CTCF-Mediated and Pax6-Associated Gene Expression in Corneal Epithelial Cell-Specific Differentiation.

BACKGROUND: The purpose of the study is to elicit the epigenetic mechanism involving CCCTC binding factor (CTCF)-mediated chromatin remodeling that regulates PAX6 gene interaction with differentiation-associated genes to control corneal epithelial differentiation. METHODS: Cell cycle progression and specific keratin expressions were measured to monitor changes of differentiation-induced primary human limbal stem/progenitor (HLS/P), human corneal epithelial (HCE) and human telomerase-immortalized corneal epithelial (HTCE) cells. PAX6-interactive and differentiation-associated genes in chromatin remodeling mediated by the epigenetic factor CTCF were detected by circular chromosome conformation capture (4C) and ChIP (Chromatin immunoprecipitation)-on-chip approaches, and verified by FISH (Fluorescent in situ hybridization). Furthermore, CTCF activities were altered by CTCF-shRNA to study the effect of CTCF on mediating interaction of Pax6 and differentiation-associated genes in corneal epithelial cell fate. RESULTS: Our results demonstrated that differentiation-induced human corneal epithelial cells expressed typical corneal epithelial characteristics including morphological changes, increased keratin12 expression and G0/G1 accumulations. Expressions of CTCF and PAX6 were suppressed and elevated following the process of differentiation, respectively. During corneal epithelial cell differentiation, differentiation-induced RCN1 and ADAM17 were found interacting with PAX6 in the process of CTCF-mediated chromatin remodeling detected by 4C and verified by ChIP-on-chip and FISH. Diminished CTCF mRNA with CTCF-shRNA in HTCE cells weakened the interaction of PAX6 gene in controlling RCN1/ADAM17 and enhanced early onset of the genes in cell differentiation. CONCLUSION: Our results explain how epigenetic factor CTCF-mediated chromatin remodeling regulates interactions between eye-specific PAX6 and those genes that are induced/associated with cell differentiation to modulate corneal epithelial cell-specific differentiation.

Nuclear targeting of IGF-1 receptor in orbital fibroblasts from Graves' disease: apparent role of ADAM17.

Insulin-like growth factor-1 receptor (IGF-1R) comprises two subunits, including a ligand binding domain on extra- cellular IGF-1Rα and a tyrosine phosphorylation site located on IGF-1Rß. IGF-1R is over-expressed by orbital fibroblasts in the autoimmune syndrome, Graves' disease (GD). When activated by IGF-1 or GD-derived IgG (GD-IgG), these fibroblasts produce RANTES and IL-16, while those from healthy donors do not. We now report that IGF-1 and GD-IgG provoke IGF-1R accumulation in the cell nucleus of GD fibroblasts where it co-localizes with chromatin. Nuclear IGF-1R is detected with anti-IGF-1Rα-specific mAb and migrates to approximately 110 kDa, consistent with its identity as an IGF-1R fragment. Nuclear IGF-1R migrating as a 200 kDa protein and consistent with an intact receptor was undetectable when probed with either anti-IGF-1Rα or anti-IGF-1Rß mAbs. Nuclear redistribution of IGF-1R is absent in control orbital fibroblasts. In GD fibroblasts, it can be abolished by an IGF-1R-blocking mAb, 1H7 and by physiological concentrations of glucocorticoids. When cell-surface IGF-1R is cross-linked with (125)I IGF-1, (125)I-IGF-1/IGF-1R complexes accumulate in the nuclei of GD fibroblasts. This requires active ADAM17, a membrane associated metalloproteinase, and the phosphorylation of IGF-1R. In contrast, virally encoded IGF-1Rα/GFP fusion protein localizes equivalently in nuclei in both control and GD fibroblasts. This result suggests that generation of IGF-1R fragments may limit the accumulation of nuclear IGF-1R. We thus identify a heretofore-unrecognized behavior of IGF-1R that appears limited to GD-derived fibroblasts. Nuclear IGF-1R may play a role in disease pathogenesis.

CTCF mediates effect of insulin on glucagon expression.

Pancreatic islet α-cell development and glucagon production are mainly regulated by Pax6 in the homeobox gene families. However, the molecular mechanism fine-tuning the regulation of these events in α-cell still remains unclear. In ocular cells, Pax6 transcription is regulated by CTCF through its binding to specific sites in Pax6 promoter. In this study, CTCF-mediated regulations of islet α-cell development and glucagon production were investigated in both CTCF transgenic mice and α-TC-1-6 cells. Over-expression of CTCF in transgenic mice affected development of pancreatic islets by significantly suppressing α-cell population in both embryonic and adult pancreases. The effect of CTCF on Pax6 gene expression and subsequently, on pro-glucagon production was however, examined in pancreatic islet α-cells. Over-expression and knock-down of CTCF directly affected Pax6 expression. More importantly, the CTCF binding sites upstream from Pax6 p0 promoter were required for regulating p0 promoter activity in islet α-cells. Stimulation of α-cells with insulin resulted in a significant increase in CTCF expression and a decrease in Pax6 expression, and consequently suppressed pro-glucagon expression. In contrast, these insulin-induced effects were blocked by knockdown of CTCF mRNA with specific siRNA in α-cells. Altogether, our results demonstrated for the first time that CTCF functions as a switch-like molecule between the insulin signaling and the regulations of Pax6 and glucagon expression in pancreatic islet α-cells.

Immunomodulation by imiquimod in patients with high-risk primary melanoma.

Imiquimod is a synthetic Toll-like receptor 7 (TLR7) agonist approved for the topical treatment of actinic keratoses, superficial basal cell carcinoma, and genital warts. Imiquimod leads to an 80-100% cure rate of lentigo maligna; however, studies of invasive melanoma are lacking. We conducted a pilot study to characterize the local, regional, and systemic immune responses induced by imiquimod in patients with high-risk melanoma. After treatment of the primary melanoma biopsy site with placebo or imiquimod cream, we measured immune responses in the treated skin, sentinel lymph nodes (SLNs), and peripheral blood. Treatment of primary melanomas with 5% imiquimod cream was associated with an increase in both CD4+ and CD8+ T cells in the skin, and CD4+ T cells in the SLN. Most of the CD8+ T cells in the skin were CD25 negative. We could not detect any increases in CD8+ T cells specifically recognizing HLA-A(*)0201-restricted melanoma epitopes in the peripheral blood. The findings from this small pilot study demonstrate that topical imiquimod treatment results in enhanced local and regional T-cell numbers in both the skin and SLN. Further research into TLR7 immunomodulating pathways as a basis for effective immunotherapy against melanoma in conjunction with surgery is warranted.

Orbital fibrosis in a mouse model of Graves' disease induced by genetic immunization of thyrotropin receptor cDNA.

The TSH receptor (TSHR) is the critical target for antibody production in Graves' disease (GD). Insulin-like growth factor 1 receptor (IGF1R) has been proposed as a second autoantigen in complications of GD such as orbitopathy. We attempted to induce orbital tissue remodeling in mice undergoing immunizations with plasmids encoding TSHR and IGF1R delivered by in vivo skeletal muscle electroporation, a procedure known to give a sustained, long-term antibody response. Female BALB/c mice were challenged with TSHR A-subunit or IGF1Rα subunit plasmid by injection and electroporation. Mice challenged with TSHR A-subunit plasmid resulted in high frequency (75%) of hyperthyroidism and thyroid-stimulating antibodies. But strikingly, immunization with TSHR A-subunit plasmid also elicited antibody to IGF1Rα subunit. Mice challenged in the same manner with IGF1Rα subunit plasmid produced strong antibody responses to IGF1R, but did not undergo any changes in phenotype. Simultaneous challenge by double antigen immunization with the two plasmids in distant anatomical sites reduced the incidence of hyperthyroidism, potentially as a consequence of antigenic competition. Thyroid glands from the TSHR A-subunit plasmid-challenged group were enlarged with patchy microscopic infiltrates. Histological analysis of the orbital tissues demonstrated moderate connective tissue fibrosis and deposition of Masson's trichrome staining material. Our findings imply that immunization with TSHR A-subunit plasmid leads to generation of IGF1R antibodies, which together with thyroid-stimulating antibodies may precipitate remodeling of orbital tissue, raising our understanding of its close association with GD.

Divergent Sp1 protein levels may underlie differential expression of UDP-glucose dehydrogenase by fibroblasts: role in susceptibility to orbital Graves disease.

UDP-glucose dehydrogenase (UGDH) catalyzes the formation of UDP-glucuronate. Glucuronate represents an integral component of the glycosaminoglycan, hyaluronan, which accumulates in orbital Graves disease. Here we report that orbital fibroblasts express higher levels of UGDH than do those from skin. This is a consequence of greater UGDH gene promoter activity and more abundant steady-state UGDH mRNA. Six Sp1 sites located in the proximal 550 bp of the UGDH gene promoter appear to determine basal promoter activity, as does a previously unrecognized 49-bp sequence spanning -1436 nucleotides (nt) and -1388 nt that negatively affects activity. Nuclear Sp1 protein is more abundant in orbital fibroblasts, and its binding to specific sites on DNA is greater than that in dermal fibroblasts. Mutating each of these Sp1 sites in a UGDH gene promoter fragment, extending from -1387 to +71 nt and fused to a luciferase reporter, results in divergent activities when transfected in orbital and dermal fibroblasts. Reducing Sp1 attenuated UGDH gene promoter activity, lowered steady-state UGDH mRNA levels, and reduced UGDH enzyme activity. Targeting Sp1 and UGDH with specific siRNAs also lowered hyaluronan synthase-1 (HAS-1) and HAS-2 levels and reduced hyaluronan accumulation in orbital fibroblasts. These findings suggest that orbital fibroblasts express high levels of UGDH in an anatomic-specific manner, apparently the result of greater constitutive Sp1. These high UGDH levels may underlie susceptibility of the orbit to localized overproduction of hyaluronan in Graves disease.

Unique attributes of orbital fibroblasts and global alterations in IGF-1 receptor signaling could explain thyroid-associated ophthalmopathy.

Tissue remodeling associated with thyroid-associated ophthalmopathy (TAO) involves the complex interplay between resident cells (endothelium, vascular smooth muscle, extraocular muscle, and fibroblasts) and those recruited to the orbit, including members of the "professional" immune system. Inflammation early in the disease can later culminate in fibrosis and diminished extraocular muscle motility. TAO remains a poorly understood process, in large part because access to tissues early in the disease is limited and because no robust and complete animal models of Graves' disease have yet been devised. Remaining uncertainty as to the identity of a pathogenic autoantigen(s) that underlies lymphocyte trafficking to the orbit complicates matters. These limitations in our understanding of extrathyroidal Graves' disease have resulted in poorly served patients with severe TAO. Therapies have targeted symptoms rather than the underlying disease processes. Our laboratory group has focused over the last several years on defining the peculiarities of the human orbital fibroblasts as a strategy for shedding more light on the pathologies occurring in TAO. We have reasoned that unique properties of these cells might ultimately prove the basis for why the manifestations of Graves' disease occur in an anatomically selective manner. In this brief review we attempt to survey our findings. We believe that they might provide a "roadmap" for further discovery into the pathogenesis of TAO. Clearly, more questions remain than those thus far answered.

Evidence for an association between thyroid-stimulating hormone and insulin-like growth factor 1 receptors: a tale of two antigens implicated in Graves' disease.

Thyroid-stimulating hormone receptor (TSHR) plays a central role in regulating thyroid function and is targeted by IgGs in Graves' disease (GD-IgG). Whether TSHR is involved in the pathogenesis of thyroid-associated ophthalmopathy (TAO), the orbital manifestation of GD, remains uncertain. TSHR signaling overlaps with that of insulin-like grow factor 1 receptor (IGF-1R). GD-IgG can activate fibroblasts derived from donors with GD to synthesize T cell chemoattractants and hyaluronan, actions mediated through IGF-1R. In this study, we compare levels of IGF-1R and TSHR on the surfaces of TAO and control orbital fibroblasts and thyrocytes and explore the physical and functional relationship between the two receptors. TSHR levels are 11-fold higher on thyrocytes than on TAO or control fibroblasts. In contrast, IGF-1R levels are 3-fold higher on TAO vs control fibroblasts. In pull-down studies using fibroblasts, thyrocytes, and thyroid tissue, Abs directed specifically against either IGF-1Rbeta or TSHR bring both proteins out of solution. Moreover, IGF-1Rbeta and TSHR colocalize to the perinuclear and cytoplasmic compartments in fibroblasts and thyrocytes by confocal microscopy. Examination of orbital tissue from patients with TAO reveals similar colocalization to cell membranes. Treatment of primary thyrocytes with recombinant human TSH results in rapid ERK phosphorylation which can be blocked by an IGF-1R-blocking mAb. Our findings suggest that IGF-1R might mediate some TSH-provoked signaling. Furthermore, they indicate that TSHR levels on orbital fibroblasts are considerably lower than those on thyrocytes and that this receptor associates with IGF-1R in situ and together may comprise a functional complex in thyroid and orbital tissue.

Interleukin-4 induces 15-lipoxygenase-1 expression in human orbital fibroblasts from patients with Graves disease. Evidence for anatomic site-selective actions of Th2 cytokines.

Orbital fibroblasts orchestrate tissue remodeling in Graves disease, at least in part, because they exhibit exaggerated responses to proinflammatory cytokines. A hallmark of late stage orbital disease is vision-threatening fibrosis, the molecular basis of which remains uncertain. We report here that the Th2 cytokines, interleukin (IL)-4 and IL-13, can induce in these cells the expression of 15-lipoxygenase-1 (15-LOX-1) and in so doing up-regulate the production of 15-hydroxyeicosatetraenoic acid. IL-4 increases 15-LOX-1 protein levels through pretranslational actions. The increased steady-state 15-LOX-1 mRNA is independent of ongoing protein synthesis and involves very modestly increased gene promoter activity. Importantly, IL-4 substantially enhances 15-LOX-1 transcript stability, activity that localizes to a 293-bp sequence of the 3'-untranslated region. IL-4 activates Jak2 in orbital fibroblasts. Interrupting signaling through that pathway, either with the specific chemical inhibitor, AG490, or by transiently transfecting the cells with a Jak2 dominant negative mutant kinase, attenuates the 15-LOX-1 induction. Interferongamma, a Th1 cytokine, could block this induction by attenuating IL-4-dependent mRNA stabilization. 15-LOX-1 protein and its mRNA were undetectable in IL-4-treated dermal fibroblasts, despite comparable levels of cell surface IL-4 receptor and phosphorylated Jak2 and STAT6. Our findings suggest that orbital connective tissues may represent a site of localized 15-hydroxyeicosatetraenoic acid generation resulting from cell type-specific 15-LOX-1 mRNA stabilization by IL-4. These results may have relevance to the pathogenesis of orbital Graves disease, an inflammatory autoimmune condition that gives way to extensive fibrosis associated with a Th2 response.

IL-1 beta induces IL-6 expression in human orbital fibroblasts: identification of an anatomic-site specific phenotypic attribute relevant to thyroid-associated ophthalmopathy.

Human orbital fibroblasts exhibit a unique inflammatory phenotype. In the present study, we report that these fibroblasts, when treated with IL-1beta, express high levels of IL-6, a cytokine involved in B cell activation and the regulation of adipocyte metabolism. The magnitude of this induction is considerably greater than that in dermal fibroblasts and involves up-regulation of IL-6 mRNA levels. IL-1beta activates both p38 and ERK 1/2 components of the MAPK pathways. Disrupting these could attenuate the IL-6 induction. The up-regulation involves enhanced IL-6 gene promoter activity and retardation of IL-6 mRNA decay by IL-1beta. Dexamethasone completely blocked the effect of IL-1beta on IL-6 expression. Orbital fibroblasts also express higher levels of IL-6R than do skin-derived cells. When treated with rIL-6 (10 ng/ml), STAT3 is transiently phosphorylated. Thus, the exaggerated capacity of orbital fibroblasts to express high levels of both IL-6 and its receptor in an anatomic site-selective manner could represent an important basis for immune responses localized to the orbit in Graves' disease.

Synovial fibroblasts from patients with rheumatoid arthritis, like fibroblasts from Graves' disease, express high levels of IL-16 when treated with Igs against insulin-like growth factor-1 receptor.

We have reported recently that IgG from patients with Graves' disease (GD) can induce the expression of the CD4-specific T lymphocyte chemoattractant, IL-16, and RANTES, a C-C chemokine, in their fibroblasts. This induction is mediated through the insulin-like growth factor-1 receptor (IGF-1R) pathway. We now report that Abs from individuals with active rheumatoid arthritis (RA-IgG) stimulate in their synovial fibroblasts the expression of these same cytokines. IgG from individuals without known autoimmune disease fails to elicit this chemoattractant production. Furthermore, RA-IgG fails to induce IL-16 or RANTES expression in synovial fibroblasts from donors with osteoarthritis. RA-IgG-provoked IL-16 and RANTES production also appears to involve the IGF-1R because receptor-blocking Abs prevent the response. RA fibroblasts transfected with a dominant-negative mutant IGF-1R fail to respond to RA-IgG. IGF-1 and the IGF-1R-specific analog Des(1-3) also induce cytokine production in RA fibroblasts. RA-IgG-provoked IL-16 expression is inhibited by rapamycin, a specific macrolide inhibitor of the Akt/FRAP/mammalian target of rapamycin/p70(s6k) pathway, and by dexamethasone. GD-IgG can also induce IL-16 in RA fibroblasts, and RA-IgG shows similar activity in GD fibroblasts. Thus, IgGs from patients with RA, like those associated with GD, activate IGF-1R, and in so doing provoke T cell chemoattraction expression in fibroblasts, suggesting a potential common pathway in the two diseases. Immune-competent cell trafficking to synovial tissue is integral to the pathogenesis of RA. Recognition of this novel RA-IgG/fibroblast interaction and its functional consequences may help identify therapeutic targets.

Up-regulation of prostaglandin E2 synthesis by interleukin-1beta in human orbital fibroblasts involves coordinate induction of prostaglandin-endoperoxide H synthase-2 and glutathione-dependent prostaglandin E2 synthase expression.

Prostaglandin E(2) (PGE(2)) production involves the activity of a multistep biosynthetic pathway. The terminal components of this cascade, two PGE(2) synthases (PGES), have very recently been identified as glutathione-dependent proteins. cPGES is cytoplasmic, apparently identical to the hsp90 chaperone, p23, and associates functionally with prostaglandin-endoperoxide H synthase-1 (PGHS-1), the constitutive cyclooxygenase. A second synthase, designated mPGES, is microsomal and can be regulated. Here we demonstrate that mPGES and PGHS-2 are expressed at very low levels in untreated human orbital fibroblasts. Interleukin (IL)-1beta treatment elicits high levels of PGHS-2 and mPGES expression. The induction of both enzymes occurs at the pretranslational level, is the consequence of enhanced gene promoter activities, and can be blocked by dexamethasone (10 nm). SC58125, a PGHS-2-selective inhibitor, could attenuate the induction of mPGES, suggesting a dependence of this enzyme on PGHS-2 activity. IL-1beta treatment activates p38 and ERK mitogen-activated protein kinases. Induction of both mPGES and PGHS-2 was susceptible to either chemical inhibition or molecular interruption of these pathways with dominant negative constructs. These results indicate that the induction of PGHS-2 and mPGES by IL-1beta underlies robust PGE(2) production in orbital fibroblasts.