The Effect Of Inflammation On Osteoblast Differentiation And Its Molecular Mechanisms

1.BackgroundThe human skeleton is constantly renewing itself and bone mass is maintained by a balance between the osteogenic activity of osteoblasts and the bone absorptive activity of osteoclasts. This bone renewal process may be complicated by inflammatory mediators that can alter cell functions. Conditions such as traumatic open fracture and rheumatoid arthritis all involve bone formation in the context of persistent inflammation, in which tumor necrosis factor-α(TNF-α)plays an important role. Although inflammation has both positive and negative effects on bone regeneration, persistent inflammation and infection have obvious negative consequences, and possible strategies for circumventing inflammatory inhibition of tissue regeneration can be pursued once the signaling pathways involved have been elucidated. One of the major signaling pathways controlling osteogenesis is the BMP-Smad pathway. Bone morphogenetic proteins (BMPs), a subgroup of transforming growth factor beta (TGF-β) super family, are multifunctional proteins that play important roles in a broad range of cellular functions from embryogenesis, cell growth and differentiation to bone healing and fracture repair. BMPs are potent inducers of osteogenesis in vitro and are able to induce ectopic bone formation in vivo. BMPs delay cell proliferation and promote differentiation and apoptosis of bone cells, which are mediated by BMP receptors type I and type II through the Smad glycoproteins. Receptor regulated Smads (R-Smads) 1,5,and 8 are BMP-specific signaling proteins. The R-Smads are activated by BMP receptors and subsequently heterodimerize with Smad4, enter the nucleus to activate transcription.Signal transduction in the TNF-a pathway occurs partly through the activity of the NF-κB family of transcription factors. Nuclear factorκB (NF-κB), a ubiquitous transcription factor that controls the expression of genes involved in immune responses, apoptosis and cell cycle, is the central inflammatory signaling molecule. Five mammalian NF-κB family members have been identified:NF-κB1 (also called p50), NF-κB2 (also named p52), RelA (also known as p65), RelB, and c-Rel. They all share a highly conserved Rel homology domain responsible for their dimerization and binding to DNA and IκB (inhibitor of NF-κB). The transcription factor NF-κB works only when two members form a dimmer.The most abundant activated form consists of a p50 or p52 subunit and a p65 subunit. NF-κB is held in the cytoplasm by binding to inhibitory molecules known as IκB. Lipopolysaccharides and cytokines such as TNF-a and IL-1βare potent inducers of NF-κB. These cytokines initiate a cascade of events that leads to phosphorylation of IκB by IKK (IκB kinase), which triggers its degradation by the ubiquitin-proteasome pathway. Degradation of IκB unmasks NF-κB's nuclear localization signal and free NF-κB then translocates into the nucleus and activates transcription. IKK is comprised of three subunits, IKKa, IKKβ, and IKKy. Although IKKγ, often referred to as NEMO (NF-κB essential modulator), does not possess a catalytic domain, it is absolutely critical for the activation of the IKK complex. The NH2-terminus of NEMO associates with a hexapeptide sequence (Leu-Asp-Trp-Ser-Trp-Leu) within the COOH terminus of IKKa and IKKβtermed NEMO-binding domain (NBD). A short cell-permeable peptide spanning the IKKβNBD can disrupt the association of NEMO with IKKβ, block TNF-a-induced NF-κB activation in cells, and effectively ameliorate responses in distinct animal models of inflammation. Hence, NBD is an attractive target for the development of anti-inflammatory drugs aimed at disrupting the IKK complex.While the effects of inflammation on osteoclast differentiation have been well studied, its effects on osteoblast differentiation have not been adequately investigated. In this study, we investigated the effects of TNF-a, NF-κB and NBD on osteoblast differentiation using mouse myoblast C2C12 cells. This study found that activation of NF-κB inhibits BMP-2-induced osteoblast differentiation by attenuating Smadl activity. Importantly, NBD peptide ameliorated this inhibition by antagonizing the TNF-a NF-κB pathway and augmenting BMP-Smad signaling, suggesting a potential for NBD peptide to be used to treat inflammatory bone damages in rheumatoid arthritis and open bone fractures.2.Objectives(1)Study on effects of inflammatory factors TNF-a on osteoblast differentiation.(2) Study on the molecular mechanisms of inhibition of inflammatory factors on osteoblast differentiation.(3) Study on the effects of NEMO binding domain peptide NBD on osteoblast inhibition by TNF-a. 3. Materials and Methods(1)Plasmids, cytokines and peptides:12xSBE-Luc was cloned as previously described, pCMV-Smadl,pCMV-NFκB (p65) were obtained from Affymetrix/Panomics (Fremont, CA, USA) and pCMV-IκBα, pNF-κB-Luc from Clontech Laboratories (Mountain View, CA,USA). Human recombinant BMP-2 was purchased from PeproTech (Rocky Hill, NJ, USA) and human recombinant TNF-a from Sigma (St. Louis, MO, USA).Cell permeable wild-type NEMO binding domain peptide (YGRKKRRQRRR-G-TTLDWSWLQME, referred as NBD) and its control mutant type (YGRKKRRQRRR-G-TTLDASALQME, referred as mNBD) were synthesized and purified by high performance liquid chromatography according to previous report.(2) Cell culture, osteoblast differentiation and alkaline phosphatase (ALP) staining:Mouse myoblast cell line C2C12 was obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). Dulbecco's modified eagle's medium (DMEM) and penicillin-streptomycin solution were purchased from Invitrogen/Gibco (Grand Island, NY, USA). After preculture with serum-free DMEM,cells were treated with BMP-2(100 ng/ml),TNF-α(5 ng/ml),NBD(100μM) or mNBD(100μM), or at concentrations indicated in the figures. For histochemical staining, ALP detection Kit (Sigma) was used to demonstrate the ALP activity. After culturing for 7 days with medium changed every 3 days, cells were washed with PBS and incubated with a mixture of naphthol AS-BI alkaline solution with fast red violet LB.The resulting red insoluble deposit indicates site and level of ALP activity. For quantitative analysis, cells were lysed after 3 days of culture, and cellular ALP activity was measured with a fluorometric detection kit using 4-methylumbelliferyl phosphate disodium substrate (Sigma, St. Louis, MO, USA) following manufacturer's instructions. The ALP activity of each sample was normalized by protein concentration.(3) Transient transfection and reporter assays:C2C12 cells(1×105 viable cells) were cultured in 12-well plates in DMEM with 10% FBS for 24 hrs. The cells were then transiently transfected with 1μg of 12xSBE-Luc, pNF-κB-Luc, pCMV-Smadl, pCMV-NFκB (p65), or pCMV-IκBa plasmid as indicated in the experiments, with mock vector added to equalize the total amount of DNA, using Superfect reagent (Qiagen) following manufacturer's instructions. The cells were treated with the indicated concentrations of BMP-2 and TNF-a in serum-free medium. After 48 hrs of culture, cells were washed with PBS and lysed with RIPA buffer. Luciferase activity of the cell lysate was measured with the Luciferase Assay System (Promega, Madison, WI, USA) by a Victor2 Multilabel Counter (PE, Waltham, MA).β-galactosidase (β-gal) was used to normalize the transfection efficiency and was measured with theβ-Galactosidase Enzyme Assay System (Promega). The data were shown as the ratio of luciferase to P-gal activity.(4) RNA isolation and reverse transcription, real-time PCR:C2C12 cells were cultured in a 12-well plate (2x105 viable cells) and treated with BMP-2(100 ng/ml) and/or TNF-a (5 ng/ml) in serum-free DMEM. After 48 hr of culture, the medium was removed, and total cellular RNA was extracted using TRIzol (Invitrogen Corp.). Total RNA(1μg) was subjected to reverse transcription using SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen Corp.) with oligo dT per manufacturer's instructions. For Power SYBR(?) Green PCR master mix (Applied Biosystems) based real-time PCR, the mouse Smadl sequence specific primers used were GCTTCGTGAAGGGTTGGG (forward) and CGGATGAAATAGGA-TTGTGGGG (reverse). A PCR program was set up on an ABI Prism Sequence Detection System 7000 (Applied Biosystems) as follows:50 C 2 min;95 C 10 min; 95 C 15 sec,60 C 30 sec,40 cycles. A melting curve was obtained for each PCR product to verify that only one amplicon was produced per PCR. The results were analyzed with ABI Prism 7000 SDS software (Applied Biosystems) by the△△Ct method where Ct values were normalized to GAPDH expression. At least three separate experiments were conducted with triplicate wells for each group.(5) SDS-PAGE and western blotting:A total of 2×105 viable C2C12 cells were cultured in 12-well plates in DMEM containing 10% FBS for 24 hrs. After preculture, the medium was replaced with fresh serum-free medium with BMP-2(100 ng/ml) and/or TNF-α(5 ng/ml) added. After incubation for 1,3 and 6 hrs, cells were lysed by RIPA buffer. Equal amounts of total cell lysate were then subjected to SDS-PAGE and immunoblotting analysis using anti phospho-NFκB p65 (Ser536), anti phospho-Smad1 (Ser 463/Ser 465) antibody and anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA),β-actin was used as a loading control.4. ResultsThe multipotent myoblast C2C12 cells can switch from their normal myotube forming pathway into an osteoblast differentiation pathway upon BMP-2 triggering [18].They have been widely used as one of the most popular tools for studying early osteoblast differentiation. Here we describe the effects of TNF-αon osteoblast differentiation based on this model.(1)TNF-αinhibits BMP-2 induced osteoblast differentiation in a dose dependent manner. We first monitored the effects of TNF-αon BMP-2-induced osteoblast differentiation and its dosage effects. ALP is a relatively late marker for osteoblast differentiation that is used to monitor the progression of osteoblast differentiation. For morphological histochemical staining, C2C12 cells were incubated with BMP-2 (100 ng/ml) and/or TNF-a (5 ng/ml) for 7 days and stained with a mixture of naphthol AS-BI alkaline solution with fast red violet LB.C2C12 cells incubated with BMP-2 alone demonstrated strong ALP activity, and addition of TNF-a markedly reduced their ALP activity.Fluorometric quantitative analysis of C2C12 cells incubated with BMP-2(100 ng/ml) and/or TNF-a (5 ng/ml) for 3 days showed that BMP-2 increased ALP activity by～10-fold compared to the control, and addition of TNF-a decreased this to 2.4-fold.To investigate the dosage dependency of this inhibition effect, C2C12 cells were incubated with different concentrations of BMP-2 and TNF-a as indicated in Fig.1C. When BMP-2 was held at 100 ng/ml, increasing the TNF-a concentration from 0 to 10 ng/ml decreased the ALP activity from 10-fold that of the control to less than 0.1-fold. When TNF-a was kept at 5 ng/ml, increasing the BMP-2 concentration from 100 ng/ml to 300 ng/ml increased ALP activity from 1.8-fold that of the control to 4.7-fold.These results indicated the inhibition of BMP-2-induced osteoinductive signaling by TNF-a is dosage dependent and sensitive to additional BMP-2.(2) The BMP2-Smadl pathway is interrupted by TNF-a and NF-κB.To investigate the mechanism by which TNF-a inhibits osteoblast differentiation, we studied the effects of TNF-a and NF-κB on the BMP-2-Smadl signaling pathway in a stepwise manner. A promoter-reporter assay system where luciferase is driven by a promoter containing 12 repeated Smad1-responsive elements(12xSBE-Luc) was used to monitor the strength of osteoinductive signaling in C2C12 cells.C2C12 cells were first treated with exogenous BMP-2(100 ng/ml) and/or TNF-α(5 ng/ml), which were then replaced with cellular overexpression of their downstream effectors or modulators Smadl,NF-κB or IκBαby transient transfection with pCMV-Smadl, pCMV-NF-κB (p65) or pCMV-IκBα, respectively. The Smadl-activated osteoinductive responses were monitored by 12xSBE-Luc reporter. Results from three separate experiments conducted in triplicate showed that BMP-2 alone induced an osteoinductive response that was 7.12-fold that of the no BMP-2 control. Upon addition of the cytokine TNF-α, the osteoinductive activity induced by BMP-2 was reduced to 1.31-fold. Replacing the cytokine TNF-αwith overexpression of its downstream effecter NF-κB decreased the BMP-2-induced osteoinductive activity from 7.9-fold to 3.4-fold, while overexpression of IκBα, an effective inhibitor of NF-κB, resulted in a 21.8-fold increase in BMP-2-induced osteoinductive activity TNF-αwas able to directly decrease the Smadl (a downstream effector of BMP-2) induced response from 2.04-fold to 0.63-fold. Overexpression of NF-κB decreased Smadl activity from 2.04-fold to 0.39-fold, while overexpression of IκBαincreased Smadl activity from 2.04 to 3.57-fold. Through stepwise dissection of the interactions between the BMP2-Smad1 pathway and the TNF-α-NF-κB pathway, these results clearly show that activation of NF-κB by TNF-αsignificantly reduced BMP-2-induced Smadl activity.(3) TNF-αreduces Smad1 signaling but does not reduce Smad1 abundance. To map the exact site underlying the crosstalk between the BMP2-Smadl pathway and the TNF-α/NFκB pathway, we examined the effects of TNF-αon Smadl expression and phosphorylation. To measure Smadl cDNA abundance, total RNA isolated from C2C12 cells treated with BMP-2(100 ng/ml)and/or TNF-α(5 ng/ml) for various times (30,60,120,240 mins) were subjected to real-time RT-PCR with primers specific to Smadl with GAPDH as reference. Results from three separate experiments showed that Smadl cDNA abundance increased from 30 mins to 120 mins and slightly decreased at 240 mins in all four groups. Although Smad1 expression seemed to be slightly higher in the BMP-2 and BMP-2/TNF-αgroup compared to control and TNF-αalone, there were no differences between the BMP-2 and BMP-2/TNF-αgroups or the control and TNF-αgroups.For Smadl phosphorylation assay, C2C12 cells were treated with BMP-2 (100 ng/ml) and/or TNF-α(5 ng/ml) for various time(1,3,6 hrs). Cell lysates with same amount of total protein were subjected to SDS-PAGE and immunoblotting. Levels of phospho-NF-κB (p65) and phospho-Smadl on duplicate membranes were stained with anti phospho-NF-KB (p65) and anti phospho-Smadl antibodies, andβ-actin was used as loading control. The results showed that treatment with BMP-2 or TNF-αalone increased the amount of phospho-Smadl or phospho-NF-KB (p65), respectively. While the amount of phospho-NF-κB in the TNF-αtreatment groups did not show significant changes with or without BMP-2 treatment at the 1,3,or 6 hr time points, the amount of phospho-Smadl induced by BMP-2 treatment showed a significant decrease at the 3 hr time point and was nearly undetectable at the 6 hr time point when combined with TNF-αtreatment. Thus,these results demonstrate that TNF-αwas able to attenuate Smadl signaling but did not reduce Smadl abundance.(4) NBD peptide ameliorates TNF-αinhibition of osteoblast differentiation. In a search for reagents capable of antagonizing the inhibitory effects of TNF-αand NF-κB on osteoblast differentiation, we tested the NEMO binding domain peptide NBD, a known short cell-permeable peptide with the ability to disrupt the association of NEMO with IKKβand therefore block TNF-a-induced NF-κB activation and TNF-a induced inhibition of osteoblast differentiation. First, we verified the inhibitory activities of the synthesized NBD and mNBD peptide on NF-κB. Overexpression of NF-κB (p65) led to a 5.85-fold increase of NF-κB-Luc reporter signal over the control, and addition of NBD peptide decreased this to 2.1-fold while overexpression of IκBαdecreased it to 1.8-fold and addition of mNBD peptide remained at 5.7-fold. Next, we investigated the effects of NBD and mNBD peptide on osteoblast differentiation. Colorimetric staining of ALP in C2C12 cells treated with BMP-2(100 ng/ml) and/or TNF-a (5 ng/ml) with addition of either the functional NBD peptide(100μM) or the mutant non-functional mNBD peptide(100μM) showed that NBD peptide was able to block most of the TNF-a inhibition of BMP-2 activity while mNBD peptide had almost no effect. Quantitative fluorometric analysis showed that TNF-a decreased BMP-2-induced ALP activity from 10.2-fold to 2.3-fold; however, with addition of NBD peptide, ALP activity recovered back to 8.3-fold while addition of mNBD peptide remained at 2.86-fold (with basal ALP as 1).NBD peptide was able to antagonize the TNF-a inhibition of BMP-2-induced Smadl activity as measured by the reporter construct 12xSBE-Luc as well. TNF-a decreased BMP-2 activity from 7.12-fold to 1.31-fold, and NBD peptide restored it back to 6.7-fold and mNBD peptide left it at 1.4-fold. These results demonstrate that NBD can effectively ameliorate TNF-a inhibition of osteoblast differentiation.5. ConclusionsOsteogenesis associated with persistent inflammation or infection exists in a broad range of conditions including traumatic open bone fracture and rheumatoid arthritis. The mechanisms underlying these conditions are still poorly understood. New strategies to circumvent the inflammatory inhibition of osteogenesis may potentially lead to effective new therapeutic methods that will greatly benefit these diseases and other conditions.Our study revealed that TNF-a inhibits BMP-2-induced osteoblast differentiation and that the inhibition of osteoinductive signaling is dosage dependent. For the first time, we provided direct evidence that TNF-a inhibits osteoblast differentiation (at least partly) through the activation of NF-κB, which directly leads to the abrogation of Smad1 signaling.In an effort to search for new therapeutic methods to antagonize the inhibitory effect of TNF-a on osteoblast differentiation, we tested the short cell permeable form of NBD peptide because it was reported that NBD peptide was able to effectively antagonize NF-κB activity in osteoclastogenesis. Our in vitro results show for the first time that NBD peptide is able to effectively ameliorate the inhibition of osteoblast differentiation by TNF-a. The fact that NBD peptide was capable of enhancing osteoblast differentiation (as shown in this study) and also capable of abrogating osteoclast differentiation strengthens its potential for treating rheumatoid arthritis and bone fracture with infection. Taken together, this study reveals for the first time that NF-κB activation inhibits osteoblast differentiation by attenuating Smadl activity, and application of NBD peptide can ameliorate this inhibitory effect. This could lead to new approaches to treating conditions such as rheumatoid arthritis, traumatic open fracture with infection and other bone loss disorders.