Transforming growth factor-beta induces plasminogen activator inhibitor type-1 in cultured human orbital fibroblasts.

PURPOSE: The level of constitutive plasminogen activator inhibitor type-1 (PAI-1) expression in cultured human orbital fibroblasts is considerably lower than that found in dermal fibroblasts. This divergence in PAI-1 expression implies differences in the pericellular proteolytic environment and, therefore, in the turnover of extracellular matrix. In this article, the authors examine the effect of transforming growth factor-beta (TGF-beta) on PAI-1 expression in orbital fibroblasts. METHODS: Human orbital and dermal fibroblasts were grown in culture. Confluent monolayers were treated with TGF-beta. PAI-1 in the extracellular matrix was quantitated by radiolabeling the cultures and electrophoresing the cellular material on SDS-PAGE. Medium content was determined by immunoprecipitation of [35S]PAI-1 with a rabbit, anti-human, polyclonal antibody. PAI-1 mRNA was determined by Northern hybridization. RESULTS: TGF-beta increased PAI-1 levels in orbital fibroblasts in a dose-dependent manner, up to 35-fold. The induction was maximal after 16 hours of treatment. The increases in extracellular matrix PAI-1 paralleled those observed in the medium. The steady state levels of the mRNA encoding the protein were upregulated by TGF-beta up to 60-fold 8 hours after the addition of TGF-beta. The fractional increase in PAI-1 expression in orbital fibroblasts was consistently greater than that observed in dermal strains. CONCLUSIONS: Exposure to TGF-beta consistently induces PAI-1 expression in orbital fibroblasts, cells that do not express the polypeptide constitutively at high levels. The effects are mediated at the pretranslational level and involve the upregulation of PAI-1 mRNA. These results suggest that TGF-beta may exert a profound regulatory influence on the pericellular proteolytic environment in orbital connective tissue.

Leukoregulin induces plasminogen activator inhibitor type 1 in human orbital fibroblasts.

Leukoregulin, a 50-kDa T lymphocyte-derived cytokine, influences the synthesis of collagenase, stromelysin-1, collagen, and hyaluronan in human fibroblasts and is thus a determinant of extracellular matrix economy. We studied the effect of leukoregulin on the expression of plasminogen activator inhibitor type 1 (PAI-1) in human orbital and dermal fibroblasts. The lymphokine upregulated 35S-labeled PAI-1 protein expression in orbital fibroblasts in dose-dependent manner. The effect on extracellular matrix-associated PAI-1 evolved over several hours and was maximal at 10 h, when levels were 75-fold higher than controls, and then fell by 24 h. Leukoregulin treatment increased prostaglandin E2 production in orbital cultures after 24 h. When this increase was blocked with indomethacin, peak PAI-1 levels were maintained. Northern analysis demonstrated a substantial induction of steady-state PAI-1 mRNA levels within 6 h of treatment in orbital cultures. In contrast, leukoregulin lowered PAI-1 protein levels dramatically in skin fibroblasts from the abdominal wall. With regard to PAI-1 expression, it would appear that the anatomic site of origin of fibroblasts is a crucial determinant of the cellular response to leukoregulin.

Interferon-gamma is an inducer of plasminogen activator inhibitor type 1 in human orbital fibroblasts.

Expression of plasminogen activator inhibitor type 1 (PAI-1), an important determinant of the pericellular proteolytic environment, was assessed in cultured human orbital and dermal fibroblasts. Bidimensional electrophoretic separation of total cellular [35S]methionine-labeled proteins revealed multiple isoforms of the 50-kDa PAI-1 protein (isoelectric point range 5.6-6.3) expressed in dermal strains but not in orbital cultures. The addition of human recombinant interferon-gamma (100 U/ml) to the media for 48 h resulted in a marked induction of PAI-1 in the orbital cultures (greater than 50-fold above baseline), whereas the expression in dermal cultures was either attenuated or induced modestly (5-fold). The identity of the 50-kDa PAI-1 was verified by immunoprecipitation of secreted proteins. Indirect immunofluorescence localized PAI-1 to aggregates at the ventral undersurface of the monolayer. Interferon-gamma induced several other proteins in cultured cells. The observation that untreated orbital fibroblasts do not express detectable cell-associated PAI-1 suggests that the pericellular proteolytic environment of these cells is regulated ordinarily by other factors. The economy of the extracellular matrix of orbital connective tissue may be particularly susceptible to the influence of inflammatory cytokines such as interferon-gamma.

Prostaglandin E2 elicits a morphological change in cultured orbital fibroblasts from patients with Graves ophthalmopathy.

Fibroblasts derived from distinct anatomical regions appear to differ in regard to their behavior in culture. These differences may reflect functions of these cells in vivo that are tissue specific. Moreover, intrinsic differences in fibroblasts may underlie the site-specific connective tissue manifestations associated with systemic disease. We have demonstrated previously that orbital fibroblasts exhibit different cytokine response domains and protein synthetic programs when compared to those emanating from the skin. In the present communication, we demonstrate that prostaglandin E2 (PGE2) elicits in cultured human orbital fibroblasts from patients with Graves ophthalmopathy a rapid and dramatic change in cell morphology in vitro as assessed by phase-contrast and scanning electron microscopy. The central areas of the cells become elevated with respect to the plane of the substratum and are stellate, with long processes that touch neighboring cells. These changes occur within 6 hr of prostanoid addition to culture medium at an apparent concentration threshold of approximately 10 nM. Shape changes are accompanied by marked alterations in monolayer impedance as assessed by electric cell-substrate impedance sensing as described previously. Both morphologic and impedance changes elicited by PGE2 revert over 24 hr toward those found in untreated cells despite the continued presence of the prostanoid in the culture medium. In contrast, dermal fibroblasts fail to respond to PGE2. These observations define a previously unrecognized phenotypic attribute of orbital fibroblasts. Intrinsic differences in these cells may account for the anatomic site-selective vulnerability of the orbit in Graves ophthalmopathy. The culture system described here may be useful for studying the morphogenic actions of prostanoids.