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Expression of Hyaluronan Synthase Messenger Ribonucleic Acids and Their Induction by Interleukin-1ß in Human Orbital Fibroblasts: Potential Insight into the Molecular Pathogenesis of Thyroid-Associated Ophthalmopathy¹

Division of Molecular and Cellular Medicine, Departments of Medicine and Biochemistry and Molecular Biology, Albany Medical College and Samuel S. Stratton Veterans Affairs Medical Center, Albany, New York 12208

Address all correspondence and requests for reprints to: Terry J. Smith, M.D., Division of Molecular and Cellular Medicine (A-175), Albany Medical College, 47 New Scotland Avenue, Albany, New York 12208.

	Abstract

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The disordered accumulation of hyaluronan, a nonsulfated glycosaminoglycan, is a hallmark feature of the tissue remodeling observed in thyroid-associated ophthalmopathy (TAO). Orbital fibroblasts have been shown to exhibit substantial up-regulation of hyaluronan synthesis when activated with proinflammatory cytokines such as interleukin-1ß (IL-1ß). Recently, three members of the hyaluronan synthase (HAS) gene family were cloned. Here we report that IL-1ß can dramatically and consistently induce in orbital fibroblasts the expression of HAS2 in the five orbital strains examined. HAS3 messenger ribonucleic acid (mRNA) was also detectable in all these strains by RT-PCR under both control and IL-1ß-treated conditions. In contrast, HAS1 mRNA was detected by Northern blot analysis in only one of the strains treated with IL-1ß, but in three of five strains examined by RT-PCR. These HAS inductions by the cytokine were time dependent and could be attenuated with dexamethasone and cycloheximide. They were accompanied by an increased incorporation of [³H]glucosamine into hyaluronan, and dexamethasone could attenuate induction of macromolecular synthesis as well. Our observations suggest that the cytokine-dependent induction of the HAS genes in orbital fibroblasts may be the molecular basis at least in part for the increased accumulation of hyaluronan, driven by immunocompetent cells, in orbital connective tissue and the extraocular muscles in TAO.

	Introduction

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HYALURONAN accumulation in orbital connective tissue and extraocular muscles represents a hallmark feature of the tissue remodeling observed in thyroid-associated ophthalmopathy (TAO) (1). This occurs in the context of often intense inflammation and results in a volume expansion of the orbital contents, leading to proptosis. Hyaluronan is a nonsulfated glycosaminoglycan lacking a protein backbone (2). It possesses rheological properties that do not differ markedly from those of the other abundant glycosaminoglycans, all of which are sulfated. Little progress has been made concerning insight into the hyaluronan synthetic process until very recently when a family of three mammalian hyaluronan synthase (HAS) genes was cloned and partially characterized (3, 4, 5, 6). These enzymes use sugar substrates from UDP donors to form repeating disaccharides consisting of D-glucuronic acid (ß13) N-acetylglucosamine (ß14). HAS enzymes function at the plasma membrane (7). In addition, the enzyme immediately upstream from the HAS enzymes, UDP glucose dehydrogenase, has been cloned and can be regulated by cytokines in orbital fibroblasts (8). Thus, multiple, proximate components of the hyaluronan biosynthetic pathway have now been identified.

Human fibroblasts are important participants in the initiation and orchestration of tissue remodeling and wound repair (9). Orbital fibroblasts exhibit a distinctive phenotype in culture that sets them apart from the other fibroblasts studied to date. They are heterogeneous (10), and at least some orbital fibroblasts are actually preadipocytes (11). They exhibit distinctive profiles of receptors (12), responses to cytokines, hormones and prostanoids (13, 14, 15, 16), gangliosides (17, 18), and proteins (19). These fibroblasts are particularly susceptible to the up-regulatory actions of proinflammatory cytokines on PG endoperoxide H synthase-2 (PGHS-2) expression and PGE₂ production (20, 21) and on plasminogen activator inhibitor type 1 (22, 23). The inductions of the prostanoid biosynthetic and serine protease inhibitor pathways are considerably greater in magnitude than those occurring in extraorbital fibroblasts such as those derived from the skin (19, 20, 22, 23). Thus, orbital fibroblasts respond to mediators of inflammation in a manner that sets their phenotype apart from those of other fibroblasts.

Fibroblasts synthesize large amounts of hyaluronan. Unprovoked hyaluronan synthesis in orbital fibroblasts is not influenced by either thyroid hormone or glucocorticoids (14), unlike dermal fibroblasts where both classes of hormone (24, 25) as well as n-butyrate (26) and retinoic acid (27) exert inhibitory effects. In contrast, hyaluronan synthesis in orbital fibroblasts manifests particularly robust responses to proinflammatory cytokines such as interleukin-1ß (IL-1ß), interferon-, and leukoregulin, a 50-kDa protein from activated T lymphocytes (28, 29).

Orbital fibroblasts express and display CD40, a member of the tumor necrosis factor- receptor superfamily (30). When ligated with CD154 or CD40 ligand, orbital fibroblasts express high levels of PGHS-2 and synthesize PGE₂ and hyaluronan at accelerated rates (31). The mechanisms involved in the cytokine and CD40/CD154-dependent hyaluronan synthesis have not yet been identified, but the differences between macromolecular synthesis and PGHS-2 expression in orbital and nonorbital fibroblasts could help explain the propensity of the orbit to become inflamed and to accumulate hyaluronan.

In this paper, we report that IL-1ß can induce, in cultured orbital fibroblasts, messenger ribonucleic acids (mRNAs) encoding the three HAS enzyme family members. These effects suggest a mechanism by which hyaluronan synthesis can be enhanced through the signaling of fibroblasts by immunocompetent cells trafficked to the orbit in TAO. Our results define a potential target for specific therapy directed at modulating glycosaminoglycan synthesis.

	Materials and Methods

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Materials

Culture medium and FBS were purchased from Life Technologies, Inc., BRL (Grand Island, NY). IL-1ß, platelet-derived growth factor, epidermal growth factor and interferon- were supplied by Biosource Technologies, Inc. (Camarillo, CA); dexamethasone (1,4-pregnadien-9-fluoro-16-methyl-11ß,17, 21-triol-3,20-dione) and cycloheximide were purchased from Sigma Chemical Co. (St. Louis, MO). Human complementary DNAs (cDNAs) for HAS1, HAS2, and HAS3 were provided by Drs. J. Briskin (LeukoSite, Cambridge, MA), Yu Yamaguchi (La Jolla Cancer Institute, La Jolla, CA), and Andrew Spicer (Mayo Clinic, Scottsdale, AZ), respectively. [³H]Glucosamine (SA, 22.7 Ci/mmol) was purchased from New England Nuclear (Boston, MA).

Cell culture

Human orbital fibroblasts were initiated from tissue explants obtained from four patients with severe TAO and an individual without known thyroid disease, as reported previously (32). Patients had not been taking adrenal cortical steroids for several months at the time of surgery, were euthyroid, and had not received radiotherapy to the orbits. Dermal fibroblasts were harvested by punch biopsy of normal appearing skin. These activities have been approved by the institutional review board of the Albany Medical College. Tissue samples were covered with Eagle’s medium supplemented with 10% FBS, glutamine, and antibiotics. Explants were removed when fibroblasts migrated out, and the monolayers were subjected to mild treatment with trypsin-ethylenediamine tetraacetate, dispersed, and replated. Experiments were performed on confluent cultures that were maintained in a 5% CO₂, humidified environment at 37 C. Culture strains were used between the 3rd and 12th passages from initiation. None of the parameters examined was found to change as a function of advancing passage number, consistent with our previous findings concerning the synthesis of hyaluronan (29).

Isolation of fibroblast RNA, RT-PCR, and Northern blot analysis

Fibroblasts were cultivated to confluence in 100-mm diameter plastic plates in medium containing 10% FBS. They were then shifted to medium with 1% serum for 16 h, and some plates received IL-1ß (10 ng/mL) or one of the other test compounds for the treatment durations indicated in the figure legends. After the treatment periods, the monolayers were washed in phosphate-buffered saline (PBS), and the RNA was extracted using ULTRASPEC isolation solution from Biotecx (Houston, TX). The RNA was prepared as described previously (20). The integrity of the nucleic acid was verified by subjecting the electrophoresed samples to ethidium bromide staining followed by inspection under UV light and by routinely verifying the 260/280 ratio. For detection of the HAS mRNAs by RT-PCR, 1 µg cellular RNA was used to synthesize cDNA primed with hexamers in a reaction mixture containing 4 µL 5 x reverse transcriptase buffer [250 mmol/L Tris-HCl (pH 8.3), 50 mmol/L MgCl₂, 300 mmol/L KCl, and 40 mmol/L DTT), 0.5 µL ribonuclease inhibitor (40 U/mL), and 2.5 mmol/L dNTPs. The reaction was allowed to proceed for 10 min at 25 C, 1 µL Superscript II (Life Technologies, Inc.) was added, and the mixture was then incubated at 42 C for 50 min, followed by a 15-min incubation at 70 C. Two microliters of the reaction mixtures were then subjected to PCR in a model PTC 100 instrument (M. J. Research, Watertown, MA) with a 1 µmol/L primer concentration. The primers used for HAS1 and HAS2 were designed with the published sequences; HAS3 primers were generously supplied by Dr. A. Spicer. HAS1 amplification involved 30 cycles of 94 C for 1 min, 60 C for 2 min, and 72 C for 2 min; cycling for HAS2 included 95 C for 1 min, 50 C for 2 min, and 72 C for 3 min; for HAS3 the reactions were 94 C for 10 s, 67 C for 30 s, and 72 C for 1 min.

For Northern analysis, RNA was electrophoresed on 1% formaldehyde denaturing gels and then transferred to Zeta-Probe membrane (Bio-Rad Laboratories, Inc., Hercules, CA). The immobilized RNA was allowed to hybridize with [³²P]CTP-labeled cDNA probes for HAS family members, and the membranes were washed under high stringency and subjected to radioautography at -70 C. They were then stripped according to the manufacturer’s instructions and rehybridized with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe.

Assay for hyaluronan synthesis in orbital fibroblasts

The assay employed for the assessment of hyaluronan production by orbital fibroblasts is based on the technique published by Smith et al (32) using the incorporation of [³H]glucosamine into macromolecular material. Briefly, fibroblasts were allowed to proliferate to confluence in 60-mm plastic culture plates and were covered with medium supplemented with 10% FBS. These were shifted to 1% serum-enriched medium without or with IL-1ß (10 ng/mL) for 24 h. The plates were then radiolabeled with [³H]glucosamine (1 µCi/mL) for an additional 6 h. Medium was collected, and cell layers were washed with PBS, followed by solubilizing the cellular material in NaOH (0.2 N). After removal of an aliquot for protein determination, the medium and cell fractions were combined; adjusted to pH 8.0 with 100 mmol/L Tris, hyaluronan, and chondroitin sulfate added as carriers; and subjected to treatment with pronase (1 mg/mL) at 50 C overnight. Samples were cooled and precipitated with trichloroacetic acid, the acid-soluble material was dialyzed extensively against water, and an aliquot was assessed by liquid scintillation counting. Another aliquot was treated with Streptomyces hyaluronidase (50 TRU/mL), which has an absolute specificity for hyaluronan, as described previously (29).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
RT-PCR analysis of HAS mRNA expression in human orbital fibroblasts

Initial studies were directed at determining HAS expression in several orbital fibroblast strains using nonquantitative RT-PCR. As the gel pictured in Fig. 1 suggests, the pattern of expression of the three HAS isoform mRNAs differs markedly. With regard to HAS1, it would appear that none of the five strains expressed this transcript under basal culture conditions. When the cultures were treated with IL-1ß (10 ng/mL) for 6 h, three of the five strains expressed HAS1 mRNA, the product of which migrated as the expected 642-bp PCR product. Basal HAS2 mRNA was clearly detected in four of the five strains and was found in all five after IL-1ß treatment. HAS3, migrating as a 358-bp product, appears to be expressed in all strains under both basal and IL-1ß-treated conditions.

View larger version (41K): [in this window] [in a new window]   Figure 1. RT-PCR analysis of HAS expression in several strains of orbital fibroblasts. Orbital fibroblasts from five different donors were allowed to proliferate to confluence in 100-mm diameter plastic culture plates in medium containing 10% FBS. They were then shifted to medium with 1% FBS without or with IL-1ß (10 ng/mL) for 6 h. Monolayers were washed in PBS, and then the monolayers were harvested, and cellular RNA was extracted. The RNA was subjected to RT-PCR for the HAS transcripts as described in Materials and Methods. The products were subjected to electrophoresis and ethidium bromide staining and were visualized under UV light. Strains 1–4 were from individuals with TAO; strain 5 was derived from a patient without known thyroid disease.

Given the expression pattern of the HAS mRNAs determined by RT-PCR, we next assessed, by the quantitative means of Northern blot hybridization, the three HAS transcripts in multiple orbital fibroblast strains. As the blot pictured in Fig. 2 indicates, striking differences in the patterns of HAS mRNA expression, suggested by RT-PCR results, were verified. Moreover, the magnitude of the specific isoform-encoding transcript expression was strain dependent. In general, HAS2 was detected the most universally and was apparent in all strains after treatment with IL-1ß (10 ng/mL) for 6 hr. It resolved as two transcripts of 2.5 and 4.4 kb, consistent with other cell types where it was expressed (4). In addition, it was found at low levels in untreated strains 2 and 4. HAS3, the transcript detected by RT-PCR in all strains regardless of treatment status, was highly inducible in four of the five strains tested. HAS3 appeared as a single mRNA of 4.3 kb. HAS1 displayed a considerably more restricted pattern of expression, being clearly detectable in only one of the strains after IL-1ß treatment where it migrated as a single band of 2.4 kb. Thus, it would appear that the HAS transcripts are expressed at a very low abundance in orbital fibroblasts under control conditions. Of the isoforms, HAS2 and HAS3 were the most widely inducible by IL-1ß.

View larger version (27K): [in this window] [in a new window]   Figure 2. Northern blot analysis of HAS mRNA in several strains of orbital fibroblasts. Orbital strains from four individuals with severe TAO and one from a normal orbit were allowed to proliferate to confluence in medium with 10% FBS, then shifted to medium with 1% serum without or with IL-1ß (10 ng/mL) for 6 h. Monolayers were harvested, and RNA was extracted and subjected to Northern blot analysis as described in Materials and Methods using cDNA probes for the HAS family. The membrane was stripped of radioactivity and rehybridized with a GAPDH cDNA probe. Radiolabeled RNA/DNA hybrids were detected with radioautography.

The time dependence of the effects of IL-1ß on HAS mRNA expression was examined in the culture strain where all three isoforms could easily be detected by Northern analysis. As Fig. 3 suggests, IL-1ß (10 ng/mL) elicited a rapid induction of HAS1, HAS2, and HAS3 mRNAs within 3–6 h of cytokine addition to the culture medium. The duration of the inductions varied, in that HAS3 mRNA levels remained elevated for at least 24 h, the duration of the study, whereas HAS1 mRNA had decayed by 24 h, and HAS2 transcript levels were approaching those in control cultures by 12 h.

View larger version (53K): [in this window] [in a new window]   Figure 3. Time dependence of the induction by IL-1ß of the HAS mRNAs in orbital fibroblasts. Confluent cultures of orbital fibroblasts from a patient with severe TAO were shifted to medium supplemented with 1% FBS, and some plates were treated with IL-1ß (10 ng/mL) for the times (in hours) indicated in the figure. Cellular RNA was extracted and subjected to Northern blot hybridizations as described in Materials and Methods. The blot was rehybridized with a GAPDH probe to verify loading equivalency.

Hyaluronan synthesis in fibroblasts is very dependent on culture density (33). We, therefore, wanted to determine whether culture density influenced basal or cytokine-dependent HAS expression. Orbital fibroblasts were seeded at two different cell densities so that at the end of the maintenance period, one group of culture plates was 60% confluent and the other was entirely confluent. Cultures were then shifted to medium containing 1% FBS, and some were treated with IL-1ß (10 ng/mL) for 6 h. As the results of the Northern blot analysis in Fig. 4 suggest, subconfluent cultures exhibited substantially less inducibility of all three isoforms by IL-1ß than did cultures that have reached confluence.

View larger version (63K): [in this window] [in a new window]   Figure 4. Culture density influences the inducibility by IL-1ß of the HAS mRNAs in orbital fibroblasts. Orbital fibroblasts, in this case from an individual with severe TAO, were used to seed culture plates at two cell densities so that half of the plates became confluent at a time when the others were 60% confluent. Plates were shifted to medium with 1% FBS, and some of the plates were treated with nothing or with IL-1ß (10 ng/mL) for the times (in hours) indicated in the figure. Monolayers were harvested, and cellular RNA was harvested and subjected to Northern blot analysis as described in Materials and Methods.

We reported that glucocorticoids such as dexamethasone can down-regulate the production of hyaluronan in dermal fibroblasts (25), but not the unprovoked hyaluronan synthesis in orbital cultures (14). On the other hand, dexamethasone could substantially attenuate the up-regulation of hyaluronan in orbital fibroblasts by proinflammatory cytokines (29). Thus, we determined whether the glucocorticoid could influence the induction of HAS mRNA by IL-1ß. As the Northern analysis in Fig. 5 indicates, dexamethasone (10 nmol/L) can almost entirely block the induction of the three HAS isoforms when it is added to the culture medium in addition to the cytokine. Cycloheximide (10 µg/mL) at a concentration that blocks at least 90% of protein synthesis in human fibroblasts (34) can also substantially attenuate the IL-1ß up-regulation of the HAS mRNAs. Thus, it would appear that intermediate protein induction is critical to the up-regulation by IL-1ß of all three HAS mRNAs. This finding is consistent with the susceptibility of hyaluronan synthesis in fibroblasts to inhibitors of protein synthesis (34).

View larger version (63K): [in this window] [in a new window]   Figure 5. Dexamethasone and cycloheximide can attenuate the induction by IL-1ß of HAS mRNAs in orbital fibroblasts. Confluent cultures of orbital fibroblasts, in this case from an individual with severe TAO, were shifted to medium containing 1% FBS, and some received IL-1ß (10 ng/mL), dexamethasone (Dex; 10 nmol/L) or cycloheximide (Cyclo; 10 µg/mL) alone or in the combinations of test compounds indicated for 6 h. Monolayers were washed, and cellular RNA was harvested as described in Materials and Methods. Northern blot analysis was performed on the RNA with the respective cDNA probes.

We next determined whether other cytokines and growth factors can up-regulate the expression of HAS2 mRNA, as the production of hyaluronan in these cells can be enhanced by several factors. We treated confluent cultures of orbital fibroblasts with interferon- (100 U/mL), platelet-derived growth factor (10 ng/mL), or epidermal growth factor (100 ng/mL) for 6 h or 24 h; then the total cellular RNA was extracted and subjected to Northern analysis for HAS2 mRNA levels. As Fig. 6 suggests, of these cytokines, EGF elicited a strong up-regulation of the HAS2 transcript. The other compounds failed to influence levels of this mRNA appreciably. Thus, it would appear that multiple molecular signals can regulate HAS2 expression in orbital fibroblasts.

View larger version (35K): [in this window] [in a new window]   Figure 6. Effects of platelet-derived growth factor (PDGF; 10 ng/mL), EGF (100 ng/mL), and interferon- (IFN-; 100 U/mL) on steady state HAS2 mRNA levels in orbital fibroblasts. Confluent cultures were treated with the test compounds for the durations of time indicated; then the monolayers were washed, and total cellular RNA was extracted as indicated in Materials and Methods. The RNA was subjected to Northern analysis with a [32P]HAS2 cDNA probe and then with a probe for GAPDH.

We assessed the effect of IL-1ß on hyaluronan synthesis by quantitating [³H]glucosamine incorporation into hyaluronan under conditions similar to those used to demonstrate the induction of HAS mRNAs. As the data presented in Fig. 7 indicate, treatment with IL-1ß (10 ng/mL) for 24 h resulted in a severalfold increase in [³H]hyaluronan synthesis in orbital fibroblasts. We reported previously the absence of detectable hyaluronidase activity in orbital fibroblast cultures (29); thus, the increase in accumulation reflects the enhanced rate of macromolecular synthesis. Seventy-three percent of the radiolabeled macromolecule was digested with Streptomyces hyaluronidase, defining the material as hyaluronan. Addition of dexamethasone to the culture medium substantially attenuated the effect of IL-1ß on hyaluronan synthesis, consistent with the blockade of HAS mRNA induction (Fig. 5). The steroid had no effect on basal (cytokine-independent) hyaluronan synthesis, in congruence with our previous reports (14, 29).

View larger version (43K): [in this window] [in a new window]   Figure 7. IL-1ß up-regulates the synthesis of [3H]hyaluronan in orbital fibroblasts. Orbital fibroblasts were allowed to proliferate to confluence in medium supplemented with 10% FBS. They were shifted to 1% serum-enriched medium without or with IL-1ß (10 ng/mL), dexamethasone (10 nmol/L), or a combination of the two for 24 h. Cultures were radiolabeled with [3H]glucosamine (2 µCi/mL) for 6 h, and then the cultures were harvested and analyzed for [3H]glycosaminoglycan synthesis as described in Materials and Methods. Each column represents the mean ± SE of three independent determinations.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Our finding in orbital fibroblasts that HAS mRNAs can be up-regulated by IL-1ß suggests a potentially important mechanism through which the synthetic rate of hyaluronan might be influenced in states of health and pathologically up-regulated during inflammation. It would appear that the effects of IL-1ß on HAS mRNA levels represent indirect actions on some as yet unidentified intermediate protein induction on the basis of the susceptibility to cycloheximide. Although the dysregulation of glycosaminoglycans had been linked to Graves’ disease many years ago, little progress into the molecular basis for this process has been made, in large part because of the uncertainty associated with hyaluronan biosynthesis. Thus, the recent cloning of the HAS family members should provoke renewed activity into examining the pathogenesis of TAO and other diseases in which hyaluronan production and/or disposal are altered.

	Acknowledgments

 
The authors thank Ms. Heather Meekins for expert technical assistance.

	Footnotes

 
¹ This work was supported in part by NIH Grants EY-08976 and EY-11708 and a Merit Review Award from the Research Service of the Department of Veterans Affairs.

Received April 26, 1999.

Revised June 15, 1999.

Accepted July 15, 1999.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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				A. G. Gianoukakis, T. A. Jennings, C. S. King, C. E. Sheehan, N. Hoa, P. Heldin, and T. J. Smith Hyaluronan Accumulation in Thyroid Tissue: Evidence for Contributions from Epithelial Cells and Fibroblasts Endocrinology, January 1, 2007; 148(1): 54 - 62. [Abstract] [Full Text] [PDF]

				R. Han and T. J. Smith T Helper Type 1 and Type 2 Cytokines Exert Divergent Influence on the Induction of Prostaglandin E2 and Hyaluronan Synthesis by Interleukin-1{beta} in Orbital Fibroblasts: Implications for the Pathogenesis of Thyroid-Associated Ophthalmopathy Endocrinology, January 1, 2006; 147(1): 13 - 19. [Abstract] [Full Text] [PDF]

				H. Chao and A. P. Spicer Natural Antisense mRNAs to Hyaluronan Synthase 2 Inhibit Hyaluronan Biosynthesis and Cell Proliferation J. Biol. Chem., July 29, 2005; 280(30): 27513 - 27522. [Abstract] [Full Text] [PDF]

				T. J. Smith and N. Hoa Immunoglobulins from Patients with Graves' Disease Induce Hyaluronan Synthesis in Their Orbital Fibroblasts through the Self-Antigen, Insulin-Like Growth Factor-I Receptor J. Clin. Endocrinol. Metab., October 1, 2004; 89(10): 5076 - 5080. [Abstract] [Full Text] [PDF]

				M. Sussmann, M. Sarbia, J. Meyer-Kirchrath, R.M. Nusing, K. Schror, and J.W. Fischer Induction of Hyaluronic Acid Synthase 2 (HAS2) in Human Vascular Smooth Muscle Cells by Vasodilatory Prostaglandins Circ. Res., March 19, 2004; 94(5): 592 - 600. [Abstract] [Full Text] [PDF]

				K. Tanimoto, A. Suzuki, S. Ohno, K. Honda, N. Tanaka, T. Doi, K. Yoneno, M. Ohno-Nakahara, Y. Nakatani, M. Ueki, et al. Effects of TGF-{beta} on Hyaluronan Anabolism in Fibroblasts Derived from the Synovial Membrane of the Rabbit Temporomandibular Joint Journal of Dental Research, January 1, 2004; 83(1): 40 - 44. [Abstract] [Full Text] [PDF]

				B. S. Prabhakar, R. S. Bahn, and T. J. Smith Current Perspective on the Pathogenesis of Graves' Disease and Ophthalmopathy Endocr. Rev., December 1, 2003; 24(6): 802 - 835. [Abstract] [Full Text] [PDF]

				M. Kazim, R. A. Goldberg, and T. J. Smith Insights Into the Pathogenesis of Thyroid-Associated Orbitopathy: Evolving Rationale for Therapy Arch Ophthalmol, March 1, 2002; 120(3): 380 - 386. [Full Text] [PDF]

				J. Pritchard, N. Horst, W. Cruikshank, and T. J. Smith Igs from Patients with Graves' Disease Induce the Expression of T Cell Chemoattractants in Their Fibroblasts J. Immunol., January 15, 2002; 168(2): 942 - 950. [Abstract] [Full Text] [PDF]

				T. J. Smith, L. Koumas, A. Gagnon, A. Bell, G. D. Sempowski, R. P. Phipps, and A. Sorisky Orbital Fibroblast Heterogeneity May Determine the Clinical Presentation of Thyroid-Associated Ophthalmopathy J. Clin. Endocrinol. Metab., January 1, 2002; 87(1): 385 - 392. [Abstract] [Full Text] [PDF]

				J.-P. Pienimaki, K. Rilla, C. Fulop, R. K. Sironen, S. Karvinen, S. Pasonen, M. J. Lammi, R. Tammi, V. C. Hascall, and M. I. Tammi Epidermal Growth Factor Activates Hyaluronan Synthase 2 in Epidermal Keratinocytes and Increases Pericellular and Intracellular Hyaluronan J. Biol. Chem., June 1, 2001; 276(23): 20428 - 20435. [Abstract] [Full Text] [PDF]

				M. A. Simpson, J. Reiland, S. R. Burger, L. T. Furcht, A. P. Spicer, T. R. Oegema Jr., and J. B. McCarthy Hyaluronan Synthase Elevation in Metastatic Prostate Carcinoma Cells Correlates with Hyaluronan Surface Retention, a Prerequisite for Rapid Adhesion to Bone Marrow Endothelial Cells J. Biol. Chem., May 18, 2001; 276(21): 17949 - 17957. [Abstract] [Full Text] [PDF]

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