Abstract
Endoplasmic reticulum (ER) stress transducers, such as old astrocyte specifically induced substance (OASIS) and activating transcription factor 6 (ATF6), which are induced by bone morphogenetic protein 2 (BMP2), regulate bone formation and osteoblast differentiation. Here, we examined the role of cAMP response element-binding protein H (CREBH), a member of the same family of ER membrane-bound basic leucine zipper (bZIP) transcription factors as OASIS and ATF6, in osteoblast differentiation and bone formation. Proinflammatory cytokine TNFα increased CREBH expression by up-regulating the nuclear factor-κB (NF-κB) signaling pathway in osteoblasts, increased the level of N-terminal fragment of CREBH in the nucleus, and inhibited BMP2 induction of osteoblast specific gene expression. Overexpression of CREBH suppressed BMP2-induced up-regulation of the osteogenic markers runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and osteocalcin (OC) in MC3T3-E1 cells and primary osteoblasts, as well as BMP2-induced ALP activity and OC protein production. In contrast, knockdown of CREBH attenuated the inhibitory effect of TNFα on BMP2-induced osteoblast differentiation. Mechanistic studies revealed that CREBH increased the expression of Smad ubiquitination regulatory factor 1 (Smurf1), leading to ubiquitin-dependent degradation of Smad1, whereas knockdown of CREBH inhibited TNFα-mediated degradation of Smad1 by Smurf1. Consistent with these in vitro findings, administration of Ad-CREBH inhibited BMP2-induced ectopic and orthotopic bone formation in vivo. Taken together, these results suggest that CREBH is a novel negative regulator of osteoblast differentiation and bone formation.
Highlights
Severe inflammatory reactions delay wound healing of bone
We examined the effects of cAMP response element-binding protein H (CREBH) on the expression levels of the Smad1 downstream factors Runx2 and activating transcription factor 6 (ATF6), which are required for osteoblast differentiation
Inflammation is triggered by inflammatory cytokines, such as TNF␣ and LPS, which suppress bone morphogenetic protein 2 (BMP2)-induced osteoblast differentiation in vitro and contribute to bone loss in inflammatory bone diseases, such as rheumatoid arthritis [17, 35]
Summary
Severe inflammatory reactions delay wound healing of bone. Results: Tumor necrosis factor ␣ (TNF␣) inhibition of osteoblast differentiation is associated with increased cAMP response element-binding protein H (CREBH) and Smurf expression. Endoplasmic reticulum (ER) stress transducers, such as old astrocyte induced substance (OASIS) and activating transcription factor 6 (ATF6), which are induced by bone morphogenetic protein 2 (BMP2), regulate bone formation and osteoblast differentiation. BMP2 stimulates osteoblast differentiation and bone formation by activating several unfolded protein response transducers, including OASIS and ATF6, which induce osteoblast-specific gene expression [9, 10]. Activation of ERK by TNF␣ results in inhibition of the transcription factor osterix, and TNF␣-mediated induction of Smad ubiquitination regulatory factor 1 (Smurf1) and Smurf accelerates the degradation of Runx protein through the proteasomal degradation pathway [25] Despite these findings, the molecular mechanisms underlying inflammatory actions in osteoblast differentiation are not fully understood. We describe a novel signaling pathway that encourages further analyses of the relationship between ER stress and bone formation
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