Co-stimulation with bone morphogenetic protein-9 and FK506 induces remarkable osteoblastic differentiation in rat dedifferentiated fat cells

Dedifferentiated fat (DFAT) cells, which are isolated from mature adipocytes using the ceiling culture method, exhibit similar characteristics to mesenchymal stem cells, and possess adipogenic, osteogenic, chondrogenic, and myogenic potentials. Bone morphogenetic protein (BMP)-2 and -9, members of the transforming growth factor-β superfamily, exhibit the most potent osteogenic activity of this growth factor family. However, the effects of BMP-2 and BMP-9 on the osteogenic differentiation of DFAT remain unknown. Here, we examined the effects of BMP-2 and BMP-9 on osteoblastic differentiation of rat DFAT (rDFAT) cells in the presence or absence of FK506, an immunosuppressive agent. Co-stimulation with BMP-9 and FK506 induced gene expression of runx2, osterix, and bone sialoprotein, and ALP activity compared with BMP-9 alone, BMP-2 alone and BMP-2 + FK506 in rDFAT cells. Furthermore, it caused mineralization of cultures and phosphorylation of smad1/5/8, compared with BMP-9 alone. The ALP activity induced by BMP-9 + FK506 was not influenced by addition of noggin, a BMP antagonist. Our data suggest that the combination of BMP-9 and FK506 potently induces osteoblastic differentiation of rDFAT cells.     

FK506 enhanced osteoblastic differentiation in mesenchymal cells.

Bone morphogenetic protein (BMP) is a bone-derived growth factor capable of promoting the differentiation of mesenchymal cells into osteogenic lineage pathways. Recently, immunosuppressants were reported to cause a moderate increase in osteoblastic differentiation in a rat osteoblast-like osteosarcoma cell line. If immunosuppressants can induce osteoblastic differentiation, it will be useful for bone tissue transplantation. We assessed the effect of immunosuppressants with or without BMP-4 on inducing osteoblastic differentiation in osteoblast-like and other mesenchymal cells. FK506, an immunosuppressant often used clinically, induced a dose- and time-dependent increase in alkaline phosphatase (ALP) activity, one of the markers of osteoblast differentiation, in cells derived from mesenchyma. In the presence of BMP-4, ALP activity, mRNA levels of ALP and osteocalcin increased. FK506 was found to not only stimulate osteoblastic differentiation, but also to enhance BMP-4 induced osteoblastic differentiation. These results suggest that FK506 promotes differentiation of osteoblastic cells.     

Activation of caspases is required for osteoblastic differentiation

Previous studies have shown that mouse osteoblastic MC3T3-E1 cells undergo apoptosis when exposed to a mixture of proinflammatory cytokines. Bone morphogenetic protein (BMP)s are important regulators of osteoblast differentiation. Because regulation of osteoblastic differentiation is poorly understood, we sought to determine if BMP-4-induced differentiation of osteoblastic cells depends on the activity of the key apoptotic proteases, i.e. the caspases. BMP-4 induced the growth arrest and differentiation of osteoblastic cell line MC3T3-E1, as evidenced by the appearance of osteoblastic phenotypes such as alkaline phosphatase (ALP) activation and parathyroid hormone (PTH)-dependent production of cAMP. Surprisingly, BMP-4 induced transient and potent activation of caspase-8, caspase-2, and caspase-3, in this order. However, no apoptosis or necrosis in BMP-4-treated cells could be detected by FACS using annexin-V/propodium iodine double staining. Peptide inhibition of caspase activity led to a dramatic reduction in ALP activation and PTH-induced production of cAMP in BMP-4-treated cells. Although BMP-4 treatment resulted in cell-cycle G0/G1 arrest as detected by FACS cell-cycle analysis, caspase inhibitors (caspase-8, caspase-2, and caspase-3 inhibitors) could block the G0/G1 arrest in MC3T3-E1 cells. Taken together, these results confirm a unique and unanticipated role for the caspase-mediated signal cascade in the differentiation of osteoblasts.     

Canonical Wnts and BMPs cooperatively induce osteoblastic differentiation through a GSK3β-dependent and β-catenin-independent mechanism

Both BMPs and Wnts play important roles in the regulation of bone formation. We examined the molecular mechanism regulating cross-talk between BMPs and Wnts in the osteoblastic differentiation of C2C12 cells. Canonical Wnts (Wnt1 and Wnt3a) but not non-canonical Wnts (Wnt5a and Wnt11) synergistically stimulated ALP activity in the presence of BMP-4. Wnt3a and BMP-4 synergistically stimulated the expression of type I collagen and osteonectin. However, Wnt3a did not stimulate ALP activity that was induced by a constitutively active BMP receptor or Smad1. Noggin and Dkk-1 suppressed the synergistic effect of BMP-4 and Wnt3a, but Smad7 did not. Overexpression of β-catenin did not affect BMP-4-induced ALP activity. By contrast, inhibition or stimulation of GSK3β activity resulted in either stimulation or suppression of ALP activity, respectively, in the presence of BMP-4. Taken together, these findings suggest that BMPs and canonical Wnts may regulate osteoblastic differentiation, especially at the early stages, through a GSK3β-dependent but β-catenin-independent mechanism.     

Tumour necrosis factor alpha-stimulated gene-6 inhibits osteoblastic differentiation of human mesenchymal stem cells induced by osteogenic differentiation medium and BMP-2

To better understand the molecular pathogenesis of OPLL (ossification of the posterior longitudinal ligament) of the spine, an ectopic bone formation disease, we performed cDNA microarray analysis on cultured ligament cells from OPLL patients. We found that TSG-6 (tumour necrosis factor alpha-stimulated gene-6) is down-regulated during osteoblastic differentiation. Adenovirus vector-mediated overexpression of TSG-6 inhibited osteoblastic differentiation of human mesenchymal stem cells induced by BMP (bone morphogenetic protein)-2 or OS (osteogenic differentiation medium). TSG-6 suppressed phosphorylation and nuclear accumulation of Smad 1/5 induced by BMP-2, probably by inhibiting binding of the ligand to the receptor, since interaction between TSG-6 and BMP-2 was observed in vitro. TSG-6 has two functional domains, a Link domain (a hyaluronan binding domain) and a CUB domain implicated in protein interaction. The inhibitory effect on osteoblastic differentiation was completely lost with exogenously added Link domain-truncated TSG-6, while partial inhibition was retained by the CUB domain-truncated protein. In addition, the inhibitory action of TSG-6 and the in vitro interaction of TSG-6 with BMP-2 were abolished by the addition of hyaluronan. Thus, TSG-6, identified as a down-regulated gene during osteoblastic differentiation, suppresses osteoblastic differentiation induced by both BMP-2 and OS and is a plausible target for therapeutic intervention in OPLL.     

Osteogenic protein-1 differentially regulates the mRNA expression of bone morphogenetic proteins and their receptors in primary cultures of osteoblasts

The mRNA expression patterns of several bone morphogenetic proteins (BMPs) and their receptors (BMPRs) in long-term primary cultures of fetal rat calvaria (FRC) cells were examined by Northern analysis. Their temporal orders of expression were correlated with those of several biochemical markers characteristic of osteoblastic cell differentiation. Distinct temporal patterns of expression of BMPs and BMPRs during osteoblastic cell differentiation were observed. BMP-2 and BMP-7 mRNA levels did not change significantly. BMP-4 mRNA expression increased and reached a peak prior to matrix formation. BMP-5 mRNA expression increased during the mineralization phase and BMP-6 mRNA expression increased throughout all phases of cell differentiation. Effects of BMP-7 (Osteogenic Protein-1; OP-1) on the expression patterns of several other members of the BMP family and the receptors were also studied. OP-1 downregulated the BMP-4, -5, and -6 mRNA levels by a maximal of 2-fold, 1.5-fold, and 6-fold, respectively. OP-1 did not change significantly the OP-1 and BMP-2 mRNA expression. Of the three type I BMPR examined, OP-1 upregulated ActR-I and BMPR-IA mRNA expression slightly but with statistical significance. OP-1 downregulated BMPR-IB mRNA expression slightly. OP-1 upregulated BMPR-II mRNA expression by a maximum of 2-fold. Our findings demonstrate that OP-1 differentially regulates the mRNA expression of several related members of the BMP family and their receptors in osteoblasts. The observations suggest that OP-1 action on osteoblastic cells involves a complex regulation of gene expression of related members of the BMP family and their receptors in a cell differentiation stage dependent manner.     

Endogenous TGF-beta signaling suppresses maturation of osteoblastic mesenchymal cells

Transforming growth factor-beta (TGF-beta), one of the most abundant cytokines in bone matrix, has positive and negative effects on bone formation, although the molecular mechanisms of these effects are not fully understood. Bone morphogenetic proteins (BMPs), members of the TGF-beta superfamily, induce bone formation in vitro and in vivo. Here, we show that osteoblastic differentiation of mouse C2C12 cells was greatly enhanced by the TGF-beta type I receptor kinase inhibitor SB431542. Endogenous TGF-beta was found to be highly active, and induced expression of inhibitory Smads during the maturation phase of osteoblastic differentiation induced by BMP-4. SB431542 suppressed endogenous TGF-beta signaling and repressed the expression of inhibitory Smads during this period, possibly leading to acceleration of BMP signaling. SB431542 also induced the production of alkaline phosphatase and bone sialoprotein, and matrix mineralization of human mesenchymal stem cells. Thus, signaling cross-talk between BMP and TGF-beta pathways plays a crucial role in the regulation of osteoblastic differentiation, and TGF-beta inhibitors may be invaluable for the treatment of various bone diseases by accelerating BMP-induced osteogenesis.     

Functions of transforming growth factor-beta family type I receptors and Smad proteins in the hypertrophic maturation and osteoblastic differentiation of chondrocytes

We investigated the effects of bone morphogenetic protein (BMP)-2, a member of the transforming growth factor-beta superfamily, on the regulation of the chondrocyte phenotype, and we identified signaling molecules involved in this regulation. BMP-2 triggers three concomitant responses in mouse primary chondrocytes and chondrocytic MC615 cells. First, BMP-2 stimulates expression or synthesis of type II collagen. Second, BMP-2 induces expression of molecular markers characteristic of pre- and hypertrophic chondrocytes, such as Indian hedgehog, parathyroid hormone/parathyroid hormone-related peptide receptor, type X collagen, and alkaline phosphatase. Third, BMP-2 induces osteocalcin expression, a specific trait of osteoblasts. Constitutively active forms of transforming growth factor-beta family type I receptors and Smad proteins were overexpressed to address their role in this process. Activin receptor-like kinase (ALK)-1, ALK-2, ALK-3, and ALK-6 were able to reproduce the hypertrophic maturation of chondrocytes induced by BMP-2. In addition, ALK-2 mimicked further the osteoblastic differentiation of chondrocytes induced by BMP-2. In the presence of BMP-2, Smad1, Smad5, and Smad8 potentiated the hypertrophic maturation of chondrocytes, but failed to induce osteocalcin expression. Smad6 and Smad7 impaired chondrocytic expression and osteoblastic differentiation induced by BMP-2. Thus, our results indicate that Smad-mediated pathways are essential for the regulation of the different steps of chondrocyte and osteoblast differentiation and suggest that additional Smad-independent pathways might be activated by ALK-2.     

Oxysterol-induced osteoblastic differentiation of pluripotent mesenchymal cells is mediated through a PKC- and PKA-dependent pathway

Oxysterols form a large family of oxygenated derivatives of cholesterol that are present in circulation, and in human and animal tissues. The discovery of osteoinductive molecules that can induce the lineage-specific differentiation of cells into osteoblastic cells and therefore enhance bone formation is crucial for better management of bone fractures and osteoporosis. We previously reported that specific oxysterols have potent osteoinductive properties and induce the osteoblastic differentiation of pluripotent mesenchymal cells. In the present report we demonstrate that the induction of osteoblastic differentiation by oxysterols is mediated through a protein kinase C (PKC)- and protein kinase A (PKA)-dependent mechanism(s). Furthermore, oxysterol-induced-osteoblastic differentiation is marked by the prolonged DNA-binding activity of Runx2 in M2-10B4 bone marrow stromal cells (MSCs) and C3H10T1/2 embryonic fibroblastic cells. This increased activity of Runx2 is almost completely inhibited by PKC inhibitors Bisindolylmaleimide and Rottlerin, and only minimally inhibited by PKA inihibitor H-89. PKC- and PKA-dependent mechanisms appear to also regulate other markers of osteoblastic differentiation including alkaline phosphatase (ALP) activity and osteocalcin mRNA expression in response to oxysterols. Finally, osteogenic oxysterols induce osteoblastic differentiation with BMP7 and BMP14 in a synergistic manner as demonstrated by the enhanced Runx2 DNA-binding activity, ALP activity, and osteocalcin mRNA expression. Since Runx2 is an indispensable factor that regulates the differentiation of osteoblastic cells and bone formation in vitro and in vivo, its increased activity in oxysterol-treated cells further validates the potential role of oxysterols in lineage-specific differentiation of pluripotent mesenchymal cells and their potential therapeutic use as bone anabolic factors.     

BMP treatment of C3H10T1/2 mesenchymal stem cells induces both chondrogenesis and osteogenesis

The molecular mechanisms by which bone morphogenetic proteins (BMPs) promote skeletal cell differentiation were investigated in the murine mesenchymal stem cell line C3H10T1/2. Both BMP-7 and BMP-2 induced C3H10T1/2 cells to undergo a sequential pattern of chondrogenic followed by osteogenic differentiation that was dependent on both the concentration and the continuous presence of BMP in the growth media. Differentiation was determined by the expression of chondrogenesis and osteogenesis associated matrix genes. Subsequent experiments using BMP-7 demonstrated that withdrawal of BMP from the growth media led to a complete loss of skeletal cell differentiation accompanied by adipogenic differentiation of these cells. Continuous treatment with BMP-7 increased the expression of Sox9, Msx 2, and c-fos during the periods of chondrogenic differentiation after which point their expression decreased. In contrast, Dlx 5 expression was induced by BMP-7 treatment and remained elevated throughout the time-course of skeletal cell differentiation. Runx2/Cbfa1 was not detected by ribonuclease protection assay (RPA) and did not appear to be induced by BMP-7. The sequential nature of differentiation of chondrocytic and osteoblastic cells and the necessity for continuous BMP treatment to maintain skeletal cell differentiation suggests that the maintenance of selective differentiation of the two skeletal cell lineages might be dependent on BMP-7-regulated expression of other morphogenetic factors. An examination of the expression of Wnt, transforming growth factor-beta (TGF-beta), and the hedgehog family of morphogens showed that Wnt 5b, Wnt 11, BMP-4, growth and differentiation factor-1 (GDF-1), Sonic hedgehog (Shh), and Indian hedgehog (Ihh) were endogenously expressed by C3H10T1/2 cells. Wnt 11, BMP-4, and GDF-1 expression were inhibited by BMP-7 treatment in a dose-dependent manner while Wnt 5b and Shh were selectively induced by BMP-7 during the period of chondrogenic differentiation. Ihh expression also showed induction by BMP-7 treatment, however, the period of maximal expression was during the later time-points, corresponding to osteogenic differentiation. An interesting phenomenon was that BMP-7 activity could be further enhanced twofold by growing the cells in a more nutrient-rich media. In summary, the murine mesenchymal stem cell line C3H10T1/2 was induced to follow an endochondral sequence of chondrogenic and osteogenic differentiation dependent on both dose and continual presence of BMP-7 and enhanced by a nutrient-rich media. Our preliminary results suggest that the induction of osteogenesis is dependent on the secondary regulation of factors that control osteogenesis through an autocrine mechanism.     

Aromatase activity in human adipose tissue stromal cells; the effect of fetal bovine serum fractions on dexamethasone-stimulated aromatization

Aromatization in human adipose stromal cells is stimulated by dexamethasone, but only in the presence of fetal bovine serum (FBS). To determine whether there was a specific fraction of FBS responsible for this stimulation, FBS was fractionated either by a pressure-driven ultrafiltration membrane or by Sephadex gel filtration techniques. The stimulating factor(s) appeared to be in the FBS fraction of 150,000-300,000 Mw by Sephadex filtration. Conversely, FBS fractions with less than 30,000 Mw as separated by the former method inhibited the dexamethasone-stimulated aromatization of cultured adipose stromal cells. Bovine serum albumin, which constituted the major portion of FBS, had no discernible effect on the dexamethasone action on the aromatization of these cells.     

Runx2 is a common target of transforming growth factor beta1 and bone morphogenetic protein 2, and cooperation between Runx2 and Smad5 induces osteoblast-specific gene expression in the pluripotent mesenchymal precursor cell line C2C12

When C2C12 pluripotent mesenchymal precursor cells are treated with transforming growth factor beta1 (TGF-beta1), terminal differentiation into myotubes is blocked. Treatment with bone morphogenetic protein 2 (BMP-2) not only blocks myogenic differentiation of C2C12 cells but also induces osteoblast differentiation. The molecular mechanisms governing the ability of TGF-beta1 and BMP-2 to both induce ligand-specific responses and inhibit myogenic differentiation are not known. We identified Runx2/PEBP2alphaA/Cbfa1, a global regulator of osteogenesis, as a major TGF-beta1-responsive element binding protein induced by TGF-beta1 and BMP-2 in C2C12 cells. Consistent with the observation that Runx2 can be induced by either TGF-beta1 or BMP-2, the exogenous expression of Runx2 mediated some of the effects of TGF-beta1 and BMP-2 but not osteoblast-specific gene expression. Runx2 mimicked common effects of TGF-beta1 and BMP-2 by inducing expression of matrix gene products (for example, collagen and fibronectin), suppressing MyoD expression, and inhibiting myotube formation of C2C12 cells. For osteoblast differentiation, an additional effector, BMP-specific Smad protein, was required. Our results indicate that Runx2 is a major target gene shared by TGF-beta and BMP signaling pathways and that the coordinated action of Runx2 and BMP-activated Smads leads to the induction of osteoblast-specific gene expression in C2C12 cells.     

Lactacystin, a proteasome inhibitor, enhances BMP-induced osteoblastic differentiation by increasing active Smads

Proteasome inhibitors enhance bone formation and osteoblastic differentiation in vivo and in vitro. In the present study, we examined whether the molecular mechanisms of lactacystin, one of many proteasome inhibitors, stimulated the osteoblastic differentiation of C2C12 cells that is induced by bone morphogenetic proteins (BMPs). Pretreatment with lactacystin enhanced the alkaline phosphatase (ALP) activity induced by BMP2, BMP4 or BMP7, but lactacystin did not induce ALP in the absence of BMPs. In addition, lactacystin-stimulated BMP2 induced mRNA expression of ALP, type I collagen, osteonectin, osteocalcin, Id1, Osterix, and Runx2. Lactacystin maintained BMP2-induced phosphorylation of Smad1/5/8 and increased the length of time that these Smads were bound to target DNA. Moreover, lactacystin prevented BMP receptor-induced Smad degradation. This enhancement of BMP2-induced ALP activity and Smad phosphorylation by lactacystin was also observed in primary osteoblasts. These findings suggest that pretreatment with lactacystin accelerates BMP-induced osteoblastic differentiation by increasing the levels of phosphorylated Smads, which are maintained because BMP receptor-induced degradation is inhibited. We propose that optimized stimulation by proteasome inhibitors in a clinical setting may facilitate autogenous or BMP-induced bone formation in areas of defective bone.