The 5q31-linked corneal dystrophies are heterogeneous autosomal-dominant eye disorders pathologically characterized by the progressive accumulation of aggregated proteinaceous deposits in the cornea, which manifests clinically as severe vision impairment. The 5q31-linked corneal dystrophies are commonly caused by mutations in the TGFBI (transforming growth factor-β-induced) gene. However, despite the identification of the culprit gene, the cellular roles of TGFBI and the molecular mechanisms underlying the pathogenesis of corneal dystrophy remain poorly understood. Here we report the identification of periostin, a molecule that is highly related to TGFBI, as a specific TGFBI-binding partner. The association of TGFBI and periostin is mediated by the amino-terminal cysteine-rich EMI domains of TGFBI and periostin. Our results indicate that the endogenous TGFBI and periostin colocalize within the trans-Golgi network and associate prior to secretion. The corneal dystrophy-associated R124H mutation in TGFBI severely impairs interaction with periostin in vivo. In addition, the R124H mutation causes aberrant redistribution of the mutant TGFBI into lysosomes. We also find that the periostin-TGFBI interaction is disrupted in corneal fibroblasts cultured from granular corneal dystrophy type II patients and that periostin accumulates in TGFBI-positive corneal deposits in granular corneal dystrophy type II (also known as Avellino corneal dystrophy). Together, our findings suggest that TGFBI and periostin may play cooperative cellular roles and that periostin may be involved in the pathogenesis of 5q31-linked corneal dystrophies.
Numerous heat-dependent chemical reactions are involved in organismal life, and temperature is an important factor that determines whether such reactions progress. To date, ultrasound and thermotherapy techniques have been established in high-end medical treatments and are proposed to monitor temperature changes on a nanoscale of localized areas such as single cells and to induce material synthesis due to local energy conversion. In this study, a nanoprobe that can measure the local temperature on the nanoscale is designed and developed using gold nanoparticles and thermo-sensitive fluorescent materials. To support this concept, a polymer capable of controlling the physical properties of gold nanorods (AuNRs) is manufactured using light-heat conversion synthesis.
In biological systems, a few sequence differences diversify the hybridization profile of nucleotides and enable the quantitative control of cellular metabolism in a cooperative manner. In this respect, the information required for a better understanding may not be in each nucleotide sequence, but representative information contained among them. Existing methodologies for nucleotide sequence design have been optimized to track the function of the genetic molecule and predict interaction with others. However, there has been no attempt to extract new sequence information to represent their inheritance function. Here, we tried to conceptually reveal the presence of a representative sequence from groups of nucleotides. The combined application of the K-means clustering algorithm and the social network analysis theorem enabled the effective calculation of the representative sequence. First, a "common sequence" is made that has the highest hybridization property to analog sequences. Next, the sequence complementary to the common sequence is designated as a 'representative sequence'. Based on this, we obtained a representative sequence from multiple analog sequences that are 8⁻10-bases long. Their hybridization was empirically tested, which confirmed that the common sequence had the highest hybridization tendency, and the representative sequence better alignment with the analogs compared to a mere complementary.
Objective: Mitochondrial gene mutations may play a role in the development of gestational diabetes mellitus. This study has assisted to confirm the relationship between the mitochondrial DNA copy number and the GDM. Methods: Peripheral blood samples were collected from 68 patients with GDM and from 79 controls. For the quantification of mtDNA content, a comparative analysis was performed by the amplification of endogenous control (nuclear DNA, 28S rRNA). The mitochondrial A3243G mutation analysis performed. Results: The ratio of mtDNA/28S rRNA was 1.20530.8307 in GDM patients and 1.79751.1355 in control group (p=0.0004), respectively. Among 68 GDM patients, the mutation in tRNA nt 3243 was detected in only one subject. The A3243G mutation in tRNALeu gene, implicated in GDM was reported in 1 of 68 (1.47%) but not in controls. Conclusion: In this investigation, blood samples from GDM patients using the real-time polymerase chain reaction will be applied to confirm the relationship between the mitochondrial DNA copy number and the GDM. It is hypothesized that this method will help to predict GDM, and aid in developing early diagnostic methods and treatment modalities.
The magnetic properties of nanoparticles make them ideal for using in various applications, especially in biomedical applications. However, the magnetic force generated by a single nanoparticle is low. Herein, we describe the development of nanocomplexes (size of 100 nm) of many iron oxide nanoparticles (IONPs) encapsulated in poly(lactic- co-glycolic acid) (PLGA) using the simple method of emulsion solvent evaporation. The response of the IONP-encapsulated PLGA nanocomplexes (IPNs) to an external magnetic field could be controlled by modifying the amount of IONPs loaded into each nanocomplex. In a constant size of IPNs, larger loading numbers of IONPs resulted in more rapid responses to a magnetic field. In addition, nanocomplexes were coated with a silica layer to facilitate the addition of fluorescent dyes. This allowed visualization of the responses of the IPNs to an applied magnetic field corresponding to the IONP loading amount. We envision that these versatile, easy-to-fabricate IPNs with controllable magnetism will have important potential applications in diverse fields.
In this study, we fabricated a cellulose nanocrystal (CNC)-embedded aerogel-like chitosan foam and carbonized the 3D foam for electrical energy harvesting. The nanocrystal-supported cellulose foam can demonstrate a high surface area and porosity, homogeneous size ranging from various microscales, and a high quality of absorbing external additives. In order to prepare CNC, microcrystalline cellulose (MCC) was chemically treated with sulfuric acid. The CNC incorporates into chitosan, enhancing mechanical properties, crystallization, and generation of the aerogel-like porous structure. The weight percentage of the CNC was 2 wt% in the chitosan composite. The CNC/chitosan foam is produced using the freeze-drying method, and the CNC-embedded CNC/chitosan foam has been carbonized. We found that the degree of crystallization of carbon structure increased, including the CNCs. Both CNC and chitosan are degradable materials when CNC includes chitosan, which can form a high surface area with some typical surface-related morphology. The electrical cyclic voltammetric result shows that the vertical composite specimen had superior electrochemical properties compared to the horizontal composite specimen. In addition, the BET measurement indicated that the CNC/chitosan foam possessed a high porosity, especially mesopores with layer structures. At the same time, the carbonized CNC led to a significant increase in the portion of micropore.
Abstract Background This study was performed to investigate the usefulness of clinical pathway (CP) using an electronic medical record (EMR) in pediatric patients undergoing closed pinning for supracondylar fracture of the humerus, by analyzing the length of hospital stay, hospital cost and satisfaction of the medical teams. Methods This before and after comparative study included consecutive children who underwent closed pinning for supracondylar fracture of the humerus since 2009. The pre-CP group consists of 90 patients with the mean age of 5.7 years, and the post-CP group consists of 32 patients with the mean age of 6.2 years. Multidisciplinary work-team developed CP using an EMR system in March 2011. The length of hospital stay was the primary outcome variable, and hospital cost and medical team’s satisfaction score were secondary outcome variables. The non-inferiority test was used to demonstrate the efficiency of the pathway. Results The length of hospital stay decreased from 2.9 ± 0.7 days to 2.4 ± 0.7 days by 15.0%, after the implementation of CP, and the lower bound of the 95% CI of the difference (0.14 day) was within the non-inferiority margin of −0.3 days. The hospital cost decreased from 1162.2 ± 236.7 US$ to 1139.8 ± 291.1 US$ by 1.9% and the lower bound of the 95% CI of the difference was −81.3 US$, which did not exceed the non-inferiority margin of −116.2 US$. Therefore, the post-CP group was not inferior compared with the pre-CP group in term of the length of hospital stay and total hospital cost. There was significant increase in the satisfaction score for doctors after implementation of CP (p < 0.001), but, no change in the satisfaction score for nursing staffs (p = 0.793). Conclusions The development and implementation of CP, using an EMR, in pediatric patients undergoing closed pinning for supracondylar fracture of the humerus enhances the treatment efficiency by streamlining the treatment process with no increases of the length of the hospital stay and total hospital costs.
The present study investigated the use of fibrous nanoparticle-filled polarizing films. Sepiolites were selected as nanoparticles and incorporated into a PVA–iodine complex. The resulting nanocomposite film was elongated and dyed with iodine. Various properties of the nanocomposite polarizing films, including thermal, morphological, optical, and rheological features, were experimentally analyzed. The study demonstrated that an increase in sepiolite loading was accompanied by an enhancement in both the mechanical and viscoelastic properties. In particular, the incorporation of nanoparticles led to an increase in birefringence and the degree of polarization. This was attributed to the alteration of the internal structure of the PVA film caused by the embedded sepiolites. The thermal analysis showed that the composite film with a higher content of sepiolites exhibited higher crystallinity and a higher melting temperature.
The 5q31-linked corneal dystrophies are heterogeneous autosomal-dominant eye disorders pathologically characterized by the progressive accumulation of aggregated proteinaceous deposits in the cornea, which manifests clinically as severe vision impairment. The 5q31-linked corneal dystrophies are commonly caused by mutations in the TGFBI (transforming growth factor-β-induced) gene. However, despite the identification of the culprit gene, the cellular roles of TGFBI and the molecular mechanisms underlying the pathogenesis of corneal dystrophy remain poorly understood. Here we report the identification of periostin, a molecule that is highly related to TGFBI, as a specific TGFBI-binding partner. The association of TGFBI and periostin is mediated by the amino-terminal cysteine-rich EMI domains of TGFBI and periostin. Our results indicate that the endogenous TGFBI and periostin colocalize within the trans-Golgi network and associate prior to secretion. The corneal dystrophy-associated R124H mutation in TGFBI severely impairs interaction with periostin in vivo. In addition, the R124H mutation causes aberrant redistribution of the mutant TGFBI into lysosomes. We also find that the periostin-TGFBI interaction is disrupted in corneal fibroblasts cultured from granular corneal dystrophy type II patients and that periostin accumulates in TGFBI-positive corneal deposits in granular corneal dystrophy type II (also known as Avellino corneal dystrophy). Together, our findings suggest that TGFBI and periostin may play cooperative cellular roles and that periostin may be involved in the pathogenesis of 5q31-linked corneal dystrophies. The 5q31-linked corneal dystrophies are heterogeneous autosomal-dominant eye disorders pathologically characterized by the progressive accumulation of aggregated proteinaceous deposits in the cornea, which manifests clinically as severe vision impairment. The 5q31-linked corneal dystrophies are commonly caused by mutations in the TGFBI (transforming growth factor-β-induced) gene. However, despite the identification of the culprit gene, the cellular roles of TGFBI and the molecular mechanisms underlying the pathogenesis of corneal dystrophy remain poorly understood. Here we report the identification of periostin, a molecule that is highly related to TGFBI, as a specific TGFBI-binding partner. The association of TGFBI and periostin is mediated by the amino-terminal cysteine-rich EMI domains of TGFBI and periostin. Our results indicate that the endogenous TGFBI and periostin colocalize within the trans-Golgi network and associate prior to secretion. The corneal dystrophy-associated R124H mutation in TGFBI severely impairs interaction with periostin in vivo. In addition, the R124H mutation causes aberrant redistribution of the mutant TGFBI into lysosomes. We also find that the periostin-TGFBI interaction is disrupted in corneal fibroblasts cultured from granular corneal dystrophy type II patients and that periostin accumulates in TGFBI-positive corneal deposits in granular corneal dystrophy type II (also known as Avellino corneal dystrophy). Together, our findings suggest that TGFBI and periostin may play cooperative cellular roles and that periostin may be involved in the pathogenesis of 5q31-linked corneal dystrophies. Corneal dystrophies are characterized by the progressive loss of corneal transparency as a result of extracellular amyloid and non-amyloid deposits, which accumulate in different layers of corneal tissues. 5q31-linked corneal dystrophies are pathologically heterogeneous, autosomal-dominant disorders caused by mutations in the TGFBI (transforming growth factor-β-induced) gene, which encodes the TGFBI protein (also known as keratoepithelin or Big-H3) (1.Stone E.M. Mathers W.D. Rosenwasser G.O. Holland E.J. Folberg R. Krachmer J.H. Nichols B.E. Gorevic P.D. Taylor C.M. Streb L.M. Nat. Genet. 1994; 6: 47-51Crossref PubMed Scopus (155) Google Scholar, 2.Munier F.L. Korvatska E. Djemaï A. Le Paslier D. Zografos L. Pescia G. Schorderet D.F. Nat. Genet. 1997; 15: 247-251Crossref PubMed Scopus (530) Google Scholar). To date, more than 30 different mutations leading to corneal dystrophies have been attributed to mutations in TGFBI, the most frequent of which are mutations within exons 4 and 12, which result in amino acid substitutions in Arg124 and Arg555, respectively (3.Kannabiran C. Klintworth G.K. Hum. Mutat. 2006; 27: 615-625Crossref PubMed Scopus (127) Google Scholar, 4.Korvatska E. Munier F.L. Djemaï A. Wang M.X. Frueh B. Chiou A.G. Uffer S. Ballestrazzi E. Braunstein R.E. Forster R.K. Culbertson W.W. Boman H. Zografos L. Schorderet D.F. Am. J. Hum. Genet. 1998; 62: 320-324Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). The different mutations in TGFBI cause clinically distinct types of corneal dystrophies, which are classified according to the accumulation patterns of the deposits, including lattice corneal dystrophies type I and IIIA, deep stromal lattice corneal dystrophy, granular corneal dystrophies (GCDs) 2The abbreviations used are: GCDgranular corneal dystrophyFAS1fasciclin 1EMINH2-terminal cysteine-rich domain of the EMILIN familyECMextracellular matrixERendoplasmic reticulumHCFhuman corneal fibroblastPBSphosphate-buffered salineNPCFprimary cultured corneal fibroblast from normal humanHCEhuman corneal epithelialTGNtrans-Golgi networkGFPgreen fluorescent protein. type I and II (also known as Avellino corneal dystrophy), Reis-Bucklers corneal dystrophy (also known as corneal dystrophy of Bowman's layer type I), or Thiel-Behnke corneal dystrophy (also known as corneal dystrophy of Bowman's layer type II) (reviewed in Refs. 5.Pieramici S.F. Afshari N.A. Curr. Opin. Ophthalmol. 2006; 17: 361-366Crossref PubMed Scopus (22) Google Scholar and 6.Poulaki V. Colby K. Semin. Ophthalmol. 2008; 23: 9-17Crossref PubMed Scopus (48) Google Scholar). Histological examinations of corneal tissues demonstrate the presence of amyloid deposits in lattice corneal dystrophies and GCD II, hyaline accumulations in GCDs, and subepithelial fibrous material in Reis-Bucklers corneal dystrophy and Thiel-Behnke corneal dystrophy (7.Korvatska E. Munier F.L. Chaubert P. Wang M.X. Mashima Y. Yamada M. Uffer S. Zografos L. Schorderet D.F. Invest. Ophthalmol. Vis. Sci. 1999; 40: 2213-2219PubMed Google Scholar, 8.Mashima Y. Nakamura Y. Noda K. Konishi M. Yamada M. Kudoh J. Shimizu N. Arch. Ophthalmol. 1999; 117: 90-93Crossref PubMed Scopus (69) Google Scholar, 9.Okada M. Yamamoto S. Tsujikawa M. Watanabe H. Inoue Y. Maeda N. Shimomura Y. Nishida K. Quantock A.J. Kinoshita S. Tano Y. Am. J. 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Vis. 2006; 12: 461-466PubMed Google Scholar). granular corneal dystrophy fasciclin 1 NH2-terminal cysteine-rich domain of the EMILIN family extracellular matrix endoplasmic reticulum human corneal fibroblast phosphate-buffered saline primary cultured corneal fibroblast from normal human human corneal epithelial trans-Golgi network green fluorescent protein. TGFBI was originally identified as a gene induced by transforming growth factor-β stimulation in adenocarcinoma cells and is expressed in many tissues (15.Skonier J. Neubauer M. Madisen L. Bennett K. Plowman G.D. Purchio A.F. DNA Cell Biol. 1992; 11: 511-522Crossref PubMed Scopus (505) Google Scholar). The human TGFBI consists of 683 amino acids, with the mature protein predicted to have a molecular mass of ∼68 kDa. As shown in Fig. 1A, TGFBI contains an NH2-terminal signal peptide that targets it for insertion into the lumen of the endoplasmic reticulum (ER) for eventual secretion, a cysteine-rich EMI domain, four tandem repeats of fasciclin-1 like (FAS1) domains, and a COOH-terminal RGD sequence (15.Skonier J. Neubauer M. Madisen L. Bennett K. Plowman G.D. Purchio A.F. DNA Cell Biol. 1992; 11: 511-522Crossref PubMed Scopus (505) Google Scholar, 16.Thapa N. Lee B.H. Kim I.S. Int. J. Biochem. Cell Biol. 2007; 39: 2183-2194Crossref PubMed Scopus (151) Google Scholar, 17.Doliana R. Bot S. Bonaldo P. Colombatti A. FEBS Lett. 2000; 484: 164-168Crossref PubMed Scopus (93) Google Scholar, 18.Runager K. Enghild J.J. Klintworth G.K. Exp. Eye Res. 2008; 87: 298-299Crossref PubMed Scopus (41) Google Scholar, 19.Rawe I.M. Zhan Q. Burrows R. Bennett K. Cintron C. Invest. Ophthalmol. Vis. Sci. 1997; 38: 893-900PubMed Google Scholar). The FAS1 domains of TGFBI display homology to the cell adhesion protein fasciclin-I in Drosophila, an axon guidance protein that is involved in neuronal development (20.Bastiani M.J. Harrelson A.L. Snow P.M. Goodman C.S. Cell. 1987; 48: 745-755Abstract Full Text PDF PubMed Scopus (292) Google Scholar). Based on the presence of multiple FAS1 domains, TGFBI has been assigned to a larger family of proteins, which includes periostin, stabilin-1, and stabilin-2 (16.Thapa N. Lee B.H. Kim I.S. Int. J. Biochem. Cell Biol. 2007; 39: 2183-2194Crossref PubMed Scopus (151) Google Scholar, 21.Lindsley A. Li W. Wang J. Maeda N. Rogers R. Conway S.J. Gene Expr. Patterns. 2005; 5: 593-600Crossref PubMed Scopus (39) Google Scholar). To date, many TGFBI homologues have been reported in various vertebrates, including mouse, chicken, pig, and zebrafish, but no homologues have been identified in invertebrates (16.Thapa N. Lee B.H. Kim I.S. Int. J. Biochem. Cell Biol. 2007; 39: 2183-2194Crossref PubMed Scopus (151) Google Scholar, 19.Rawe I.M. Zhan Q. Burrows R. Bennett K. Cintron C. Invest. Ophthalmol. Vis. Sci. 1997; 38: 893-900PubMed Google Scholar, 21.Lindsley A. Li W. Wang J. Maeda N. Rogers R. Conway S.J. Gene Expr. Patterns. 2005; 5: 593-600Crossref PubMed Scopus (39) Google Scholar). TGFBI has been shown to interact with a number of extracellular matrix (ECM) proteins, including fibronectin, biglycan, decorin, and several types of collagen (19.Rawe I.M. Zhan Q. Burrows R. Bennett K. Cintron C. Invest. Ophthalmol. Vis. Sci. 1997; 38: 893-900PubMed Google Scholar, 22.Billings P.C. Whitbeck J.C. Adams C.S. Abrams W.R. Cohen A.J. Engelsberg B.N. Howard P.S. Rosenbloom J. J. Biol. Chem. 2002; 277: 28003-28009Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 23.Reinboth B. Thomas J. Hanssen E. Gibson M.A. J. Biol. Chem. 2006; 281: 7816-7824Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 24.Hanssen E. Reinboth B. Gibson M.A. J. Biol. Chem. 2003; 278: 24334-24341Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 25.Kim J.E. Park R.W. Choi J.Y. Bae Y.C. Kim K.S. Joo C.K. Kim I.S. Invest. Ophthalmol. Vis. Sci. 2002; 43: 656-661PubMed Google Scholar). Furthermore, TGFBI also functions as a ligand for several integrins, including α3β1, αvβ5, αvβ3, and αmβ2 (26.Kim J.E. Jeong H.W. Nam J.O. Lee B.H. Choi J.Y. Park R.W. Park J.Y. Kim I.S. J. Biol. Chem. 2002; 277: 46159-46165Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 27.Nam J.O. Kim J.E. Jeong H.W. Lee S.J. Lee B.H. Choi J.Y. Park R.W. Park J.Y. Kim I.S. J. Biol. Chem. 2003; 278: 25902-25909Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 28.Nam E.J. Sa K.H. You D.W. Cho J.H. Seo J.S. Han S.W. Park J.Y. Kim S.I. Kyung H.S. Kim I.S. Kang Y.M. Arthritis Rheum. 2006; 54: 2734-2744Crossref PubMed Scopus (46) Google Scholar, 29.Kim H.J. Kim I.S. Int. J. Biochem. Cell Biol. 2008; 40: 991-1004Crossref PubMed Scopus (32) Google Scholar). The COOH-terminal RGD domain of TGFBI is the putative integrin-binding motif. However, several studies have suggested that the interactions between TGFBI and integrins are mediated via the YH (tyrosine-histidine) motifs and DI (aspartate-isoleucine) motifs present in the TGFBI FAS1 domains (30.Park S.J. Park S. Ahn H.C. Kim I.S. Lee B. J Peptides. 2004; 25: 199-205Crossref PubMed Scopus (16) Google Scholar). Although the precise roles of TGFBI are not fully understood yet, emerging evidence suggests a role for TGFBI as a secreted factor involved in cell adhesion, proliferation, and migration. TGFBI and periostin show a high degree of similarity in amino acid sequence and in overall domain structure, diverging primarily at the COOH terminus (Fig. 1A) (16.Thapa N. Lee B.H. Kim I.S. Int. J. Biochem. Cell Biol. 2007; 39: 2183-2194Crossref PubMed Scopus (151) Google Scholar, 21.Lindsley A. Li W. Wang J. Maeda N. Rogers R. Conway S.J. Gene Expr. Patterns. 2005; 5: 593-600Crossref PubMed Scopus (39) Google Scholar). Similar to TGFBI, periostin contains a NH2-terminal secretory signal peptide followed by a cysteine-rich EMI domain, four tandem repeats of FAS1 domains, and a hydrophilic region in its COOH terminus (Fig. 1A) (16.Thapa N. Lee B.H. Kim I.S. Int. J. Biochem. Cell Biol. 2007; 39: 2183-2194Crossref PubMed Scopus (151) Google Scholar, 17.Doliana R. Bot S. Bonaldo P. Colombatti A. FEBS Lett. 2000; 484: 164-168Crossref PubMed Scopus (93) Google Scholar, 31.Takeshita S. Kikuno R. Tezuka K. Amann E. Biochem. J. 1993; 294: 271-278Crossref PubMed Scopus (551) Google Scholar, 32.Kudo Y. Siriwardena B.S. Hatano H. Ogawa I. Takata T. Histol. Histopathol. 2007; 22: 1167-1174PubMed Google Scholar). Periostin has been found to be ubiquitously expressed in multiple tissues in mammals (31.Takeshita S. Kikuno R. Tezuka K. Amann E. Biochem. J. 1993; 294: 271-278Crossref PubMed Scopus (551) Google Scholar, 33.Horiuchi K. Amizuka N. Takeshita S. Takamatsu H. Katsuura M. Ozawa H. Toyama Y. Bonewald L.F. Kudo A. J. Bone Miner. Res. 1999; 14: 1239-1249Crossref PubMed Scopus (812) Google Scholar, 34.Yoshioka N. Fuji S. Shimakage M. Kodama K. Hakura A. Yutsudo M. Inoue H. Nojima H. Exp. Cell Res. 2002; 279: 91-99Crossref PubMed Scopus (64) Google Scholar). In addition, the expression of periostin has been implicated in the development of variety of cancers, including neuroblastoma, head and neck cancer, and non-small cell lung cancer, possibly by regulating the metastatic growth (32.Kudo Y. Siriwardena B.S. Hatano H. Ogawa I. Takata T. Histol. Histopathol. 2007; 22: 1167-1174PubMed Google Scholar, 35.Bao S. Ouyang G. Bai X. Huang Z. Ma C. Liu M. Shao R. Anderson R.M. Rich J.N. Wang X.F. Cancer Cell. 2004; 5: 329-339Abstract Full Text Full Text PDF PubMed Scopus (474) Google Scholar). Periostin is also associated with epithelial-mesenchymal transition during cardiac development (36.Kühn B. del Monte F. Hajjar R.J. Chang Y.S. Lebeche D. Arab S. Keating M.T. Nat. Med. 2007; 13: 962-969Crossref PubMed Scopus (538) Google Scholar) and is induced during the proliferation of cardiomyocytes, thereby promoting cardiac repair after heart failure (37.Butcher J.T. Norris R.A. Hoffman S. Mjaatvedt C.H. Markwald R.R. Dev. Biol. 2007; 302: 256-266Crossref PubMed Scopus (149) Google Scholar, 38.Shimazaki M. Nakamura K. Kii I. Kashima T. Amizuka N. Li M. Saito M. Fukuda K. Nishiyama T. Kitajima S. Saga Y. Fukayama M. Sata M. Kudo A. J. Exp. Med. 2008; 205: 295-303Crossref PubMed Scopus (368) Google Scholar). In addition, interlukin-4 and -13 have been found to induce the secretion of periostin from lung fibroblasts, implicating periostin in subepithelial fibrosis in bronchial asthma (39.Takayama G. Arima K. Kanaji T. Toda S. Tanaka H. Shoji S. McKenzie A.N. Nagai H. Hotokebuchi T. Izuhara K. J. Allergy Clin. Immunol. 2006; 118: 98-104Abstract Full Text Full Text PDF PubMed Scopus (541) Google Scholar). Despite the similarities between TGFBI and periostin, it is not known whether periostin is involved in the pathogenesis of 5q31-linked corneal dystrophies. In this study, we find that periostin specifically interacts with TGFBI via the NH2-terminal cysteine-rich EMI domain and colocalizes with TGFBI in the trans-Golgi network of COS-7 and corneal fibroblast cells. In addition, corneal dystrophy-linked mutations in TGFBI disrupt its subcellular localization and impair its interaction with periostin. Furthermore, we find that periostin accumulates in extracellular corneal deposits in GCD II patients bearing homozygous R124H mutations in TGFBI. These findings provide new insights into the pathogenic mechanisms of TGFBI mutations in 5q31-linked corneal dystrophies and have important implications for understanding and treating corneal dystrophies. pcDNA3-Periostin-GFP (34.Yoshioka N. Fuji S. Shimakage M. Kodama K. Hakura A. Yutsudo M. Inoue H. Nojima H. Exp. Cell Res. 2002; 279: 91-99Crossref PubMed Scopus (64) Google Scholar) and pcDNA3.1- Periostin-His (35.Bao S. Ouyang G. Bai X. Huang Z. Ma C. Liu M. Shao R. Anderson R.M. Rich J.N. Wang X.F. Cancer Cell. 2004; 5: 329-339Abstract Full Text Full Text PDF PubMed Scopus (474) Google Scholar) constructs were kind gifts from Dr. Hirokazu Inoue (Siga University of Medical Science, Japan) and Dr. Xiao-Fan Wang (Duke University, Durham, NC). Full-length human TGFBI cDNA was cloned into the pcDNA3.1 mammalian expression vector (Invitrogen) with a V5 and His6 tag at the COOH terminus of TGFBI. Deletion and point mutation mutants of TGFBI and periostin were generated in using conventional PCR methods and the QuikChange site-directed mutagenesis kit (Stratagene), following the manufacturer's instructions. The sequences of all constructs were verified by direct sequencing. The primary antibodies used in this study were as follows: mouse monoclonal anti-TGFBI antibody, a kind gift from Dr. In-San Kim (Kyoung Pook University, Korea) (48.Lee S.H. Bae J.S. Park S.H. Lee B.H. Park R.W. Choi J.Y. Park J.Y. Ha S.W. Kim Y.L. Kwon T.H. Kim I.S. Kidney Int. 2003; 64: 1012-1021Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar); goat polyclonal anti-TGFBI (R&D Systems); rabbit polyclonal anti-periostin (ab14041; Abcam); mouse monoclonal anti-V5 (Invitrogen); goat polyclonal anti-periostin (C-20), anti-actin (A-19), mouse monoclonal anti-tenascin-C (300-3), anti-GFP(B-2), mouse monoclonal anti-Myc(9E10), rabbit polyclonal anti-collagen type VI, and TGN38 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); mouse monoclonal anti-Lamp2 (H5C6; BD Pharminogen); horseradish peroxidase-conjugated anti-mouse (GE Healthcare); horseradish peroxidase-conjugated anti-rabbit (GE Healthcare); and anti-goat IgG (Santa Cruz Biotechnology, Inc.). The secondary antibodies used for immunofluorescence were as follows: goat anti-mouse or goat anti-rabbit conjugated to Alex Fluor 488 or 594 (Invitrogen). HeLa, COS-7, HEK293, and human corneal fibroblast (HCF) cell lines were grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 2 mm l-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, and 10% (w/v) fetal bovine serum (Invitrogen) at 37 °C in a 5% CO2 incubator. The human corneal epithelial (HCE) cell line was grown in Dulbecco's modified Eagle's medium and F-12 (1:1) media supplemented with 2 mm l-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, 10% (w/v) fetal bovine serum, 10 ng/ml recombinant human epidermal growth factor (R&D Systems) at 37 °C in a 5% CO2 incubator. Human corneal epithelial and fibroblast cell lines were a kind gift from Dr. Shigeru Kinoshita (Kyoto Prefectural University of Medicine, Japan) and Dr. James V. Jester (University of California, Irvine, CA). Primary corneal fibroblasts were cultured from corneal buttons obtained from a 60-year-old control and a 27-year-old homozygous GCD II patient during penetrating keratoplasty. The endothelial and epithelial layers were removed from the corneas, and stroma was used as explants to initiate corneal fibroblast cultures. The cells were maintained in Dulbecco's modified Eagle's medium supplemented with 2 mm l-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, and 10% (w/v) fetal bovine serum at 37 °C in a 5% CO2 incubator. Donor confidentiality was maintained according to the Declaration of Helsinki and was approved by the Severance Hospital IRB Committee (CR04124). Transfections were performed using GeneJammer (Stratagene) according to the manufacturer's instructions, analyses were conducted 24 h post-transfection, and immunoprecipitations were carried out as described previously (49.Kim B.Y. Krämer H. Yamamoto A. Kominami E. Kohsaka S. Akazawa C. J. Biol. Chem. 2001; 276: 29393-29402Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Cells were washed with PBS, and extracts were obtained by passing the suspension through a 26-gauge needle in ice-cold lysis buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% Nonidet P-40, 0.1% Triton X-100 supplemented with protease inhibitor mixtures (Applied Biological Materials Inc.)). Soluble supernatants were analyzed by SDS-PAGE under reducing conditions and transferred to nitrocellulose membranes (Millipore). The membrane was then blocked with 5% skim milk (Difco) in 1× TBST buffers (20 mm Tris-HCl, 137 mm NaCl, pH 7.6, 0.1% Tween 20) and incubated with the indicated antibodies. The SuperSignal West Pico chemiluminescent substrate Kit (Thermo Scientific) was used for protein detection. The band intensities were quantified using the ImageJ program (version 1.38). Normal human corneal epithelial cells were obtained by scraping the epithelial layer during photorefractive keratectomy. Patient corneal epithelial cells from a GCD II patient were obtained by scraping the epithelial layer during deep lamellar corneal transplantation. After scraping the corneal surface using a blunt blade, samples were immediately placed into ice-cold lysis buffer, and proteins were extracted. For His tag pull-down assays, COOH-terminal His-tagged wild type of TGFBI was purified as described previously (25.Kim J.E. Park R.W. Choi J.Y. Bae Y.C. Kim K.S. Joo C.K. Kim I.S. Invest. Ophthalmol. Vis. Sci. 2002; 43: 656-661PubMed Google Scholar), and NH2-terminal His-tagged periostin was purchased from BioVendor. Twenty micrograms of His-tagged recombinant TGFBI or periostin was immobilized on nickel-agarose resin (Applied Biological Materials) and incubated overnight at 4 °C with 500 μg of HCF cell lysates. Bound proteins were resolved by SDS-PAGE and detected by Western blotting with the indicated antibodies. For immunofluorescence microscopy, cells were grown on coverslips, fixed in cold methanol/acetone (1:1, v/v) for 10 min at −20 °C, and blocked with 2% bovine serum albumin for 30 min. Cells were incubated with primary antibodies in 2% bovine serum albumin for 1 h at room temperature. Cells were washed with PBS and subsequently incubated with secondary antibodies in 2% bovine serum albumin for 1 h at room temperature. After washing with PBS, cells were mounted using Vectashield (Vector Laboratories, Inc.). Images were acquired using a TCS SP5 confocal microscope (Leica). For immunohistochemistry analyses, corneas from normal human, R124H mutated heterozygous and homozygous GCD II patients were fixed in 10% neutral-buffered formalin and embedded in paraffin. The paraffin-embedded samples were sectioned on a microtome at a thickness of 5 μm, mounted on slide glasses, deparaffinized in xylene, and rehydrated in ethanol. The sections were incubated in 0.3% H2O2 for 30 min and blocked with 2.5% normal horse serum for 20 min. The sections were then incubated with normal rabbit IgG serum and/or rabbit polyclonal anti-periostin antibody (1:500, v/v) in 2.5% normal horse serum and 0.1% bovine serum albumin for 1 h at room temperature. The sections were washed with PBS and incubated in ImPress universal reagent (Vector Laboratories) for 30 min. After washing with PBS for 5 min, the sections were incubated with DAB solution and visualized according to the manufacturer's instructions (Vector Laboratories). Sections were washed with PBS three times and mounted using Vectashield (Vector Laboratories). Masson's trichrome stains were used to confirm the mutated TGFBI deposits in the corneal stroma. Images were acquired using a BX 40 light microscope (Olympus). Despite the fact that TGFBI and periostin share several similarities in structure and expression patterns (Fig. 1A) (15.Skonier J. Neubauer M. Madisen L. Bennett K. Plowman G.D. Purchio A.F. DNA Cell Biol. 1992; 11: 511-522Crossref PubMed Scopus (505) Google Scholar, 33.Horiuchi K. Amizuka N. Takeshita S. Takamatsu H. Katsuura M. Ozawa H. Toyama Y. Bonewald L.F. Kudo A. J. Bone Miner. Res. 1999; 14: 1239-1249Crossref PubMed Scopus (812) Google Scholar), little is known about the roles of periostin in corneal tissues. To examine the expression of periostin in cornea and cornea-derived cells, we first performed Western blot analysis with specific anti-periostin antibodies (C-20 and ab14041) (Fig. 1B). Western blot analysis revealed expression of periostin in all of the tested cells and tissues, including COS-7, HeLa, HEK293, and HCF (40.Jester J.V. Huang J. Fisher S. Spiekerman J. Chang J.H. Wright W.E. Shay J.W. Invest. Ophthalmol. Vis. Sci. 2003; 44: 1850-1858Crossref PubMed Scopus (113) Google Scholar); primary cultured corneal fibroblast from normal human (NPCF) and HCE cell lines (41.Araki-Sasaki K. Ohashi Y. Sasabe T. Hayashi K. Watanabe H. Tano Y. Handa H. Invest. Ophthalmol. Vis. Sci. 1995; 36: 614-621PubMed Google Scholar); and normal human corneal epithelium (Fig. 1B, top, lanes 1–7). In HeLa, COS-7, HEK293, HCF, and NPCF, endogenous periostin was detected primarily as a single band that migrated with an apparent molecular mass of ∼85 kDa, consistent with the predicted molecular weight (Fig. 1B, lanes 1–5). A second high molecular mass band of ∼170 kDa was observed in some cell lines. This band may represent the previously reported covalently linked periostin multimer (42.Gillan L. Matei D. Fishman D.A. Gerbin C.S. Karlan B.Y. Chang D.D. Cancer Res. 2002; 62: 5385-5394Google Scholar) or perhaps some other covalent postranslational modification. However, in HCE and corneal epithelium, periostin was detected as a single band of ∼60 kDa (Fig. 1B, top, lanes 6 and 7). The C-20 anti-periostin antibody was raised against a COOH-terminal periostin peptide (amino acids 725–775), and preabsorption with a periostin peptide completely abolished the immunoreactivity of anti-periostin antibody (C-20), confirming the specificity of this antibody (Fig. 1B, second panel). To further determine the identity of the periostin-immunoreactive band, we performed additional Western blot analyses using an independent anti-periostin antibody generated against a separate epitope (ab14041, amino acids 22–669) and found that this antibody also recognized the ∼60 kDa band in human corneal epithelium (supplemental Fig. 1). Together, these results suggest that periostin is expressed in cornea-derived fibroblast and epithelial cell lines as well as in corneal epithelium. In addition, the detection of a form of periostin of reduced molecular weight with two anti-periostin antibodies that recognize separate periostin epitopes raises the possibility of cell type-specific proteolytic processing of periostin or cell type-specific periostin splice variants. Periostin has previously been shown to form dimers (42.Gillan L. Matei D. Fishman D.A. Gerbin C.S. Karlan B.Y. Chang D.D. Cancer Res. 2002; 62: 5385-5394Google Scholar), and given the structural similarity between periostin and TGFBI, we next sought to determine whether the two proteins interact. We first performed pull-down assays using immobilized His-tagged TGFBI or periostin with HCF cell lysates. Bound proteins were separated by SDS-PAGE and visualized by Western blotting. As shown in Fig. 2A, His-tagged TGFBI efficiently pulled down endogenous periostin from HFC cell lysates (Fig. 2A). Consistent with previous reports (19.Rawe I.M. Zhan Q. Burrows R. Bennett K. Cintron C. Invest. Ophthalmol. Vis. Sci. 1997; 38: 893-900PubMed Google Scholar, 23.Reinboth B. Thomas J. Hanssen E. Gibson M.A. J. Biol. Chem. 2006; 281: 7816-7824Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 24.Hanssen E. Reinboth B. Gibson M.A. J. Biol. Chem. 2003; 278: 24334-24341Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), we found that collagen VI was readily pulled down from HFC cell lysates by His-tagged recombinant TGFBI (Fig. 2A). In addition, His-tagged TGFBI did not pull down the cytoskeletal protein actin, confirming the specificity of this experiment. In the reciprocal experiment, we found that His-tagged periostin efficiently pulled down endogenous TGFBI, but not actin or collagen VI. These in vitro binding studies indicate that periostin is able to interact with TGFBI. These results also show that periostin does not interact with the TGFBI-binding partner collagen VI (Fig. 2B), indicating that despite the large degree of sequence similarity, periostin and TGFBI are not interchangeable. To verify that the periostin-TGFBI interaction occurs in vivo, we performed co-immunoprecipitation experiments using antibodies specific for periostin and TGFBI. As shown in Fig. 2C, anti-TGFBI antibodies, but not the IgG control, efficiently co-immunoprecipitated endogenous periostin from HCF cell lysates. Furthermore, anti-periostin antibodies specifically co-immunoprecipitated endogenous TGFBI from HCF cell lysates (Fig. 2D, lane 3). Taken together, the pull-down assays and co-immunoprecipitation experiments demonstrate that periostin interacts with TGFBI in vit
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