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The multiple facets of periostin in bone metabolism
B. Merle & P. Garnero
Received: 18 October 2011 / Accepted: 22 December 2011 # International Osteoporosis Foundation and National Osteoporosis Foundation 2012
Abstract Periostin is a matricellular glutamate-containing protein expressed during ontogenesis and in adult connective tissues submitted to mechanical strains including bone and, more specifically, the periosteum, periodontal ligaments, tendons, heart valves, or skin. It is also expressed in neoplastic tissues, cardiovascular and fibrotic diseases, and during wound repair. Its biological functions are extensively investigated in fields such as cardiovascular physiology or oncology. Despite its initial identification in bone, investigations of periostin functions in bone-related physiopathology are less abundant. Recently, several studies have analyzed the potential role of periostin in bone biology and suggest that periostin may be an important regulator of bone formation. The aim of this article is to provide an extensive review on the implications of periostin in bone biology and its potential use in benign and metabolic bone diseases. Keywords Bone quality . Bone markers . Bone metabolism . Bone metastasis . Periosteum Abbreviations βig-H3 protein
BMP CRS dpc DPD DHLNL ECM FAK FGF Gla Glu HLNL LOX PDL PDGF PHPT PLF PTH PTHrP PYD TNFα TRAP5b
transforming growth factor-βinducible protein
bone morphogenetic protein carboxylase recognition sites days postconception deoxypyridinoline dihydroxylysinonorleucine extracellular matrix focal adhesion kinase basic fibroblast growth factors γ-carboxyglutamate glutamate hydroxylysinonorleucine lysyl oxidase periodontal ligament platelet-derived growth factor primary hyperparathyroidism periostin-like factor parathyroid hormone parathyroid hormone-related peptide pyridinoline tumor necrosis factor α tartrate resistant acid phosphatase
B. Merle (*) : P. Garnero INSERM Research Unit 1033, Pavillon F, Hopital E. Herriot, Place d'Arsonval, 69437 Lyon cédex 03, France e-mail: firstname.lastname@example.com B. Merle : P. Garnero INSERM U 1033 and Universite Lyon 1, Lyon, France P. Garnero Cisbio Bioassays, Codolet, France
Introduction Originally known as osteoblast-specific factor, periostin was first identified in a mouse osteoblastic cell line as a putative cell adhesion protein for preosteoblasts [1]. It was then renamed periostin due to its preferential location in the periosteum. Periosteum covers the outer surface of bone and contributes to intramembranous bone embryogenesis and growth in diameter of bone. Although periosteum is particularly active during growth, it also plays a crucial role in adults
4 repeated and conserved FAS1 domains. 17]. chicken. which present themselves as six cassette exons.187 bp long and contains an 18-bp 5′ untranslated region. its preferential expression in collagenrich tissues submitted to mechanical stress such as periosteum from development to perinatal life. thus. periostin belongs to the fasciclin family and shows a structure similar to the insect axon guidance fasciclin and Transforming growth factor-β-inducible protein (βig-H3) involved in cell attachment [14]. also known as periostin-like factor. The presence of integrin binding motifs in the second and fourth FAS-1 domains suggests that periostin is implicated in cell adhesion. Due to the presence of FAS-1 domains. the four FAS-1 domains. giving rise to different isoforms [3]. Periostin also possesses four putative N-glycosylation sites and a heparin-binding domain (arginine-rich consensus sequence) at its C-terminal end creating a potential binding site for glycoproteins. The protein is composed of a signal sequence. specific roles of certain isoforms have been reported in in vitro cell invasiveness according to their differential expression patterns [19. Its expression during development prevails over that in the adult organism [7–9]. Periostin expression is not restricted to bone as it is predominantly expressed in collagen-rich fibrous connective tissues subjected to constant mechanical stress. Even though this C-terminal hydrophilic region is devoided of known protein domains. and proteoglycans [3. However. The periostin isoforms seem to be differentially expressed during development or following stress such as infarct or neoplasm [4. 1 Periostin protein sequence showing the signal sequence. 19]. which has been localized in the nucleus and the cytosol of cells also bears a signal sequence and is secreted. In addition. and tendons [6]. cassette e is missing [9]. periostin is reexpressed in adult rats after myocardial. whereas in isoform 3. suggests that periostin isoforms can have intracellular and extracellular functions as recently reviewed [20]. rat. Periostin isoform 1 contains the six cassettes. Periostin expression is also increased in a large variety of tumors including colon. as these domains have been shown to mediate βig-H3 adhesion to α3β1 [13]. The aim of this review is to discuss the role of periostin in bone metabolism. 9]. 11]. These cassettes can be present or deleted in mature mRNA in various combinations. a to f.Osteoporos Int as it contributes to the determination of bone diameter and. vascular. and an open reading frame of 2. Periostin up-regulation usually correlates with aggressiveness and/or poor survival [12]. Structure of periostin Periostin gene has been cloned in several species (mouse. periostin can also form disulfide-bonded dimers through its EMI domains [16. Additionally. suggests that it may play a crucial role in bone maintenance and regeneration. glycosaminoglycans. The mouse periostin cDNA is 3. However. bladder. The predicted amino acid sequence analysis of the C-terminal end identified a nuclear localization sequence implying that some isoforms will be localized in the nucleus. followed by an Emilin-like (EMI) domain rich in cysteine. or CRS) responsible for the post-translational modification of glutamic residues (Glu) to γ-carboxyglutamate (Gla). etc. specific of a tissue type.) and is located at locus 13q13. bovine. as it has been shown for isoform 3 [8. Alternative splicing of the C-terminal domain gives rise to at least five different human isoforms [1. A large body of information about periostin functions comes from investigations of cardiac development or tumor expansion. and a C-terminal hydrophilic and variable domain. 21].2% homology. bone strength. 13]. according to the denomination established by Horiuchi and colleagues [3]. head and neck. non-small cell lung cancer. 5]. heart valves [2. 9. oral. a variant lacking both b and e cassettes has been described to be preferentially expressed in the periosteum and PDL as well as in heart tissues after myocardial infarction [20]. The C-terminal part of periostin is encoded by exons 15 to 21. each FAS-1 domain is rich in glutamate residues and contains an N-terminal recognition site for the vitamin K-dependent enzyme γ-glutamyl carboxylase (γ-carboxylase recognition sites. xenopus. and the C-terminal variable region with the six different cassettes (A–F) whose combination gives rise to different isoforms. periostin is highly conserved between human and mouse with 89. Originally cloned from both human placental and osteosarcoma tissue. and skeletal muscle injuries and bone fracture [10. the EMI domain. 8. The fact that periostin. a development stage or a disease. as well as up-regulation during fracture healing. a 733-bp 3′ untranslated region. breast.3 in human and 3C in mouse. Each FAS-1 domain contains an N-terminal recognition site for the γ-glutamyl carboxylase and a cell adhesion site . An additional variability caused by single nucleotide polymorphism was also reported National Center for Biotechnology Information (NCBI database). Finally. 1).436 bp corresponding to a protein precursor of 838 amino acids (Fig. 15]. such as periodontal ligament (PDL) [2–4]. Fig. 18. Alternative splicing of periostin gives rise to different isoforms. and pancreas.
25]. it is of interest to report the expression of periostin in fibroblast-like synoviocytes from rheumatoid arthritis [30] and in cartilage biopsies from osteoarthritis patients [31]. periostin mRNA was detected as soon as 9. at least in part. 27]. Periosteal apposition occurs throughout life: it is high during growth and decreases in adult life. a tri-dimensional structural model for periostin has been proposed: the four FAS domains of human periostin have a secondary structure consisting of a helix-turn-helix motif. As in bone. and osterix pathways are essential in the commitment of pluripotent mesenchymal cells to the . articular cartilage. 24. which develop into osteoblasts. Furthermore. that is. 16. in dental pulp at the sites of hard/soft tissue interfaces in mouse and human tooth [32. they should. or lining cell of the underlying bone [26]. osteoclasts.5 dpc with an expression more or less important over time [4. young. 9. 8]. isoform 3 showed both nuclear and cytoplasmic expressions in accordance with the presence of a nuclear localization sequence. a highly specialized connective tissue that links teeth to the alveolar bone. they will miss variants lacking cassettes b and e. The concomitant stimulation of periostin and inhibition of mineralization by activin A and the presence of ectopic mineral deposit on bone in periostin KO mice strengthen the association of periostin with the early stages of osteoblast differentiation and bone formation [16. immunohistochemistry on human bone with an antibody recognizing the N-terminal part of periostin common to all variants revealed that periostin was largely expressed in the extracellular matrix (ECM) of the cambial layer of the periosteum of long bones and calvaria. 28]. 20]. especially in cancer in which periostin is largely implicated. Fetal development of the skeleton implies two processes: intramembranous ossification involves mesenchymal stem cells.5 days postconception (dpc) in mouse embryos. osteocytes.5 dpc and all along the differentiation process. a cartilage template first formed by chondrocytes is then replaced by bone. and adult rodents bone [3. which covers a large majority of bones. 13. in neonates. In situ hybridization and immunohistochemistry showed that periostin is expressed in the PDL. Additionally. In adult. Beyond its crucial importance during embryogenesis. and osteocytes in a time and spatial localization pattern [24. these antibodies may detect more than one isoform and miss variants lacking b and e cassettes. At 16. As in embryonic bone. periostin is secreted. Wnt/β-catenin.Osteoporos Int Finally. preosteoblasts and osteoblasts of the periosteum and in the osteoblasts lining trabecular bone [3. Similarly. Among the transcription factors involved in osteoblast differentiation. whereas in endochondral ossification. Periostin is secreted by fibroblasts and osteoblasts in PDL. whereas variant 2 was only found in cytoplasm [9]. By contrast. 9]. As mentioned earlier. have shown that many factors can modify periostin expression. Periostin protein (at least isoform 3) was detected in mesenchymal cells and alkaline phosphatase expressing cells. but to a lesser extent. Finally. and bone size is related to bone strength. multiple protein variants of periostin are expressed. In developing bone. expression of both periostin variants was induced by chronic overload in the cellular periosteum. Periostin is expressed in bone and teeth Periostin is preferentially expressed in the periosteum. suggesting different roles for these variant proteins during osteogenesis. immunolocalization showed that periostin isoforms 2 and 3 were localized to proliferating and hypertrophied chondrocytes from 12. Additionally. using two antibodies recognizing isoform 2 or 3. it is reexpressed after mechanical stress and fracture where bone formation is important. Overall. these data suggest that periostin is preferentially expressed in the periosteum at a high level during embryogenesis and bone growth. Overall. it is noticeably absent from terminally differentiated mineralized tissues (dentin.5 dpc.5 dpc [4. Periosteum is responsible for changes in bone diameter and cortical thickness. These observations are consistent with the inhibition of periostin expression by inorganic phosphate as described previously [29]. described to be expressed in periosteum and PDL [8. as the 2 antibodies used in this study have been raised against a peptide arising from cassette b or e to recognize isoform 3 or isoform 2. while the C-terminal region presents beta-strands [1. in principle. Periostin mRNA was detected in periosteum cells but not in osteoblasts. periosteum is largely involved in fracture healing [23]. 22]. one group showed that these isoforms were not expressed in the periosteum nor epiphyseal plate of adult normal rat bone [27]. 24]. alveolar bone) [32]. also detect all the isoforms bearing one of these cassettes. respectively. isoform 3 expression appeared in mesenchymal preosteoblasts cells from the periosteum and in ameloblasts and odontoblasts in developing teeth. in the periosteum as in the infarcted myocardium. and in the PDL of the teeth. Runx2/cbfa1. as a cleaved form lacking its C-terminal domain [16]. osteoblasts. Further analyses are needed to localize and characterize all the isoforms to have a more complete view of the periostin functions. whereas isoform 2 was primarily localized in the mesenchymal cells of the perichondrium till 19. However. During embryogenesis. In the developing long bone. 33]. at least four protein isoforms could be detected from 12 to 19. these data show that there is a spatial and temporal expression and localization of periostin isoforms. However. Periostin expression is transcriptionally regulated A number of studies.
a potent antagonist of bone formation through inhibition of Wnt and BMP signaling. at least in pathological conditions resulting in altered collagen fibrillogenesis and deposition.Osteoporos Int osteoblastic lineage [34]. and retinoic acid all stimulate periostin expression in osteoblasts [3. inhibits periostin expression while stimulating mineralization and transition from osteoblasts into preosteocytes [50]. 54]. associated with periostin up-regulation [38]. 28. Additionally. 38. These results are in agreement with in vivo data. Twist-1 and periostin are coexpressed by differentiating osteoblasts and fibroblasts at the osteogenic front of calvaria and at the alveolar bone surfaces in PDL [32. whereas normal osteoblasts did not. periostin expression may be directly up-regulated by PTH treatment in mouse calvaria osteoblasts [45]. there is a reduction of twist-1 level with an increase in the ratio of twist-1 homodimers versus heterodimers. Finally. 47]. whereas heterodimers binding will lead to inhibition of periostin expression and osteoblast differentiation [38. suggesting a local regulation loop [45]. In human PDL cells culture.or BMP-2-induced actions suggesting a possible different regulation of periostin [25]. Blocking TGF-β receptor activation or the noncanonical focal adhesion kinase (FAK) pathway reduced periostin mRNA expression. leading to an upregulation of periostin expression and a premature osteoblast differentiation and sutures closure [38]. and Wnt-dependent signaling pathways. bone morphogenetic protein (BMP)-. In craniosynostosis characterized by premature closure of cranial sutures due to twist-1 haploinsufficiency. BMP-2. Wnt-3. It may result in a switch from inhibition of osteoblast differentiation to induction. 39]. Additionally. suggesting that c-Fos pathway might represent one mechanism for periostin up-regulation. an osteocytespecific protein [46. suggesting its involvement in early bone cell differentiation [35]. transgenic mice overexpressing cFos develop sclerotic lesions with transformed osteoblasts expressing high levels of periostin. has not yet been demonstrated in bone [36]. a factor involved in the commitment of stem cells to preosteoblasts. estrogens were reported to enhance cell proliferation and periostin mRNA expression through binding to estrogen receptor B. Twist-1 homodimers can bind a response element in the 5′ flanking region of the periostin promoter and up-regulate its transcription in undifferentiated osteoblasts. Leptin. PTH can stimulate osteoblastogenesis through inhibition of sclerostin. an adipocyte-derived hormone. TGF-β. it is possible that PTH effect on periostin is mediated through one of these pathways [25]. 33. periostin knockdown induced a significant reduction of PTHrP mRNA and protein expression by osteoblasts in vitro. activin. These data suggest that there is a dynamic regulation of twist-1 dimers expression at the osteogenic front. Twist-1 is another transcription factor that has been shown to play both positive and negative roles in cell differentiation. FAK/src inhibition in human PDL fibroblasts reduces nuclear translocation of twist and periostin mRNA levels [33]. 52]. 39]. Both PTH and estrogen seem also to protect undifferentiated periosteal cells from apoptosis [25]. By contrast. leading to twist-1 translocation to the nucleus and subsequent periostin activation [33. Among growth factors and cytokines. which controls body weight through its effects on food intake and energy expenditure. As it has been shown that intermittent PTH promotes periosteal cells differentiation and early progenitors proliferation in vitro and in vivo through ERK-. 47]. expressed high levels of periostin [26]. and mineralized nodules formation [48]. Periostin expression is regulated by hormones and growth factors Intermittent injections of parathyroid hormone (1-34) (PTH) and sex steroids are both anabolic agents that preferentially target periosteum in bone [41. Others showed that estrogens promote undifferentiated periosteal cell proliferation but inhibit their differentiation in young rodents by attenuating PTH. FAK activates signaling molecules. is also down-regulated by periostin [46. Similarly. 44]. associated with increased expression of c-Fos. TGF-β and BMP are well-known regulators of bone development by stimulating the differentiation of osteoblast progenitors [51. occlusal hypofunction in mouse PDL induced a significant but transient decrease in twist-1 and periostin expression [32]. Sclerostin. In MC3T3-E1 osteoblast-like cells overexpressing Runx2. showing that estrogens inhibit periosteal bone growth while being anabolic on endosteal and trabecular bone [49]. All these data suggest that PTH may influence osteoblast synthesis and subsequent bone remodeling through periostin via different ways. periostin was positively regulated. Two potential binding sites for c-Fos/AP1 have been described in the periostin promoter [26]. periostin expression was shown to be regulated by several members of the TGF-β superfamily. osteocalcin production. c-Fos/AP-1 is a transcription factor that plays an important role in bone cell proliferation and differentiation [40]. These data demonstrate that TGF-β can regulate periostin expression through activation of TGF-β receptors but also through FAK/src signaling pathway. 53. maybe as an adaptation to environmental changes. was shown to downregulate periostin expression in an epithelial cell model [37]. but also to stimulate osteoblastic differentiation by increasing alkaline phosphatase activity. The fact that activin and TGF-β inhibit mineralization and that periostin . In vitro. One of this pathway. Periostin regulation by Wnt pathways. Gene expression profiles studies on PHPT patients and rodents submitted to continuous infusion of PTH show increased bone turnover associated with higher periostin mRNA levels [43. 39]. Human bone tissues from patients with fibrous dysplasia. critical factors of bone mass regulation required in response to mechanical loading. 42].
strengthening the association of periostin with the early stages of osteoblast differentiation and bone formation [28. studied in bone. To increase periostin expression in response to an environmental stress may represent an adaptative cell strategy to maintain cell survival. to regulate cell migration and survival [26. suggesting that periostin could recruit and attach osteoblasts to bone matrix. These data confirm that periostin is expressed early in osteoblast and is involved in the differentiation process. 60. basic fibroblast growth factors (FGF-1 and FGF-2). and angiotensin II in cancer cell lines. Indeed. and matrix formation [18. because PDGF and FGF have been shown to stimulate bone formation in vivo. elevation of phosphate as mineralization begins leads to a down-regulation of periostin gene expression in vitro. Ras/ MEK. and metastasis [62–65]. interleukin (IL)-4 and IL-13. studies on cardiac and cancer cells have extensively reported that periostin can bind through its FAS-1 domains to the integrins αvβ3.e. 61]. Periostin was shown to be up-regulated by plateletderived growth factor (PDGF). we can speculate that they also act. leading to inhibition of cell death. at least in part. which induce migration. 29]. especially in stress conditions. 67]. have been shown to induce periostin in an in vivo mouse model of fibrosis [17]. 59]. possibly through integrin binding. Table 1 Factors involved in positive or negative regulation of periostin expression Regulation Transcription factors Positive Twist Runx-2 C-Fos/AP1 Hormones. Finally. A reduced expression of Notch1 and its downstream effector Bcl-xl has been described in PDL and femur of periostin null mice compared to normal mice [56. Periostin and αvβ3 are concomitantly expressed in human bone tissue. 62. two antiinflammatory cytokines that inhibit osteoblast proliferation but enhance their differentiation and matrix production. periostin up-regulates expression of Notch and Bcl-xl. through different pathways including RTK/PI3K. αvβ5. Rho/PI3-kinase. 69]. All these mechanisms are particularly important in periostin-induced response to environmental stress (i. However..Osteoporos Int expression is negatively regulated as mineralization proceeds suggests that TGF-β and activin may control the onset of mineralization. transducing periostin signals is also achieved by nonintegrin receptors. it is interesting to note that. inorganic phosphate can affect cell functions and gene expression. at least αvβ3 and αvβ5. However. cytokines. However. proliferation. differentiation. and Akt/PKB signaling pathways. one of most potent prosurvival signaling pathways that has been demonstrated in UMR-106 osteoblast-like cells. Besides its function in the formation of hydroxyapatite. so far. as βig-H3. In bone. Cytokine regulation has not been. 56. and Ras/p38MAPK [54–57]. 68. Tumor necrosis factor α increased periostin expression levels by osteoblasts from adult rat bones submitted to chronic overload [59]. which have been shown to be implicated in βig-H3 adhesion to α3β1 expressing corneal epithelial cells [13]. which may mediate periostin signaling in bone. 65. 64–66]. However. migration. Notch proteins are transmembrane receptors that affect osteoblast differentiation and play important roles in anti-cell death function under mechanical stress [52]. via the up-regulation of periostin expression [28]. and survival In MC3T3 and primary rat osteoblasts. periostin inhibition abrogated cell proliferation and differentiation and reduced the expression of cbfa1 [9. through periostin regulation [58]. at least in part. All these data demonstrate that periostin is regulated by factors acting on preosteoblasts rather than on fully differentiated cells and underlines its importance in cell proliferation and early stages of osteoblast differentiation (Table 1). Downstream effectors of periostin binding to integrins include FAK. Although the mechanisms involved in periostin/integrin binding in bone have not been analyzed in details. . periostin binding to αvβ3 may also activate the downstream FAK and Akt/PKB pathway. mechanical loading and hypoxia) in order to maintain cell survival and functions in bone [54–56. osteoblasts and osteoclasts express different integrins. Their effect on periostin expression has not yet been studied in bone. and growth factors PTH Estrogens TGF-β Activin BMP-2 Retinoic acid IL-4 IL-13 TNFα PDGF FGF Angiotensin Physicochemical factors Mechanical stress Hypoxia Phosphate Microgravity Leptin Negative Wnt Periostin promotes osteoblast adhesion. periostin contains dimers of Asp and Ile in its second and fourth FAS-1 domains. By binding to Notch. and α6β4 and enhance cell proliferation and survival.
tendons. Similar experiments showed that cellular fibronectin interacts with BMP-1 and the N-terminal EMI domain of periostin. Many studies have shown that periostin could bind to matrix proteins. while sclerostin. 18. BMP-1 then induces the proteolytic activation of proLOX. Periostin binds to type I collagen and fibronectin and is a key regulator of collagen cross-linking Periostin is strongly expressed in collagen-rich fibrous connective tissues subjected to constant mechanical stress in vivo. These results indicate that inside the cell. or showing a nuclear localization sequence. By binding to integrins or cell surface receptors. it is possible that periostin regulates sclerostin secretion by osteocytes through integrin signaling. osteopontin. the same group demonstrated that the four tandem repeats of the FAS-1 domains of periostin directly interacted with the metalloproteinase domain of BMP-1 [78]. These data demonstrate that intracellular periostin acts as a scaffold that increases the deposition of BMP-1 into the fibronectin matrix. but the concentration of the enzymatic cross-links pyridinoline and deoxypyridinoline was decreased in the periostin−/− whole femur when compared to wild-type mice. We recently reported that not only the enzymatic cross-links but also the immature precursors DHLNL and HLNL were decreased in periostin-deficient mice. osteonectin. Interestingly and unlike many other matricellular proteins. 63. consistent with their partial contribution to structural integrity. periostin may also have intracellular functions [20]. 72]. A common feature of these proteins is that several animal models in which a matricellular protein has been knocked out survive embryogenesis and show only mild phenotypes at birth. and their levels correlated with bone strength (Gineyts et al. 78] (Fig. or probiglycan. Periostin colocalizes with collagen Iα1 chain in the ECM of mouse periosteum and binds collagen Iα1 through its N-term EMI domains [17. periostin is also present in adult animal in collagen-rich tissues such as PDL and bone. largely involved in cell response to environmental stress [61. Corroborating these data. and tenascin-X. periostin interacts with BMP-1 to increase its deposition in the fibronectin matrix in close proximity of proLOX. Calvarial osteoblast cells from periostin null mice show a decreased in LOX expression [78]. a potent antagonist of bone formation through inhibition of Wnt and BMP signaling. the fact that periostin presents variants that are hardly secreted. periostin can activate intracellular signaling pathways leading to inhibition of apoptosis through inactivation of caspases 3 and 9 and prosurvival signaling resulting in increased bone formation [12. these data show that periostin is a prosurvival protein. The fact that periostin null mice had their bone biomechanical properties restored by injection of a sclerostin-blocking antibody further lends to support this hypothesis [67]. However. tenascin-C. 77]. suggesting its involvement in their structure and integrity. to activate pro-LOX and promote collagen cross-linking [20]. Using intact and domain-deletions forms of periostin and BMP-1. which is catalyzed by lysyl oxidase (LOX). Under mechanical stress. but also cardiac tissue viscosity. abstract ASBMR 2011). suggesting a key role in connective tissue homeostasis. The covalent cross-linking of collagen is an essential step in its fibrillogenesis. overexpression of periostin in rats increased bone formation and bone mass. and heart [16. 68]. Mechanical loading experiments on mouse bone have shown that periostin is upregulated. a measure of collagen cross-linking [24]. 77]. 71]. proteins that regulate cell functions and cell–matrix interactions rather than contributing directly to the formation of structural elements [74. 67. is down-regulated [46. 45. and promotes collagen cross-linking as assessed by the measurement of pyridinoline and deoxypyridinoline content in collagen [20. However. 61. strongly suggests that besides its involvement in cell survival and ECM organization through binding to cell surface receptors and ECM proteins. particularly during wound healing. which colocalizes with periostin inside the cell [78]. . 70]. 47.. 17. laminin 5. Additionally. In addition to periostin. that is. as described for intact periostin. to stimulate osteoblast activity and maintain bone integrity [67. 67. Indeed. periostin has been shown to bind to collagen type V [17]. periostin may stimulate bone formation directly through Wnt/β-catenin signaling [36. 2). 66. LOX is synthesized in an inactive form and activated by cleavage of its propeptide by BMP-1.Osteoporos Int Other pathways still have to be elucidated to further explain periostin action. these findings suggest that periostin may have a wider role in matrix organization [78]. the collagen content of femur was not affected. 75]. Periostin is involved in ECM structure and organization Periostin belongs to the matricellular proteins family. Because BMP-1 is involved in the activation—via proteolytic maturation—of other extracellular proteins present in bone including different types of collagens. this family includes thrombospondin-1. and that endogenous BMP-1 and fibronectin coimmunoprecipitate in cell culture models [16. In summary. Alternatively. 79]. 73]. thanks to its four central FAS-1 domains characteristic of the FAS-1 family of adhesion proteins and its N-term EMI-domain. these proteins still play a critical role in collagen assembly. which could be involved in protein–protein interactions [76]. Periosteum from periostin null mice exhibited aberrant collagen fibrillogenesis with an alteration in fibrils diameter and collagen crosslinking as observed in the skin.
promoting the deposition of BMP-1 into the matrix. 20]. tenascin. implying that these two proteins are essential in the extracellular architecture of periosteum [75. 3]. This increases bifurcations of the matrix fibrils to support the meshwork architecture of the ECM [16. tenascin-C forms a disulfide-linked hexamer called "hexabrachion" [16. As suggested by Kudo [20] in his recent review. periostin also possesses a heparin-binding domain at its C-terminal end creating a potential binding site for glycoproteins. suggesting that the C-terminal domain prevents aggregation of periostin with tenascin in the endoplasmic reticulum [16. Fas-1 domains with Gla residues Cell adhesion and protein interactions Periostin is involved in bone strength and response to mechanical stress First studies on periostin have highlighted its preferential expression in periosteum [1. Periostin associates with other matricellular proteins such as Connective tissue growth factor. Even if small changes in periosteal apposition occur throughout life. glycosaminoglycans. periostin can also form disulfide-bonded dimers through its EMI domains [16. However. corroborating its central role in ECM organization [82]. by its involvement in collagen fibril assembly and cross-linking. and BMP-1. and Nephroblastoma overexpressed gene (CCN) family members and thrombospondin known to be involved in some aspect of osteogenesis or chondrogenesis [20]. 81]. However in situ proximity ligation assays showed that both periostin and tenascin colocalize in the periphery or inside the cell. Additionally. Periostin null mice showed an inhibition of the deposition of tenascin-C and a disorganization of the ECM at the lower tibia similar to the Table 2 Periostin domains involved in interaction with ECM proteins Periostin domain N-terminal EMI domains Functions Protein interactions and protein multimerization ECM protein Collagen I Fibronectin Periostin BMP-1 Tenascin Calcium Integrins Heparin phenotype observed in the periostitis in tenascin-C null mice [16]. and proteoglycans [15]. 17] (Table 2). periostin has recently been shown to colocalize with laminin and fibronectin at the basement membrane in hair follicles. By binding to endogenous fibronectin. All these data clearly demonstrate that periostin may support intracellular and extracellular functions. periostin is essential for proper collagen folding and maintenance of mechanical properties of collagen-rich tissues like bones (Table 2).Osteoporos Int Fig. 80]. BMP-1 activates LOX precursor (pro-LOX) to mature active form (LOX). Secreted as a cleaved form in the periosteum. it is conceivable that periostin anchors tenascin in the intracellular network with fibronectin and BMP-1 to further induce collagen cross-linking. 2 Periostin is a key regulator of collagen cross-linking. Finally. Cystein rich protein. with cellular or secreted isoforms that are differentially expressed [20]. suggesting that periostin may be involved in extracellular collagen fibrillogenesis and meshwork architecture to support mechanical strength in periosteum. these data imply that. a growing C-terminal end Potential binding site for glycoproteins . periostin may support the incorporation of tenascin-C hexabrachions in the ECM by binding to tenascin and other proteins like fibronectin. cellular periostin is a key element in collagen cross-linking [78]. Indeed. further linking collagen fibers Overall. Periostin (POSTN) binds fibronectin (FN) through its EMI domain and to BMP1 through its FAS-1 domain. coimmunoprecipitation assays demonstrated that tenascin interacts with the FAS-1 domains of periostin lacking its C-terminal domain but not with intact periostin. indicating a possible interaction of both proteins intracellularly or in the matricellular space. all these proteins are also expressed into the ECM. Periostin binds tenascin-C Expressed in bone and myocardium and inducible by mechanical stimulation. Periostin and tenascin-C present similarities in their expression profiles and are both induced by mechanical stress. 17. 17]. Furthermore. LOX is responsible for the synthesis of pyridinium cross-links.
Mechanical properties of long bones are more profoundly affected by modifications of the periosteal surface than the endocortical compartment [83. Periostin and response to mechanical stress Physical activity has a positive effect on skeletal growth and development. that is. Mechanical forces stimulate bone formation and suppress bone resorption. without any significant differences in osteoblast and osteoclast number per bone surface [67]. periostin−/− mice showed a higher baseline expression of sclerostin than periostin+/+. elevated expression of periostin mRNA in bone after axial compression in periostin+/+ mice was associated with an inhibition of sclerostin mRNA expression by osteocytes [67. particularly within the regions exhibiting the highest strain. . cytoskeletal changes. In teeth. in the alteration of the bone response to mechanical stress in periostin null mice. it is possible that alteration of tendons disrupts the mechanical transduction of strain resulting from compression or physical activity and. By contrast. Periostin+/− mice exhibited intermediate biomechanical characteristics. 60. Periostin+/− mice showed a similar response to axial compression. These defects in cortical microarchitecture were associated with reduced bone strength and turnover. As described earlier. In periostin+/+ mice. However. and dwarfism persisted throughout life. These stimulating effects are correlated with an elevated expression of periostin in the periosteum. to maintain bone structure in stress condition. corroborating previous data in skin [77]. with periostin−/− adult mice consistently smaller than their +/− and +/+ littermates. Mechanical stimulation induces an increase in bone strength by targeting new bone formation to the periosteum [85]. responsible for the lower bone mass of periostin-deficient mice [67]. in part. 60. 88]. leading to disorganized enamel and dentin and increased tooth wear [2. axial compression and moderate physical activity significantly improve bone mineral density. is involved. Indeed. However. but at 3 months. Compression also increases bone formation. trabecular network was restricted. thus. periostin may down-regulate sclerostin secretion by osteocytes through integrin signaling. 86]. whereas some showed a decline in growth rate. By contrast. whereas periostin response to stress was abrogated by TGF-β neutralizing antibodies [33. bone-forming indices were unchanged at the endocortical surfaces in periostin null mice compared to normal littermates. Growth retardation was detectable 3 to 4 weeks after birth. Furthermore. this does not exclude a direct action of periostin via Wnt/β-catenin. a known activator of periostin expression. Indeed. bone mass. cartilaginous growth plates were disrupted. but they were less affected by physical activity. Furthermore. overexpression of periostin in femur of 6week-old rats increased bone formation and bone mass at least by increasing osteoblast activity [24]. periostin mRNA and protein expression are increased in rodent and human PDL fibroblasts. 88]. external root resorption. its high vascularity and support for tendons and muscle attachment. leading to an overall increase in bone mass. they show alveolar bone destruction. As tendons are involved in the transmission of mechanical stress to bone. in accordance with the absence of periostin expression in endosteum [3]. suggesting that sclerostin was. Under mechanical strain. Their bones were undersized. trabecular and cortical microarchitecture. The preferential expression of periostin in periosteum suggests that it may be involved in bone microarchitecture and bone strength. their bone architecture and response to axial compression were restored by injection of a sclerostin-blocking antibody. the proximal tibial midshaft. Furthermore. By contrast. and microarchitecture Periostin null mice are viable in utero and appear normal at birth. recent data have shown a reduced collagen cross-linking in tendons of periostin null mice [7]. periosteal osteoblasts show greater mechanosensivity and responsiveness to osteogenic compounds than endocortical ones. the periosteum may be more sensitive to mechanical and hormonal stimuli and largely involved in bone strength maintenance. 70]. Loss of periostin resulted in altered cancellous and cortical bone microarchitecture and lower bone mineral density [2. periostin−/− littermates responded neither to axial compression nor to exercise and showed a disorganized collagen matrix [67]. All these data show that loss of periostin results in loss of integrity of bone and PDL matrix and suggest that periostin is a key regulator of cortical and cancellous bone microarchitecture and bone strength. Cells from bone and PDL respond to stimulation by activation of mechanosensory signaling systems. and incisor enamel defects. 84]. compared to nonstimulated cells [33. and ECM architecture reorganization to withstand these loads without damage [87]. 67]. particularly in the periosteum. Periostin. Loss and gain of functions studies have clarified its importance in bone strength. and biomechanical properties of long bones. some embryos died before weaning (14%). 60. suggesting that periostin is not essential for in utero survival but may be rather required for events occurring later [2].Osteoporos Int number of evidence that periosteum is a major contributor to bone strength are arising. at least in part. Due to its external localization on bone. Periostin null mice appear to have normal developing teeth and periodontium at birth. which remained unchanged following axial compression. By contrast. PDL is involved in the transmission of physical forces resulting from mastication. periostin overexpression was preceded by an up-regulation of TGF-β.
In experimental rodent . Osteocalcin and Matrix Gla-protein contain 3 and 5 Gla sites. osteopontin and sialoprotein. and result in dwarfism [2. periostin mRNA expression was inversely related to the ability of cells to differentiate and mineralize. articular cartilage. subjected to mechanical forces suggests a role in the regulation of mineralization. periostin expression is up-regulated and activates different cellular pathways to support cell survival. it can be speculated that the different isoforms of periostin would be carboxylated [22]. Indeed. two acidic and glutamate-rich proteins of bone. osteoclasts. 90. respectively. In vitro. A tightly regulated cascade of molecular events is triggered soon after fracture with hematoma formation and inflammation. As the overload continued. to ensure a correct collagen fibrillogenesis and matrix organization to preserve tissue integrity and function. which plays a central role in bone formation during embryogenesis but also during fracture healing and bone repair. involved in TGF-β activation. and a modulation in the degree of carboxylation may lead to regulation of periostin–hydroxyapatite binding [22]. periostin null mice submitted to experimental tooth movement showed an increased PDL cells death associated with a reduced expression of Notch-1 and its downstream effector Bcl-xl. Fracture healing occurs through direct or indirect repair. All these data are in favor of a role of periostin in regulating premature and ectopic bone mineralization. As γ-carboxylation is a posttranslational mechanism. 91]. Additionally. removal of occlusal forces caused a concomitant and transient inhibition of periostin and twist mRNA expression in PDL [32]. are rather responsible of nucleation of hydroxyapatite during the late maturation and mineralization phase of bone formation. and muscle. periostin is largely expressed in embryos. bone and tooth.Osteoporos Int Similarly. 86]. inhibition of FAK/src pathway. These results suggest that periostin associates with Notch-1 to maintain its expression and subsequent signaling in order to enhance cell tolerance against the stress and protect them from death [68]. part of which is carboxylated and abundantly localized in mineralized bone nodules. periostin may play a role in the regulation of ECM mineralization [89]. two other bone Gla-proteins of bone. the absence of periostin in genetically modified mice is associated with an early ossification. that is. indicative of adaptive bone formation. and osteocytes in long bone. This suggests that periostin could have a greater affinity for calcium. various degrees of carboxylation may. variant 3 was expressed at the beginning of overload primarily in cellular periosteum. the effect of γ-carboxylation of periostin on its properties requires further investigations. Periostin is a Gla-containing protein involved in mineralization Localization of periostin in two mineralized tissues. In response to mechanical stress. and is reexpressed in pathological conditions [75]. Periostin mRNA is among the most strongly up-regulated transcripts after tissue injury in the bone. In fact. thus. Like osteocalcin and Matrix Glaprotein. but also suppressed 3D collagen gel contraction induced by TGF-β [33]. that mimic early developmental processes. whereas variant 2 was induced at a constant high level all along the loading. By contrast. it has been shown that βig-h3 protein. 67]. respectively. impair tooth eruption. human bone marrow-derived stromal cells secrete periostin. inhibits bone nodule formation of osteoblasts in vitro [14]. Gla residues have the ability to bind divalent cations such as calcium and to incorporate them into hydroxyapatite crystals. cartilage formation. heart. 60. periostin knockout mice display a unique phenotype demonstrating that periostin is essential for bone and PDL development and functions in postnatal animals and highlight its crucial role in mechanotransduction [2. and bone remodeling. These data corroborate the results obtained with a rat model of chronic overload showing that. Periostin is involved in bone repair Periostin is preferentially expressed in periosteum. In vitro. which will disturb the normal growth. a molecule-bearing fasciclin domains and highly homologous to periostin. known to play an important role in cell survival under stress condition [67. This rapid increase is concomitant to a transient increase in serum osteocalcin. Of interest. As a matricellular protein. Clearly. These data suggest that mechanical strain will activate latent TGF-β and twist-1 nuclear translocation. declines during the course of skeletal growth. 53. osteoblasts. In conclusion. paralleling with an increase in serum tartrate resistance acid phosphatase (TRAP5b)—a biological marker of bone resorption— thinning of the growth plate and reduced cortical thickness indicative of a pathological response to excess loading [27]. and suggests that periostin protein expression is associated with anabolic bone changes in response to loading. followed by the reparative phases. Additionally. where osteoblasts are embedded [22]. periostin is a vitamin Kdependent Gla-protein containing a consensus CRS. that is. 68]. also affect periostin interaction with ECM proteins and integrins. resulting in periostin activation. Finally. reduced nuclear translocation of twist and periostin expression in PDL fibroblasts. intramembranous ossification or endochondral bone formation. angiogenesis. 11. and many Glu-residues embedded within each of the four fasciclin-like domains of the protein. immunoexpression of variant 3 decreased. suggesting a role in tissue regeneration [10. whereas there are 28 potential sites in periostin and more than one functional CRS. suggesting that periostin may be necessary to retard premature mineralization [45].
These results suggest that periostin is involved in early events of fracture repair. CTR. a Relative changes of serum periostin levels in control mice (n07) and vehicle. Periostin is involved in several cellular and extracellular functions resulting in stimulation of bone formation during development. during the recruitment of progenitors into the callus and the early stages of osteoblast differentiation and bone formation.05 vs. periostin mRNA was up-regulated in proliferating preosteoblastic and undifferentiated mesenchymal cells of the soft callus compared to unfractured bone. Similar expression of periostin has been demonstrated in the remodeling events occurring after vascular remodeling and infarct where periostin may induce reentry into the cell cycle leading to proliferation [66]. Fig.05 vs. *p<0. UNT. 3 Modulation of periostin expression levels in control animals (CTR) and vehicle (UNT). p<0. periostin may be a potential marker of bone metastasis 24]. Overexpressed in tumors.(n08) and zoledronic acid-treated (n08) metastatic mice. d Higher magnification of the marrow immunostaining image depicted in b. e Higher magnification of a field containing marrow not affected by metastatic tissue shown in c. Its expression peaked at day 7 in the hard callus and decreased thereafter: it could still be detected in periosteal osteoblasts by day 14. f Higher magnification of metastatic tissue depicted in c. b Representative immunostaining of periostin in hind limbs from control mice and c mice bearing bone metastases (asterisks). [97] # models of fractures on long bone. From Contie et al.Osteoporos Int Fig.and zoledronic acid (ZOL)-treated metastatic mice on day 32 after tumor cell inoculation. . 4 Putative functions of periostin in bone. response to mechanical stress. when the hard callus has been replaced by woven bone [11. Values are the mean (±SD) relative change compared to control group. or bone fracture. it was shown that on day 3 postfracture.
and periostin null mice show a phenotype similar to Marfan syndrome caused by a defect in fibrillin synthesis. Periostin stimulates metastatic growth by promoting cell survival. periostin could be an earlier marker of bone metastasis reflecting the change in bone marrow stroma under the influence of metastasis. as well as cell–matrix interaction. it can modulate cell adhesion. These data suggest that periostin does not reflect bone remodeling but. Markers available to assess bone metastases are associated with the process of bone formation and resorption. Kikuno R. inducible by TGF-β and able to bind similar integrins. Kruzynska-Frejtag A. bone strength (Fig. Thus. e). Tezuka K. References 1. and elastin disorders. Kudo A (1999) Identification and characterization of a novel protein. we have shown that serum levels of periostin decrease with skeletal growth and stabilize as adult age has been reached. rather. no data were reported on the expression of periostin in bone metastases. in a mouse model of human breast cancer bone metastases using specific mRNA probes. and angiogenesis. This is supportive of the importance of periostin in the maturation of collagen and ECM proteins. Lindsley A. may play an active role in fracture repair. d. The finding that periostin influences metastatic potential of tumors including breast and prostate tumors raises the possibility that it could be used as a molecular target in antimetastasis therapy. 3b.Osteoporos Int Data on systemic circulating levels of periostin are scarce. therefore. and differentiation. Toyama Y. it will be interesting to analyze their differential expression according to time and site of bone formation. Periostin is a new Gla-protein identified in bone whose role in regulating mineralization has to be clarified. Wang J. They cannot detect changes before the metastatic bone has proliferated and caused enough disruption of the balance between formation and resorption. It is interesting to notice that βig-H3 protein. Katsuura M. with its preferential location to periosteum. Takamatsu H. Furthermore. a strong expression of periostin has been shown in the stroma of prostate cancer in patients with bone metastases [99. Bonewald LF. By binding to cell surface receptors. Wang H. However. fibrillin. 100]. 4). incisor enamel defects. it is of interest to report the overexpression of periostin in fibrous dysplasia. Finally. Conflicts of interest None. Moreover. ontogenic ossification processes [92]. Chen Z. Periostin and bone metastasis Cancer metastasis to the bone site represents a final stage of tumor progression. Periostin levels do not correlate with bone formation or bone resorption markers in growthstabilized animals and are not modulated by the bisphosphonate zoledronic acid. Using a mouse-specific enzyme-linked immunosorbent assay that we recently developed in our laboratory and recognizing all periostin isoforms. Its involvement in collagen folding is crucial for matrix assembly and. periostin may be a potential target in a therapy to treat ectopic fibro-osseous tissue formation in fibrous dysplasia and possibly joint diseases such as rheumatoid arthritis and osteoarthritis. Amann E (1993) Osteoblastspecific factor 2: cloning of a putative bone adhesion protein with homology with the insect protein fasciclin I. Zhou HM. Overexpression of periostin has been described in the stroma of many cancers and is associated with metastasis and poor prognosis in various cancers [93–95]. The different periostin isoforms may contribute to a broad range of biological functions in both normal and pathological tissues. 3a) [97]. Recently. Rios H. Acknowledgements The authors would like to thank Dr. as these disease commonly involve collagen. and an elevated circulating levels of periostin (Fig. 3c. Biochem J 294(Pt 1):271–278 2. is expressed in bone [14]. bone quality. Ozawa H. periostin. leading to bone fragility [26]. Horiuchi K. another FAS-1 protein rich in glutamate residues. Conclusions Periostin is a matricellular protein playing a fundamental role as an anabolic factor in bone and tooth tissue development and repair. Conway SJ (2005) periostin null mice exhibit dwarfism. Finally. with restricted expression to periosteum and periodontal ligament and increased expression by transforming growth factor beta. Periostin. Further studies are necessary to understand the regulation of periostin isoform expression and their biological activities in bone. J Bone Miner Res 14:1239–1249 . Mol Cell Biol 25:11131–11144 3. Takeshita S. Maeda M. Therefore. compared to healthy bone (Fig. f). proliferation. Amizuka N. which are a frequent complication of solid tumor cancers. and an early-onset periodontal disease-like phenotype. Feng JQ. The development of specific and sensitive assay for serum measurement will also be of crucial importance to investigate the potential of periostin as a biomarker of periosteal activity. Koushik SV. Sylvain Contie for helpful discussion on this manuscript. invasion. especially in the early events of bone regeneration. especially from prostate and breast origin [96]. Takeshita S. increased levels of circulating periostin were reported in women with breast cancer and bone metastases [98]. Similarly. Rogers R. periostin possible involvement in connective tissue diseases is under scrutiny. and/or bone metastases. a hypothesis that would need to be confirmed by longitudinal studies. we showed an overexpression of periostin in the stroma surrounding the bone metastases (Fig. a bone disorder in which fibro-osseous tissue develops in place of normal bone.
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Liu M. Kurland ES. Keating MT (2007) Periostin induces proliferation of differentiated cardiomyocytes and promotes cardiac repair. Bellido T. Gotoh M. Lemoine NR (2007) Periostin promotes invasiveness and resistance of pancreatic cancer cells to hypoxia-induced cell death: role of the beta4 integrin and the PI3k pathway. Endocrinology 146:4577–4583 47. Markwald RR (2007) Periostin promotes atrioventricular mesenchyme matrix invasion and remodeling mediated by integrin signaling through Rho/PI 3-kinase. Kulkarni NH. Mao Y. apoptosis. 70. Chen Q. Wang XF (2004) Periostin potently promotes metastatic growth of colon cancer by augmenting cell survival via the Akt/PKB pathway. Yu S. 66. Katsube K. Mikuni-Takagaki Y (2006) AlphaVbeta3 integrin 56. Xu L. Tamasi J. Wang Q.
Osteoporos Int ligands enhance volume-sensitive calcium influx in mechanically stretched osteocytes. Wen X. Yu CY. Chen LB (2005) Periostin induction in tumor cell line explants and inhibition of in vitro cell growth by antiperiostin antibodies. Sarasa-Renedo A. Clin Cancer Res 12:6243s–6249s 97. J Bone Miner Res 20:2159– 2168 Kii I. Kasyanov V. Chenu C (2005) Sympathetic nervous system does not mediate the loadinduced cortical new bone formation. Ornstein DK. Hill AA. J Biol Chem 281:31720–31728 Tai IT. Babij P. Jung K. Chiquet M (2008) Tenascin-C induction by cyclic strain requires integrin-linked kinase. Trusk T. 84. 86. Leatherman JL. Chen LB. Brasseur F. Schraml P. Bugel S (2008) Vitamin K and bone health in adult humans. Tam C. Kim B. Nah HD. Recker RR. Hermanns T. Mjaatvedt CH. Schreiner GF. Lanyon LE. Conway SJ. Dai M. 83. Shirasawa S (2009) The increased expression of periostin during early stages of prostate cancer and advanced stages of cancer stroma. 74. Johnson ML. Kikuchi Y. Saga Y. Pacifici M (1996) Expression of syndecan-3 and tenascin-C: possible involvement in periosteum development. tumors and melanoma: evidences for periostin production by both stromal and melanoma cells. Protter AA. Seifert HH. 81. Contie S. 73. Cell Tissue Res 312:345–351 89. PLoS One 6:e18410 Seeman E (2007) The periosteum—a surface for all seasons. Tischler V. Ramamurthi A. Kudo A (2010) Interaction between periostin and BMP-1 promotes proteolytic activation of lysyl oxidase. J Cell Commun Signal 2:9–17 Callebaut I. Marshall SJ. Grashoff C. Moch H. Zhang Y. Contie S. Sasaki H. Dobi A. Kern CB. 82. Komm BS. Loda M. Biochem Biophys Res Commun 342:766–772 87. Stephan C. Shimazaki M. Elias A (2003) Elevated serum periostin levels in patients with bone metastases from breast but not lung cancer. Ohazama A. Sugi Y. Goodwin RL. Kitajima S. Kudo A. Forgacs G. Richardson JA. Davis J. Ogawa I. Delmas PD (2006) Bone fragility: failure of periosteal apposition to compensate for increased endocortical resorption in postmenopausal women. Kii I. Fritzsche FR. Schenk S. Goetsch SC. Terai K. Circ Res 86:939–945 91. Litvin J. Garrard LJ. Markwald RR (2007) Periostin regulates collagen fibrillogenesis and the biomechanical properties of connective tissues. Seeman E. Breast Cancer Res Treat 77:245–252 99. Garnero P (2010) Increased expression and serum levels of the stromal cell-secreted protein periostin in breast cancer bone metastases. Utispan K. Ryder MI. Sauter L. J Orthop Res 14:403–412 Nishiyama T. Bonnet N. Tsunoda T. Golden EB. Kudo A (2011) Delayed re-epithelialization in periostin-deficient mice during cutaneous wound healing. Garnero P (2010) Development of a new ELISA for serum periostin: evaluation of growth-related changes and bisphosphonate treatment in mice. Annis DS. Decottignies A (2007) Human periostin gene expression in normal tissues. Yaworsky PJ. Damon B. 75. Mornon JP (2003) EMI domains are widespread and reveal the probable orthologs of the Caenorhabditis elegans CED-1 protein. Souchet M. Biochim Biophys Acta 1783:1150–1162 Koyama E. White RT (2000) Altered patterns of gene expression in response to myocardial infarction. Oka T. Int J Cancer 128:352–360 98. Yokozeki M. Carcinogenesis 26:908–915 Alford AI. Brown EL. Biomaterials 28:5238–5245 88. Lutz R. Moreno-Rodriguez R. Riener MO. and regeneration. Coleman RE (2006) Clinical features of metastatic bone disease and risk of skeletal morbidity. Osteoporos Int 18:123–128 Szulc P. Ravulapalli S. Gelman L. 76. Prostate 69:1398–1403 100. Ge G. J Biol Chem 285:13294–13303 Huang G. Stanton LW. Physiol Genomics 14:261–271 92. Soltermann A. Mosher DF. BMC Cancer 10:273 72. Pitsillides AA. Chatterjee-Kishore M. Kharode Y. Mol Cancer 6:80 95. Litvin J. Chau-in S. Mignotte V. Moriyama K (2003) The divergent expression of periostin mRNA in the periodontal ligament during experimental tooth movement. Skerry TM. Bone 38:749–757 Hamilton DW (2008) Functional role of periostin in development and wound repair: implications for connective tissue disease. Abiko Y. Srivastava S. chemical composition and mechanical properties of collagen fibers in the periodontium. Saito M. Marshall GW (2007) The tooth attachment mechanism defined by structure. Siriwardena BS. van Baren N. 78. J Cell Biochem 101:695–711 Maruhashi T. Hatano H. Mironov V. Paupairoj A. Sornay-Rendu E. Ho SP. Bex FJ (2006) Wnt/beta-catenin signaling is a normal physiological response to mechanical loading in bone. Ferrari S. Histol Histopathol 22:1167–1174 94. Thuwajit P. 85. Mortezavi A. Fukayama M. J Biol Chem 284:25879–25888 Maier S. 77. Kristiansen G (2010) Periostin is upregulated in high grade and high stage prostate cancer. Kii I. Calcif Tissue Int 87:341–350 93. Kapoun AM. Turner DK. Cullen DM. Akhter MP. Gallardo TD. Lam A. Clezardin P. Clezardin P. Auclair D. Molkentin JD. Sesterhenn IA. Tilman G. Biochem Biophys Res Commun 300:619–623 Norris RA. Li C. Furusato B. J Bone Miner Res 21:1856–1863 de Souza RL. Minqi L. Kashima TG. McLeod DG. Potts JD. Voorzanger-Rousselot N. Kudo A (2006) Periostin is an extracellular matrix protein required for eruption of incisors in mice. Thuwajit C (2010) Gene expression profiling of cholangiocarcinoma-derived fibroblast reveals alterations related to tumor progression and indicates periostin as a poor prognostic marker. J Bone Miner Metab 24:498–504 Robinson JA. Garrick BL. Garry DJ (2003) Transcriptional profiling and regulation of the extracellular matrix during muscle regeneration. Voorzanger-Rousselot N. Wild PJ. Takata T (2007) Periostin: novel diagnostic and therapeutic target for cancer. Duboeuf F. Dai M. Hankenson KD (2006) Matricellular proteins: extracellular modulators of bone development. Shimazu A. . 80. Charngkaew K. Mattiussi M. Damm D. Amizuka N. Vitam Horm 78:393–416 90. Greenspan DS (2009) Fibronectin binds and enhances the activity of bone morphogenetic protein 1. Gerhardt J. Takashima Y. 79. Zheng Q. Zhao W. Mol Cancer 9:13 96. Kudo Y. Wilde J. remodeling. Hawke TJ. Hoffman S.
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Anti gay marriage website shut down
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A website dedicated to opposing gay marriage has been removed from the internet the day it was launched after it was the target of one of the "largest unprecedented attacks" on a website in New Zealand.
The "Protect Marriage" website, launched today by Family First, crashed in a matter of hours as a result of a "large-scale denial of service attack" according to the site's webhost.
It was back up and running at 1pm, but had been removed completely by the webhosting company later this afternoon, to "protect its own servers".
"Due to large scale Denial of Service attacks against this domain it has been decided to ensure the stability and security of our servers and network, this account has been removed," said a message from 24/7 Hosting.
Launched to oppose MP Louisa Wall's same-sex marriage bill drawn recently from the ballot, Family First director Bob McCroskrie said the site was apparently the target of "the largest unprecedented attack on a website in New Zealand history".
McCroskrie said he had expected "a good amount of debate" on the issue, but the attack was "obviously a desperate attempt" at shutting down one side of the argument.
He said Family First were now working with an international webhosting company with advanced security and the website would be live again by tomorrow.
In another setback for the Family First campaign, US band Train has also reportedly requested the website cease using their song "Marry Me".
Train caught wind their song was being used by Family First after a Twitter user alerted the band their song was being used on an "anti gay marriage website".
A user named @Mikey_J_S6 tweeted the band last night saying: "Why does your music video appear on a homophobic lobby group's website?". Train responded saying "Didn't know. Getting it off asap. Tnx 4 tip".
McCroskrie said they had not yet heard from Train, but if they were asked to take the song down they would.
"We're not going to go by some post on Twitter, but if the band contact us then we will certainly take it down."
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text: Beyonce and Jay-Z – Celebrity Couples Now + Then by Cristin Maher May 24, 2013 5:56 PM SHARE TWEET EMAIL REDDIT EMAIL REDDIT Now: Beyonce and Jay-Z are one of the most famous and affluent couples around. On top of managing their insane, multi-dimensional careers, they are also now the proud parents of daughter Blue Ivy, born in Jan. 2012. They've been living in marital bliss these past few years, and here, they're looking stunning at the Feb. 2013 premiere of Bey's HBO documentary, 'Life Is But a Dream.' Larry Busacca, Getty Images Then: Although they've always kept somewhat of a low-profile, rumors began circulating that Bey and Jay-Z were dating after they collaborated on "03 Bonnie & Clyde,' with even more romance buzz generated after 'Crazy in Love' dropped. Pretty soon, they got tired of hiding, but still managed to keep their April 2008 wedding in New York City a complete secret. Check them out on the set of 'SNL' in 2002 below — this was probably at the very start of their relationship. Mary Ellen Matthews, Getty Images See the Next Celebrity Couple Now + Then Category: Special Features SHARE TWEET EMAIL REDDIT More EMAIL Print this page Pin it! REDDIT Share on Tumblr! Recommended for You ZergNet Best of the Web ZergNet Best of PopCrush Kylie Jenner's Lip Evolution Into the Woods With Kerli: Cover Story Selena Gomez's Hottest Instagram Photos Nick Jonas' Hottest Instagram Photos


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- File Size: 1593 KB
- Print Length: 356 pages
- Publication Date: November 21, 2014
- Sold by: Amazon Digital Services LLC
- Language: English
- Text-to-Speech: Enabled
- Word Wise: Enabled
- Lending: Enabled
Amazon Best Sellers Rank:
#97,513 Paid in Kindle Store (See Top 100 Paid in Kindle Store)
- #1176 in Kindle Store > Kindle eBooks > Literature & Fiction > Humor & Satire > General Humor
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- #4578 in Kindle Store > Kindle eBooks > Romance > Romantic Comedy
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A Reason To Kill (Reason Series Book 2) Kindle Edition
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- Book 2 of 3 in Reason Series (3 Book Series)
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Top Customer Reviews
Stumbling off the plane, she catches the eye of Max Hunter. He is a lumberjack and superhero rolled into one brawny, scowling hulk of a man. He is immediately drawn to her. In the synopsis, the author as a disclaimer about inta-love and soul mates....well, that is what happens. Mia has Max at her first trip and fall. It being a small town, they can't help but run into each other. He makes assumptions about her based on his past relationships and his previous experience with Mia's boss, who was the leader of previous trips, but she proves his assumptions wrong.
The setting of a small town in northern Alaska is amazing. I can just see the magnificent vistas and shimmering lakes. It also allows for the things only present in small towns, the gossips, the fast spread of rumors. The side characters are what also give it the small town feel. Max's mother Maxine and her best friend Martha who sell sex toys and do secret criminal investigations on the side. Jake, the handsome sexy lumberjack for Mia's friend. Mia's quirky aunt Jesse. The Bears being researched are a story unto themselves with Nala and her cub Booboo and the hulking Cowboy.
I love Mia. She accepts her limitations, but doesn't let them stop her. She has spunk. And, Max, well who wouldn't love Thor, God of Thunder in flannel.
This book had me from page one and I couldn't put it down. It is very well written, suspenseful and unique. It can be read as a standalone, but we get a glimpse of Jack and Jenn from A Reason To Breathe which I loved as well.
The hero has had a friend with benefits for like two years. He breaks it off with her when he starts things with the h. Yet when the ex gets drunk the hero goes to her, leaves the bar with her without even looking at, or speaking to the heroine. Stays with the ex for hours. Doesn't call the heroine or try to see her to put her mind at ease, explain. NOTHING!!!!
The h leaves. Instead of telling the H what a stupid thing he did she sends a text saying the "Hey, I was ready to go. I need more excitement, shopping malls, bla bla bla." Really?!?! STUPID!!!!!
The sex was good enough but they hardly had any meaningful conversations. The whole book the heroine was so UNSURE about EVERYTHING!!! It got so tiring. Open your month & ask him the questions your worried about. Dang!!! Really the H & h didn't even seem to be together that much.
I have to admit the sex party was funny. But the irritating things just outweighed the good. First & last time reading a book by this author.
A Reason to Kill is book two in C.P. Smith's Reason series. I am a huge fan of this author and this is a book that I was very much highly anticipating. I was not disappointed in the least. C.P. Smith has penned yet another wonderfully entertaining and suspense-filled book where our growly and over-protective hero meets his match in a sassy and adorably clumsy heroine. And ladies, if you fell in love with Jack from A Reason to Breathe then you best prepare yourself for his cousin, a sexy lumberjack who will leave you wanting to pick up and move to Alaska!
Thirty year old Mia Roberts is a zoologist for the Seattle Institute of Zoology. She is an analyzer and keynote speaker with absolutely no field experience. None whatsoever. When her boss is unable to go out on the field himself, Mia suddenly finds herself on a small plane on her way to Trails End, Alaska to continue with their study of the grizzlies of the Alaskan frontier. Mia is not exactly known for her athletic aptitude nor is she known for her ability to stay upright for an indeterminate amount of time. Field work means getting out into nature and the bear's natural habitat. It means climbing, camping and all those things that Mia has managed to stay away from until now. Mia has a feeling that this trip will be a disaster in the making and when she finds herself at the center of a murder mystery in the small town as well as the focus of a very ruggedly male lumberjack who resembles "Thor in flannel", she decides that she is not wrong. Epic disaster. Oh yes!
"You've seen him, he's, he's, well, he's Thor in flannel."
"...perfection in flannel."
"He was primal, almost dangerous, the sexiest and most fascinating man I'd ever met.Read more ›
Most Recent Customer Reviews
I couldn't put this book down! It was so entertaining. I loved it and I can't wait to read another by CP Smith!Published 22 days ago by Cindylou
This was a fun, engaging and enjoyable book to read. Love does not always have to be tragic to be sincere. Read morePublished 2 months ago by PaMochaMom
Just like the first in the series, a great read mixed with suspense and laughter! I love all of the supporting characters! Read morePublished 4 months ago by Sarah P
This book did not disappoint!! I love how everything tied in together! I can't wait for your next series!!Published 4 months ago by Amazon Customer
Lumberjack. Alpha-Male. Thor… Oh My!
"Women who read romance novels dream of men like that. Read more
"With me, he'd been flirty, bossy, arrogant, giving sexually compatible, and I'd been… a pain in his ss according him. Read morePublished 5 months ago by Melissa Mendoza with Alpha Book Club
"Why me? Why are you so h*** bent on this?" "Because my lungs locked up when I laid eyes on you, and I didn't breathe again until I kissed you. Read morePublished 5 months ago by M*RcR
Read my he first book A Reason to Breath and loved it. Loved this one too. Looking forward to the next.Published 5 months ago by Myrt
Laughed a lot!
Mia has only worked from behind a desk and suddenly is on her way to a remote Alaskan town to observe grizzly bears. Read more
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text:
Which sex is the worst about washing up? Why is it so important? We've got the dirty truth on how and when to wash your hands.
Conflict of Interest Checkup
April 10, 2000 (San Francisco) -- The long-standing relationship between doctors and drug companies is intended to serve mutual interests: Doctors need tested remedies with which to treat patients, and drug companies need a way to sell their tested remedies.
Meanwhile, the interests of the third party who actually pays for the drugs -- you, the patient -- are not well represented. Here are ways you can ensure your concerns will be addressed when you go to the doctor:
Recommended Related to Drugs and Herbs
Q: What should I expect to be told about any newly prescribed medication? A: When it comes to medication, what you don't know can definitely hurt you. Almost half of all Americans regularly take at least one prescription drug, and half of older patients take three or more, according to a report released in by the National Center on Health Statistics. But doctors don't always communicate critical information about the drugs they prescribe. And prescription drug misuse is a growing problem,...
Read the New Prescriptions article > >
1. Research your illness to see what types of drugs are used to treat it so that you can ask about alternative medications and even non-pharmaceutical remedies.
2. If your doctor prescribes a name-brand medication, ask whether generic versions are available. If your doctor voices reservations about generics, ask to be directed to research that discusses the drug and addresses these reservations.
3. Consider asking your doctor if he or she has any involvement with the company that makes the prescribed drug, particularly if you question the choice of medications. Keep in mind that some doctors direct clinical trials for drug companies. Others may accept research money from drug companies, serve as paid consultants, own stock in, or even sit on the boards of biotechnology and drug companies.
4. Be wary of drug samples. Free samples do serve as a quick and inexpensive way to allow patients to try a drug, but they also serve to introduce new, and perhaps more expensive, medications. Ask if equally effective treatments are already available.
Kristi Coale is a San Francisco-based freelance journalist who specializes in science and medical issues. Her work has appeared in Salon,Wired, and The Nation.
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Khaki Straight Leg Capri School Uniform Pants Plus Size Girls 8.5A great pair of pants for your school age girl by Classroom.
A great pair of pants for your school age girl by Classroom. Mid-rise girls plus size Capri with tab-over waist has fly front, slash pockets, center back elastic, and belt loops. Stretch cotton/poly/spandex twill has supple fit and feel with wash-and-wear ease.
Fabric and Care: 65%Polyester, 35% Cotton Wash Before Wearing Machine Wash Warm With Similar Colors Use Only Non-Chlorine Bleach When Needed Tumble Dry Low Warm Iron If Needed
Brand: Classroom Uniforms
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text:
Sateen Sunday: Take 15% off 1+ Yards of Organic Cotton Sateen Ultra fabric through 11:59 p.m. EDT July 24th.
Preview scale: 21.0 x 18.0 inches

Change DPI Base size is 150
Collection: Mayflower Collection
pink (26445),
whimsical (23500),
animals (20099),
cat (5083),
kawaii (3566),
spoonflower (1031),
squid (736),
sushi (203),
staff (91),
samurai (37)
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stephanie says:
I like how your squids are eating other squids. I'm in favor of cannibalism.
Posted over 4 years ago.
chad says:
I was happy with how it ended up. Thanks for all the support guys. :-) I think a throw pillow is definitely in the future.
Posted over 5 years ago.
lucindawei says:
So sad this didn't win. I was hoping to see this on the couch. Maybe a throw pillow to adorn the couch?
uzumakijo says:
hahhaaa!!! love it! i'm also loving mr. ninja chef and his fantastic knife wielding antics, i love a bit of creepy cute..wicked!!
chad says:
Thanks for all the compliments guys. Truly appreciated. :-)
paragonstudios says:
Chad, so cute I love the samurai chef adorable
ceanirminger says:
It's a cephalo-party of cannibalistic cuteness! I'm happy and hungry just looking at it. Great work!
lucindawei says:
Before reading the title, I was hoping it was an octopus eating squid sushi. Kawaii noir. So cute but so dark. Like it. It would make for a fun couch.
thirdhalfstudios says:
This reminds me of the "Fruity Oaty Bar" commercial - great job!
pixeldust says:
I just came back to say, I know he's supposed to be a sushi chef but with all the knives, I'm still sticking with ninja. I'm glad I'm not the only one who found him ninja-riffic:)
inktreepress says:
HAHAHA, I love this!
victorialasher says:
I'm laughing at the ninja, and the kitty, and ...well the whole thing actually. This would be sooo funny on the couch.
vo_aka_virginiao says:
Gotta love the pepto bismol color and the streak of cannibalism.
happysewlucky says:
This is just hilarious! Happy, pink cannibal squid. Gotta love it.
pixeldust says:
LOL I love the ninja squid/octopus!
saraink says:
Oh how I want to see a couch recovered in this print! Very original!
© 2008-2015 Spoonflower Inc


text:
I want to download a single frame of a video file from a server from my android application. I do not wat to download the full video at front. Use that frame as a thumbnail to display to the user, so that if choosen by user it can be downloaded.
Announcing Stack Overflow Documentation
We started with Q&A. Technical documentation is next, and we need your help.
Whether you're a beginner or an experienced developer, you can contribute.
To get
It will be easier if your server provided thumbnails for each video like youtube/dailymotion does. You could create this thumbnail when video is added to the server.
Ex: http://img.youtube.com/vi/


text:
Foursquare © 2016 Lovingly made in NYC & SF
Italian · $$
Jalan Danau Tamblingan No. 228 (Jalan Danau Poso)
"Probably the best pizza in Bali! Place is beautiful and relatively cheap. Free bread balls they give are awesome too"
Luhtu's Coffee Shop
Café · $$
Sindu Beach (Sanur)
"Tasty simple food and cozy atmosphere. You can sit outside with an ocean view. Fresh food and good service for reasonable prices. Tuna steak was great. Recommend it for breakfast or lunch."
Sanur Beach Groove
Food Court
"Good place and nice food also drink, recommended for hangout with friend ^_^"
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