Induction of the hyaluronic acid-binding protein, tumor necrosis factor-stimulated gene-6, in cervical smooth muscle cells by tumor necrosis factor-alpha and prostaglandin E(2)

Abstract

Immediately before parturition the cervix undergoes striking changes in structure (ripening) that facilitate dilatation and effacement. Cervical ripening shares many features in common with inflammation-associated tissue remodeling, making it a valuable process to explore with respect to the biochemical events in extracellular matrix restructuring. Cervical ripening can be pharmacologically induced with prostaglandin E(2) (PGE(2)). Among the biochemical changes in the cervix at parturition is a marked increase in the hyaluronic acid (HA) content. HA and HA-binding proteins have been implicated in tissue hydration, release of collagenase, and leukocyte migration, but their roles in cervical ripening have not been explored. In the present study we examined the ability of PGE(2) to induce expression of the HA-binding protein, tumor necrosis factor-stimulated gene (TSG)-6, in human cervical smooth muscle cells (hCSMCs) and compared the PGE(2) response to that of tumor necrosis factor-alpha (TNF-alpha), an established inducer of TSG-6. TNF-alpha stimulated TSG-6 mRNA accumulation in a dose- and time-dependent manner, with the maximal response observed at 10 ng/ml after 6 hours of incubation. PGE(2) stimulated TSG-6 mRNA expression, but the magnitude of response was substantially less than that produced by TNF-alpha, and it was maximal only after 24 hours of incubation. Quantitative real-time polymerase chain reaction was performed to assess the induction of TSG-6 mRNA and nascent transcripts at 24 hours of treatment. Induction of TSG-6 mRNA and nascent transcripts in response to 10 micromol/L of PGE(2) was 5.7-fold and 6.3-fold greater than control values, respectively, whereas TNF-alpha (10 ng/ml) induced TSG-6 mRNA and nascent transcripts by 80-fold and 134-fold, respectively. TNF-alpha and PGE(2) stimulated secretion of TSG-6 into the culture medium as detected by Western blotting. The effects of PGE(2) on secretion of TSG-6 were delayed compared to TNF-alpha. A 1.3-kb fragment of the human TSG-6 proximal promoter drove luciferase expression in transfected hCSMCs. PGE(2) increased TSG-6 promoter activity 1.75-fold. Paradoxically, TNF-alpha reduced TSG-6 promoter activity by 50%. We conclude that hCSMCs express the hyaladherin TSG-6; that TSG-6 expression in these cells is regulated by PGE(2) as well as proinflammatory cytokines; responses of hCSMCs to TNF-alpha and PGE(2) are distinct in terms of magnitude and the time course; and PGE(2) and TNF-alpha exert different effects on the TSG-6 proximal promoter.

Figure 1.

Effect of TNF-α on expression of TSG-6 mRNA. A Northern blot analysis of total RNA (40 μg/lane) extracted from hCSMCs cultured in the absence (control) or presence of different concentrations of TNF-α for 48 hours is shown. The blot was sequentially probed to detect 28S rRNA.

Figure 2.

Time course of TNF-α-induced expression of TSG-6 mRNA. A: Northern blot analysis of total RNA (40 μg/lane) extracted from hCSMCs cultured in the absence (control) or presence of TNF-α (10 ng/ml) for the indicated time periods is shown. The blot was also probed for 28S rRNA. B: Histogram showing induction of TSG-6 mRNA normalized to 28S rRNA relative to the control value that was arbitrarily set to 1.0. The results are representative of two separate studies.

Figure 3.

Effect of PGE₂ on expression of TSG-6 mRNA. A Northern blot analysis of total RNA (40 μg/lane) extracted from hCSMCs cultured in the absence (control) or presence of different concentrations of PGE₂ or TNF-α (10 ng/ml) for 24 hours is shown. The blot was sequentially probed with 28S rRNA.

Figure 4.

Time course of PGE₂-induced TSG-6 mRNA expression. A: Northern blot analysis of total RNA (40 μg/lane) extracted from hCSMCs cultured in the absence (control) or presence of PGE₂ (10 μmol/L) for up to 72 hours is shown. The blots were sequentially probed for 28S rRNA. B: Histogram showing induction of TSG-6 mRNA normalized to 28S rRNA relative to the control value that was arbitrarily set to 1.0. Values are means ± SE from five separate experiments.

Figure 5.

TSG-6 production by hCSMCs treated with TNF-α or PGE₂. hCSMCs were incubated with TNF-α at 10 ng/ml or PGE₂ at 10 μmol/L for 24 hours in serum-free medium and aliquots of conditioned media (80 μl/lane) were subjected to SDS-polyacrylamide gel electrophoresis and then Western blotting.

Figure 6.

Time course of TSG-6 production by hCSMCs treated with PGE₂. hCSMCs were incubated without or with PGE₂ (10 μmol/L) for up to 72 hours in serum-free medium and aliquots of conditioned media (80 μl/lane) were subjected to SDS-polyacrylamide gel electrophoresis and then Western blotting.

Figure 7.

TSG-6 production by hCSMCs treated with different concentrations of PGE₂. hCSMCs were incubated in the absence or presence of different concentrations of PGE₂ for 72 hours in serum-free medium and aliquots of conditioned media (80 μl/lane) were subjected to SDS-polyacrylamide gel electrophoresis and then Western blotting.

Figure 8.

Effect of CHX and Act D on TNF-α and PGE₂ induction of TSG-6 mRNA in hCSMCs. A: Northern blot analysis of total RNA (50 μg/lane) extracted from hCSMCs treated with 10 ng/ml of TNF-α, 10 μmol/L of PGE₂, 50 μg/ml of CHX, 2 μg/ml of Act D, or the combination of these for 6 hours is shown. The blot was sequentially probed for 28S rRNA. B: hCSMCs were exposed to the same treatments for a period of 24 hours and Northern blot analysis was conducted as described above.

Figure 9.

Analysis of TSG-6 promoter function. The indicated promoter constructs were transfected into hCSMCs and incubated with 0.1% ethanol or 10 μmol/L of PGE₂ as described in Materials and Methods. The relative light units (RLU) presented are means ± SE from four independent experiments, each conducted with triplicate cultures.