated reviously (11,13,14). However, the obtained results are controversial, with some studies suggesting a stimulatory role of JNKs in osteogenic differentiation and bone formation (11,13), and others proposing that JNKs is inhibitory (14). Although these studies about the precise role of JNKs in skeletal development didn’t lead to complete unanimity, it is well accepted that JNKs plays a functional role in osteogenesis and bone metabolism.…”

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

“…(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. The phosphorylation within the linker-region of Smad1 by MAPK has been reported to desensitize cells to BMP2 and inhibit both nuclear accumulation and transcriptional activity of Smad1.…”

“…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.…”

“…(25) Studies in jnk1 À/À mice or jnk2 À/À mice have shown that whereas reduction of bone volume occurs in jnk1 À/À mice, the absence of jnk2 does not affect bone volume, suggesting that JNK1, rather than JNK2, plays an important role in regulating bone development. (26) Moreover, JNK1 not JNK2, has been reported to be involved in BMP2-induced osteoblastic differentiation in MC3T3-L1 cells (27) ; also activated by TAK1, a BMP2-activated kinase, it thereby inhibits TNF-a-induced apoptosis in MCF7 cells. (28,29) These studies indicate that JNK1 is important in BMP2-induced osteoblastic differentiation.…”