Cyclic AMP Response Element-binding Protein H (CREBH) Mediates the Inhibitory Actions of Tumor Necrosis Factor α in Osteoblast Differentiation by Stimulating Smad1 Degradation*

Background: Severe inflammatory reactions delay wound healing of bone. Results: Tumor necrosis factor α (TNFα) inhibition of osteoblast differentiation is associated with increased cAMP response element-binding protein H (CREBH) and Smurf1 expression. Conclusion: CREBH mediates the inhibitory actions of TNFα in bone regeneration. Significance: CREBH is identified as a new mediator of inflammation-dependent bone degradation and a potential therapeutic target. 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.

BMP2 5 is an important regulator of osteoblast differentiation, bone development, and the repair of bone defects (1,2). Recently, it was reported that the BMP2 signaling pathway activates unfolded protein response molecules during osteogenesis. ER stress is caused by the accumulation of excessive amounts of unfolded proteins in the ER (3,4). The physiological significance of ER stress has been definitively associated with the activation of three major unfolded protein response transducers: protein kinase RNA-like endoplasmic reticulum kinase (PERK), inositol-requiring kinase 1 (IRE-1), and ATF6 (5)(6)(7)(8). 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). However, the ability of BMPs to induce bone formation in clinical settings is limited, and the repair of bone defects by BMPs is negatively regulated by other factors (11).
OASIS and ATF6 are structurally similar to CREBH; these three proteins belong to the ER membrane-bound bZIP tran-scription factor family and are collectively referred to as OASIS family members (12). However, the mechanisms by which CREBH and ATF6 sense unfolded proteins and translocate from the ER to the Golgi differ from those of other OASIS family members (13). In addition, OASIS family members have distinct expression patterns, suggesting that these transcription factors may be associated with cell-or tissue-specific physiological responses. CREBH is induced and activated by the proinflammatory cytokines TNF␣, IL-6, and LPS (14). Hence, it integrates the proinflammatory response and regulation of ER stress, thereby underlining its importance to inflammatory responses. Although these transcriptional factors have a structural similarity, they can produce an opposite effect on biological systems. In glucose metabolism, CREBH promotes gluconeogenic activity in a CRTC2-independent manner (15), and ATF6 inhibits hepatic glucose output by competing with CREB for interaction with CRTC2 (16).
Inflammatory cytokines inhibit BMP-induced osteogenesis and bone formation (17,18); in fact, TNF␣ is a major inflammatory mediator responsible for bone loss in a number of bonerelated inflammatory diseases (19,20). TNF␣ inhibits BMP signaling by interfering with the DNA-binding ability of Smads via activation of the nuclear factor-B (NF-B) pathway, regulating Runx2 expression, and inhibiting BMP-induced osteoblast differentiation (18,21,22). In addition, TNF␣ induces the expression of Smad7 and Msx2, which also inhibit BMP signaling and related osteogenesis (23,24). 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 Smurf2 accelerates the degradation of Runx2 protein through the proteasomal degradation pathway (25). Despite these findings, the molecular mechanisms underlying inflammatory actions in osteoblast differentiation are not fully understood.
Here, we examined the effect of CREBH on osteoblast differentiation in vitro and ectopic and orthotopic bone formation in vivo. The results indicate that CREBH functions as a modulator of TNF␣-mediated inhibition of osteogenesis, and that this action is mediated mainly by Smurf1-induced degradation of Smad1. Overall, we describe a novel signaling pathway that encourages further analyses of the relationship between ER stress and bone formation.
Cell Culture, Transient Transfection, and Viral Infection-The pre-osteoblast MC3T3-E1 cells and primary osteoblasts have been described previously (27). The cells were cultured in ␣-minimal essential medium (Gibco-BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; GIBCO-BRL) and antibiotics, and incubated at 37°C in a humidified atmosphere containing 5% CO 2 . Transient transfections were performed as described previously (28). For viral infection, the cells were treated with the indicated viruses at the designated multiplicity of infection (m.o.i.) under serum-free conditions. After 4 h, the medium was replaced with an equivalent volume of medium containing 10% FBS, and the cells were incubated for an additional 24 -48 h.
qRT-PCR Analysis-Total RNA was isolated from the cultured cells using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. RT-PCR was performed using 1 g of total RNA. For quantification of gene transcription, cDNA was generated with the Maxime RT PreMix Kit (iNtRon, Sungnam, Korea), and then amplified on the StepOnePlus Realtime PCR System (ABI, Abilene, TX) using the QuantiTect SYBR PCR Kit (Qiagen, Valencia, CA) and specific primers. Cycling conditions were 95°C for 10 min, followed by 40 cycles of 95°C for 30 s, 58°C for 1 min, and 72°C for 30 s. Post-run samples were analyzed using ABI software and the relative expression was quantified using the 2 Ϫ⌬⌬Ct method with endogenous ␤-actin levels. The primer sequences have been described previously (29).
Western Blotting-Total cells or nuclear fractions were harvested in lysis buffer (Cell Signaling Technology, Cambridge, MA) and centrifuged at 12,000 ϫ g for 15 min at 4°C. The nuclear and cytoplasmic fractions were prepared using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Pierce Biotechnology), according to the manufacturer's instructions. Quantification of total protein was performed using the BCA Protein Assay Kit (Bio-Rad). Proteins were resolved by 10% SDS-PAGE and transferred to a PVDF membrane. After blocking using Tris-buffered saline (TBS) containing 0.1% Tween 20 and 5% milk, the membrane was incubated with specific primary antibodies. Signals were detected using an enhanced chemiluminescence reagent (Santa Cruz Biotechnology) according to the manufacturer's instructions. Densitometric analysis of the membrane was performed using a LAS-4000 lumino-image analyzer (Fujifilm, Tokyo, Japan).
ChIP Assay-MC3T3-E1 cells were treated with TNF␣ for the designated times and ChIP assays were performed as described previously (30). The DNA samples were quantified by qPCR using two pairs of primers. The primer sequences for the p50 and p65 binding regions of the CREBH promoter were 5Ј-CCAACTCTCAAGAATCAGTCAGC-3Ј (forward) and 5Ј-GCTTTGCATCTGTGACAGGATG-3Ј (reverse). The control primer sequences were 5Ј-GTTCTTGCATAGACCAG-GCCA-3Ј (forward) and 5Ј-TGGCCTGGTCTATGCAA-GAAC-3Ј (reverse). For quantitative comparison with qPCR, the ⌬C T method was applied. A ⌬C T value was calculated by subtracting the C T value of the input from that of the immunoprecipitated sample. A ⌬⌬C T value was then obtained by subtracting the ⌬C T value of the sample immunoprecipitated with p65 or p50 antiserum from that of the corresponding control sample with normal rabbit IgG. Fold-differences were determined by raising 2 to the ⌬⌬C T power.
Alkaline Phosphatase Staining and Osteocalcin Production Assay-For detection of alkaline phosphatase (ALP), the cultured cells were fixed with 70% ethanol, rinsed three times with deionized water, and treated with BCIP/nitro blue tetrazolium solution (Sigma) for 15 min. The stained cultures were then documented on an Epson Perfection V700 photo scanner (Seiko Epson, Nagano, Japan). For quantitative comparison, color intensities were measured from scanned images using Image J software and normalized to the value of the untreated control group. The level of osteocalcin (OC) secreted into the culture medium was determined using a mouse osteocalcin ELISA kit (Biomedical Technologies, Stoughton, MA), according to the manufacturer's instructions.
Animals and Surgical Procedure-The study was performed in accordance with the guidelines of the Chonnam National University Animal Care and Use Committee. C57BL/6 mice were purchased from Daehan Biolink (Eumsung, Korea) and 6-week-old male mice were randomly assigned to each experimental group. The animals were anesthetized by an intraperitoneal injection of a mixture of Zoletil (30 mg/kg; Virbac, Carros Cedex, France) and Rompun (10 mg/kg; Bayer Korea, Seoul, Korea). For ectopic bone formation, a sagittal incision (0.8 -1.0 cm) was made on the back of each mouse and the subcutaneous pocket was formed by blunt dissecting. Absorbable collagen sponges (Colladerm, Bioland, Ochang, Korea) containing Ad-BMP2 and Ad-CREBH were implanted into the pocket. For an orthotopic model, a sagittal incision was made on the scalp, and the calvarium was exposed by blunt dissection. A critical-sized defect was created by means of a 5-mm diameter trephine bur (Fine Science Tools, Foster City, CA) under low speed drilling and copious phosphate-buffered saline irrigation. Ad-BMP2 and Ad-CREBH were administered into the defect with absorbable collagen sponges. The total amount of viruses was adjusted by adding Ad-GFP control virus. Three weeks after the implantation, bone formation was evaluated using a three-dimensional micro-computed tomography (CT) system (model 1172, Skyscan, Aartselaar, Belgium). For the CT analysis, the scanned images were collected at 50 kV and 200 A and were reconstructed using the NRecon and CT analyzer software (Skyscan). For histology study, the implant or calvarial specimens were harvested, fixed in 10% neutral-buffered formalin, decalcified in Calci-Clear Rapid (National Diagnostics, Atlanta, GA), embedded in paraffin, and then cut into sections of 4 m thickness. The sections were stained with hematoxylin/eosin, and evaluated for general tissue response and bone formation.
Statistical Analyses-All experiments were repeated at least three times and statistical analyses were performed using a Student's t test or analysis of variance followed by Duncan's multiple comparison test. p Ͻ 0.05 was considered significant. The results are expressed as the mean Ϯ S.E. of triplicate independent experiments.

TNF␣ Increases the Expression and Activation of CREBH in
Osteoblasts-The proinflammatory cytokines TNF␣ and IL-6 increase Crebh mRNA expression in hepatocytes (31). Therefore, we examined whether CREBH expression is also regulated by TNF␣ in osteoblasts. Treatment of MC3T3-E1 cells with TNF␣ increased Crebh mRNA expression significantly, but did not affect the expression of the Atf6 and Oasis mRNAs (Fig. 1A). In contrast, the bone-forming cytokine BMP2 induced Atf6 and Oasis mRNA, as described in other studies (9, 32), but did not alter Crebh expression (Fig. 1B). Western blot analyses using total or nuclear protein extracts showed that TNF␣ also increased the CREBH protein level in a dose-dependent manner. Notably, both TNF␣ and tunicamycin, a strong ER stress inducer, increased the amount of the cleaved (nuclear) form of CREBH protein (Fig. 1, C and D).
In response to ER stress, membrane-bound transcription factors, such as ATF6 and OASIS, are cleaved and a part of the cytosolic component is translocated to the nucleus to function as a transcription factor. Therefore, we determined whether TNF␣ can promote the translocation of the cleaved (active) form of CREBH protein to the nucleus using a GAL4-based luciferase reporter assay. Exposure of TNF␣ increased the luciferase activity of GAL4-fused full-length CREBH in a dose-dependent manner (Fig. 1E). Taken together, these results suggest that TNF␣ increases CREBH expression and may enhance its transcriptional activity in the nucleus.
NF-B Signaling Pathway Is Involved in TNF␣-induced CREBH Expression-TNF␣ regulates the expression of several genes related to inflammation, by activation of the NF-B pathway (33). To determine whether this pathway is involved in TNF␣-induced CREBH expression in osteoblasts, MC3T3-E1 cells were co-transfected with a Crebh reporter construct (CREBH-Luc), and a plasmid expressing the p50 and/or p65 subunits of NF-B. The overexpression of p50 and p65 increased the luciferase activity of CREBH-Luc ( Fig. 2A). Treatment of BAY11-7082, a chemical inhibitor of the IKK/NF-B pathway, decreased the TNF␣-induced Crebh promoter activity in a dose-dependent manner (Fig. 2B). The compound also abrogated the TNF␣ induction of CREBH protein expression with the reduced phosphorylation of IB (Fig. 2C).
To more confirm the involvement of specific IKK/IB␣/ NF-B pathway in the TNF␣-induced CREBH expression, we examined the effects of the dnIB␣ (S32A/S36A) mutant, which is unable to be phosphorylated and proteolytically degraded (23). Transfection of dnIB␣ effectively blocked TNF␣-increased p65 and CREBH protein levels in nucleus and Crebh mRNA expression in the cells (Fig. 2, D and E). On the other hand, TNF␣ inhibited BMP2-induced Alp and Oc mRNA expressions and treatment of BAY-11-7082 was partially rescued the TNF␣ action (Fig. 2F). An in silico analysis revealed a consensus NF-B binding site at nucleotides Ϫ112 to Ϫ103 relative to the transcription initiation site of the Crebh gene. TNF␣ failed to activate the luciferase activity of a mutant form of the Crebh promoter (M-CREBH-Luc) in which four nucleotides in the NF-B binding site were substituted, compared with the WT-CREBH-Luc activity (Fig. 2G). In addition, ChIP-qPCR analysis showed treatment of TNF␣ enhanced the binding of NF-B (p50 or p65) on the Crebh promoter (Fig. 2H). Taken together, these results suggest that TNF␣ stimulates CREBH expression by activating the NF-B pathway.
TNF␣ Suppresses Osteoblast Differentiation by Stimulating CREBH Expression-TNF␣ suppresses osteoblast differentiation by inhibiting the BMP signaling pathway (21). Therefore, we determined whether CREBH is involved in this process using gain-or loss-of-function experiments. TNF␣ inhibited BMP2-induced ALP activity and OC protein production (Figs.  3, A and B). In this condition, overexpression of CREBH using adenovirus encoding the nuclear form of CREBH (Ad-CREBH-N) dramatically inhibited BMP2 induction of ALP activity and OC production, as well as expression of osteogenic markers in a dose-dependent manner (Fig. 3, C-E). TNF␣ still stimulated the activity of the Crebh promoter to some extent in the presence of BMP2 (Fig. 3F). However, overexpression of CREBH did not affect the expression of the mRNAs encoding osteoprotegerin (Opg) and receptor activator of NF-B ligand (Rankl) in primary osteoblasts, indicating that the expression of CREBH in osteoblasts might not be directly related to osteoclastogenesis (Fig. 3G).
Next, we examined the effect of knockdown of CREBH using adenoviruses encoding an unspecific control shRNA (Ad-USi) or a CREBH-specific shRNA (Ad-CREBHi) on BMP2-induced osteoblast differentiation. Western blot analysis confirmed that treatment of Ad-CREBHi decreased the level of CREBH protein in MC3T3-E1 cells (Fig. 4A). Knockdown of CREBH attenuated the TNF␣-mediated suppression of BMP2-induced expression of the Alp and Oc mRNAs (Fig. 4B), as well as OC protein production (Fig. 4C) and ALP activity (Fig. 4D). These results indicate that CREBH negatively regulates BMP2-induced osteoblast differentiation.
CREBH Promotes Smad1 Degradation by Inducing Smurf1 Expression-Smurf1 is a negative regulator of BMP signaling that suppresses osteoblast function by promoting Smad1 degradation via a proteosomal-dependent mechanism (34). To determine whether TNF␣ or CREBH affects Smurf1 expression, MC3T3-E1 cells were treated with TNF␣ or infected with Ad-CREBH-N, and then Smurf1 mRNA expression was measured by qRT-PCR. TNF␣ and Ad-CREBH-N increased the expression of Smurf1 mRNA significantly, whereas infection with a control adenovirus (Ad-GFP) did not (Fig. 5A). However, TNF␣ and Ad-CREBH-N did not affect the expression of Smurf2 mRNA (Fig. 5A). In addition, introduction of CREBH-N increased the Smurf1 promoter activity in a dose-dependent manner (Fig. 5B).  Next, we determined whether CREBH affects the expression level of Smad1 protein by regulating Smurf1. Western blot analyses of MC3T3-E1 cells that were transfected with an expres-sion vector harboring Myc-Smad1 revealed that exposure to the potent proteasome inhibitor MG132 resulted in the production of small amounts of ubiquitinated Smad1. However, the level of ubiquitinated Smad1 was increased markedly by overexpression of Ad-CREBH-N (Fig. 5C), indicating that CREBH induces ubiquitination of Smad1, leading to its breakdown through proteasomal degradation. Moreover, TNF␣ decreased the level of Smad1 protein with the increased Smurf1, whereas inhibition of CREBH with Ad-CREBHi blocked TNF␣-mediated Smad1 degradation and reduced Smurf1 expression (Fig. 5D). In addition, a luciferase reporter assay revealed that inhibition of CREBH reduced TNF␣-mediated Smurf1 promoter activation in a dose-dependent manner (Fig. 5E).
In the study, we also examined the effects of CREBH on the expression levels of the Smad1 downstream factors Runx2 and ATF6, which are required for osteoblast differentiation. Inhibition of CREBH attenuated TNF␣-mediated suppression of Runx2 and Atf6 mRNA levels, as well as the TNF␣-mediated increase in Smurf1 expression (Fig. 5F). In contrast, overexpression of CREBH reduced BMP2-mediated induction of Smad1, Runx2, and ATF6 protein levels, and increased Smurf1 protein expression (Fig. 5G), suggesting that CREBH is involved in TNF␣-mediated suppression of BMP2 downstream signals. Overall, these results indicate that CREBH negatively regulates BMP2-induced osteoblast differentiation by inducing Smurf1related degradation of Smad1.
CREBH Suppresses BMP2-induced Bone Formation in Vivo-The results described above suggested that CREBH suppresses BMP2-induced osteoblast differentiation. Therefore, we investigated the role of CREBH in BMP2-induced bone formation in vivo. Radiographic analyses showed that administration of BMP2 strongly induced ectopic and orthotopic bone formation, and co-administration with CREBH significantly reduced BMP2-induced bone formation at both ectopic subcutaneous and orthotopic calvarial defect models (Fig. 6, A and C). A quantitative CT analysis also confirmed that co-administration of CREBH decreased the BMP2 action (Fig. 6, B and D). However, administration of Ad-CREBH-N or the control Ad-GFP did not produce any significant changes (Fig. 6, B and D). Histology analysis consistently showed that BMP2 regenerated newly mineralized tissues in the administered regions, whereas co-administration of BMP2 and CREBH produced less or immature mineralized tissues (Fig. 6, E and F). Taken together, these findings suggest that CREBH has an inhibitory role in BMP2-induced bone formation in vivo.

Discussion
Inflammation is triggered by inflammatory cytokines, such as TNF␣ and LPS, which suppress BMP2-induced osteoblast differentiation in vitro and contribute to bone loss in inflammatory bone diseases, such as rheumatoid arthritis (17,35). Therefore, BMP2 and inflammatory cytokines have opposing roles in osteoblast differentiation. However, other cytokines, such as IL-6, IL-1␤, and nitric oxide, are also secreted into inflammatory environments and may play different roles in osteogenesis (36 -39). Moreover, local inhibitors of inflammation such as triptolide and BMP-binding peptide, can enhance the osteoinductive efficacy of BMP-2 in vivo (40,41). However, direct evidence supporting an inhibitory effect of inflammation on BMP2-induced osteoblast differentiation is currently lacking. This study examined the effect of the inflammatory mediator CREBH on BMP2-induced osteoblast differentiation.
ER membrane-bound bZIP transcription factors ATF6, OASIS, and CREBH can be cleaved by cellular stresses, such as ER stress, to provide important signals for regulating cellular physiology (42). OASIS Ϫ/Ϫ mice exhibit severe osteopenia with  . CREBH suppresses BMP2-induced ectopic and orthotopic bone formation. Ad-GFP (5 ϫ 10 10 particle number, PN), Ad-BMP2 (5 ϫ 10 10 PN), and/or Ad-CREBH-N (5 ϫ 10 10 or 10 ϫ 10 10 PN) with absorbable collagen sponges were subcutaneously implanted into the backs or critical sized cranial defects of mice. The total amount of implanted virus was adjusted by adding Ad-GFP virus. Three weeks after implantation, the implants were harvested and ectopic or orthotopic bone formation was evaluated. A and B, two-and three-dimensional CT reconstructions (A) and volume (B) of subcutaneously formed ectopic bones. *, p Ͻ 0.05 versus the Ad-MP2treated group. C and D, CT reconstructions (C) and volume (D) of newly formed orthotopic bones in critical-sized calvarial defects. *, p Ͻ 0.05 versus the Ad-BMP2-treated group. E and F, histology of ectopic (E) or orthotopic (F) regenerated bones. Lower panels are magnified images of the squared areas in upper panel, respectively. Black arrowheads indicate newly formed mineralized tissues. Representative data are shown (n ϭ 4). G, an overview of the role of CREBH in BMP2-induced osteoblast differentiation. Under normal conditions, BMP2 stimulates osteoblast differentiation via Smad1/ 5/8-dependent intracellular signaling. In the presence of severe inflammation, TNF␣ induces the expression of CREBH and Smurf1 by NF-B pathway, leading to the suppression of BMP2-induced osteoblast differentiation through Smurf1-dependent degradation of Smad1. reduced levels of collagen type ␣I in the bone matrix and reduced activity of osteoblasts (9). In our previous study, ATF6 stimulated osteoblast differentiation by regulating osteocalcin gene expression directly (32). However, the mechanisms involved in the sensing of unfolded proteins and translocation from the ER to the Golgi differ between OASIS family members. In addition, OASIS family members have unique cell-or tissue-specific expression patterns, suggesting that these transcription factors may be activated by, and associated with, distinct physiological responses that are dependent on particular environments (31). The results presented here demonstrate that the expression properties of CREBH differ from those of OASIS and ATF6; specifically, Crebh mRNA expression in osteoblasts was not affected by BMP2 but was increased in response to TNF␣, whereas the expression levels of Oasis and Atf6 mRNAs were up-regulated by BMP2 but were not affected by TNF␣ (Fig. 1, A and B). Although CREBH belongs to the same family of transcription factors as OASIS and ATF6, it seems to play a different role in osteoblast differentiation due to differences in its activating stimuli, tissue distribution, and response element binding.
Here, we established that overexpression and knockdown of CREBH enhance and inhibit TNF␣-mediated inhibition of BMP2-induced osteoblast differentiation, respectively. Furthermore, we sought to clarify the molecular mechanism by which TNF␣ regulates CREBH expression. Luciferase reporter assays and Western blot analysis showed that TNF␣ increased the CREBH promoter activity and the protein levels. Treatment of BAY-11-7082 or dnIB␣ (S32A/S36A) consistently inhibited the TNF␣-induced CREBH expression with the decreased phosphorylation of IB or level of p65 subunit. In addition, one particular NF-B binding site was crucial for TNF␣-mediated stimulation of Crebh promoter activity (Fig. 2G). NF-B subunits p50 and p65 bound to a consensus NF-B site in the Crebh gene promoter and may utilize this cis-acting element to regulate promoter activity (Fig. 2H). In this study, we demonstrate for the first time in osteoblasts that TNF␣ may regulate CREBH expression via the IKK/IB␣/NF-B signaling pathway.
TNF␣ suppresses BMP2-induced osteoblast differentiation and increases the expression of Smurf1, leading to the subsequent degradation of Smad1 and Runx2, which are critical mediators of BMP2 signaling (25,43). Smurf1 interacts directly with Smad1 and Runx2, and stimulates the degradation of these proteins in ubiquitin-and proteasome-dependent manners (44). However, the factors downstream of TNF␣ that regulate Smurf expression were previously unknown. The results presented here suggest that CREBH mediates TNF␣-induced Smurf1 expression in osteoblasts. Treatment of cells with TNF␣ or overexpression of CREBH stimulated ubiquitin-mediated degradation of Smad1. In addition, BMP2 increased the levels of the Smad1, Runx2, and ATF6 proteins in osteoblasts (Fig. 5G), as described previously (32,45), and overexpression of CREBH attenuated these BMP2 effects with the increased Smurf1 level. These indicate that TNF␣ may suppress BMP2induced osteoblast differentiation through the CREBH/Smurf/ Smad1 regulatory system.
We also examined the in vivo effects of CREBH overexpression on BMP2-induced bone formation, using a subcutaneous ectopic model and a critical-sized calvarial defect model in mice. Consistent with the results of in vitro cell experiments, BMP2 strongly regenerated new bones within defected calvariae and subcutaneous spaces, and overexpression of CREBH significantly reduced the BMP2 effects. These in vivo findings firmly support that CREBH has an inhibitory role in BMP2induced bone formation.
Overall, our findings reveal a novel mechanism by which TNF␣ inhibits BMP2-induced osteogenesis, namely the up-regulation of CREBH and subsequent stimulation of the Smurf1 E3 ligase to promote Smad1 degradation. Fig. 6G summarizes our proposed model of the inhibitory role of CREBH in osteoblast differentiation. Our results suggest that this interplay network might regulate several biological and pathological processes and provide valuable insights into why the repair of bone defects associated with severe inflammation is delayed.