Mineral tessellation in bone and the stenciling principle for extracellular matrix mineralization

We review here the Stenciling Principle for extracellular matrix mineralization that describes a double-negative process (inhibition of inhibitors) that promotes mineralization in bone and other mineralized tissues, whereas the default condition of inhibition alone prevents mineralization elsewhere in soft connective tissues. The stenciling principle acts across multiple levels from the macroscale (skeleton/dentition vs soft connective tissues), to the microscale (for example, entheses, and the tooth attachment complex where the soft periodontal ligament is situated between mineralized tooth cementum and mineralized alveolar bone), and to the mesoscale (mineral tessellation). It relates to both small-molecule (e.g. pyrophosphate) and protein (e.g. osteopontin) inhibitors of mineralization, and promoters (enzymes, e.g. TNAP, PHEX) that degrade the inhibitors to permit and regulate mineralization. In this process, an organizational motif for bone mineral arises that we call crossfibrillar mineral tessellation where mineral formations – called tesselles – geometrically approximate prolate ellipsoids and traverse multiple collagen fibrils (laterally). Tesselle growth is directed by the structural anisotropy of collagen, being spatially restrained in the shorter transverse tesselle dimensions (averaging 1.6 × 0.8 × 0.8 μm, aspect ratio 2, length range 1.5–2.5 μm). Temporo-spatially, the tesselles abut in 3D (close ellipsoid packing) to fill the volume of lamellar bone extracellular matrix. Poorly mineralized interfacial gaps between adjacent tesselles remain discernable even in mature lamellar bone. Tessellation of a same, small basic unit to form larger structural assemblies results in numerous 3D interfaces, allows dissipation of critical stresses, and enables fail-safe cyclic deformations. Incomplete tessellation in osteomalacia/odontomalacia may explain why soft osteomalacic bones buckle and deform under loading.     

TNF-α and IL-1β inhibit RUNX2 and collagen expression but increase alkaline phosphatase activity and mineralization in human mesenchymal stem cells

AimsJoint inflammation leads to bone erosion in rheumatoid arthritis (RA), whereas it induces new bone formation in spondyloarthropathies (SpAs). Our aims were to clarify the effects of tumour necrosis factor α (TNF-α) and interleukin 1β (IL-1β) on osteoblast differentiation and mineralization in human mesenchymal stem cells (MSCs).Main methodsIn MSCs, expression of osteoblast markers was assessed by real-time PCR and ELISA. Activity of tissue-nonspecific alkaline phosphatase (TNAP) and mineralization were determined by the method of Lowry and alizarin red staining respectively. Involvement of RUNX2 in cytokine effects was investigated in osteoblast-like cells transfected with a dominant negative construct.Key findingsTNF-α (from 0.1 to 10 ng/ml) and IL-1β (from 0.1 to 1 ng/ml) stimulated TNAP activity and mineralization in MSCs. Addition of 50 ng/ml of IL-1 receptor antagonist in TNF-α-treated cultures did not reverse TNF-α effects, indicating that IL-1 was not involved in TNF-α-stimulated TNAP activity. Both TNF-α and IL-1β decreased RUNX2 expression and osteocalcin secretion, suggesting that RUNX2 was not involved in mineralization. This hypothesis was confirmed in osteoblast-like cells expressing a dominant negative RUNX2, in which TNAP expression and activity were not reduced. Finally, since mineralization may merely rely on increased TNAP activity in a collagen-rich tissue, we investigated cytokine effects on collagen expression, and observed that cytokines decreased collagen expression in osteoblasts from MSC cultures.SignificanceThe different effects of cytokines on TNAP activity and collagen expression may therefore help explain why inflammation decreases bone formation in RA whereas it induces ectopic ossification from collagen-rich entheses during SpAs.     

Discovery of 5-((5-chloro-2-methoxyphenyl)sulfonamido)nicotinamide (SBI-425), a potent and orally bioavailable tissue-nonspecific alkaline phosphatase (TNAP) inhibitor

Tissue-nonspecific alkaline phosphatase (TNAP) is an ectoenzyme crucial for bone matrix mineralization via its ability to hydrolyze extracellular inorganic pyrophosphate (ePP_i), a potent mineralization inhibitor, to phosphate (P_i). By the controlled hydrolysis of ePP_i, TNAP maintains the correct ratio of P_i to ePP_i and therefore enables normal skeletal and dental calcification. In other areas of the body low ePP_i levels lead to the development of pathological soft-tissue calcification, which can progress to a number of disorders. TNAP inhibitors have been shown to prevent these processes via an increase of ePP_i. Herein we describe the use of a whole blood assay to optimize a previously described series of TNAP inhibitors resulting in 5-((5-chloro-2-methoxyphenyl)sulfonamido)nicotinamide (SBI-425), a potent, selective and oral bioavailable compound that robustly inhibits TNAP in vivo.     

Osteoblast tissue-nonspecific alkaline phosphatase antagonizes and regulates PC-1

Tissue-nonspecific alkaline phosphatase (TNAP) is essential for bone matrix mineralization, but the central mechanism for TNAP action remains undefined. We observed that ATP-dependent (45)Ca precipitation was decreased in calvarial osteoblast matrix vesicle (MV) fractions from TNAP-/- mice, a model of infantile hypophosphatasia. Because TNAP hydrolyzes the mineralization inhibitor inorganic pyrophosphate (PP(i)), we assessed phosphodiesterase nucleotide pyrophosphatase (PDNP/NTPPPH) activity, which hydrolyzes ATP to generate PP(i). Plasma cell membrane glycoprotein-1 (PC-1), but not the isozyme B10 (also called PDNP3) colocalized with TNAP in osteoblast MV fractions and pericellular matrix. PC-1 but not B10 increased MV fraction PP(i) and inhibited (45)Ca precipitation by MVs. TNAP directly antagonized inhibition by PC-1 of MV-mediated (45)Ca precipitation. Furthermore, the PP(i) content of MV fractions was greater in cultured TNAP-/- than TNAP+/+ calvarial osteoblasts. Paradoxically, transfection with wild-type TNAP significantly increased osteoblast MV fraction NTPPPH. Specific activity of NTPPPH also was twofold greater in MV fractions of osteoblasts from TNAP+/+ mice relative to TNAP-/- mice. Thus TNAP attenuates PC-1/NTPPPH-induced PP(i) generation that would otherwise inhibit MV-mediated mineralization. TNAP also paradoxically regulates PC-1 expression and NTPPPH activity in osteoblasts.     

Inhibitors of tissue-nonspecific alkaline phosphatase: Design,synthesis, kinetics,biomineralization and cellular tests

Chronic kidney disease (CKD) is associated with numerous metabolic and endocrine disturbances, including abnormalities of calcium and phosphate metabolism and an inflammatory syndrome. The latter occurs early in the course of CKD and contributes to the development and progression of vascular calcification. A few therapeutic strategies are today contemplated to target vascular calcification in patients with CKD: vitamin K2, calcimimetics and phosphate binders. However, none has provided complete prevention of vascular calcification and there is an urgent need for alternate efficient treatments. Recent findings indicate that tissue-nonspecific alkaline phosphatase (TNAP) may represent a very promising drug target due to its participation in mineralization by vascular smooth muscle cells. We report the synthesis of four levamisole derivatives having better inhibition property on TNAP than levamisole. Their IC₅₀, K_i and water solubility have been determined. We found that the four inhibitors bind to TNAP in an uncompetitive manner and are selective to TNAP. Indeed, they do not inhibit intestinal and placental alkaline phosphatases. Survival MTT tests on human MG-63 and Saos-2 osteoblast-like cells have been performed in the presence of inhibitors. All the inhibitors are not toxic at concentrations that block TNAP activity. Moreover, they are able to significantly reduce mineralization in MG63 and Saos-2 osteoblast-like cells, indicating that they are promising molecules to prevent vascular calcification.     

Mir24-2-5p suppresses the osteogenic differentiation with Gnai3 inhibition presenting a direct target via inactivating JNK-p38 MAPK signaling axis

Background: Congenital anomalies are increasingly becoming a global pediatric health concern, which requires immediate attention to its early diagnosis, preventive strategies, and efficient treatments. Guanine nucleotide binding protein, alpha inhibiting activity polypeptide 3 (Gnai3) gene mutation has been demonstrated to cause congenital small jaw deformity, but the functions of Gnai3 in the disease-specific microRNA (miRNA) upregulations and their downstream signaling pathways during osteogenesis have not yet been reported. Our previous studies found that the expression of Mir24-2-5p was significantly downregulated in the serum of young people with overgrowing mandibular, and bioinformatics analysis suggested possible binding sites of Mir24-2-5p in the Gnai3 3''UTR region. Therefore, this study was designed to investigate the mechanism of Mir24-2-5p-mediated regulation of Gnai3 gene expression and explore the possibility of potential treatment strategies for bone defects.Methods: Synthetic miRNA mimics and inhibitors were transduced into osteoblast precursor cells to regulate Mir24-2-5p expression. Dual-luciferase reporter assay was utilized to identify the direct binding of Gnai3 and its regulator Mir24-2-5p. Gnai3 levels in osteoblast precursor cells were downregulated by shRNA (shGnai3). Agomir, Morpholino Oligo (MO), and mRNA were microinjected into zebrafish embryos to control mir24-2-5p and gnai3 expression. Relevant expression levels were determined by the qRT-PCR and Western blotting. CCK-8 assay, flow cytometry, and transwell migration assays were performed to assess cell proliferation, apoptosis, and migration. ALP, ARS and Von Kossa staining were performed to observe osteogenic differentiation. Alcian blue staining and calcein immersions were performed to evaluate the embryonic development and calcification of zebrafish.Results: The expression of Mir24-2-5p was reduced throughout the mineralization process of osteoblast precursor cells. miRNA inhibitors and mimics were transfected into osteoblast precursor cells. Cell proliferation, migration, osteogenic differentiation, and mineralization processes were measured, which showed a reverse correlation with the expression of Mir24-2-5p. Dual-luciferase reporter gene detection assay confirmed the direct interaction between Mir24-2-5p and Gnai3 mRNA. Moreover, in osteoblast precursor cells treated with Mir24-2-5p inhibitor, the expression of Gnai3 gene was increased, suggesting that Mir24-2-5p negatively targeted Gnai3. Silencing of Gnai3 inhibited osteoblast precursor cells proliferation, migration, osteogenic differentiation, and mineralization. Promoting effects of osteoblast precursor cells proliferation, migration, osteogenic differentiation, and mineralization by low expression of Mir24-2-5p was partially rescued upon silencing of Gnai3. In vivo, mir24-2-5p Agomir microinjection into zebrafish embryo resulted in shorter body length, smaller and retruded mandible, decreased cartilage development, and vertebral calcification, which was partially rescued by microinjecting gnai3 mRNA. Notably, quite similar phenotypic outcomes were observed in gnai3 MO embryos, which were also partially rescued by mir24-2-5p MO. Besides, the expression of phospho-JNK (p-JNK) and p-p38 were increased upon Mir24-2-5p inhibitor treatment and decreased upon shGnai3-mediated Gnai3 downregulation in osteoblast precursor cells. Osteogenic differentiation and mineralization abilities of shGnai3-treated osteoblast precursor cells were promoted by p-JNK and p-p38 pathway activators, suggesting that Gnai3 might regulate the differentiation and mineralization processes in osteoblast precursor cells through the MAPK signaling pathway.Conclusions: In this study, we investigated the regulatory mechanism of Mir24-2-5p on Gnai3 expression regulation in osteoblast precursor cells and provided a new idea of improving the prevention and treatment strategies for congenital mandibular defects and mandibular protrusion.     

Direct Determination of Phosphatase Activity from Physiological Substrates in Cells

A direct and continuous approach to determine simultaneously protein and phosphate concentrations in cells and kinetics of phosphate release from physiological substrates by cells without any labeling has been developed. Among the enzymes having a phosphatase activity, tissue non-specific alkaline phosphatase (TNAP) performs indispensable, multiple functions in humans. It is expressed in numerous tissues with high levels detected in bones, liver and neurons. It is absolutely required for bone mineralization and also necessary for neurotransmitter synthesis. We provided the proof of concept that infrared spectroscopy is a reliable assay to determine a phosphatase activity in the osteoblasts. For the first time, an overall specific phosphatase activity in cells was determined in a single step by measuring simultaneously protein and substrate concentrations. We found specific activities in osteoblast like cells amounting to 116 ± 13 nmol min^-1 mg^-1 for PPi, to 56 ± 11 nmol min^-1 mg^-1 for AMP, to 79 ± 23 nmol min^-1 mg^-1 for beta-glycerophosphate and to 73 ± 15 nmol min^-1 mg^-1 for 1-alpha-D glucose phosphate. The assay was also effective to monitor phosphatase activity in primary osteoblasts and in matrix vesicles. The use of levamisole – a TNAP inhibitor- served to demonstrate that a part of the phosphatase activity originated from this enzyme. An IC₅₀ value of 1.16 ± 0.03 mM was obtained for the inhibition of phosphatase activity of levamisole in osteoblast like cells. The infrared assay could be extended to determine any type of phosphatase activity in other cells. It may serve as a metabolomic tool to monitor an overall phosphatase activity including acid phosphatases or other related enzymes.     

Design,synthesis and evaluation of benzoisothiazolones as selective inhibitors of PHOSPHO1

We report the discovery and characterization of a series of benzoisothiazolone inhibitors of PHOSPHO1, a newly identified soluble phosphatase implicated in skeletal mineralization and soft tissue ossification abnormalities. High-throughput screening (HTS) of a small molecule library led to the identification of benzoisothiazolones as potent and selective inhibitors of PHOSPHO1. Critical structural requirements for activity were determined, and the compounds were subsequently derivatized and measured for in vitro activity and ADME parameters including metabolic stability and permeability. On the basis of its overall profile the benzoisothiazolone analogue 2q was selected as MLPCN probe ML086.     

Osteogenic Differentiation of Human Mesenchymal Stem Cells in Mineralized Alginate Matrices

Mineralized biomaterials are promising for use in bone tissue engineering. Culturing osteogenic cells in such materials will potentially generate biological bone grafts that may even further augment bone healing. Here, we studied osteogenic differentiation of human mesenchymal stem cells (MSC) in an alginate hydrogel system where the cells were co-immobilized with alkaline phosphatase (ALP) for gradual mineralization of the microenvironment. MSC were embedded in unmodified alginate beads and alginate beads mineralized with ALP to generate a polymer/hydroxyapatite scaffold mimicking the composition of bone. The initial scaffold mineralization induced further mineralization of the beads with nanosized particles, and scanning electron micrographs demonstrated presence of collagen in the mineralized and unmineralized alginate beads cultured in osteogenic medium. Cells in both types of beads sustained high viability and metabolic activity for the duration of the study (21 days) as evaluated by live/dead staining and alamar blue assay. MSC in beads induced to differentiate in osteogenic direction expressed higher mRNA levels of osteoblast-specific genes (RUNX2, COL1AI, SP7, BGLAP) than MSC in traditional cell cultures. Furthermore, cells differentiated in beads expressed both sclerostin (SOST) and dental matrix protein-1 (DMP1), markers for late osteoblasts/osteocytes. In conclusion, Both ALP-modified and unmodified alginate beads provide an environment that enhance osteogenic differentiation compared with traditional 2D culture. Also, the ALP-modified alginate beads showed profound mineralization and thus have the potential to serve as a bone substitute in tissue engineering.     

Ectonucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1) protein regulates osteoblast differentiation

ENPP1 (ectonucleotide pyrophosphatase/phosphodiesterase-1) is an established regulator of tissue mineralization. Previous studies demonstrated that ENPP1 is expressed in differentiated osteoblasts and that ENPP1 influences matrix mineralization by increasing extracellular levels of inorganic pyrophosphate. ENPP1 is also expressed in osteoblastic precursor cells when stimulated with FGF2, but the role of ENPP1 in preosteoblastic and other precursor cells is unknown. Here we investigate the function of ENPP1 in preosteoblasts. We find that ENPP1 expression is critical for osteoblastic differentiation and that this effect is not mediated by changes in extracellular concentration levels of phosphate or pyrophosphate or ENPP1 catalytic activity. MC3T3E1(C4) preosteoblastic cells, in which ENPP1 expression was suppressed by ENPP1-specific shRNA, and calvarial cells isolated from Enpp1 knock-out mice show defective osteoblastic differentiation upon stimulation with ascorbate, as indicated by a lack of cellular morphological change, a lack of osteoblast marker gene expression, and an inability to mineralize matrix. Additionally, MC3T3E1(C4) cells, in which wild type or catalytic inactive ENPP1 expression was increased, exhibited an increased tendency to differentiate, as evidenced by increased osteoblast marker gene expression and increased mineralization. Notably, treatment of cells with inorganic phosphate or pyrophosphate inhibited, as opposed to enhanced, expression of multiple genes that are expressed in association with osteoblast differentiation, matrix deposition, and mineralization. Our results indicate that ENPP1 plays multiple and distinct roles in the development of mineralized tissues and that the influence of ENPP1 on osteoblast differentiation and gene expression may include a mechanism that is independent of its catalytic activity.     

Estradiol influences the mechanical properties of human fetal osteoblasts through cytoskeletal changes

Estrogen is known to have a direct effect on bone forming osteoblasts and bone resorbing osteoclasts. The cellular and molecular effects of estrogen on osteoblasts and osteoblasts-like cells have been extensively studied. However, the effect of estrogen on the mechanical property of osteoblasts has not been studied yet. It is important since mechanical property of the mechanosensory osteoblasts could be pivotal to its functionality in bone remodeling. This is the first study aimed to assess the direct effect of estradiol on the apparent elastic modulus (E¹) and corresponding cytoskeletal changes of human fetal osteoblasts (hFOB 1.19). The cells were cultured in either medium alone or medium supplemented with β-estradiol and then subjected to Atomic Force Microscopy indentation (AFM) to determine E¹. The underlying changes in cytoskeleton were studied by staining the cells with TRITC-Phalloidin. Following estradiol treatment, the cells were also tested for proliferation, alkaline phosphatase activity and mineralization. With estradiol treatment, E¹ of osteoblasts significantly decreased by 43–46%. The confocal images showed that the changes in f-actin network observed in estradiol treated cells can give rise to the changes in the stiffness of the cells. Estradiol also increases the inherent alkaline phosphatase activity of the cells. Estradiol induced stiffness changes of osteoblasts were not associated with changes in the synthesized mineralized matrix of the cells. Thus, a decrease in osteoblast stiffness with estrogen treatment was demonstrated in this study, with positive links to cytoskeletal changes. The estradiol associated changes in osteoblast mechanical properties could bear implications for bone remodeling and its mechanical integrity.     

TGF-β1 modifications in nuclear matrix proteins of osteoblasts during differentiation

Nuclear matrix protein (NMP) composition of osteoblasts shows distinct two-dimensional gel electrophoretic profiles of labeled proteins as a function of stages of cellular differentiation. Because NMPs are involved in the control of gene expression, we examined modifications in the representation of NMPs induced by TGF-β1 treatment of osteoblasts to gain insight into the effects of TGF-β on development of the osteoblast phenotype. Exposure of proliferating fetal rat calvarial derived primary cells in culture to TGF-β1 for 48 h (day 4–6) modifies osteoblast cell morphology and proliferation and blocks subsequent formation of mineralized nodules. Nuclear matrix protein profiles were very similar between control and TGF-β–treated cultures until day 14, but subsequently differences in nuclear matrix proteins were apparent in TGF-β–treated cultures. These findings support the concept that TGF-β1 modifies the final stage of osteoblast mineralization and alters the composition of the osteoblast nuclear matrix as reflected by selective and TGF-β–dependent modifications in the levels of specific nuclear matrix proteins. The specific changes induced by TGF-β in nuclear matrix associated proteins may reflect specialized mechanisms by which TGF-β signalling mediates the alterations in cell organization and nodule formation and/or the consequential block in extracellular mineralization. J. Cell. Biochem. 69:291–303, 1998. © 1998 Wiley-Liss, Inc.     

mTORC1 Signaling Promotes Osteoblast Differentiation from Preosteoblasts

Preosteoblasts are precursor cells that are committed to the osteoblast lineage. Differentiation of these cells to mature osteoblasts is regulated by the extracellular factors and environmental cues. Recent studies have implicated mTOR signaling in the regulation of osteoblast differentiation. However, mTOR exists in two distinct protein complexes (mTORC1 and mTORC2), and the specific role of mTORC1 in regulating the progression of preosteoblasts to mature osteoblastis still unclear. In this study, we first deleted Raptor, a unique and essential component of mTORC1, in primary calvarial cells. Deletion of Raptor resulted in loss of mTORC1 but an increase in mTORC2 signaling without overtly affecting autophagy. Under the osteogenic culture condition, Raptor-deficient cells exhibited a decrease in matrix synthesis and mineralization. qPCR analyses revealed that deletion of Raptor reduced the expression of late-stage markers for osteoblast differentiation (Bglap, Ibsp, and Col1a), while slightly increasing early osteoblast markers (Runx2, Sp7, and Alpl). Consistent with the findings in vitro, genetic ablation of Raptor in osterix-expressing cells led to osteopenia in mice. Together, our findings have identified a specific role for mTORC1 in the transition from preosteoblasts to mature osteoblasts.     

Experimental acute pancreatitis is enhanced in mice with tissue nonspecific alkaline phoshatase haplodeficiency due to modulation of neutrophils and acinar cells

Tissue nonspecific alkaline phosphatase (TNAP) has a well established role in bone homeostasis and in hepatic/biliary conditions. In addition, TNAP is expressed in the inflamed intestine and is relevant to T and B lymphocyte function. TNAP KO mice are only viable for a few days, but TNAP^+/? haplodeficient mice are viable. Acute pancreatitis was induced by repeated caerulein injection in WT and TNAP^+/? mice. TNAP^+/? mice presented an increased expression of Cxcl2, Ccl2, Selplg (P-selectin ligand), Il6 and Il1b in the pancreas. Freshly isolated acinar cells showed a dramatic upregulation of Cxcl1, Cxcl2, Ccl2, Il6, Selpg or Bax in both pancreatitis groups. TNAP^+/? cells displayed a 2-fold higher expression of Cxcl2, and a smaller increase in Il6. These findings could be partly replicated by in vitro treatment of primary acinar cells with caerulein. Furthermore, the proinflammatory effect on acinar cells could be partially reproduced in wild type cells treated with the TNAP inhibitor levamisole. TNAP mRNA levels were also markedly upregulated by pancreatitis in acinar cells. Neutrophil infiltration (MRP8+ cells) and activation (IL-6 and TNF production in LPS treated primary neutrophils) were increased in TNAP^+/? vs WT mice. Neutrophil depletion greatly attenuated inflammation, indicating that this cell type is mainly responsible for the higher inflammatory status of TNAP^+/? mice. In conclusion, our results show that altered TNAP expression results in heightened pancreatic inflammation, which may be explained by an augmented response of neutrophils and by a higher sensitivity of acinar cells to caerulein injury.     

Apoptosis during bone-like tissue development in vitro

We present evidence of cell death by apoptosis during the development of bone-like tissue formation in vitro. Fetal rat calvaria-derived osteoblasts differentiate in vitro, progressing through three stages of maturation: a proliferation period, a matrix maturation period when growth is downregulated and expression of the bone cell phenotype is induced, and a third mineralization stage marked by the expression of bone-specific genes. Here we show for the first time that cells differentiating to the mature bone cell phenotype undergo programmed cell death and express genes regulating apoptosis. Culture conditions that modify expression of the osteoblast phenotype simultaneously modify the incidence of apoptosis. Cell death by apoptosis is directly demonstrated by visualization of degraded DNA into oligonucleosomal fragments after gel electrophoresis. Bcl-X_L, an inhibitor of apoptosis, and Bax, which can accelerate apoptosis, are expressed at maximal levels 24 h after initial isolation of the cells and again after day 25 in heavily mineralized bone tissue nodules. Bcl-2 is expressed in a reciprocal manner to its related gene product Bcl-X_L with the highest levels observed during the early post-proliferative stages of osteoblast maturation. Expression of p53, c-fos, and the interferon regulatory factors IRF-1 and IRF-2, but not cdc2 or cdk, were also induced in mineralized bone nodules. The upregulation of Msx-2 in association with apoptosis is consistent with its in vivo expression during embryogenesis in areas that will undergo programmed cell death. We propose that cell death by apoptosis is a fundamental component of osteoblast differentiation that contributes to maintaining tissue organization. J. Cell. Biochem. 68:31–49, 1998. © 1998 Wiley-Liss, Inc.     

Validation of a simple and fast method to quantify in vitro mineralization with fluorescent probes used in molecular imaging of bone

Alizarin Red S staining is the standard method to indicate and quantify matrix mineralization during differentiation of osteoblast cultures. KS483 cells are multipotent mouse mesenchymal progenitor cells that can differentiate into chondrocytes, adipocytes and osteoblasts and are a well-characterized model for the study of bone formation. Matrix mineralization is the last step of differentiation of bone cells and is therefore a very important outcome measure in bone research. Fluorescently labelled calcium chelating agents, e.g. BoneTag and OsteoSense, are currently used for in vivo imaging of bone. The aim of the present study was to validate these probes for fast and simple detection and quantification of in vitro matrix mineralization by KS483 cells and thus enabling high-throughput screening experiments.     

Regulation of expression of collagenase-3 in normal, differentiating rat osteoblasts.

We investigated the regulation of collagenase-3 expression in normal, differentiating rat osteoblasts. Fetal rat calvarial cell cultures showed an increase in alkaline phosphatase activity reaching maximal levels between 7-14 days post-confluence, then declining with the onset of mineralization. Collagenase-3 mRNA was just detectable after proliferation ceased at day 7, increased up to day 21, and declined at later ages. Postconfluent cells maintained in non-mineralizing medium expressed collagenase-3 but did not show the developmental increase exhibited by cells switched to mineralization medium. Cells maintained in non-mineralizing medium continued to proliferate; cells in mineralization medium ceased proliferation. In addition, collagenase-3 mRNA was not detected in subcultured cells allowed to remineralize. These results suggest that enhanced accumulation of collagenase-3 mRNA is triggered by cessation of proliferation or acquisition of a mineralized extracellular matrix and that other factors may also be required. After initiation of basal expression, parathyroid hormone (PTH) caused a dose-dependent increase in collagenase-3 mRNA. Both the cyclic adenosine monophosphate (cAMP) analogue, 8-bromo-cAMP (8-Br-cAMP), and the protein kinase C (PKC) activator, phorbol myristate acetate, increased collagenase-3 expression, while the calcium ionophore, ionomycin, did not, suggesting that PTH was acting through the protein kinase A (PKA) and PKC pathways. Inhibition of protein synthesis with cycloheximide caused an increase in basal collagenase-3 expression but blocked the effect of PTH, suggesting that an inhibitory factor prevents basal expression while an inductive factor is involved with PTH action. In summary, collagenase-3 is expressed in mineralized osteoblasts and cessation of proliferation and initiation of mineralization are triggers for collagenase-3 expression. PTH also stimulates expression of the enzyme through both PKA and PKC pathways in the mineralizing osteoblast.     

Sprouty2 is involved in the control of osteoblast proliferation and differentiation through the FGF and BMP signaling pathways

Fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) play essential roles in bone formation and osteoblast activity through the extracellular signal‐regulated kinase 1/2 (ERK1/2) and Smad pathways. Sprouty family members are intracellular inhibitors of the FGF signaling pathway, and four orthologs of Sprouty have been identified in mammals. In vivo analyses have revealed that Sprouty2 is associated with bone formation. However, the mechanism by which the Sprouty family controls bone formation has not been clarified. In this study, we investigated the involvement of Sprouty2 in osteoblast proliferation and differentiation. We examined Sprouty2 expression in MC3T3‐E1 cells, and found that high levels of Sprouty2 expression were induced by basic FGF stimulation. Overexpression of Sprouty2 in MC3T3‐E1 cells resulted in suppressed proliferation compared with control cells. Sprouty2 negatively regulated the phosphorylation of ERK1/2 after basic FGF stimulation, and of Smad1/5/8 after BMP stimulation. Furthermore, Sprouty2 suppressed the expression of osterix, alkaline phosphatase, and osteocalcin mRNA, which are markers of osteoblast differentiation. Additionally, Sprouty2 inhibited osteoblast matrix mineralization. These results suggest that Sprouty2 is involved in the control of osteoblast proliferation and differentiation by downregulating the FGF‐ERK1/2 and BMP‐Smad pathways, and suppresses the induction of markers of osteoblast differentiation.