调控骨骼形成的信号通路

骨骼形成后会处于不断的分解与重建中.通过骨骼形成与骨骼吸收之间的动态平衡来维持骨量.如果二者间的平衡被打破,骨吸收大于骨形成时,骨量会减少,骨骼微环境随之发生改变,脆性增加,进而引发骨质疏松、骨折等疾病.其中,骨骼形成是成骨细胞的重要功能.成骨细胞由间充质干细胞(mesenchymal stem cells,MSCs)...     

Membrane trafficking in osteoblasts and osteoclasts: new avenues for understanding and treating skeletal diseases

The endocytic and exocytic/secretory pathways are two major intracellular membrane trafficking routes that regulate numerous cellular functions in a variety of cell types. Osteoblasts and osteoclasts, two major bone cells responsible for bone remodeling and homeostasis, are no exceptions. During the past few years, emerging evidence has pinpointed a critical role for endocytic and secretory pathways in osteoblast and osteoclast differentiation and function. The endosomal membrane provides a platform to integrate bone tropic signals of hormones and growth factors in osteoblasts. In osteoclasts, endocytosis, followed by transcytosis, of degraded bone matrix promotes bone resorption. Secretory pathways, especially lysosome secretion, not only participate in bone matrix deposition by osteoblasts and degradation of mineralized bone matrix by osteoclasts; they may also be involved in the coupling of bone resorption and bone formation during bone remodeling. More importantly, mutations in genes encoding regulatory factors within the endocytic and secretory pathways have been identified as causes for bone diseases. Identification of the molecular mechanisms of these genes in bone cells may provide new therapeutic targets for skeletal disorders.     

Roles of sphingosine-1-phosphate (S1P) receptors in malignant behavior of glioma cells. Differential effects of S1P2 on cell migration and invasiveness

Sphingosine-1-phosphate (S1P) is a bioactive lipid that signals through a family of five G-protein-coupled receptors, termed S1P(1-5). S1P stimulates growth and invasiveness of glioma cells, and high expression levels of the enzyme that forms S1P, sphingosine kinase-1, correlate with short survival of glioma patients. In this study we examined the mechanism of S1P stimulation of glioma cell proliferation and invasion by either overexpressing or knocking down, by RNA interference, S1P receptor expression in glioma cell lines. S1P(1), S1P(2) and S1P(3) all contribute positively to S1P-stimulated glioma cell proliferation, with S1P(1) being the major contributor. Stimulation of glioma cell proliferation by these receptors correlated with activation of ERK MAP kinase. S1P(5) blocks glioma cell proliferation, and inhibits ERK activation. S1P(1) and S1P(3) enhance glioma cell migration and invasion. S1P(2) inhibits migration through Rho activation, Rho kinase signaling and stress fiber formation, but unexpectedly, enhances glioma cell invasiveness by stimulating cell adhesion. S1P(2) also potently enhances expression of the matricellular protein CCN1/Cyr61, which has been implicated in tumor cell adhesion, and invasion as well as tumor angiogenesis. A neutralizing antibody to CCN1 blocked S1P(2)-stimulated glioma invasion. Thus, while S1P(2) decreases glioma cell motility, it may enhance invasion through induction of proteins that modulate glioma cell interaction with the extracellular matrix.     

Osteoclast-derived activity in the coupling of bone formation to resorption

The cells of bone and the immune system communicate by means of soluble and membrane-bound cytokines and growth factors. Through local signalling mechanisms, cells of the osteoblast lineage control the formation and activity of osteoclasts and, therefore, the resorption of bone. Both T and B lymphocytes produce activators and inhibitors of osteoclast formation. A local 'coupling factor' linking bone resorption to subsequent formation in the bone multicellular unit (BMU) has long been proposed as the key regulator of the bone remodelling process, but never identified. There is evidence in support of the view that the coupling mechanism is dependent on growth factors released from the bone matrix during resorption, or is generated from maturing osteoblasts. We argue that osteoclasts contribute in important ways to the transiently activated osteoclast, and stimulate osteoblast lineage cells to begin replacing the resorbed bone in each BMU.     

Sphingosine‐1‐phosphate (S1P) receptors: Promising drug targets for treating bone‐related diseases

Sphingosine‐1‐phosphate (S1P) is a natural bioactive lipid molecule and a common first or second messenger in the cardiovascular and immune systems. By binding with its receptors, S1P can serve as mediator of signalling during cell migration, differentiation, proliferation and apoptosis. Although the predominant role of S1P in bone regeneration has been noted in many studies, this role is not as well‐known as its roles in the cardiovascular and immune systems. In this review, we summarize previous research on the role of S1P receptors (S1PRs) in osteoblasts and osteoclasts. In addition, S1P is regarded as a bridge between bone resorption and formation, which brings hope to patients with bone‐related diseases. Finally, we discuss S1P and its receptors as therapeutic targets for treating osteoporosis, inflammatory osteolysis and bone metastasis based on the biological effects of S1P in osteoclastic/osteoblastic cells, immune cells and tumour cells.     

Resorption Controls Bone Anabolism Driven by Parathyroid Hormone (PTH) Receptor Signaling in Osteocytes

The contribution of remodeling-based bone formation coupled to osteoclast activity versus modeling-based bone formation that occurs independently of resorption, to the anabolic effect of PTH remains unclear. We addressed this question using transgenic mice with activated PTH receptor signaling in osteocytes that exhibit increased bone mass and remodeling, recognized skeletal effects of PTH elevation. Direct inhibition of bone formation was accomplished genetically by overexpressing the Wnt antagonist Sost/sclerostin; and resorption-dependent bone formation was inhibited pharmacologically with the bisphosphonate alendronate. We found that bone formation induced by osteocytic PTH receptor signaling on the periosteal surface depends on Wnt signaling but not on resorption. In contrast, bone formation on the endocortical surface results from a combination of Wnt-driven increased osteoblast number and resorption-dependent osteoblast activity. Moreover, elevated osteoclasts and intracortical/calvarial porosity is exacerbated by overexpressing Sost and reversed by blocking resorption. Furthermore, increased cancellous bone is abolished by Wnt inhibition but further increased by blocking resorption. Thus, resorption induced by PTH receptor signaling in osteocytes is critical for full anabolism in cortical bone, but tempers bone gain in cancellous bone. Dissecting underlying mechanisms of PTH receptor signaling would allow targeting actions in different bone compartments, enhancing the therapeutic potential of the pathway.     

Coupling factors and exosomal packaging microRNAs involved in the regulation of bone remodelling

Bone remodelling is a continuous process by which bone resorption by osteoclasts is followed by bone formation by osteoblasts to maintain skeletal homeostasis. These two forces must be tightly coordinated not only quantitatively, but also in time and space, and its malfunction leads to diseases such as osteoporosis. Recent research focusing on the cross‐talk and coupling mechanisms associated with the sequential recruitment of osteoblasts to areas where osteoclasts have removed bone matrix have identified a number of osteogenic factors produced by the osteoclasts themselves. Osteoclast‐derived factors and exosomal‐containing microRNA (miRNA) can either enhance or inhibit osteoblast differentiation through paracrine and juxtacrine mechanisms, and therefore may have a central coupling role in bone formation. Entwined with angiocrine factors released by vessel‐specific endothelial cells and perivascular cells or pericytes, these factors play a critical role in angiogenesis–osteogenesis coupling essential in bone remodelling.     

Sphingosine-1-phosphate signaling controlling osteoclasts and bone homeostasis

Bone is a dynamic organ that is continuously turned over during growth, even in adults. During bone remodeling, homeostasis is regulated by the balance between bone formation by osteoblasts and bone resorption by osteoclasts. However, in pathological conditions such as osteoporosis, osteopetrosis, arthritic joint destruction, and bone metastasis, this equilibrium is disrupted. Since osteoclasts are excessively activated in osteolytic diseases, the inhibition of osteoclast function has been a major therapeutic strategy. It has recently been demonstrated that sphingosine-1-phosphate (S1P), a biologically active lysophospholipid that is enriched in blood, controls the trafficking of osteoclast precursors between the circulation and bone marrow cavities via G protein-coupled receptors, S1PRs. While S1PR1 mediates chemoattraction toward S1P in bone marrow, where S1P concentration is low, S1PR2 mediates chemorepulsion in blood, where the S1P concentration is high. The regulation of precursor recruitment may represent a novel therapeutic strategy for controlling osteoclast-dependent bone remodeling. By means of intravital multiphoton imaging of bone tissues, we have recently revealed that the reciprocal action of S1P controls the migration of osteoclast precursors between bone tissues and blood stream. Imaging technologies have enabled us to visualize the in situ behaviors of different cell types in intact tissues. In this review we also discuss future perspectives on this new method in the field of bone biology and medical sciences in general. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.     

Memo Has a Novel Role in S1P Signaling and Crucial for Vascular Development

Memo is a conserved protein that was identified as an essential mediator of tumor cell motility induced by receptor tyrosine kinase activation. Here we show that Memo null mouse embryonic fibroblasts (MEFs) are impaired in PDGF-induced migration and this is due to a defect in sphingosine-1-phosphate (S1P) signaling. S1P is a bioactive phospholipid produced in response to multiple stimuli, which regulates many cellular processes. S1P is secreted to the extracellular milieu where it exerts its function by binding a family of G-protein coupled receptors (S1PRs), causing their activation in an autocrine or paracrine manner. The process, termed cell-autonomous S1PR signaling, plays a role in survival and migration. Indeed, PDGF uses cell-autonomous S1PR signaling to promote cell migration; we show here that this S1P pathway requires Memo. Using vascular endothelial cells (HUVECs) with Memo knock-down we show that their survival in conditions of serum-starvation is impaired. Furthermore, Memo loss in HUVECs causes a reduction of junctional VE-cadherin and an increase in sprout formation. Each of these phenotypes is rescued by S1P or S1P agonist addition, showing that Memo also plays an important role in cell-autonomous S1PR signaling in endothelial cells. We also produced conventional and endothelial cell-specific conditional Memo knock-out mouse strains and show that Memo is essential for embryonic development. Starting at E13.5 embryos of both strains display bleeding and other vascular problems, some of the phenotypes that have been described in mouse strains lacking S1PRs. The essential role of Memo in embryonic vascular development may be due in part to alterations in S1P signaling. Taken together our results show that Memo has a novel role in the S1P pathway and that Memo is needed to promote cell-autonomous S1PR activation.     

Calcineurin/NFAT signaling in osteoblasts regulates bone mass

Development and repair of the vertebrate skeleton requires the precise coordination of bone-forming osteoblasts and bone-resorbing osteoclasts. In diseases such as osteoporosis, bone resorption dominates over bone formation, suggesting a failure to harmonize osteoclast and osteoblast function. Here, we show that mice expressing a constitutively nuclear NFATc1 variant (NFATc1(nuc)) in osteoblasts develop high bone mass. NFATc1(nuc) mice have massive osteoblast overgrowth, enhanced osteoblast proliferation, and coordinated changes in the expression of Wnt signaling components. In contrast, viable NFATc1-deficient mice have defects in skull bone formation in addition to impaired osteoclast development. NFATc1(nuc) mice have increased osteoclastogenesis despite normal levels of RANKL and OPG, indicating that an additional NFAT-regulated mechanism influences osteoclastogenesis in vivo. Calcineurin/NFATc signaling in osteoblasts controls the expression of chemoattractants that attract monocytic osteoclast precursors, thereby coupling bone formation and bone resorption. Our results indicate that NFATc1 regulates bone mass by functioning in both osteoblasts and osteoclasts.     

Fingolimod inhibits PDGF-B-induced migration of vascular smooth muscle cell by down-regulating the S1PR1/S1PR3 pathway

Platelet Derived Growth Factor (PDGF) and sphingosine-1-phosphate (S1P) pathways play a key role in mural cell recruitment during tumor growth and angiogenesis. Fingolimod, a S1P analogue, has been shown to exert antitumor and antiangiogenic properties. However, molecular targets and modes of action of fingolimod remain unclear. In this study, we confirmed the antagonizing action of S1P and PDGF-B on rat vascular smooth muscle cell (VSMCs) growth and migration. We then compared siRNA and/or fingolimod (100 nM) treatments on PDGFR-β, S1PR1 S1PR2 and S1PR3 expression. Fingolimod induced a 50% reduction in S1PR3 protein expression which was cumulative with that obtained with anti-S1PR3 siRNA. We found that siRNA-induced inhibition of both PDGFR-β and S1PR3 was the most effective means to block VSMC migration induced by PDGF-B. Finally, we observed that fingolimod treatment associated with anti-S1PR1 siRNA principally inhibited VSMC growth while in combination with anti-S1PR3 siRNA it strongly inhibited VSMC migration. These results suggest that for rat VSMCs, the PDGFR-S1PR1 pathway is predominantly dedicated to cell growth while PDGFR-S1PR3 stimulates cell migration. As an S1P analogue, fingolimod is considered a potent activator of S1PR1 and S1PR3. However, its action on the PDGFR-S1PR platform appears to be dependent on S1PR1 and S1PR3 specific downregulation. Considering that the S1P pathway has already been shown to exert various crosstalks with tyrosine kinase pathways, it seems of great interest to evaluate fingolimod potential in combination with the numerous tyrosine kinase inhibitors used in oncology.     

Sphingosine 1-Phosphate (S1P) Receptor Agonists Mediate Pro-fibrotic Responses in Normal Human Lung Fibroblasts via S1P2 and S1P3 Receptors and Smad-independent Signaling

Synthetic sphingosine 1-phosphate receptor 1 modulators constitute a new class of drugs for the treatment of autoimmune diseases. Sphingosine 1-phosphate (S1P) signaling, however, is also involved in the development of fibrosis. Using normal human lung fibroblasts, we investigated the induction of fibrotic responses by the S1P receptor (S1PR) agonists S1P, FTY720-P, ponesimod, and SEW2871 and compared them with the responses induced by the known fibrotic mediator TGF-β1. In contrast to TGF-β1, S1PR agonists did not induce expression of the myofibroblast marker α-smooth muscle actin. However, TGF-β1, S1P, and FTY720-P caused robust stimulation of extracellular matrix (ECM) synthesis and increased pro-fibrotic marker gene expression including connective tissue growth factor. Ponesimod showed limited and SEW2871 showed no pro-fibrotic potential in these readouts. Analysis of pro-fibrotic signaling pathways showed that in contrast to TGF-β1, S1PR agonists did not activate Smad2/3 signaling but rather activated PI3K/Akt and ERK1/2 signaling to induce ECM synthesis. The strong induction of ECM synthesis by the nonselective agonists S1P and FTY720-P was due to the stimulation of S1P₂ and S1P₃ receptors, whereas the weaker induction of ECM synthesis at high concentrations of ponesimod was due to a low potency activation of S1P₃ receptors. Finally, in normal human lung fibroblast-derived myofibroblasts that were generated by TGF-β1 pretreatment, S1P and FTY720-P were effective stimulators of ECM synthesis, whereas ponesimod was inactive, because of the down-regulation of S1P₃R expression in myofibroblasts. These data demonstrate that S1PR agonists are pro-fibrotic via S1P₂R and S1P₃R stimulation using Smad-independent pathways.     

Cell-specific paracrine actions of IL-6 family cytokines from bone,marrow and muscle that control bone formation and resorption

Bone renews itself and changes shape throughout life to account for the changing needs of the body; this requires co-ordinated activities of bone resorbing cells (osteoclasts), bone forming cells (osteoblasts) and bone’s internal cellular network (osteocytes). This review focuses on paracrine signaling by the IL-6 family of cytokines between bone cells, bone marrow, and skeletal muscle in normal physiology and in pathological states where their levels may be locally or systemically elevated. These functions include the support of osteoclast formation by osteoblast lineage cells in response to interleukin 6 (IL-6), interleukin 11 (IL-11), oncostatin M (OSM) and cardiotrophin 1 (CT-1). In addition it will discuss how bone-resorbing osteoclasts promote osteoblast activity by secreting CT-1, which acts as a “coupling factor” on osteocytes, osteoblasts, and their precursors to promote bone formation. OSM, produced by osteoblast lineage cells and macrophages, stimulates bone formation via osteocytes. IL-6 family cytokines also mediate actions of other bone formation stimuli like parathyroid hormone (PTH) and mechanical loading. CT-1, OSM and LIF suppress marrow adipogenesis by shifting commitment of pluripotent precursors towards osteoblast differentiation. Ciliary neurotrophic factor (CNTF) is released as a myokine from skeletal muscle and suppresses osteoblast differentiation and bone formation on the periosteum (outer bone surface in apposition to muscle). Finally, IL-6 acts directly on marrow-derived osteoclasts to stimulate release of “osteotransmitters” that act through the cortical osteocyte network to stimulate bone formation on the periosteum. Each will be discussed as illustrations of how the extended family of IL-6 cytokines acts within the skeleton in physiology and may be altered in pathological conditions or by targeted therapies.     

Follicular fluid high-density lipoprotein-associated sphingosine 1-phosphate (S1P) promotes human granulosa lutein cell migration via S1P receptor type 3 and small G-protein RAC1

Coordinated migration and progesterone production by granulosa cells is critical to the development of the corpus luteum, but the underlying mechanisms remain obscure. Sphingosine 1-phosphate (S1P), which is associated with follicular fluid high-density lipoprotein (FF-HDL), was previously shown to regulate ovarian angiogenesis. We herein examined the effects of S1P and FF-HDL on the function of granulosa lutein cells. Both FF-HDL and S1P induced migration of primary human granulosa lutein cells (hGCs) and the granulosa lutein cell line HGL5. In addition, FF-HDL but not S1P promoted progesterone synthesis, and neither of the two compounds stimulated proliferation of granulosa lutein cells. Polymerase chain reaction and Western blot experiments demonstrated the expression of S1P receptor type 1 (S1PR1), S1PR2, S1PR3, and S1PR5 but not S1PR4 in hGCs and HGL5 cells. The FF-HDL- and S1P-induced granulosa lutein cell migration was emulated by FTY720, an agonist of S1PR1, S1PR3, S1PR4, and S1PR5, and by VPC24191, an agonist of S1PR1 and S1PR3, but not by SEW2871 and phytosphingosine 1-phosphate, agonists of S1PR1 and S1PR4, respectively. In addition, blockade of S1PR3 with CAY1044, suramine, or pertussis toxin inhibited hGC and HGL5 cell migration toward FF-HDL or S1P, while blockade of S1PR1 and S1PR2 with W146 and JTE013, respectively, had no effect. Both FF-HDL and S1P triggered activation of small G-protein RAC1 and actin polymerization in granulosa cells, and RAC1 inhibition with Clostridium difficile toxin B or NSC23766 abolished FF-HDL- and S1P-induced migration. The FF-HDL-associated S1P promotes granulosa lutein cell migration via S1PR3 and RAC1 activation. This may represent a novel mechanism contributing to the development of the corpus luteum.     

High Density Lipoprotein Stimulated Migration of Macrophages Depends on the Scavenger Receptor Class B,Type I,PDZK1 and Akt1 and Is Blocked by Sphingosine 1 Phosphate Receptor Antagonists

HDL carries biologically active lipids such as sphingosine-1-phosphate (S1P) and stimulates a variety of cell signaling pathways in diverse cell types, which may contribute to its ability to protect against atherosclerosis. HDL and sphingosine-1-phosphate receptor agonists, FTY720 and SEW2871 triggered macrophage migration. HDL-, but not FTY720-stimulated migration was inhibited by an antibody against the HDL receptor, SR-BI, and an inhibitor of SR-BI mediated lipid transfer. HDL and FTY720-stimulated migration was also inhibited in macrophages lacking either SR-BI or PDZK1, an adaptor protein that binds to SR-BI''s C-terminal cytoplasmic tail. Migration in response to HDL and S1P receptor agonists was inhibited by treatment of macrophages with sphingosine-1-phosphate receptor type 1 (S1PR1) antagonists and by pertussis toxin. S1PR1 activates signaling pathways including PI3K-Akt, PKC, p38 MAPK, ERK1/2 and Rho kinases. Using selective inhibitors or macrophages from gene targeted mice, we demonstrated the involvement of each of these pathways in HDL-dependent macrophage migration. These data suggest that HDL stimulates the migration of macrophages in a manner that requires the activities of the HDL receptor SR-BI as well as S1PR1 activity.     

Phorbaketal A stimulates osteoblast differentiation through TAZ mediated Runx2 activation

Osteoporosis arises from an imbalance between osteoblastic bone formation and osteoclastic bone resorption. In this study, we screened molecules from marine natural products that stimulate osteoblast differentiation. We found that phorbaketal A significantly stimulates osteoblast differentiation in mesenchymal cells. Increased interaction of TAZ and Runx2 stimulated phorbaketal A-induced expression of osteoblastic marker genes. The activation of ERK was important for the stimulation of differentiation because an inhibitor of ERK blocked phorbaketal A-induced osteogenic differentiation. Taken together, the results showed that phorbaketal A stimulates TAZ-mediated osteoblast differentiation through the activation of ERK.     

Consequences of daily administered parathyroid hormone on myeloma growth, bone disease, and molecular profiling of whole myelomatous bone

Background

Induction of osteolytic bone lesions in multiple myeloma is caused by an uncoupling of osteoclastic bone resorption and osteoblastic bone formation. Current management of myeloma bone disease is limited to the use of antiresorptive agents such as bisphosphonates.

Methodology/Principal Findings

We tested the effects of daily administered parathyroid hormone (PTH) on bone disease and myeloma growth, and we investigated molecular mechanisms by analyzing gene expression profiles of unique myeloma cell lines and primary myeloma cells engrafted in SCID-rab and SCID-hu mouse models. PTH resulted in increased bone mineral density of myelomatous bones and reduced tumor burden, which reflected the dependence of primary myeloma cells on the bone marrow microenvironment. Treatment with PTH also increased bone mineral density of uninvolved murine bones in myelomatous hosts and bone mineral density of implanted human bones in nonmyelomatous hosts. In myelomatous bone, PTH markedly increased the number of osteoblasts and bone-formation parameters, and the number of osteoclasts was unaffected or moderately reduced. Pretreatment with PTH before injecting myeloma cells increased bone mineral density of the implanted bone and delayed tumor progression. Human global gene expression profiling of myelomatous bones from SCID-hu mice treated with PTH or saline revealed activation of multiple distinct pathways involved in bone formation and coupling; involvement of Wnt signaling was prominent. Treatment with PTH also downregulated markers typically expressed by osteoclasts and myeloma cells, and altered expression of genes that control oxidative stress and inflammation. PTH receptors were not expressed by myeloma cells, and PTH had no effect on myeloma cell growth in vitro.

Conclusions/Significance

We conclude that PTH-induced bone formation in myelomatous bones is mediated by activation of multiple signaling pathways involved in osteoblastogenesis and attenuated bone resorption and myeloma growth; mechanisms involve increased osteoblast production of anti-myeloma factors and minimized myeloma induction of inflammatory conditions.