Protein kinase C-dependent upregulation of N-cadherin expression by phorbol ester in human calvaria osteoblasts

Cell-cell adhesion mediated by cadherins is believed to play an essential role in the control of cell differentiation and tissue formation. Our recent studies indicate that N-cadherin is involved in human osteoblast differentiation. However, the signalling molecules that regulate cadherins in osteoblasts are not known. We tested the possibility that N-cadherin expression and function may be regulated by direct activation of protein kinase C (PKC) in human osteoblasts. Treatment of immortalized human neonatal calvaria (IHNC) cells with phorbol 12,13-dibutyrate (100 nM) transiently increased PKC activity. RT-PCR analysis showed that transient treatment with phorbol ester transiently increased N-cadherin mRNA levels at 4-12 h. Western blot analysis showed that N-cadherin protein levels were increased by phorbol ester at 24-48 h, and this was confirmed by immunocytochemical analysis. In contrast, E-cadherin expression was not affected. Transient treatment of IHNC cells with phorbol ester increased cell-cell aggregation, which was suppressed by neutralizing N-cadherin antibody, showing that the increased N-cadherin induced by phorbol ester was functional. Finally, phorbol ester dose-dependently increased alkaline phosphatase activity, an early marker of osteoblast differentiation. This effect was comparable to the promoting effect of BMP-2, a potent activator of osteoblast differentiation. These data show that direct activation of PKC by phorbol ester increases N-cadherin expression and function, and promotes ALP activity in human calvaria osteoblasts, which provides a signaling mechanism by which N-cadherin is regulated and suggests a role for PKC in N-cadherin-mediated control of human osteoblast differentiation.     

N-Cadherin Negatively Regulates Osteoblast Proliferation and Survival by Antagonizing Wnt,ERK and PI3K/Akt Signalling

Background

Osteoblasts are bone forming cells that play an essential role in osteogenesis. The elucidation of the mechanisms that control osteoblast number is of major interest for the treatment of skeletal disorders characterized by abnormal bone formation. Canonical Wnt signalling plays an important role in the control of osteoblast proliferation, differentiation and survival. Recent studies indicate that the cell-cell adhesion molecule N-cadherin interacts with the Wnt co-receptors LRP5/6 to regulate osteoblast differentiation and bone accrual. The role of N-cadherin in the control of osteoblast proliferation and survival remains unknown.

Methods and Principal Findings

Using murine MC3T3-E1 osteoblastic cells and N-cadherin transgenic mice, we demonstrate that N-cadherin overexpression inhibits cell proliferation in vitro and in vivo. The negative effect of N-cadherin on cell proliferation results from decreased Wnt, ERK and PI3K/Akt signalling and is restored by N-cadherin neutralizing antibody that antagonizes N-cadherin-LRP5 interaction. Inhibition of Wnt signalling using DKK1 or Sfrp1 abolishes the ability of N-cadherin blockade to restore ERK and PI3K signalling and cell proliferation, indicating that the altered cell growth in N-cadherin overexpressing cells is in part secondary to alterations in Wnt signalling. Consistently, we found that N-cadherin overexpression inhibits the expression of Wnt3a ligand and its downstream targets c-myc and cyclin D1, an effect that is partially reversed by N-cadherin blockade. We also show that N-cadherin overexpression decreases osteoblast survival in vitro and in vivo. This negative effect on cell survival results from inhibition of PI3K/Akt signalling and increased Bax/Bcl-2, a mechanism that is rescued by Wnt3a.

Conclusion

The data show that N-cadherin negatively controls osteoblast proliferation and survival via inhibition of autocrine/paracrine Wnt3a ligand expression and attenuation of Wnt, ERK and PI3K/Akt signalling, which provides novel mechanisms by which N-cadherin regulates osteoblast number.     

Differential regulation of cadherins by dexamethasone in human osteoblastic cells

Human osteoblasts express a repertoire of cadherins, including N-cadherin (N-cad), cadherin-11 (C11), and cadherin-4 (C4). We have previously shown that direct cell-cell adhesion via cadherins is critical for BMP-2-induced osteoblast differentiation. In this study, we have analyzed the regulation of cadherin expression in normal human trabecular bone osteoblasts (HOB), and osteoprogenitor marrow stromal cells (BMC), during exposure to dexamethasone, another inducer of human bone cell differentiation. Dexamethasone inhibited the expression of both C11 and N-cad mRNA in both BMC and HOB, although the effect was much more pronounced on N-cad than on C11. This action of the steroid was dose dependent, was maximal at 10(-7) M concentration, and occurred as early as after 1 day of incubation. By contrast, expression of C4 mRNA and protein was strongly induced by dexamethasone in BMC and was stimulated in HOB. This stimulatory effect lasted for at least 2 weeks of incubation. A cadherin inhibitor, HAV-containing decapeptide only partially ( approximately 50%) prevented dexamethasone-induced stimulation of alkaline phosphatase activity by BMC, which instead was not altered by incubation with a neutralizing antibody against C4. Therefore, the pattern of cadherin regulation by dexamethasone radically differs form that observed with BMP-2. Dexamethasone effects on certain osteoblast differentiated features, such as induction of alkaline phosphatase activity are not strictly dependent on cadherin function.     

Premature osteoblast clustering by enamel matrix proteins induces osteoblast differentiation through up-regulation of connexin 43 and N-cadherin

In recent years, enamel matrix derivative (EMD) has garnered much interest in the dental field for its apparent bioactivity that stimulates regeneration of periodontal tissues including periodontal ligament, cementum and alveolar bone. Despite its widespread use, the underlying cellular mechanisms remain unclear and an understanding of its biological interactions could identify new strategies for tissue engineering. Previous in vitro research has demonstrated that EMD promotes premature osteoblast clustering at early time points. The aim of the present study was to evaluate the influence of cell clustering on vital osteoblast cell-cell communication and adhesion molecules, connexin 43 (cx43) and N-cadherin (N-cad) as assessed by immunofluorescence imaging, real-time PCR and Western blot analysis. In addition, differentiation markers of osteoblasts were quantified using alkaline phosphatase, osteocalcin and von Kossa staining. EMD significantly increased the expression of connexin 43 and N-cadherin at early time points ranging from 2 to 5 days. Protein expression was localized to cell membranes when compared to control groups. Alkaline phosphatase activity was also significantly increased on EMD-coated samples at 3, 5 and 7 days post seeding. Interestingly, higher activity was localized to cell cluster regions. There was a 3 fold increase in osteocalcin and bone sialoprotein mRNA levels for osteoblasts cultured on EMD-coated culture dishes. Moreover, EMD significantly increased extracellular mineral deposition in cell clusters as assessed through von Kossa staining at 5, 7, 10 and 14 days post seeding. We conclude that EMD up-regulates the expression of vital osteoblast cell-cell communication and adhesion molecules, which enhances the differentiation and mineralization activity of osteoblasts. These findings provide further support for the clinical evidence that EMD increases the speed and quality of new bone formation in vivo.     

N-cadherin-dependent cell-cell contact regulates Rho GTPases and beta-catenin localization in mouse C2C12 myoblasts

N-cadherin, a member of the Ca(2+)-dependent cell-cell adhesion molecule family, plays an essential role in skeletal muscle cell differentiation. We show that inhibition of N-cadherin-dependent adhesion impairs the upregulation of the two cyclin-dependent kinase inhibitors p21 and p27, the expression of the muscle-specific genes myogenin and troponin T, and C2C12 myoblast fusion. To determine the nature of N-cadherin-mediated signals involved in myogenesis, we investigated whether N-cadherin-dependent adhesion regulates the activity of Rac1, Cdc42Hs, and RhoA. N-cadherin-dependent adhesion decreases Rac1 and Cdc42Hs activity, and as a consequence, c-jun NH2-terminal kinase (JNK) MAPK activity but not that of the p38 MAPK pathway. On the other hand, N-cadherin-mediated adhesion increases RhoA activity and activates three skeletal muscle-specific promoters. Furthermore, RhoA activity is required for beta-catenin accumulation at cell-cell contact sites. We propose that cell-cell contacts formed via N-cadherin trigger signaling events that promote the commitment to myogenesis through the positive regulation of RhoA and negative regulation of Rac1, Cdc42Hs, and JNK activities.     

Plasticity of cadherin-catenin expression in the melanocyte lineage

Cadherins are calcium-dependent cell adhesion receptors with strong morphoregulatory functions. To mediate functional adhesion, cadherins must interact with actin cytoskeleton. Catenins are cytoplasmic proteins that mediate the interactions between cadherins and the cytoskeleton. In addition to their role in cell-cell adhesion, catenins also participate in signaling pathways that regulate cell growth and differentiation. Cadherins and catenins appear to be involved in melanocyte development and transformation. Here, we investigated the function of cadherin-catenin complexes in the normal development and transformation of melanocytes by studying the patterns of expression of the cell-cell adhesion molecules, E-, N- and P-cadherin, and the expression of their cytoplasmic partners, alpha-, beta- and gamma-catenin during murine development. Similar analyses were performed in vitro using murine melanoblast, melanocyte, and melanoma cell lines in the presence and absence of keratinocytes, the cells with which melanocytes interact in vivo. Overall, the results suggest that the expression of cadherins and catenins is very plastic and depends on their environment as well as the transformation status of the cells. This plasticity is important in fundamental cellular mechanisms associated with normal and pathological ontogenesis, as well as with tumorigenesis.     

Periostin 蛋白与成骨相关性的研究进展

Peiostin又称为成骨细胞特异因子2(OSF-2),是一种具有多种功能的细胞外基质蛋白,其在物种形成过程中高度保守,已知其在骨、肿瘤、心血管、呼吸系统以及组织修复中均有作用。研究表明,periostin的表达受多种生长因子和环境因子的调控,通过与整合素受体αvβ3或者Notch 1蛋白的结合,激活FAK和Akt/PKB或者c-Fos等相关信号通路,进而调控多种细胞(成纤维细胞、内皮细胞、上皮细胞、成骨细胞和平滑肌细胞)的分化、粘附、迁移和存活。在既往的研究中,学者们主要探讨了Periostin在肿瘤发生和转移中的作用。尽管其最先发现于骨,但是periostin与骨相关的研究相对较少。近几年,部分研究分析了periostin在骨生物学中的作用,并指出periostin可能参与了骨的形成。本文就已有的periostin在成骨中的研究作一综述。     

Lack of evidence that hematopoietic stem cells depend on N-cadherin-mediated adhesion to osteoblasts for their maintenance

Recent studies have proposed that bone marrow hematopoietic stem cells (HSCs) are maintained via N-cadherin-mediated homophilic adhesion with osteoblasts. However, there is not yet any evidence that N-cadherin-expressing cells have HSC activity or that osteoblasts are required for HSC maintenance. We were unable to detect N-cadherin expression in highly purified HSCs by polymerase chain reaction, by using commercial anti-N-cadherin antibodies, or by beta-galactosidase staining of N-cadherin gene trap mice. Only N-cadherin-negative bone marrow cells exhibited HSC activity in irradiated mice. Finally, biglycan-deficient mice had significant reductions in trabecular bone and osteoblasts but showed no defects in hematopoiesis, HSC frequency, or function. Thus, reductions in osteoblasts do not necessarily lead to reductions in HSCs. Most bone marrow HSCs in wild-type and biglycan-deficient mice localized to sinusoids, and few localized within five cell diameters of the endosteum. These results question whether significant numbers of HSCs depend on N-cadherin-mediated adhesion to osteoblasts.     

Regulation of human cranial osteoblast phenotype by FGF-2, FGFR-2 and BMP-2 signaling

The formation of cranial bone requires the differentiation of osteoblasts from undifferentiated mesenchymal cells. The balance between osteoblast recruitment, proliferation, differentiation and apoptosis in sutures between cranial bones is essential for calvarial bone formation. The mechanisms that control human osteoblasts during normal calvarial bone formation and premature suture ossification (craniosynostosis) begin to be understood. Our studies of the human calvaria osteoblast phenotype and calvarial bone formation showed that premature fusion of the sutures in non-syndromic and syndromic (Apert syndrome) craniosynostoses results from precocious osteoblast differentiation. We showed that Fibroblast Growth Factor-2 (FGF-2), FGF receptor-2 (FGFR-2) and Bone Morphogenetic Protein-2 (BMP-2), three essential factors involved in skeletal development, regulate the proliferation, differentiation and apoptosis in human calvaria osteoblasts. Mechanisms that induce the differentiated osteoblast phenotype have also been identified in human calvaria osteoblasts. We demonstrated the implication of molecules (N-cadherin, Il-1) and signaling pathways (src, PKC) by which these local factors modulate human calvaria osteoblast differentiation and apoptosis. The identification of these essential signaling molecules provides new insights into the pathways controlling the differentiated osteoblast phenotype, and leads to a more comprehensive view in the mechanisms that control normal and premature cranial ossification in humans.     

Cellular and molecular alterations of osteoblasts in human disorders of bone formation

Osteogenesis is a complex process characterized sequentially by the commitment of precursor cells, the proliferation of osteoprogenitor cells, the differentiation of pre-osteoblasts into mature osteoblasts and the apposition of a calcified bone matrix. Recent advances in cell and molecular biology have improved our knowledge of the cellular and molecular mechanisms controlling the different steps of bone formation in humans. Using ex vivo/in vitro studies of disorders of bone formation, we showed that the recruitment of osteoprogenitor cells is the most important step controlling the rate of bone formation in both rodents and humans. Accordingly, treatments stimulating osteoblast recruitment were found to increase bone formation in experimental models of osteopenic disorders. Using models of human osteoblastic cells, we identified the profile of phenotypic markers expressed during osteoblast differentiation, and found that hormones and growth factors control osteoblastic cell proliferation and differentiation in a sequential and coordinate manner during osteogenesis in vitro. Our recent evaluation of the phenotypic osteoblast abnormalities induced by genetic mutations in the Gs alpha and FGFR-2 genes led to the characterization of the role of these genes in the alterations of osteoblast proliferation and differentiation in humans. These studies at the histological, cellular and molecular levels provided new insight into the mechanisms that are involved in pathological bone formation in humans. It is expected that further determination of the pathogenic pathways in metabolic and genetic abnormalities in human osteoblasts will help to identify novel target genes and to conceive new therapeutic tools to stimulate bone formation in osteopenic disorders.     

Cadherins in development and cancer

Proper embryonic development is guaranteed under conditions of regulated cell-cell and cell-matrix adhesion. The cells of an embryo have to be able to distinguish their neighbours as being alike or different. Cadherins, single-pass transmembrane, Ca(2+)-dependent adhesion molecules that mainly interact in a homophilic manner, are major contributors to cell-cell adhesion. Cadherins play pivotal roles in important morphogenetic and differentiation processes during development, and in maintaining tissue integrity and homeostasis. Changes in cadherin expression throughout development enable differentiation and the formation of various organs. In addition to these functions, cadherins have strong implications in tumourigenesis, since frequently tumour cells show deregulated cadherin expression and inappropriate switching among family members. In this review, I focus on E- and N-cadherin, giving an overview of their structure, cellular function, importance during development, role in cancer, and of the complexity of Ecadherin gene regulation.     

Rescuing the N-cadherin knockout by cardiac-specific expression of N- or E-cadherin

Cell-cell adhesion mediated by some members of the cadherin family is essential for embryonic survival. The N-cadherin-null embryo dies during mid-gestation, with multiple developmental defects. We show that N-cadherin-null embryos expressing cadherins using muscle-specific promoters, alpha- or beta-myosin heavy chain, are partially rescued. Somewhat surprisingly, either N-cadherin or E-cadherin was effective in rescuing the embryos. The rescued embryos exhibited an increased number of somites, branchial arches and the presence of forelimb buds; however, in contrast, brain development was severely impaired. In rescued animals, the aberrant yolk sac morphology seen in N-cadherin-null embryos was corrected, demonstrating that this phenotype was secondary to the cardiac defect. Dye injection studies and analysis of chimeric animals that have both wild-type and N-cadherin-null cells support the conclusion that obstruction of the cardiac outflow tract represents a major defect that is likely to be the primary cause of pericardial swelling seen in null embryos. Although rescued embryos were more developed than null embryos, they were smaller than wild-type embryos, even though the integrity of the cardiovascular system appeared normal. The smaller size of rescued embryos may be due, at least in part, to increased apoptosis observed in tissues not rescued by transgene expression, indicating that N-cadherin-mediated cell adhesion provides an essential survival signal for embryonic cells. Our data provide in vivo evidence that cadherin adhesion is essential for cell survival and for normal heart development. Our data also show that E-cadherin can functionally substitute for N-cadherin during cardiogenesis, suggesting a critical role for cadherin-mediated cell-cell adhesion, but not cadherin family member-specific signaling, at the looping stage of heart development.     

N-cadherin as a key regulator of collective cell migration in a 3D environment

Cell migration is a critical step of normal developmental processes and disease progression. Often, migrating cells interact and maintain contact with neighboring cells. However, the precise roles of cell-cell adhesion in cell migration have thus far been poorly defined. Often in aggressive cancers, N-cadherin is prominently upregulated, yet, these highly motile cells have limited cell-cell adhesion when plated on a stiff 2D substrate. But, the same cells in a 3D matrix migrate as a multicellular cluster. This new observation suggests that N-cadherin-mediated cell-cell adhesion supports cell interactions between migrating cells in a more physiologically relevant 3D matrix, but not on a 2D substrate. While N-cadherin is an integral part of neural synapses, the ectopic expression of N-cadherin in transformed epithelial cells plays an equally important part in initiating pro-migratory signaling, and providing strong yet flexible cell cohesion essential for persistent cell migration in a 3D matrix. The 3D cell migration analysis for studying cell-to-cell interactions exposes the roles of N-cadherin in multicellular migration, and reveals novel insights into cell migration-dependent normal and pathological processes.     

Transforming growth factor-beta-mediated chondrogenesis of human mesenchymal progenitor cells involves N-cadherin and mitogen-activated protein kinase and Wnt signaling cross-talk

The multilineage differentiation potential of adult tissue-derived mesenchymal progenitor cells (MPCs), such as those from bone marrow and trabecular bone, makes them a useful model to investigate mechanisms regulating tissue development and regeneration, such as cartilage. Treatment with transforming growth factor-beta (TGF-beta) superfamily members is a key requirement for the in vitro chondrogenic differentiation of MPCs. Intracellular signaling cascades, particularly those involving the mitogen-activated protein (MAP) kinases, p38, ERK-1, and JNK, have been shown to be activated by TGF-betas in promoting cartilage-specific gene expression. MPC chondrogenesis in vitro also requires high cell seeding density, reminiscent of the cellular condensation requirements for embryonic mesenchymal chondrogenesis, suggesting common chondro-regulatory mechanisms. Prompted by recent findings of the crucial role of the cell adhesion protein, N-cadherin, and Wnt signaling in condensation and chondrogenesis, we have examined here their involvement, as well as MAP kinase signaling, in TGF-beta1-induced chondrogenesis of trabecular bone-derived MPCs. Our results showed that TGF-beta1 treatment initiates and maintains chondrogenesis of MPCs through the differential chondro-stimulatory activities of p38, ERK-1, and to a lesser extent, JNK. This regulation of MPC chondrogenic differentiation by the MAP kinases involves the modulation of N-cadherin expression levels, thereby likely controlling condensation-like cell-cell interaction and progression to chondrogenic differentiation, by the sequential up-regulation and progressive down-regulation of N-cadherin. TGF-beta1-mediated MAP kinase activation also controls WNT-7A gene expression and Wnt-mediated signaling through the intracellular beta-catenin-TCF pathway, which likely regulates N-cadherin expression and subsequent N-cadherin-mediated cell-adhesion complexes during the early steps of MPC chondrogenesis.     

N-cadherin as a key regulator of collective cell migration in a 3D environment

Cell migration is a critical step of normal developmental processes and disease progression. Often, migrating cells interact and maintain contact with neighboring cells. However, the precise roles of cell-cell adhesion in cell migration have thus far been poorly defined. Often in aggressive cancers, N-cadherin is prominently upregulated, yet, these highly motile cells have limited cell-cell adhesion when plated on a stiff 2D substrate. But, the same cells in a 3D matrix migrate as a multicellular cluster. This new observation suggests that N-cadherin-mediated cell-cell adhesion supports cell interactions between migrating cells in a more physiologically relevant 3D matrix, but not on a 2D substrate. While N-cadherin is an integral part of neural synapses, the ectopic expression of N-cadherin in transformed epithelial cells plays an equally important part in initiating pro-migratory signaling, and providing strong yet flexible cell cohesion essential for persistent cell migration in a 3D matrix. The 3D cell migration analysis for studying cell-to-cell interactions exposes the roles of N-cadherin in multicellular migration, and reveals novel insights into cell migration-dependent normal and pathological processes.