Dual effects of 17beta-oestradiol on interleukin 1beta-induced proteoglycan degradation in chondrocytes

Abstract

Objective: To determine whether 17beta-oestradiol (E2) modulates interleukin (IL) 1beta-induced proteoglycan degradation in chondrocytes, and to analyse the part played by metalloproteinases (MMPs) in this process.

Methods: Primary cultured rabbit articular chondrocytes were prepared and treated with 10 ng/ml IL1beta combined or not with 0.1-10 nM E2. Neosynthesised proteoglycans (PGs) were evaluated after incorporation of [(35)SO(4)]sulphate and further analysed after chromatography on a Sepharose 2B column. Chondrocyte mRNA levels of aggrecan, MMP-1, -3, -13, and tissue inhibitor of metalloproteinase-1 (TIMP-1) were studied by northern blot. MMP-1 activity was measured by zymography. MMP-1 gene transcription was studied by transient transfection of chondrocytes with an MMP-1-luciferase construct.

Results: E2 modulated the IL1beta-induced total sulphated PGs in rabbit articular chondrocytes, which decreased as the E2 concentration was increased. At a low concentration (0.1 nmol/l) E2 counteracts the IL1beta-induced decrease in sulphated PG, while at high concentration (10 nmol/l) E2 enhances the IL1beta effects. A biphasic E2 effect was also observed on IL1beta-induced disaggregation of PG, 53-58 kDa gelatinolytic activity, and MMP-1, -3, and -13 mRNA levels. In contrast, E2 did not modify the level of aggrecan mRNA and had no effect on TIMP-1 mRNA expression. Finally, simultaneous addition of IL1beta and E2 (0.1-10 nmol/l) did not modify IL1beta-induced MMP-1-luciferase activity, suggesting that E2 effects probably occur at the post-transcriptional level of MMP gene expression.

Conclusion: Oestrogen concentration may have an inverse effect on IL1beta stimulated proteoglycan degradation and MMP production by chondrocytes.

Figure 1

(A) [³⁵SO₄]Sulphate incorporation into PGs synthesised and secreted by chondrocytes treated or not with 10 ng/ml IL1ß combined or not with E2 (0.1–10 nmol/l). Articular chondrocytes were cultured until confluency. After 24 hours without FCS, cells were incubated with 1.5 µCi/ml [³⁵SO₄]sulphate for 20 hours with or without effectors. [³⁵SO₄]Sulphated proteoglycans were extracted as described in "Materials and methods". In each experiment six similarly treated flasks were prepared, three being used for the sulphation assay and the other three for DNA measurement. Radioactivity incorporated into [³⁵SO₄]sulphated PG was calculated as mean (SD) dpm/10 µg DNA from four experiments performed with different animal donors and expressed as a percentage of controls incubated without effectors (*p⩽0.05; **p<0.01). (B) Northern blot showing aggrecan mRNA hybridisation signal in chondrocytes treated or not with 10 ng/ml IL1ß combined or not with E2 (0.1–10 nmol/l). Total mRNA was extracted after 20 hours' incubation with or without effectors. The upper part shows the hybridisation signal to the rabbit aggrecan probe and to a human GAPDH probe which indicates the relative amount of RNA loaded in each lane. These data are representative of one of three independent experiments. The lower part shows the densitometric quantification of the autoradiogram expressed as the aggrecan/GAPDH band density ratio. Results are the mean (SD) of three experiments performed with different animal donors.

Figure 2

(A) Sepharose 2B elution profile of [³⁵SO₄]sulphated proteoglycans synthesised and secreted by chondrocytes treated or not with IL1ß combined or not with E2 (0.1–10 nmol/l). Two similarly treated flasks were prepared in each experiment. Confluent chondrocytes were incubated with [³⁵SO₄]sulphate for 20 hours with or without effectors as described in fig 1. [³⁵SO₄]Sulphated PGs were extracted in associative conditions with protease inhibitors as described in "Materials and methods". Aliquots were applied to a column of Sepharose 2B. Eluates were collected and the radioactivity measured by scintillation counting. Vo is the void volume and Vt is the total volume of the column. In each experimental condition, LMW radioactive material (hatched grey) eluted between Kav 0.3 and 0.6, is expressed as a percentage of total radioactivity eluted on the column between Vo and Vt (plain grey). The yield of the column is >90%. Data are representative of a series of flasks examined in one of three independent experiments. (B). For each column, radioactivity eluted at Kav 0.3–0.6 (LMW PG) was measured and expressed as a percentage of the total radioactivity eluted on the column (Vo to Vt). In each experiment two similarly treated flasks were analysed. Results are expressed as the mean (SD) of three experiments performed with different animal donors (*p⩽0.05).

Figure 3

Northern blot showing the effects of E2 on IL1ß-induced MMP-1, -3, and -13 mRNA hybridisation signals. Confluent chondrocytes were incubated for 20 hours with or without effectors as described above. After 20 hours, total RNA was extracted and analysed as in fig 1B. The upper part shows the hybridisation signal to the rabbit MMP-1, -3, -13 probes and to the human GAPDH probe. Data are representative of one of four independent experiments. The lower part shows the densitometric quantification of the autoradiogram expressed as the MMP/GAPDH band density ratio. Data are the mean (SD) from four independent experiments (*p⩽0.05, **p<0.01).

Figure 4

(A) Gelatinolytic activities and (B) TIMP-1 mRNA levels in chondrocytes treated or not with 10 ng/ml IL1ß combined or not with E2 (0.1–10 nmol/l). Confluent chondrocytes were incubated for 20 hours with or without effectors as described in fig 1. Culture medium was collected and subjected to gelatin zymography as described in "Materials and methods". Total RNA was extracted from the corresponding cultured flasks and analysed as in fig 1B for TIMP-1 hybridisation signal. (A) Upper part: the gelatinolytic bands are representative of a series of flasks from one of four independent experiments. Lower part: the level of gelatinolytic bands was determined by scanning video-densitometry. Because the 72 kDa band densitometry was not modified by IL1ß or E2, this band was used as internal control. The densitometric quantification of the IL1ß-induced 53–58 kDa band was thus expressed as the 53–58 kDa/72 kDa band ratio. Data are the mean (SD) of results obtained from four independent experiments performed with different animals. (B) Northern blot analysis of TIMP-1 mRNA hybridisation signal. The upper part shows the hybridisation signal to the rabbit TIMP-1 probe and to a human GAPDH probe. Data are representative of a series of flasks from one of four independent experiments. The lower part shows the densitometric quantification of the autoradiogram expressed as mean (SD) of TIMP-1/GAPDH band density ratio measured in four independent experiments.

Figure 5

MMP-1 gene promoter activity. The upper part represents the design of the 3.45 kb of the 5' flanking region linked to the luciferase reported gene used in DNA transfection experiments. Chondrocytes were transiently cotransfected with MMP-1 gene promoter and with a ß-galactosidase expression vector and further treated with each effector for 20 hours. Each point was measured in triplicate. The lower part shows the relative luciferase activity calculated as fold induction in treated cells with respect to the basal activity measured in untreated cells. Values for the luciferase activity were normalised to the ß-galactosidase activity. Results are the mean (SD) of three independent experiments.