Periostin in fibrillogenesis for tissue regeneration: periostin actions inside and outside the cell
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More than 10 years have passed since the naming of periostin derived from its expression sites in the periosteum and periodontal ligament. Following this finding, we have accumulated more data on the expression patterns of periostin, and, finally, with the generation of periostin-deficient mice, have revealed functions of periostin in the regeneration of tissues in bone, tooth, heart, and skin, and its action in cancer invasion. Since periostin is a matricellular protein, the first investigation of periostin function showed its enhancement of cell migration by acting outside the cell. On the other hand, recent observations have demonstrated that periostin functions in fibrillogenesis in association with extracellular matrix molecules inside the cell.
KeywordsPeriostin Fibrillogenesis Bone Type I collagen Tooth Heart Matricellular protein ECM
Periostin directly interacts with type I collagen [8, 9, 10], fibronectin  and Notch1  through its EMI domain and interacts with tenascin-C  and BMP-1  through its fas I domains. These periostin interactions with mainly extracellular matrix (ECM) molecules firstly occur intracellularly . Furthermore, periostin serves as a ligand of integrins such as αvβ3 and αvβ5 and promotes cell motility  by acting outside the cell. In light of these properties of protein interactions, I have classified periostin action into two major functional categories, i.e., fibrillogenesis, which occurs inside the cell, and cell migration outside the cell, involving its extracellular action.
After cloning the periostin gene, the establishment of 3 independent periostin-knockout mice provided the first insights into periostin function [8, 15, 16, 17]. These studies demonstrated that periostin functions in regeneration of tissues such as bone, tooth, and heart, and in cancer invasion and wound healing.
Several reviews on the involvement of periostin in cardiac development , tumorigenesis , and wound healing  have already been published. Therefore, I would like to focus on recent findings that support a new concept of periostin function, i.e., its participation in fibrillogenesis.
Periostin in bone and fibrillogenesis
What is the molecular mechanism for generating this collagen structure? In tendon, bone, and skin, type I collagen is the major component of these fibrils. In fibrillogenesis, collagen fibril formation in cell culture is dependent on the prior assembly of fibronectin into fibrils. Therefore, collagen fibrillogenesis that occurs on the cell surface is downstream of fibronectin assembly, and it is mediated by interactions with cell-surface integrins . Tenascin-C forms a typical disulfide-linked hexamer, called the hexabrachion, in which six flexible arms emanate from a central globular particle, which possibly catches and stabilizes a bifurcation of the ECM fibrils composed of fibronectin and type I collagen to underlie the extracellular meshwork architecture. Periostin and tenascin-C expression are coordinately induced by mechanical stress, with transcription of these genes being regulated by cytoskeletal actin , indicating that periostin-mediated incorporation of tenascin-C into the ECM architecture maintains structural homeostasis when adapting to a changing mechanical environment.
Periostin in teeth
The first observed phenotype of periostin −/− mice was fragility of the teeth, mainly caused by defective periodontal ligaments [8, 16]. Based on expression analysis of periostin in embryonic teeth of the mouse mandible, the periostin protein was localized to the interface between the inner enamel epithelium and preodontoblasts as well as in the mesenchymal tissues around the cervical loop . In juvenile mice at postnatal day 7, periostin protein is restricted to the fibrous bundles in the periodontal ligament in accordance with the organization of periodontal fibers . These findings suggest that periostin is involved at sites of cell-to-matrix interaction, serving as adhesive equipment for bearing mechanical forces including tooth eruption, and transducing the occlusal force that activates latent TGFβ to enhance periostin expression . Consistently, periostin −/− mice show defective eruption of their incisors  and degradation of the periodontal ligament of molars during experimental tooth movement . The abnormal presence of non-digested collagen fibrils in the shear zone in the periostin −/− periodontal ligament  is now explained by the low activity of MMPs, which are efficiently secreted following their induction by periostin .
Periostin in mechanotransduction
It still remains to be investigated whether periostin is directly connected with the mechanosensor. From the specific expression of periostin in the periosteum and periodontal ligament, we expected that periostin would be linked to mechanical stress. To search for this linkage, we looked for an upstream signal regulating periostin expression that would act in a transcriptional manner. Investigating the periostin promoter, we found that twist, a basic helix-loop-helix transcription factor, binds to up-regulate its transcription . To further demonstrate the involvement of periostin and twist in mechanical stress, we prepared an experimental model in which teeth were subjected to movement. This forced mechanical stress up-regulated periostin transcription . In contrast, occlusal hypofunction decreases the expression of both periostin and twist . Although the up-stream signals that regulate twist remain unknown, periostin is very closely involved with the mechanosensing signal. Analysis of the periostin promoter sequences revealed predicted c-fos-binding sites, which may provide for the ectopic expression of periostin in the bone marrow of c-fos transgenic mice and in that of patients with fibrous dysplasia (FD) .
Specific transcription of the periostin gene in embryonic fascias of muscles that receive mechanical stress in mice provides further evidence of the involvement of periostin in mechanical sensing . Another animal model using zebrafish showed that periostin is expressed in the myoseptum between muscles. This tissue senses mechanical stress and delivers that signal from one muscle to another . The knock-down of periostin with morpholino antisense oligonucleotide leads to defects in myoseptum formation, a delay in the differentiation of myofibers, and disorder of the connection between myofibrils and myoseptum . Similar to the myoseptum in zebrafish, tendons in mammals are a uniaxial connective tissue that functions in the transmission of forces from the musculature to the skeletal system . The relationship between periostin and the mechanical integrity of tendons needs to be further investigated.
In another study involving periostin in mechanotransduction, periostin was shown to negatively regulate the action of sclerostin, which is a receiver of bone-related mechanical stress signals. Consequently, mechanical stimuli have no effect on the skeletal properties in periostin −/− mice . All these results indicate that periostin is very closely related to the mechanosensor.
Periostin in heart disease
Periostin is expressed in the heart valves throughout life from embryo to adult. However, we could not observe any significant abnormalities in the formation of heart valves, which would indicate that periostin mainly functions in heart diseases in association with tissue repair. The expression of periostin in the embryonic heart has already been reviewed by Norris et al. . Therefore, I will focus here on periostin function in heart diseases.
In humans, periostin is abundantly expressed in the infarct border after a myocardial infarction. In mice, periostin expression is induced by TGFβ following inflammation at the infarct border . To further study these expression patterns, we assessed the regeneration of heart tissues after a myocardial infarction in periostin −/− mice . Consistent with the findings made by Oka et al. , we demonstrated that periosin functions in the migration of cardiac fibroblasts through engaging integrin αvβ3, which induces phosphorylation of the downstream kinase FAK (focal adhesion kinase), leading to the production of type I collagen. Thereafter, periostin acts to promote the collagen cross-linking in ECM. Interestingly, one splice variant (ΔbΔe: deletion of exons b and e at the 3′ end of periostin mRNA) is preferentially expressed in response to TGFβ at the beginning of regeneration of heart tissues and activates the integrin signals. This ΔbΔe variant form is also dominantly observed in the early stage of valve development  and in periodontal tissues in mice .
In the later chronic stage of a myocardial infarction, fibrillogenesis is accelerated by periostin and proceeds to generate a tight scar, in a similar fashion as in cardiac hypertrophy . Furthermore, in an experiment in which rat cardiomyocytes were manipulated, Kuhn et al.  showed that periostin induced the proliferation of differentiated cardiomyocytes in the rat to improve ventricular remodeling and myocardial infarction. However, this proliferation was not observed in mice . This discrepancy needs to be resolved in the future.
The regulatory hierarchies active in the development of cartilage, tendon, and bone are also important in heart valve maturation and ECM remodeling. In diseased heart valves, there is a distinct loss of ECM organization associated with changes in mechanical properties that ultimately lead to cardiac dysfunction . In this respect, periostin is a good candidate to be tested for involvement in valve diseases. Hakuno et al.  reported that periostin enhances the degeneration of atherosclerotic and rheumatic cardiac valves. In mice fed with a high-fat diet, aortic valve thickening and annular fibrosis are increased, concomitant with increased periostin expression, which is correlated with tight scar formation after a myocardial infarction. Periostin enhances fibrillogenesis at the valve. Interestingly, this report additionally found a new periostin function, i.e., that periostin induces the secretion of MMP-2 and MMP-9 from human epithelial cells and mouse macrophages, respectively. This finding suggests that periostin probably interacts with the precursor of MMP-2 or MMP-9 inside the cell for efficient secretion following proteolytic digestion to generate mature MMPs, which scenario would be consistent with Notch1 maturation by periostin after interaction of periostin and precursor Notch1 inside the cell .
Now, it is important to consider the two periostin functions, cell migration outside the cell and fibrillogenesis through protein association inside the cell, separately in heart diseases.
Periostin in tumor fibrillogenesis
Although other functions of periostin in tumorigenesis have been reviewed recently by Ruan et al. , I wish to address a new concept regarding periostin in tumor fibrillogenesis. During tumorigenesis, periostin is a potential contributor to tumor metastasis and epithelial–mesenchymal transition (EMT). In colon cancer, periostin is expressed in cancer-associated fibroblasts , and in pancreatic cancer, periostin is deposited in the periductal stroma . In these cancers, however, periostin expression is not observed in the cancer cells themselves.
In contrast, periostin plays a role as a suppressor of invasion and metastasis in the progression of human bladder cancers . Our observations on cancer cells in periostin −/− mice show the importance of endogenous periostin in suppressing tumor size through activation of fibroblasts that produce ECM molecules for capsule formation . By considering different actions in tumor malignancy, periostin-promoted cell migration or capsule formation suppressing tumor growth, we can precisely justify the periostin function in each tumor.
Actions of the C-terminal region of periostin
Alternative splicing at the 3′ end of periostin generates 3 major variant forms: deletion of exon b, exon e, and exons b and e (ΔbΔe) in addition to the full form in mice. This splicing event at the 3′ end of periostin is evolutionarily conserved among mouse, human and zebrafish, suggesting an important function. The ΔbΔe variant of periostin is preferentially and commonly present in the periodontal ligament, periosteum, and heart tissues after a myocardial infarction. This form is also preferentially found in the corresponding human tissues (unpublished data). The major difference between the full form and the ΔbΔe form of periostin is recognizable at the moment of their secretion. Although intact periostin is hardly secreted, the ΔbΔe form is effectively secreted, indicating that this variant form is preferentially localized at the outside of cells and on the ECM to interact with integrins. Myofibroblasts produce periostin, and they also express the splice variants of fibronectin and tenascin-C induced by the action of TGFβ, suggesting the presence of a functional complex involving these three molecules. Han et al.  reported that TGFβ1 regulates fibronectin isoform expression and splicing factor SRp40 expression, suggesting the involvement of a common splicing system in the same cell and developmental stage that is organized by the same splicing factor.
Periostin protein is often cleaved by proteolysis in heart disease , periosteum , and during wound healing . This cleavage occurs preferentially from the C-terminal end, although almost no functional observations regarding cleaved truncated periostins were reported. However, recently, we found that specific cleavage at a region near the C-terminus of periostin, which is a heparin-binding site , is essential for association of periostin with tenascin-C , indicating that proteolytic cleavage is functional in a developmental or pathological stage-dependent manner. Further investigations regarding the function of proteolytic cleavage of periostin are required.
Regarding extracellular collagen fibril growth after secretion of the type I collagen meshwork, the initial stage of ECM deposition results in arrays of narrow fibrils of uniform diameter, which diameter can increase by about tenfold. In the ECM, collagen fibril growth is thought to occur by accretion as well as by lateral and end-to-end fusion of collagen fibrils . Thus, we need to further investigate the function of periostin in fibrillogenesis outside the cell and the new possible functions of periostin associated with matricellular proteins such as thrombospondins and CCN family members in the ECM. In our recent analysis, we found that periostin can associate with CCN3 to function in periodontal ligament (Tanabe et al., in preparation). Moreover, Hakuno et al.  have shown some interesting data indicating overlapping expression of periostin with elastin in the adult human cardiac valve, suggesting the involvement of periostin in elastin cross-linking outside the cell. Furthermore, in recent observations on periostin action outside the cell, periostin is found to associate with laminin on the basement membrane in hair follicles to induce keratinocyte proliferation during wound healing .
Finally, regarding pathological fibrillogenesis induced by periostin, over-protective reactions by periostin in pathogenesis accelerate fibrillogenesis, which gives rise to fibrosis. In an experimental model of lung fibrosis initiated using bleomycin, a high expression of periostin is induced, indicating a possible important action of periostin in tight collagen fiber formation (Kondo et al., unpublished data).
I would like to thank the following collaborators at Tokyo Institute of Technology:Isao Kii, Masashi Shimazaki, Takashi Nishiyama, Takumi Maruhashi, Issei Takayama, and Hideyuki Tanabe. This work was supported by grants-in-aid for scientific research from the Ministry of Education, Science, Culture, and Sports of Japan and by grants from the Ground-based Research Program for Space Utilization promoted by Japan Space Forum.
This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.
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