Signaling involved in osteoblastic cell differentiation remains largely unknown. This study further investigates mechanisms involved in BMP-2-induced osteoblastic cell differentiation. We report that BMP-2 can activate JNK and p38 in osteoblastic cells and provide evidences that these MAP kinases have distinct roles in regulating alkaline phosphatase and osteocalcin expression. Introduction: Bone morphogenetic protein (BMP)-2 exerts many of its biological effects through activation of the Smad pathway. Cooperative interactions between the Smads and the stress-activated protein kinase (SAPK) p38 and c-Jun-NH2-terminal kinase (JNK) pathways have recently been observed in TGF-β signaling. Materials and Methods: Activation of mitogen-activated protein (MAP) kinases by BMP-2 and the role of these signaling pathways for cell differentiation induced by BMP-2 was investigated in mouse MC3T3-E1 and primary cultured calvaria-derived osteoblastic cells using immunoprecipitation, in vitro kinase assay and Western blot analysis, as well as specific MAP kinase inhibitors. Results: Associated with the rapid activation of Smads, BMP-2 barely affected extracellular-signal regulated kinase (ERK) activity, whereas it induced a transient activation of p38 and JNK. The role of p38 and JNK in mediating BMP-2-induced stimulation of osteoblastic cell differentiation was evaluated using the respective specific inhibitors SB203580 and SP600125. Inhibition of p38 by SB203580 was mainly associated with decreased alkaline phosphatase (ALP) activity, whereas inhibition of JNK by SP600125 was associated with a marked reduction in osteocalcin (OC) production induced by BMP-2. Corresponding alterations in ALP and OC mRNA levels were found in cells treated with BMP-2 and inhibitors, suggesting an implication of p38 and JNK pathways in BMP-2-induced osteoblastic cell differentiation at a transcriptional level. Conclusion: Data presented in this study describe p38 and JNK as new signaling pathways involved in BMP-2-induced osteoblastic cell differentiation with evidences for a distinct role of each MAP kinase in the control of alkaline phosphatase and osteocalcin expression.

Pi handling by osteogenic cells is important for bone mineralization. The role of Pi transport in BMP‐2–induced matrix calcification was studied. BMP‐2 enhances Pit‐1 Pi transporters in osteogenic cells. Experimental analysis suggest that this response is required for bone matrix calcification. Introduction: Bone morphogenetic proteins (BMPs) are produced by osteogenic cells and play an important role in bone formation. Inorganic phosphate (Pi) is a fundamental constituent of hydroxyapatite, and its transport by osteogenic cells is an important function for primary calcification of the bone matrix. In this study, we investigated the role of Pi transport in BMP‐2–induced matrix mineralization. Materials and Methods: Confluent MC3T3‐E1 osteoblast‐like cells were exposed to BMP‐2 for various time periods. Pi and alanine transport was determined using radiolabeled substrate, Pit‐1 and Pit‐2 expression by Northern blot analysis, cell differentiation by alkaline phosphatase activity, matrix mineralization by alizarin red staining, and the characteristics of mineral deposited in the matrix by transmission electron microscopy, electron diffraction analysis, and Fourier transformed infrared resolution (FTIR). Results: BMP‐2 time‐ and dose‐dependently stimulated Na‐dependent Pi transport in MC3T3‐E1 cells by increasing the V_max of the transport system. This effect was preceded by an increase in mRNA encoding Pit‐1 but not Pit‐2. BMP‐2 also dose‐dependently enhanced extracellular matrix mineralization, an effect blunted by either phosphonoformic acid or expression of antisense Pit‐1. Enhanced Pi transport and matrix mineralization induced by BMP‐2 were blunted by a specific inhibitor of the c‐Jun‐N‐terminal kinase (JNK) pathway. Conclusions: Results presented in this study indicate that, in addition to its well‐known effect on several markers of the differentiation of osteoblastic cells, BMP‐2 also stimulates Pi transport activity through a selective increase in expression of type III Pi transporters Pit‐1. In MC3T3‐E1 cells, this effect is mediated by the JNK pathway and plays an essential role in bone matrix calcification induced by BMP‐2.

In the last three decades, all efforts in bone tissue engineering were driven by the dogma that the ideal pore size in bone substitutes lies between 0.3 and 0.5 mm in diameter. Newly developed additive manufacturing methodologies for ceramics facilitate the total control over pore size, pore distribution, bottleneck size, and bottleneck distribution. Therefore, this appears to be the method of choice with which to test the aforementioned characteristics of an ideal bone substitute. To this end, we produced a library of 15 scaffolds with diverse defined pore/bottleneck dimensions and distributions, tested them in vivo in a calvarial bone defect model in rabbits, and assessed the clinically most relevant parameters: defect bridging and bony regenerated area. Our in vivo data revealed that the ideal pore/bottleneck dimension for bone substitutes is in the range of 0.7–1.2 mm, and appears therefore to be twofold to fourfold more extended than previously thought. Pore/bottleneck dimensions of 1.5 and 1.7 mm perform significantly worse and appear unsuitable in bone substitutes. Thus, our results set the ideal range of pore/bottleneck dimensions and are likely to have a significant impact on the microarchitectural design of future bone substitutes for use in orthopedic, trauma, cranio-maxillofacial and oral surgery.

Interleukin-1β (IL-1β) is a pleiotropic cytokine that was shown to inhibit the biosynthesis of articular cartilage components. Here we demonstrate that IL-1β inhibits the production of newly synthesized collagens in proliferating rabbit articular chondrocytes and that this effect is accompanied by a decrease in the steady-state levels of type II collagen mRNA. IL-1β down-regulates COL2A1 gene transcription through a –41/–33 bp sequence that binds a multimeric complex including Sp1 and Sp3 transcription factors. Specificity of IL-1β effects on COL2A1 promoter activity was demonstrated in experiments in which transfection of a wild type –50/+1 sequence of COL2A1 promoter as a decoy oligonucleotide abolished the IL-1β inhibition of a –63/+47 COL2A1-mediated transcription. By contrast, transfection of the related oligonucleotide harboring a targeted mutation in the –41/–33 sequence did not modify the negative effect the cytokine. Because we demonstrated previously that Sp1 was a strong activator of COL2A1 gene expression via the –63/+1 promoter region, whereas Sp3 overexpression blocked Sp1-induced promoter activity and inhibited COL2A1 gene transcription, we conclude that IL-1β down-regulation of that gene, as we found previously for transforming growth factor-β1, is mediated by an increase in the Sp3/Sp1 ratio. Moreover, IL-1β increased steady-state levels of Sp1 and Sp3 mRNAs, whereas it enhanced Sp3 protein expression and inhibited Sp1 protein biosynthesis. Nevertheless, IL-1β decreased the binding activity of both Sp1 and Sp3 to the 63-bp short COL2A1 promoter, suggesting that the cytokine exerts a post-transcriptional regulatory mechanism on Sp1 and Sp3 gene expressions. Altogether, these data indicate that modulation of Sp3/Sp1 ratio in cartilage could be a potential target to prevent or limit the tissue degradation.

An important role for JNK* and p38 has recently been discovered in the differentiating effect of bone morphogenetic protein 2 (BMP-2) on osteoblastic cells. In this study, we investigated the molecular mechanism by which BMP-2 activates JNK and p38 in MC3T3-E1 osteoblastic cells. Activation of JNK and p38 induced by BMP-2 was blocked by the protein kinase C/protein kinase D (PKC/PKD) inhibitor Go6976 but not by the related compound, Go6983, a selective inhibitor of conventional PKCs. Associated with this inhibitory effect of Go6976, BMP-2 induced a selective and a dose-dependent Ser⁹¹⁶ phosphorylation/activation of PKD, which was also blocked by Go6976. In contrast to the recently described PKC-dependent molecular mechanism involved in activation of PKD by G protein-coupled receptor agonists, BMP-2 did not induce a phosphorylation of PKD on Ser^744/748. To further document an implication of PKD in activation of JNK and p38 induced by BMP-2, we constructed MC3T3-E1 cells stably expressing PKD antisense oligonucleotide (AS-PKD). In AS-PKD clones having low PKD levels, activation of JNK and p38 by BMP-2, but not of Smad1/5, was markedly impaired compared with empty vector transfected (V-PKD) cells. Analysis of osteoblastic cell differentiation in AS-PKD compared with V-PKD cells showed that mRNA and protein expressions of alkaline phosphatase and osteocalcin induced by BMP-2 were markedly reduced in AS-PKD. In conclusion, results presented in this study indicate that BMP-2 can induce activation of PKD in osteoblastic cells by a PKC-independent mechanism and that this kinase is involved in activation of JNK and p38 induced by BMP-2. Thus, this pathway, in addition to Smads, appears to be essential for the effect of BMP-2 on osteoblastic cell differentiation.

Epigenetics describes mechanisms which control gene expression and cellular processes without changing the DNA sequence. The main mechanisms in epigenetics are DNA methylation in CpG-rich promoters, histone modifications and non-coding RNAs (ncRNAs). DNA methylation modifies the function of the DNA and correlates with gene silencing. Histone modifications including acetylation/deacetylation and phosphorylation act in diverse biological processes such as transcriptional activation/inactivation and DNA repair. Non-coding RNAs play a large part in epigenetic regulation of gene expression in addition to their roles at the transcriptional and post-transcriptional level. Osteoporosis is the most common skeletal disorder, characterized by compromised bone strength and bone micro-architectural deterioration that predisposes the bones to an increased risk of fracture. It is most often caused by an increase in bone resorption that is not sufficiently compensated by a corresponding increase in bone formation. Nowadays it is well accepted that osteoporosis is a multifactorial disorder and there are genetic risk factors for osteoporosis and bone fractures. Here we review emerging evidence that epigenetics contributes to the machinery that can alter DNA structure, gene expression, and cellular differentiation during physiological and pathological bone remodeling.