Willem De Boer

Chronic obstructive pulmonary disease is associated with enhanced bronchial expression of FGF‐1, FGF‐2, and FGFR‐1

An important feature of chronic obstructive pulmonary disease (COPD) is airway remodelling, the molecular mechanisms of which are poorly understood. In this study, the role of fibroblast growth factors (FGF‐1 and FGF‐2) and their receptor, FGFR‐1, was assessed in bronchial airway wall remodelling in patients with COPD (FEV1 < 75%; n = 15) and without COPD (FEV1 > 85%; n = 16). FGF‐1 and FGFR‐1 were immunolocalized in bronchial epithelium, airway smooth muscle (ASM), submucosal glandular epithelium, and vascular smooth muscle. Quantitative digital image analysis revealed increased cytoplasmic expression of FGF‐2 in bronchial epithelium (0.35 ± 0.03 vs 0.20 ± 0.04, p < 0.008) and nuclear localization in ASM (p < 0.0001) in COPD patients compared with controls. Elevated levels of FGFR‐1 in ASM (p < 0.005) and of FGF‐1 (p < 0.04) and FGFR‐1 (p < 0.001) in bronchial epithelium were observed. In cultured human ASM cells, FGF‐1 and/or FGF‐2 (10 ng/ml) induced cellular proliferation, as shown by [3H]thymidine incorporation and by cell number counts. Steady‐state mRNA levels of FGFR‐1 were elevated in human ASM cells treated with either FGF‐1 or FGF‐2. The increased bronchial expression of fibroblast growth factors and their receptor in patients with COPD, and the mitogenic response of human ASM cells to FGFs in vitro suggest a potential role for the FGF/FGFR‐1 system in the remodelling of bronchial airways in COPD. Copyright

Epidermal Growth Factor Receptor Regulates Normal Urothelial Regeneration

Members of the epidermal growth factor (EGF) family and their receptors are involved in many cellular processes, including proliferation, migration, and differentiation. We have previously reported that these growth factors are expressed and have specific regulatory functions in an organ-like culture model of normal human urothelial cells. Here, we used this model to investigate the involvement of EGF receptor (EGFR) in human urothelial regeneration. Three 4-mm-diameter damaged areas were made in confluent normal human urothelial cell cultures with a biopsy punch. Regeneration was measured, on fixed stained cultures, with an image analyzer, at 4, 24, and 48 hours after injury. Cell proliferation was assessed by 5-bromo-2-deoxyuridine incorporation. To identify EGF family factors potentially involved in the healing process, we studied the effect of these factors on damaged confluent cultures and the level of expression of mRNAs extracted from these cultures. EGFR inhibition of the proliferation and migration of urothelial cells was tested with (1) a specific tyrosine kinase inhibitor (AG1478) and (2) a blocking anti-EGFR antibody (LA22). Exogenously added amphiregulin, EGF, transforming growth factor-α and heparin-binding EGF (HB-EGF) stimulated urothelial regeneration. The damaged areas were repaired by regrowth within 48 hours. Both AG1478 and LA22 inhibited the repair (by 50% and 30%, respectively), as well as proliferation and migration. This regeneration was accompanied by increased HB-EGF mRNA expression in cultures of cells from four of six subjects, but no corresponding change in EGFR protein level was observed. These results indicate that the EGFR signaling pathway is involved in urothelial regeneration. Our data support an autocrine role of HB-EGF in this process and suggest that the EGFR pathway is a potential therapeutic target for modulating urothelial cell proliferation.

Analysis of steroid- and DNA-binding domains of the calf uterine androgen receptor by limited proteolysis

The DNA-binding form of the calf uterine androgen receptor (AR) was subjected to limited protease digestion using chymotrypsin, trypsin and a rat prostate cytosol protease. The properties of the generated polypeptide fragments were identified and compared with those of the intact AR. Physicochemical characterization was achieved through sedimentation analysis, gel filtration chromatography and DEAE anion exchange chromatography. Intactness of functional binding domains was evaluated by measuring the retention of steroid- and DNA-binding capacity. Under non-denaturing conditions the intact AR is a highly asymmetrical molecule with a Stokes radius (RS) of 45A, a sedimentation coefficient of 4.3S and a relative molecular mass of 80,000 daltons. This form of AR has an intrinsic binding affinity for DNA and was eluted from DNA-cellulose with 9 mM MgCl2. Chymotrypsin produced a more globular polypeptide (RS: 31A; 3.1S; 41,000 daltons) with a decreased net negative charge. This fragment also displayed DNA-binding affinity but required a higher concentration of MgCl2 (14 mM) for DNA-cellulose elution, indicating an increased affinity for DNA. The observed reduction in molecular size upon chymotrypsin treatment was confirmed when analysed by SDS-polyacrylamide gel electrophoresis after covalently labelling of the AR with [3H]R1881. Rat prostate cytosol contains a protease which is very active in generating an AR polypeptide with an increased affinity for DNA, without changing the AR net negative charge (RS: 33A; 3.7S; 51,000 daltons). The specificity of this protease remained unknown since none of a large number of inhibitors was able to inactivate this enzyme. The fragment generated is different from that obtained with chymotrypsin since significant differences in size as well as in charge were measured. Trypsin treatment generated a much smaller polypeptide (RS: 25A; 2.9S; 30,000 daltons) which had lost its DNA-binding capacity, but not its steroid binding site. This form probably represents the so-called meroreceptor. When intact AR was treated sequentially with prostate cytosol and trypsin, a polypeptide fragment with identical properties was obtained, indicating the spatial separation of two of the proteolytic cleavage sites. These studies provide evidence for the distinct nature of the molecular domains for androgen and DNA interaction on the calf uterine AR.

Oestradiol-receptor complexes in subnuclear fractions of rat uterine tissue

The subnuclear distribution of 3H-oestradiol-receptor complexes was studied in uterine tissue of ovariectomized adult rats. Nuclei were sonically disrupted and 8 different subnuclear fractions were isolated by discontinuous sucrose density gradient centrifugation. 3H-Oestradiol-receptor complexes, measured by hydroxylapatite column chromatography, were localized in a light chromatin fraction as well as in a heavy chromatin fraction. Using the hydroxylapatite chromatography technique it was possible to demonstrate three classes of oestradiol-receptor complexes which differ in affinity for the chromatin. Oestradiol-receptor complexes with a high affinity for the chromatin were predominantly localized in the heavy chromatin fraction, whereas complexes with a lower affinity for their acceptor sites were present in the lighter chromatin fraction.

Differential effects of molybdate on the hydrodynamic and DNA-binding properties of the non-activated and activated forms of the androgen receptor in calf uterus

Calf uterine cytosol contains an androgen receptor with a relative molecular mass of approx. 90,000. In this study we have analysed the structure and aggregation properties of the androgen receptor, using sucrose density gradient centrifugation on a vertical rotor (VTi65). In the presence of 10 mM NaCl the androgen receptor in whole cytosol sedimented at 8 S irrespective of the presence of molybdate. In 400 mM NaCl the receptor dissociated to a 4.3 S entity. In whole cytosol molybdate promoted a partial shift of the 4.3 S receptor into the aggregated 8 S state. The time of exposure of the receptor to molybdate and NaCl determined the proportion of receptor sedimentating at 8 S and 4.3 S. The DNA-binding form of the uterine androgen receptor when analysed under the conditions of the DNA-cellulose binding assay, sedimented at 6.5 S. Increasing concentrations of molybdate shifted its sedimentation coefficient gradually from 6.5 S to 4.5 S and in parallel reduced the DNA-binding capacity. Molybdate added to a partially purified, DNA-binding form of the androgen receptor did not promote receptor aggregation to faster sedimentating forms. This suggests that such preparations are devoid of an androgen receptor-aggregation factor. Indirect evidence for such a factor was obtained from reconstitution experiments with whole cytosol. Our results indicate that the DNA-binding form of the androgen receptor interacts with a cytosol factor to form the 8 S receptor complex. Molybdate has diverse effects: in the presence of the cytosol factor it stabilizes the 8S complex; in its absence molybdate prevents in a concentration-dependent way DNA-binding as well as reaggregation of the monomeric 4.3 S form.

Kinetics of in vitro binding of oestradiol in subcellular fractions of testicular and uterine tissue; characterization of oestradiol binding in testicular nuclei.

Abstract 1. 1. Oestradiol in rat testicular and uterine tissue is specifically bound to nuclear receptor sites, which can be separated in KCl-extractable nuclear and nuclear residual (a nuclear fraction which resists KC1 extraction) receptor sites. 2. 2. The amount of “extractable” nuclear binding sites for oestradiol in testis could be increased by mild trypsin treatment. Treatment of testicular nuclei with deoxycholate or DNAse resulted in a decrease of residual receptor sites and a concomitant increase of unbound oestradiol in the “extractable” nuclear fraction. 3. 3. The presence of KCN in vitro resulted in a relative increase in the number of oestradiol binding sites in the nuclear residual fraction in both uterine and testicular tissue; the number of binding sites in the KCl-extractable fraction was not affected by KCN. 4. 4. During in vitro incubations of testicular tissue the number of oestradiol binding sites in the KCl-extractable nuclear fraction reached a maximum and remained constant after 30min of incubation; the number of binding sites in the nuclear residual fraction decreased after incubation periods longer than 30 min. 5. 5. During in vitro incubations of uterine tissue the number of oestradiol binding sites in the KCl-extractable nuclear fraction and the nuclear residual fraction after an initial increase decreased to 50% of the maximal value between 30 and 60 min of incubation. 6. 6. It is concluded, that the testicular oestradiol receptor shows certain characteristics comparable with those of the uterine receptor. However, regarding the differences in retention time of steroids in the nucleus, it seems very unlikely that the oestradiol effect in uterus and the oestradiol effect in testis, if present, are mediated by identical receptor mechanisms.