Characterization of Five Monoclonal Antibodies Raised against Domain E of the Porcine Estradiol Receptor

Thole, Hubert; Jakob, Franz

doi:10.1055/s-0029-1211216

Cited by 15 publications

(2 citation statements)

References 16 publications

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“…Endogenous peroxidases were blocked in 0.6% H 2 O 2 -methanol for 20 min. After washing with TBS, specimens were incubated with primary mouse monoclonal antibodies (see Table 2) against collagen types I and II (Col-I and -II), estrogen receptor α (ERα; Thole and Jakob 1993), matrix metalloproteinases-1, -3 and -13 (MMP-1, -3, -13) and tissue inhibitors of metalloproteinases-1 and -2 (TIMP-1, -2) at 37°C for 60 min. Paraffin sections of growth plate (collagen types I and II), uterine tissue (ERα) and placentar tissue (MMP-1, -3, -13, TIMP-1, -2) were used as positive controls.…”

Section: Methodsmentioning

confidence: 99%

17β-estradiol reduces expression of MMP-1, -3, and -13 in human primary articular chondrocytes from female patients cultured in a three dimensional alginate system

Claassen

Steffen

Hassenpflug³

et al. 2010

Cell Tissue Res

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Clinical observations have suggested a relationship between osteoarthritis and a changed sex-hormone metabolism, especially in menopausal women. This study analyzes the effect of 17β-estradiol on expression of matrix metalloproteinases-1, -3, -13 (MMP-1, -3, -13) and tissue inhibitors of metalloproteinases-1, -2 (TIMP-1, -2) in articular chondrocytes. An imbalance of matrix metalloproteinases (MMPs) specialized on degradation of articular cartilage matrix over the respective inhibitors of these enzymes (TIMPs) that leads to matrix destruction was postulated in the pathogenesis of osteoarthritis. Primary human articular chondrocytes from patients of both genders were cultured in alginate beads at 5% O(2) to which 10(-11)M-10(-5)M 17β-estradiol had been added and analyzed by means of immunohistochemistry, immunocytochemistry and real-time RT-PCR. Since articular chondrocytes in vivo are adapted to a low oxygen tension, culture was performed at 5% O(2). Immunohistochemical staining in articular cartilage tissue from patients and immunocytochemical staining in articular chondrocytes cultured in alginate beads was positive for type II collagen, estrogen receptor α, MMP-1, and -13. It was negative for type I collagen, MMP-3, TIMP-1 and -2. Using real-time RT-PCR, it was demonstrated that physiological and supraphysiological doses of 17β-estradiol suppress mRNA levels of MMP-3 and -13 significantly in articular chondrocytes of female patients. A significant suppressing effect was also seen in MMP-1 mRNA after a high dose of 10(-5)M 17β-estradiol. Furthermore, high doses of this hormone led to tendentially lower TIMP-1 levels whereas the TIMP-2 mRNA level was not influenced. In male patients, only incubations with high doses (10(-5)M) of 17β-estradiol were followed by a tendency to suppressed MMP-1 and TIMP-1 levels while TIMP-2 mRNA level was decreased significantly. There was no effect on MMP-13 expression of cells from male patients. Taken together, application of 17β-estradiol in physiological doses will improve the imbalance between the amounts of MMPs and TIMPs in articular chondrocytes from female patients. Downregulation of TIMP-2 by 17β-estradiol in male patients would not be articular cartilage protective.

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Section: Methodsmentioning

confidence: 99%

17β-estradiol reduces expression of MMP-1, -3, and -13 in human primary articular chondrocytes from female patients cultured in a three dimensional alginate system

Claassen

Steffen

Hassenpflug³

et al. 2010

Cell Tissue Res

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“…After being washed three times in TBS (0.14 M NaCl in 20 mM TRIS/NaCl buffer, pH 7.4), cells were fixed in −20°C cold acetone for 5 min, blocked in 0.6% H 2 O 2 -methanol for 20 min, and then incubated for 1 h with the primary monoclonal antibody against estrogen receptor α (clone 13H2, according to Thole and Jakob 1993;diluted 1:75 in TBS). Cryostat sections of uterine tissue were used as positive controls.…”

Section: Immunocytochemistry For Estrogen Receptor αmentioning

confidence: 99%

The phytoestrogens daidzein and genistein enhance the insulin-stimulated sulfate uptake in articular chondrocytes

et al. 2008

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Clinical observations have suggested a relationship between osteoarthritis and a changed estrogen metabolism in menopausal women. Phytoestrogens have been shown to ameliorate various menopausal symptoms. Proteoglycans (PG) consisting of low and high sulfated glycosaminoglycans (GAG) are the main components of articular cartilage matrix, and their synthesis is increased by insulin in growth plate cartilage. We have investigated whether GAG synthesis and sodium [35S]sulfate incorporation in female bovine articular chondrocytes are affected by daidzein, genistein, and/or insulin. For comparative purposes, estradiol incubations were performed. Articular chondrocytes were cultured in monolayers at 5% O2 and 5% CO2 in medium containing serum for 7 days followed by the addition of 10(-11) M-10(-4) M daidzein, genistein, 17beta-estradiol, or 5 microg/ml insulin in a serum-free culture phase of 2 days. Photometrically analyzed GAG synthesis was significantly suppressed by high doses (10(-5) M-10(-4) M) of daidzein, genistein, and 17beta-estradiol. Although insulin raised the sodium [35S]sulfate uptake significantly, different concentrations of daidzein, genistein, or 17beta-estradiol showed no significant effects. However, the stimulating effect of insulin on sulfate incorporation was enhanced significantly after preincubation of cells with 10(-11) M-10(-5) M daidzein or 10(-9) M-10(-5) M genistein but not by 17beta-estradiol. In view of the risks of long-term estrogen replacement therapy, further experiments should clarify the potential benefit of phytoestrogens and insulin in articular cartilage metabolism.

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Immunogold labelling of estradiol receptor in MCF7 cells

Sierralta¹,

Bönig²,

Thole³

1995

Cell Tissue Res

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The distribution of estradiol receptor in serial sections of estradiol-deprived and estradiol-stimulated MCF7 cells was studied by using mouse monoclonal antibodies reacting with different domains of the receptor and goat-antimouse IgG/6 nm gold. In the nucleus and the cytoplasm of estradiol-deprived cells, the receptor was detected by all three monoclonals (13H2, HT 65 and MA1-310). The antibodies 13H2 and MA1-310 detected receptor associated to the microfilament bundles in the cytoplasm. Higher densities of antireceptor attachment to the nuclear areas were accompanied by a reduction in the attachment to the cytoplasm after estradiol stimulation of the cells. The results confirm earlier observations on the presence of cytoplasmic estrogen receptor in estradiol-deprived cells and support the premise of an estradiol-induced translocation of this ligand-dependent transcription regulator.

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Characterization of Five Monoclonal Antibodies Raised against Domain E of the Porcine Estradiol Receptor

Cited by 15 publications

References 16 publications

17β-estradiol reduces expression of MMP-1, -3, and -13 in human primary articular chondrocytes from female patients cultured in a three dimensional alginate system

17β-estradiol reduces expression of MMP-1, -3, and -13 in human primary articular chondrocytes from female patients cultured in a three dimensional alginate system

The phytoestrogens daidzein and genistein enhance the insulin-stimulated sulfate uptake in articular chondrocytes

Immunogold labelling of estradiol receptor in MCF7 cells

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