- Email: [email protected]
Fibromodulin gene is expressed in human epidermal keratinocytes in culture and in human epidermis in vivo
Biochemical and Biophysical Research Communications 371 (2008) 420–424
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Fibromodulin gene is expressed in human epidermal keratinocytes in culture and in human epidermis in vivo Cristina Vélez-delValle a, Meytha Marsch-Moreno a, Federico Castro-Muñozledo a, Yesid Jaime Bolivar-Flores b, Walid Kuri-Harcuch a,* a b
Department of Cell Biology, Centro de Investigación y de Estudios Avanzados del IPN, Apdo. Postal 14-740, México City 07000, Mexico Hospital Medica Sur, Puente de Piedra 150, Suite 726, Mexico City 14050, Mexico
a r t i c l e
i n f o
Article history: Received 9 April 2008 Available online 28 April 2008
Keywords: Cultured epidermal keratinocytes Human epidermis Fibromodulin Small leucine rich proteoglycan
a b s t r a c t Fibromodulin is a small leucine-rich proteoglycan that has a central role in the maintenance of collagen fibrils structure, and in regulation of TGF-b biological activity. Although, it is mainly found in cartilage and tendon, little is known regarding the expression of the fibromodulin gene in other cell types. By RT-PCR, real time PCR and immunohistochemistry, we describe the expression of the fibromodulin gene and the presence of the protein in human epidermal keratinocytes (HEK), both in culture and in normal human epidermis. Our results show, for the first time, that fibromodulin gene is constantly expressed in HEK during culture time. Immunostaining showed that fibromodulin is located intracytoplasmically in basal and stratified keratinocytes of the growing colonies, confluent cultures, and epidermis in vivo. The expression and intracellular localization of fibromodulin in HEK is a new finding and opens new possible biological roles for the SLRP family. Ó 2008 Elsevier Inc. All rights reserved.
Fibromodulin was found in cartilage and tendon and it was isolated as a 59 kDa protein from articular cartilage [1]; the protein belongs to a family of small leucine-rich proteoglycans/glycoproteins (SLRP’s). Proteoglycans (PG’s) are macromolecules consisting of a protein core covalently bound to one or more glycosaminoglycan (GAG) side chains [2]. PGs are important components of mammalian extracellular matrices (ECM’s), and they are divided in the SLRP’s, the hyalectans, and the basement-membrane proteoglycans [3]. The core proteins of SLRPs have a central domain containing 6– 10 leucine-rich repeats (LRR’s), flanked by N-terminal and C-terminal regions with conserved Cys residues [4]. The LRR motif has been found in a number of proteins of diverse origin and function and varies in length from 20 to 29 amino acids [5]. The functions of SLRPs are still somewhat controversial; fibromodulin, along with decorin, has a central role in organization of type I and II collagen fibrils structure, and it is a ubiquitous protein most prominent in articular cartilage, tendon, ligament [6–9], and dermal tissues [10,11]. On the other hand, the isoforms of TGF-b interact not only with the core proteins of decorin and biglycan, but also with fibromodulin [12] modulating the cellular fibrotic response in many tissues including lung, kidney, and skin [13–17]. Studies in fibromodulin have been carried out mainly in connective tissue [18] and endothelial cells [19], but little is known regarding its gene expression in other cell types such as epithelial * Corresponding author. Fax: +52(55)5747 3393. E-mail address: [email protected] (W. Kuri-Harcuch). 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.04.095
cells, with the exception of corneal epithelium in which the protein is found at a very low level, but nothing about its gene expression or protein localization is known [20]. Therefore, we thought of interest to study the expression of the fibromodulin gene in epithelial cells; we chose the human epidermal keratinocytes under culture conditions that support differentiation and stratification of the epidermal layers [21]. Since basement membrane separates the basal keratinocytes of the epidermis from the dermis, which contains large amounts of the SLRP’s, human epidermis would be a suitable model to study fibromodulin gene expression and its protein function in epithelial cells in vivo. Materials and methods Materials. Thermo ScriptTM RT-PCR System with PlatinumÒ Taq DNA Polimerase High Fidelity, and the TOPO TA Cloning Kit were from Invitrogen-Life Technologies (Carlsbad, CA). The LightCycler FastStart DNA MasterPLUS SYBR Green I kit, the biotinylated antibody against mouse IgG and the FITC-streptavidin were from Roche Applied Science (Indianapolis, IN). Fetal bovine (FBS) and bovine calf (BCS) sera were from HyClone Laboratories (Logan, UT). Monoclonal antibody against human fibromodulin was purchased from Kamiya Biomedical Company (Seattle, WA). All other reagents were analytical grade. Cell culture. The human epidermal keratinocytes (cHEK), strain HE-123, were isolated from newborn foreskin as previously described [21,22]. For experiments, 3rd to 5th passage keratinocytes
C. Vélez-delValle et al. / Biochemical and Biophysical Research Communications 371 (2008) 420–424
were plated together with mitomycin C treated 3T3 feeder cells in DMEM–F12 (3:1) supplemented with 4%(v/v) FBS, 0.4 lg/ml hydrocortisone, 2 109 M L-T3, 5 lg/ml transferrin, 5 lg/ml insulin, 24.3 mg/l adenine, and 10 ng/ml EGF, as previously described [21–23]. The human dermal fibroblasts (strain hFib-132) were isolated in our laboratory from human skin and cultured as above. Cell cultures were incubated at 37 °C in humidified incubator equilibrated with 10% CO2; medium was changed every other day. RNA extraction, RT-PCR, and sequencing. Total RNA was isolated with Trizol reagent (Invitrogen, CA) and stored at 70 °C as an ethanol precipitate until further use. For the end point PCR assays, reverse transcription was carried out with Thermo ScriptTM reverse transcriptase, using 1.0 lg total RNA, and oligo(dT) primer. Primers used for PCR reactions were 50 -GGTCGCCACCATGCAGTGGGC GTCCCT (forward) and 30 -GCTGCTCAGATCTCGATGA (reverse), which were specific for exon 1 and exon 3 at the fibromodulin encoding gene, respectively. The amplification products were separated in 2%(w/v) agarose gel, cloned into pCRII cloning vector, and then sequenced with both T7 and SP6 sequencing primers, and with fibromodulin-specific primers. Sequencing was carried out using an ABI PRISMTM 310 DNA Sequencer (Perkin-Elmer) and the Big Dye Terminator kit v3.1 (Perkin-Elmer). Sequencing data were compared with the published fibromodulin gene sequence using the BLASTN 2.2 search program [24]. The relative expression of the mRNA encoding fibromodulin was quantified by Real-time PCR. PCR reactions were carried out using LightCycler FastStart DNA MasterPLUS SYBR Green I according to the manufacturer’s instructions. Gene expression was analyzed with the 7500 real time thermal cycler (Applied Biosystems). Fibromodulin mRNA levels were normalized to the levels of the mRNA encoding the housekeeping enzyme GAPDH. The specificity of the PCR product was analyzed by agarose gel electrophoresis and melting curve. Immunodetection of human fibromodulin. Cells were grown on glass coverslips; after 7 days in culture, they were fixed with 3.5%(w/v) para-formaldehyde in PBS. Cells, permeabilized or not with 0.1% Triton X-100, were stained for fibromodulin; the primary antibody was detected with a biotinylated secondary antibody and streptavidin-FITC. Slides were mounted with VECTASHIELDÒ Mounting Medium (Vector Laboratories, Burlingame, CA). For
421
immunodetection in cultured epithelia, the cultured epidermal sheets were detached from the dish using Dispase II (2.5 mg/ml). Both cultured epithelia and normal skin were embedded with tissue freezing medium (Jung) and frozen at –70 °C until further use. Afterwards, 8-lm tissue cryosections in salinized glass slides were air-dried and then hydrated in phosphate-buffered saline (PBS) for 5 min prior to antibody detection. Results Fibromodulin expression in cultured human epidermal keratinocytes Total RNA was extracted from growing or 2 days post-confluent HEK cultures, and PCR amplification was carried out with primers specific to exons 1 and 3 of the human fibromodulin gene [9]; we amplified the ORF of the core protein and part of the 50 untranslated region. By end point PCR we obtained a 1150-bp fragment (Fig. 1A, lane 4) corresponding to the expected amplification product of fibromodulin; we obtained the same product from total RNA from cultured human dermal fibroblasts, as control (Fig. 1A, lane 3). The primer set was specific for human fibromodulin mRNA since no amplification products were identified from the murine 3T3 cells RNA (Fig. 1A, lane 2). We verified the integrity and functionality of the cDNA, by testing the same samples for amplification of GAPDH. In order to avoid any gap or uncertain nucleotides in the obtained sequence, we cloned and sequenced twice in both directions the 1150-bp fragment from HEK, using universal sequencing primers or the fibromodulin specific primers (see Materials and methods). BLAST analysis of the cloned fragment showed complete matching with the published human cDNA sequence (Gene Bank reference NM_002023). These data strongly evidenced that the fibromodulin gene is expressed in cultured human epidermal keratinocytes. By relative quantification of fibromodulin mRNA using primers previously described [25], we determined by real time PCR the expression levels of fibromodulin mRNA in HEK at various times of culture, after washing out the murine 3T3 feeder cells. The mean amplification plots show that fibromodulin expression was constant during culture and considerably lower in cHEK than in hFib (Fig. 1B) this was quantified (Table 1) by normalizing the data to
Fig. 1. PCR amplification of fibromodulin mRNA. (A, upper panel) End point PCR of fibromodulin encoding mRNA. The 1150 bp product, that corresponds to the ORF of hFMOD. (A, lower panel) GAPDH mRNA was used as an amplification control. (B) Real time PCR amplification of fibromodulin mRNA expression in total RNA from HEK at different times in culture, and in proliferative human fibroblasts (hFib).
422
C. Vélez-delValle et al. / Biochemical and Biophysical Research Communications 371 (2008) 420–424
Table 1 Fibromodulin mRNA expression in HEK at different culture days Culture condition
Expression level (folds) (±)SD
hFIB cHEK cHEK cHEK cHEK
1.00 ± 0.08 0.006 ± 0.001 0.006 ± 0.002 0.006 ± 0.005 0.007 ± 0.008
5d* 7d* 12d* 14d
Total RNA was isolated from cultures as described. After real time-PCR experiments, the relative expression of fibromodulin encoding mRNA was analyzed. Results were normalized against the GAPDH expression levels. Columns represent the relative proportion of fibromodulin mRNA, referred to that found in hFIB cultures. * p < 0.05.
as it is seen by the dark boundary showing the intercellular space. Cryosections of the confluent epithelium, confirmed the protein was inside the cells of the 2–3 layers of the cultured epidermis (Fig. 2C and D). These results raised the possibility that keratinocytes in normal human epidermis should also express fibromodulin. Frozen sections from adult human skin showed that fibromodulin was localized in the basal and suprabasal keratinocytes of the epidermis, but not in the stratum corneum (Fig. 2E and F), and it seemed also to be inside the keratinocytes. As an internal control of the antibody, fibromodulin was also immunodetected around some dermal cells, most likely fibroblasts (Fig. 2E and F). Discussion
the expression of the housekeeping gene GAPDH, using the 2DDC T aproach [26]. These data clearly give evidence that the HEK in culture have the ability to express the fibromodulin gene, however, at lower levels as compared to the cultured human fibroblasts. Immunolocalization of fibromodulin in cultured human epidermal keratinocytes, and in human epidermis in vivo By immunohistochemistry with an antibody specific against fibromodulin we studied the distribution of this protein in cultured HEK. Fibromodulin was inmunodetected in basal and stratified keratinocytes of the growing colonies (Fig. 2A and B); it seemed to be located intracytoplasmically, with a pericellular distribution,
Fibromodulin remains as the least explored PG from the SLRP family; it is mainly described as a structural molecule related to collagen fibrillogenesis [10,11,27,28]. However, the most relevant functional activity described for fibromodulin is its binding of TGFb isoforms in vitro [12]. Biglycan is the only described SLRP in human epidermis [29,30], but nothing is known about its function in this tissue. Lumican has been described in epithelial cells during corneal wound healing, and the data suggested that lumican null-animals had delayed re-epithelization of the central cornea [30,31]. While we were doing this work, fibromodulin and lumican have been described in basal cells of human epithelial gingiva [32], but the mesenchymal or epithelial origin of these proteoglycans, as
Fig. 2. Immunolocalization of fibromodulin in cHEK and human skin. (A and B) Five day HEK growing colonies were immunostained with antibodies raised against human fibromodulin. (C and D) 8-lm cryosections of confluent cultured epidermal sheets were stained with the anti-hFMOD antibody, the PG was observed both in basal and in stratified cells, with no preferential distribution in the cell layers. (E and F) In similar experiments, cryosections of normal human skin showed staining for the PG from the basal layer to the stratum spinosum, but not in the stratum corneum. (A, C, and E) display the phase contrast micrographs of the immunofluorescence fields shown in (B, D and F). (B and D), scale bar = 100 lm, (F), scale bar = 50 lm.
C. Vélez-delValle et al. / Biochemical and Biophysical Research Communications 371 (2008) 420–424
well as the expression of their encoding genes, remain unknown. Our results are the first to show fibromodulin expression in epidermal keratinocytes in vivo and in culture. Expression of fibromodulin mRNA, and the presence of fibromodulin protein support the conclusion that human keratinocytes can synthesize and accumulate this protein. Our evidence shows that keratinocytes are a novel source of fibromodulin in normal human skin in vivo. Previous studies, by Northern blot in total RNA extracts, have failed to detect fibromodulin in human keratinocytes cultured under serum free medium conditions [7]. We think that our approach has two main advantages that can explain these differences. First, co-cultivation of human epidermal keratinocytes with 3T3 cells as a feeder layer [21] supports the proliferative potential of the keratinocytes, the expression of differentiation markers, as well as the deposition of a more complete extracellular matrix [21,33,34]. Second, the use of RT-PCR, and of real time PCR, for the detection of mRNAs have more sensitivity than Northern blot analysis [35]. Unfortunately, we could not assay if mRNA expression correlates with the content of the protein, because our antibody did not recognize it in western blot analysis of the cell extracts. Also, it remains necessary to establish the glycoprotein or proteoglycan nature of the fibromodulin found in cHEK. The intracellular localization of fibromodulin is a new finding; its extensive intracellular distribution is concurrent with previous evidence of keratan sulfate epitopes in the cytoplasm of HEK [36]; it was shown that this keratan sulfate co-migrates with keratins in SDS-polyacrilamide gels [13]. Interestingly, the migration pattern of the keratins with associated keratan sulfate comprises the same range of molecular weights of the recombinant fibromodulin migration pattern [37], raising the possibility that some interaction between keratins and the proteoglycan, might stabilize the intermediate filament structure. This remains as an interesting and challenging possibility that we are pursuing in further investigation. Elucidation of other biological roles for fibromodulin remain open, mainly those that could resemble similar functions to those found for other members of this SLRP family, such as lumican [30,38,39], or decorin [40–42]. The transitory expression of lumican and its association with cellular migration during wound healing in corneal epithelial cells [30], raises an interesting possibility for a similar role of fibromodulin in epidermal or other epithelial keratinocytes. Acknowledgments This work was supported in part by CONACyT Grants # G28272N, 39690-Q and 46453. We thank M en C. Erika Sánchez for their technical assistance. We also thank Ms. María Elena Rojano for her secretarial assistance and Mr. Alberto Rodriguez and Mrs. Columba Guadarrama for preparing all materials. References [1] D. Heinegard, T. Larsson, Y. Sommarin, A. Franzen, M. Paulsson, E. Hedbom, Two novel matrix proteins isolated from articular cartilage show wide distributions among connective tissues, J. Biol. Chem. 261 (1986) 13866– 13872. [2] L. Kjellen, U. Lindahl, Proteoglycans: structures and interactions, Annu. Rev. Biochem. 60 (1991) 443–475. [3] R.V. Iozzo, Matrix proteoglycans: from molecular design to cellular function, Annu. Rev. Biochem. 67 (1998) 609–652. [4] R.V. Iozzo, The family of the small leucine-rich proteoglycans: key regulators of matrix assembly and cellular growth, Crit. Rev. Biochem. Mol. Biol. 32 (1997) 141–174. [5] R.V. Iozzo, A.D. Murdoch, Proteoglycans of the extracellular environment: clues from the gene and protein side offer novel perspectives in molecular diversity and function, FASEB J. 10 (1996) 598–614. [6] G. Westergren-Thorsson, P. Antonsson, A. Malmstrom, D. Heinegard, A. Oldberg, The synthesis of a family of structurally related proteoglycans in fibroblasts is differently regulated by TFG-beta, Matrix 11 (1991) 177–183.
423
[7] J.B. Petri, O. Rott, T. Wetzig, K. Herrmann, U.F. Haustein, The small proteoglycan fibromodulin is expressed in mitotic, but not in postmitotic fibroblasts, Mol. Cell. Biol. Res. Commun. 1 (1999) 59–65. [8] P.J. Roughley, R.J. White, G. Cs-Szabo, J.S. Mort, Changes with age in the structure of fibromodulin in human articular cartilage, Osteoarthr. Cartilage 4 (1996) 153–161. [9] P. Antonsson, D. Heinegard, A. Oldberg, Structure and deduced amino acid sequence of the human fibromodulin gene, Biochim. Biophys. Acta 1174 (1993) 204–206. [10] E. Hedbom, D. Heinegard, Binding of fibromodulin and decorin to separate sites on fibrillar collagens, J. Biol. Chem. 268 (1993) 27307–27312. [11] K. Noyori, H.E. Jasin, Inhibition of human fibroblast adhesion by cartilage surface proteoglycans, Arthritis Rheum. 37 (1994) 1656–1663. [12] A. Hildebrand, M. Romaris, L.M. Rasmussen, D. Heinegard, D.R. Twardzik, W.A. Border, E. Ruoslahti, Interaction of the small interstitial proteoglycans biglycan, decorin and fibromodulin with transforming growth factor beta, Biochem. J. 302 (Pt 2) (1994) 527–534. [13] I.A. Schafer, J.M. Sorrell, Human keratinocytes contain keratin filaments that are glycosylated with keratan sulfate, Exp. Cell Res. 207 (1993) 213–219. [14] N. Venkatesan, T. Ebihara, P.J. Roughley, M.S. Ludwig, Alterations in large and small proteoglycans in bleomycin-induced pulmonary fibrosis in rats, Am. J. Respir. Crit. Care Med. 161 (2000) 2066–2073. [15] G. Westergren-Thorsson, J. Hernnas, B. Sarnstrand, A. Oldberg, D. Heinegard, A. Malmstrom, Altered expression of small proteoglycans, collagen, and transforming growth factor-beta 1 in developing bleomycin-induced pulmonary fibrosis in rats, J. Clin. Invest. 92 (1993) 632–637. [16] T. Ebihara, N. Venkatesan, R. Tanaka, M.S. Ludwig, Changes in extracellular matrix and tissue viscoelasticity in bleomycin-induced lung fibrosis. Temporal aspects, Am. J. Respir. Crit. Care Med. 162 (2000) 1569–1576. [17] C.C. Reed, R.V. Iozzo, The role of decorin in collagen fibrillogenesis and skin homeostasis, Glycoconj. J. 19 (2002) 249–255. [18] M. Benjamin, J.R. Ralphs, Biology of fibrocartilage cells, Int. Rev. Cytol. 233 (2004) 1–45. [19] L. Schaefer, H.J. Grone, I. Raslik, H. Robenek, J. Ugorcakova, S. Budny, R.M. Schaefer, H. Kresse, Small proteoglycans of normal adult human kidney: distinct expression patterns of decorin, biglycan, fibromodulin, and lumican, Kidney Int. 58 (2000) 1557–1568. [20] E. Schonherr, C. Sunderkotter, L. Schaefer, S. Thanos, S. Grassel, A. Oldberg, R.V. Iozzo, M.F. Young, H. Kresse, Decorin deficiency leads to impaired angiogenesis in injured mouse cornea, J. Vasc. Res. 41 (2004) 499–508. [21] J.G. Rheinwald, H. Green, Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells, Cell 6 (1975) 331–343. [22] J. Bolivar-Flores, E. Poumian, M. Marsch-Moreno, G. Montes de Oca, W. KuriHarcuch, Use of cultured human epidermal keratinocytes for allografting burns and conditions for temporary banking of the cultured allografts, Burns 16 (1990) 3–8. [23] F. Castro-Munozledo, M. Hernandez-Quintero, M. Marsch-Moreno, W. KuriHarcuch, Cultivation, serial transfer, and differentiation of epidermal keratinocytes in serum-free medium, Biochem. Biophys. Res. Commun. 236 (1997) 167–172. [24] S.F. Altschul, T.L. Madden, A.A. Schaffer, J. Zhang, Z. Zhang, W. Miller, D.J. Lipman, Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, Nucleic Acids Res. 25 (1997) 3389–3402. [25] F. Barry, R.E. Boynton, B. Liu, J.M. Murphy, Chondrogenic differentiation of mesenchymal stem cells from bone marrow: differentiation-dependent gene expression of matrix components, Exp. Cell Res. 268 (2001) 189–200. [26] K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using realtime quantitative PCR and the 2(-Delta Delta C(T)) method, Methods 25 (2001) 402–408. [27] K.G. Vogel, M. Paulsson, D. Heinegard, Specific inhibition of type I and type II collagen fibrillogenesis by the small proteoglycan of tendon, Biochem. J. 223 (1984) 587–597. [28] E. Hedbom, D. Heinegard, Interaction of a 59-kDa connective tissue matrix protein with collagen I and collagen II, J. Biol. Chem. 264 (1989) 6898–6905. [29] P. Bianco, L.W. Fisher, M.F. Young, J.D. Termine, P.G. Robey, Expression and localization of the two small proteoglycans biglycan and decorin in developing human skeletal and non-skeletal tissues, J. Histochem. Cytochem. 38 (1990) 1549–1563. [30] S. Saika, A. Shiraishi, C.Y. Liu, J.L. Funderburgh, C.W. Kao, R.L. Converse, W.W. Kao, Role of lumican in the corneal epithelium during wound healing, J. Biol. Chem. 275 (2000) 2607–2612. [31] L.K. Yeh, W.L. Chen, W. Li, E.M. Espana, J. Ouyang, T. Kawakita, W.W. Kao, S.C. Tseng, C.Y. Liu, Soluble lumican glycoprotein purified from human amniotic membrane promotes corneal epithelial wound healing, Invest. Ophthalmol. Vis. Sci. 46 (2005) 479–486. [32] S. Chakravarti, Functions of lumican and fibromodulin: lessons from knockout mice, Glycoconj. J. 19 (2002) 287–293. [33] P.P. Parnigotto, V. Bassani, S. Pastore, F. Valenti, M.T. Conconi, Fibroblastkeratinocyte co-cultures in vitro: growth, morphometry and nutrient exchange, Ital. J. Anat. Embryol. 99 (1994) 17–30. [34] K. Alitalo, E. Kuismanen, R. Myllyla, U. Kiistala, S. Asko-Seljavaara, A. Vaheri, Extracellular matrix proteins of human epidermal keratinocytes and feeder 3T3 cells, J. Cell Biol. 94 (1982) 497–505. [35] K. Reue, mRNA quantitation techniques: considerations for experimental design and application, J. Nutr. 128 (1998) 2038–2044.
424
C. Vélez-delValle et al. / Biochemical and Biophysical Research Communications 371 (2008) 420–424
[36] J.M. Sorrell, B. Caterson, Monoclonal antibodies specific for keratan sulfate detect epithelial-associated carbohydrates, Histochemistry 94 (1990) 269–275. [37] L. Svensson, I. Narlid, A. Oldberg, Fibromodulin and lumican bind to the same region on collagen type I fibrils, FEBS Lett. 470 (2000) 178–182. [38] N. Vij, L. Roberts, S. Joyce, S. Chakravarti, Lumican suppresses cell proliferation and aids Fas–Fas ligand mediated apoptosis: implications in the cornea, Exp. Eye Res. 78 (2004) 957–971. [39] N. Vij, L. Roberts, S. Joyce, S. Chakravarti, Lumican regulates corneal inflammatory responses by modulating Fas–Fas ligand signaling, Invest. Ophthalmol. Vis. Sci. 46 (2005) 88–95. [40] M.L. D’Antoni, C. Torregiani, P. Ferraro, M.C. Michoud, B. Mazer, J.G. Martin, M.S. Ludwig, Effects of decorin and biglycan on human airway smooth muscle
cell proliferation and apoptosis, Am. J. Physiol. Lung Cell Mol. Physiol. (2008), doi:10.1152/ajplung.00436.2007. [41] L. Schaefer, K. Macakova, I. Raslik, M. Micegova, H.J. Grone, E. Schonherr, H. Robenek, F.G. Echtermeyer, S. Grassel, P. Bruckner, R.M. Schaefer, R.V. Iozzo, H. Kresse, Absence of decorin adversely influences tubulointerstitial fibrosis of the obstructed kidney by enhanced apoptosis and increased inflammatory reaction, Am. J. Pathol. 160 (2002) 1181–1191. [42] J.G. Tralhao, L. Schaefer, M. Micegova, C. Evaristo, E. Schonherr, S. Kayal, H. Veiga-Fernandes, C. Danel, R.V. Iozzo, H. Kresse, P. Lemarchand, In vivo selective and distant killing of cancer cells using adenovirus-mediated decorin gene transfer, FASEB J. 17 (2003) 464–466.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Fibromodulin gene is expressed in human epidermal keratinocytes in culture and in human epidermis in vivo Cristina Vélez-delValle a, Meytha Marsch-Moreno a, Federico Castro-Muñozledo a, Yesid Jaime Bolivar-Flores b, Walid Kuri-Harcuch a,* a b
Department of Cell Biology, Centro de Investigación y de Estudios Avanzados del IPN, Apdo. Postal 14-740, México City 07000, Mexico Hospital Medica Sur, Puente de Piedra 150, Suite 726, Mexico City 14050, Mexico
a r t i c l e
i n f o
Article history: Received 9 April 2008 Available online 28 April 2008
Keywords: Cultured epidermal keratinocytes Human epidermis Fibromodulin Small leucine rich proteoglycan
a b s t r a c t Fibromodulin is a small leucine-rich proteoglycan that has a central role in the maintenance of collagen fibrils structure, and in regulation of TGF-b biological activity. Although, it is mainly found in cartilage and tendon, little is known regarding the expression of the fibromodulin gene in other cell types. By RT-PCR, real time PCR and immunohistochemistry, we describe the expression of the fibromodulin gene and the presence of the protein in human epidermal keratinocytes (HEK), both in culture and in normal human epidermis. Our results show, for the first time, that fibromodulin gene is constantly expressed in HEK during culture time. Immunostaining showed that fibromodulin is located intracytoplasmically in basal and stratified keratinocytes of the growing colonies, confluent cultures, and epidermis in vivo. The expression and intracellular localization of fibromodulin in HEK is a new finding and opens new possible biological roles for the SLRP family. Ó 2008 Elsevier Inc. All rights reserved.
Fibromodulin was found in cartilage and tendon and it was isolated as a 59 kDa protein from articular cartilage [1]; the protein belongs to a family of small leucine-rich proteoglycans/glycoproteins (SLRP’s). Proteoglycans (PG’s) are macromolecules consisting of a protein core covalently bound to one or more glycosaminoglycan (GAG) side chains [2]. PGs are important components of mammalian extracellular matrices (ECM’s), and they are divided in the SLRP’s, the hyalectans, and the basement-membrane proteoglycans [3]. The core proteins of SLRPs have a central domain containing 6– 10 leucine-rich repeats (LRR’s), flanked by N-terminal and C-terminal regions with conserved Cys residues [4]. The LRR motif has been found in a number of proteins of diverse origin and function and varies in length from 20 to 29 amino acids [5]. The functions of SLRPs are still somewhat controversial; fibromodulin, along with decorin, has a central role in organization of type I and II collagen fibrils structure, and it is a ubiquitous protein most prominent in articular cartilage, tendon, ligament [6–9], and dermal tissues [10,11]. On the other hand, the isoforms of TGF-b interact not only with the core proteins of decorin and biglycan, but also with fibromodulin [12] modulating the cellular fibrotic response in many tissues including lung, kidney, and skin [13–17]. Studies in fibromodulin have been carried out mainly in connective tissue [18] and endothelial cells [19], but little is known regarding its gene expression in other cell types such as epithelial * Corresponding author. Fax: +52(55)5747 3393. E-mail address: [email protected] (W. Kuri-Harcuch). 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.04.095
cells, with the exception of corneal epithelium in which the protein is found at a very low level, but nothing about its gene expression or protein localization is known [20]. Therefore, we thought of interest to study the expression of the fibromodulin gene in epithelial cells; we chose the human epidermal keratinocytes under culture conditions that support differentiation and stratification of the epidermal layers [21]. Since basement membrane separates the basal keratinocytes of the epidermis from the dermis, which contains large amounts of the SLRP’s, human epidermis would be a suitable model to study fibromodulin gene expression and its protein function in epithelial cells in vivo. Materials and methods Materials. Thermo ScriptTM RT-PCR System with PlatinumÒ Taq DNA Polimerase High Fidelity, and the TOPO TA Cloning Kit were from Invitrogen-Life Technologies (Carlsbad, CA). The LightCycler FastStart DNA MasterPLUS SYBR Green I kit, the biotinylated antibody against mouse IgG and the FITC-streptavidin were from Roche Applied Science (Indianapolis, IN). Fetal bovine (FBS) and bovine calf (BCS) sera were from HyClone Laboratories (Logan, UT). Monoclonal antibody against human fibromodulin was purchased from Kamiya Biomedical Company (Seattle, WA). All other reagents were analytical grade. Cell culture. The human epidermal keratinocytes (cHEK), strain HE-123, were isolated from newborn foreskin as previously described [21,22]. For experiments, 3rd to 5th passage keratinocytes
C. Vélez-delValle et al. / Biochemical and Biophysical Research Communications 371 (2008) 420–424
were plated together with mitomycin C treated 3T3 feeder cells in DMEM–F12 (3:1) supplemented with 4%(v/v) FBS, 0.4 lg/ml hydrocortisone, 2 109 M L-T3, 5 lg/ml transferrin, 5 lg/ml insulin, 24.3 mg/l adenine, and 10 ng/ml EGF, as previously described [21–23]. The human dermal fibroblasts (strain hFib-132) were isolated in our laboratory from human skin and cultured as above. Cell cultures were incubated at 37 °C in humidified incubator equilibrated with 10% CO2; medium was changed every other day. RNA extraction, RT-PCR, and sequencing. Total RNA was isolated with Trizol reagent (Invitrogen, CA) and stored at 70 °C as an ethanol precipitate until further use. For the end point PCR assays, reverse transcription was carried out with Thermo ScriptTM reverse transcriptase, using 1.0 lg total RNA, and oligo(dT) primer. Primers used for PCR reactions were 50 -GGTCGCCACCATGCAGTGGGC GTCCCT (forward) and 30 -GCTGCTCAGATCTCGATGA (reverse), which were specific for exon 1 and exon 3 at the fibromodulin encoding gene, respectively. The amplification products were separated in 2%(w/v) agarose gel, cloned into pCRII cloning vector, and then sequenced with both T7 and SP6 sequencing primers, and with fibromodulin-specific primers. Sequencing was carried out using an ABI PRISMTM 310 DNA Sequencer (Perkin-Elmer) and the Big Dye Terminator kit v3.1 (Perkin-Elmer). Sequencing data were compared with the published fibromodulin gene sequence using the BLASTN 2.2 search program [24]. The relative expression of the mRNA encoding fibromodulin was quantified by Real-time PCR. PCR reactions were carried out using LightCycler FastStart DNA MasterPLUS SYBR Green I according to the manufacturer’s instructions. Gene expression was analyzed with the 7500 real time thermal cycler (Applied Biosystems). Fibromodulin mRNA levels were normalized to the levels of the mRNA encoding the housekeeping enzyme GAPDH. The specificity of the PCR product was analyzed by agarose gel electrophoresis and melting curve. Immunodetection of human fibromodulin. Cells were grown on glass coverslips; after 7 days in culture, they were fixed with 3.5%(w/v) para-formaldehyde in PBS. Cells, permeabilized or not with 0.1% Triton X-100, were stained for fibromodulin; the primary antibody was detected with a biotinylated secondary antibody and streptavidin-FITC. Slides were mounted with VECTASHIELDÒ Mounting Medium (Vector Laboratories, Burlingame, CA). For
421
immunodetection in cultured epithelia, the cultured epidermal sheets were detached from the dish using Dispase II (2.5 mg/ml). Both cultured epithelia and normal skin were embedded with tissue freezing medium (Jung) and frozen at –70 °C until further use. Afterwards, 8-lm tissue cryosections in salinized glass slides were air-dried and then hydrated in phosphate-buffered saline (PBS) for 5 min prior to antibody detection. Results Fibromodulin expression in cultured human epidermal keratinocytes Total RNA was extracted from growing or 2 days post-confluent HEK cultures, and PCR amplification was carried out with primers specific to exons 1 and 3 of the human fibromodulin gene [9]; we amplified the ORF of the core protein and part of the 50 untranslated region. By end point PCR we obtained a 1150-bp fragment (Fig. 1A, lane 4) corresponding to the expected amplification product of fibromodulin; we obtained the same product from total RNA from cultured human dermal fibroblasts, as control (Fig. 1A, lane 3). The primer set was specific for human fibromodulin mRNA since no amplification products were identified from the murine 3T3 cells RNA (Fig. 1A, lane 2). We verified the integrity and functionality of the cDNA, by testing the same samples for amplification of GAPDH. In order to avoid any gap or uncertain nucleotides in the obtained sequence, we cloned and sequenced twice in both directions the 1150-bp fragment from HEK, using universal sequencing primers or the fibromodulin specific primers (see Materials and methods). BLAST analysis of the cloned fragment showed complete matching with the published human cDNA sequence (Gene Bank reference NM_002023). These data strongly evidenced that the fibromodulin gene is expressed in cultured human epidermal keratinocytes. By relative quantification of fibromodulin mRNA using primers previously described [25], we determined by real time PCR the expression levels of fibromodulin mRNA in HEK at various times of culture, after washing out the murine 3T3 feeder cells. The mean amplification plots show that fibromodulin expression was constant during culture and considerably lower in cHEK than in hFib (Fig. 1B) this was quantified (Table 1) by normalizing the data to
Fig. 1. PCR amplification of fibromodulin mRNA. (A, upper panel) End point PCR of fibromodulin encoding mRNA. The 1150 bp product, that corresponds to the ORF of hFMOD. (A, lower panel) GAPDH mRNA was used as an amplification control. (B) Real time PCR amplification of fibromodulin mRNA expression in total RNA from HEK at different times in culture, and in proliferative human fibroblasts (hFib).
422
C. Vélez-delValle et al. / Biochemical and Biophysical Research Communications 371 (2008) 420–424
Table 1 Fibromodulin mRNA expression in HEK at different culture days Culture condition
Expression level (folds) (±)SD
hFIB cHEK cHEK cHEK cHEK
1.00 ± 0.08 0.006 ± 0.001 0.006 ± 0.002 0.006 ± 0.005 0.007 ± 0.008
5d* 7d* 12d* 14d
Total RNA was isolated from cultures as described. After real time-PCR experiments, the relative expression of fibromodulin encoding mRNA was analyzed. Results were normalized against the GAPDH expression levels. Columns represent the relative proportion of fibromodulin mRNA, referred to that found in hFIB cultures. * p < 0.05.
as it is seen by the dark boundary showing the intercellular space. Cryosections of the confluent epithelium, confirmed the protein was inside the cells of the 2–3 layers of the cultured epidermis (Fig. 2C and D). These results raised the possibility that keratinocytes in normal human epidermis should also express fibromodulin. Frozen sections from adult human skin showed that fibromodulin was localized in the basal and suprabasal keratinocytes of the epidermis, but not in the stratum corneum (Fig. 2E and F), and it seemed also to be inside the keratinocytes. As an internal control of the antibody, fibromodulin was also immunodetected around some dermal cells, most likely fibroblasts (Fig. 2E and F). Discussion
the expression of the housekeeping gene GAPDH, using the 2DDC T aproach [26]. These data clearly give evidence that the HEK in culture have the ability to express the fibromodulin gene, however, at lower levels as compared to the cultured human fibroblasts. Immunolocalization of fibromodulin in cultured human epidermal keratinocytes, and in human epidermis in vivo By immunohistochemistry with an antibody specific against fibromodulin we studied the distribution of this protein in cultured HEK. Fibromodulin was inmunodetected in basal and stratified keratinocytes of the growing colonies (Fig. 2A and B); it seemed to be located intracytoplasmically, with a pericellular distribution,
Fibromodulin remains as the least explored PG from the SLRP family; it is mainly described as a structural molecule related to collagen fibrillogenesis [10,11,27,28]. However, the most relevant functional activity described for fibromodulin is its binding of TGFb isoforms in vitro [12]. Biglycan is the only described SLRP in human epidermis [29,30], but nothing is known about its function in this tissue. Lumican has been described in epithelial cells during corneal wound healing, and the data suggested that lumican null-animals had delayed re-epithelization of the central cornea [30,31]. While we were doing this work, fibromodulin and lumican have been described in basal cells of human epithelial gingiva [32], but the mesenchymal or epithelial origin of these proteoglycans, as
Fig. 2. Immunolocalization of fibromodulin in cHEK and human skin. (A and B) Five day HEK growing colonies were immunostained with antibodies raised against human fibromodulin. (C and D) 8-lm cryosections of confluent cultured epidermal sheets were stained with the anti-hFMOD antibody, the PG was observed both in basal and in stratified cells, with no preferential distribution in the cell layers. (E and F) In similar experiments, cryosections of normal human skin showed staining for the PG from the basal layer to the stratum spinosum, but not in the stratum corneum. (A, C, and E) display the phase contrast micrographs of the immunofluorescence fields shown in (B, D and F). (B and D), scale bar = 100 lm, (F), scale bar = 50 lm.
C. Vélez-delValle et al. / Biochemical and Biophysical Research Communications 371 (2008) 420–424
well as the expression of their encoding genes, remain unknown. Our results are the first to show fibromodulin expression in epidermal keratinocytes in vivo and in culture. Expression of fibromodulin mRNA, and the presence of fibromodulin protein support the conclusion that human keratinocytes can synthesize and accumulate this protein. Our evidence shows that keratinocytes are a novel source of fibromodulin in normal human skin in vivo. Previous studies, by Northern blot in total RNA extracts, have failed to detect fibromodulin in human keratinocytes cultured under serum free medium conditions [7]. We think that our approach has two main advantages that can explain these differences. First, co-cultivation of human epidermal keratinocytes with 3T3 cells as a feeder layer [21] supports the proliferative potential of the keratinocytes, the expression of differentiation markers, as well as the deposition of a more complete extracellular matrix [21,33,34]. Second, the use of RT-PCR, and of real time PCR, for the detection of mRNAs have more sensitivity than Northern blot analysis [35]. Unfortunately, we could not assay if mRNA expression correlates with the content of the protein, because our antibody did not recognize it in western blot analysis of the cell extracts. Also, it remains necessary to establish the glycoprotein or proteoglycan nature of the fibromodulin found in cHEK. The intracellular localization of fibromodulin is a new finding; its extensive intracellular distribution is concurrent with previous evidence of keratan sulfate epitopes in the cytoplasm of HEK [36]; it was shown that this keratan sulfate co-migrates with keratins in SDS-polyacrilamide gels [13]. Interestingly, the migration pattern of the keratins with associated keratan sulfate comprises the same range of molecular weights of the recombinant fibromodulin migration pattern [37], raising the possibility that some interaction between keratins and the proteoglycan, might stabilize the intermediate filament structure. This remains as an interesting and challenging possibility that we are pursuing in further investigation. Elucidation of other biological roles for fibromodulin remain open, mainly those that could resemble similar functions to those found for other members of this SLRP family, such as lumican [30,38,39], or decorin [40–42]. The transitory expression of lumican and its association with cellular migration during wound healing in corneal epithelial cells [30], raises an interesting possibility for a similar role of fibromodulin in epidermal or other epithelial keratinocytes. Acknowledgments This work was supported in part by CONACyT Grants # G28272N, 39690-Q and 46453. We thank M en C. Erika Sánchez for their technical assistance. We also thank Ms. María Elena Rojano for her secretarial assistance and Mr. Alberto Rodriguez and Mrs. Columba Guadarrama for preparing all materials. References [1] D. Heinegard, T. Larsson, Y. Sommarin, A. Franzen, M. Paulsson, E. Hedbom, Two novel matrix proteins isolated from articular cartilage show wide distributions among connective tissues, J. Biol. Chem. 261 (1986) 13866– 13872. [2] L. Kjellen, U. Lindahl, Proteoglycans: structures and interactions, Annu. Rev. Biochem. 60 (1991) 443–475. [3] R.V. Iozzo, Matrix proteoglycans: from molecular design to cellular function, Annu. Rev. Biochem. 67 (1998) 609–652. [4] R.V. Iozzo, The family of the small leucine-rich proteoglycans: key regulators of matrix assembly and cellular growth, Crit. Rev. Biochem. Mol. Biol. 32 (1997) 141–174. [5] R.V. Iozzo, A.D. Murdoch, Proteoglycans of the extracellular environment: clues from the gene and protein side offer novel perspectives in molecular diversity and function, FASEB J. 10 (1996) 598–614. [6] G. Westergren-Thorsson, P. Antonsson, A. Malmstrom, D. Heinegard, A. Oldberg, The synthesis of a family of structurally related proteoglycans in fibroblasts is differently regulated by TFG-beta, Matrix 11 (1991) 177–183.
423
[7] J.B. Petri, O. Rott, T. Wetzig, K. Herrmann, U.F. Haustein, The small proteoglycan fibromodulin is expressed in mitotic, but not in postmitotic fibroblasts, Mol. Cell. Biol. Res. Commun. 1 (1999) 59–65. [8] P.J. Roughley, R.J. White, G. Cs-Szabo, J.S. Mort, Changes with age in the structure of fibromodulin in human articular cartilage, Osteoarthr. Cartilage 4 (1996) 153–161. [9] P. Antonsson, D. Heinegard, A. Oldberg, Structure and deduced amino acid sequence of the human fibromodulin gene, Biochim. Biophys. Acta 1174 (1993) 204–206. [10] E. Hedbom, D. Heinegard, Binding of fibromodulin and decorin to separate sites on fibrillar collagens, J. Biol. Chem. 268 (1993) 27307–27312. [11] K. Noyori, H.E. Jasin, Inhibition of human fibroblast adhesion by cartilage surface proteoglycans, Arthritis Rheum. 37 (1994) 1656–1663. [12] A. Hildebrand, M. Romaris, L.M. Rasmussen, D. Heinegard, D.R. Twardzik, W.A. Border, E. Ruoslahti, Interaction of the small interstitial proteoglycans biglycan, decorin and fibromodulin with transforming growth factor beta, Biochem. J. 302 (Pt 2) (1994) 527–534. [13] I.A. Schafer, J.M. Sorrell, Human keratinocytes contain keratin filaments that are glycosylated with keratan sulfate, Exp. Cell Res. 207 (1993) 213–219. [14] N. Venkatesan, T. Ebihara, P.J. Roughley, M.S. Ludwig, Alterations in large and small proteoglycans in bleomycin-induced pulmonary fibrosis in rats, Am. J. Respir. Crit. Care Med. 161 (2000) 2066–2073. [15] G. Westergren-Thorsson, J. Hernnas, B. Sarnstrand, A. Oldberg, D. Heinegard, A. Malmstrom, Altered expression of small proteoglycans, collagen, and transforming growth factor-beta 1 in developing bleomycin-induced pulmonary fibrosis in rats, J. Clin. Invest. 92 (1993) 632–637. [16] T. Ebihara, N. Venkatesan, R. Tanaka, M.S. Ludwig, Changes in extracellular matrix and tissue viscoelasticity in bleomycin-induced lung fibrosis. Temporal aspects, Am. J. Respir. Crit. Care Med. 162 (2000) 1569–1576. [17] C.C. Reed, R.V. Iozzo, The role of decorin in collagen fibrillogenesis and skin homeostasis, Glycoconj. J. 19 (2002) 249–255. [18] M. Benjamin, J.R. Ralphs, Biology of fibrocartilage cells, Int. Rev. Cytol. 233 (2004) 1–45. [19] L. Schaefer, H.J. Grone, I. Raslik, H. Robenek, J. Ugorcakova, S. Budny, R.M. Schaefer, H. Kresse, Small proteoglycans of normal adult human kidney: distinct expression patterns of decorin, biglycan, fibromodulin, and lumican, Kidney Int. 58 (2000) 1557–1568. [20] E. Schonherr, C. Sunderkotter, L. Schaefer, S. Thanos, S. Grassel, A. Oldberg, R.V. Iozzo, M.F. Young, H. Kresse, Decorin deficiency leads to impaired angiogenesis in injured mouse cornea, J. Vasc. Res. 41 (2004) 499–508. [21] J.G. Rheinwald, H. Green, Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells, Cell 6 (1975) 331–343. [22] J. Bolivar-Flores, E. Poumian, M. Marsch-Moreno, G. Montes de Oca, W. KuriHarcuch, Use of cultured human epidermal keratinocytes for allografting burns and conditions for temporary banking of the cultured allografts, Burns 16 (1990) 3–8. [23] F. Castro-Munozledo, M. Hernandez-Quintero, M. Marsch-Moreno, W. KuriHarcuch, Cultivation, serial transfer, and differentiation of epidermal keratinocytes in serum-free medium, Biochem. Biophys. Res. Commun. 236 (1997) 167–172. [24] S.F. Altschul, T.L. Madden, A.A. Schaffer, J. Zhang, Z. Zhang, W. Miller, D.J. Lipman, Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, Nucleic Acids Res. 25 (1997) 3389–3402. [25] F. Barry, R.E. Boynton, B. Liu, J.M. Murphy, Chondrogenic differentiation of mesenchymal stem cells from bone marrow: differentiation-dependent gene expression of matrix components, Exp. Cell Res. 268 (2001) 189–200. [26] K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using realtime quantitative PCR and the 2(-Delta Delta C(T)) method, Methods 25 (2001) 402–408. [27] K.G. Vogel, M. Paulsson, D. Heinegard, Specific inhibition of type I and type II collagen fibrillogenesis by the small proteoglycan of tendon, Biochem. J. 223 (1984) 587–597. [28] E. Hedbom, D. Heinegard, Interaction of a 59-kDa connective tissue matrix protein with collagen I and collagen II, J. Biol. Chem. 264 (1989) 6898–6905. [29] P. Bianco, L.W. Fisher, M.F. Young, J.D. Termine, P.G. Robey, Expression and localization of the two small proteoglycans biglycan and decorin in developing human skeletal and non-skeletal tissues, J. Histochem. Cytochem. 38 (1990) 1549–1563. [30] S. Saika, A. Shiraishi, C.Y. Liu, J.L. Funderburgh, C.W. Kao, R.L. Converse, W.W. Kao, Role of lumican in the corneal epithelium during wound healing, J. Biol. Chem. 275 (2000) 2607–2612. [31] L.K. Yeh, W.L. Chen, W. Li, E.M. Espana, J. Ouyang, T. Kawakita, W.W. Kao, S.C. Tseng, C.Y. Liu, Soluble lumican glycoprotein purified from human amniotic membrane promotes corneal epithelial wound healing, Invest. Ophthalmol. Vis. Sci. 46 (2005) 479–486. [32] S. Chakravarti, Functions of lumican and fibromodulin: lessons from knockout mice, Glycoconj. J. 19 (2002) 287–293. [33] P.P. Parnigotto, V. Bassani, S. Pastore, F. Valenti, M.T. Conconi, Fibroblastkeratinocyte co-cultures in vitro: growth, morphometry and nutrient exchange, Ital. J. Anat. Embryol. 99 (1994) 17–30. [34] K. Alitalo, E. Kuismanen, R. Myllyla, U. Kiistala, S. Asko-Seljavaara, A. Vaheri, Extracellular matrix proteins of human epidermal keratinocytes and feeder 3T3 cells, J. Cell Biol. 94 (1982) 497–505. [35] K. Reue, mRNA quantitation techniques: considerations for experimental design and application, J. Nutr. 128 (1998) 2038–2044.
424
C. Vélez-delValle et al. / Biochemical and Biophysical Research Communications 371 (2008) 420–424
[36] J.M. Sorrell, B. Caterson, Monoclonal antibodies specific for keratan sulfate detect epithelial-associated carbohydrates, Histochemistry 94 (1990) 269–275. [37] L. Svensson, I. Narlid, A. Oldberg, Fibromodulin and lumican bind to the same region on collagen type I fibrils, FEBS Lett. 470 (2000) 178–182. [38] N. Vij, L. Roberts, S. Joyce, S. Chakravarti, Lumican suppresses cell proliferation and aids Fas–Fas ligand mediated apoptosis: implications in the cornea, Exp. Eye Res. 78 (2004) 957–971. [39] N. Vij, L. Roberts, S. Joyce, S. Chakravarti, Lumican regulates corneal inflammatory responses by modulating Fas–Fas ligand signaling, Invest. Ophthalmol. Vis. Sci. 46 (2005) 88–95. [40] M.L. D’Antoni, C. Torregiani, P. Ferraro, M.C. Michoud, B. Mazer, J.G. Martin, M.S. Ludwig, Effects of decorin and biglycan on human airway smooth muscle
cell proliferation and apoptosis, Am. J. Physiol. Lung Cell Mol. Physiol. (2008), doi:10.1152/ajplung.00436.2007. [41] L. Schaefer, K. Macakova, I. Raslik, M. Micegova, H.J. Grone, E. Schonherr, H. Robenek, F.G. Echtermeyer, S. Grassel, P. Bruckner, R.M. Schaefer, R.V. Iozzo, H. Kresse, Absence of decorin adversely influences tubulointerstitial fibrosis of the obstructed kidney by enhanced apoptosis and increased inflammatory reaction, Am. J. Pathol. 160 (2002) 1181–1191. [42] J.G. Tralhao, L. Schaefer, M. Micegova, C. Evaristo, E. Schonherr, S. Kayal, H. Veiga-Fernandes, C. Danel, R.V. Iozzo, H. Kresse, P. Lemarchand, In vivo selective and distant killing of cancer cells using adenovirus-mediated decorin gene transfer, FASEB J. 17 (2003) 464–466.