Expression of collagen and aggrecan genes in normal and osteoarthritic murine knee joints.

OBJECTIVE: The STR/ort mouse strain develops osteoarthritis (OA) of the medial tibial cartilage whilst CBA mice do not develop this disease. We investigated whether changes occur in the expression of genes encoding major extracellular matrix proteins in the connective tissue of the murine knee joint in OA. DESIGN: Expression of the genes encoding collagens II (Col2alpha1), X (Col10alpha1), alpha2(XI) (Col11alpha2) and aggrecan (Agc) was detected in skeletally mature and immature male mice of the CBA and STR/ort strains by in situ hybridization. RESULTS: Col2alpha1 was expressed by chondrocytes of the tibial and patella-femoral cartilage and by the meniscal cartilage in all young mice (4-9 weeks) but only in the patella-femoral cartilage in older mice of both strains (36-45 weeks). In contrast Col2alpha1 was expressed by growth plate chondrocytes of both species at all ages. Similarly, Col2alpha1 transcripts were detected in cruciate ligament cells in both strains at all ages. Col10alpha1 transcripts were detected in cruciate ligament cells in both strains at all ages. Col10alpha1 expression was evident in the hypertrophic chondrocytes in the growth plate of young CBA and STR mice, but was not active in these cells in mature animals. However, Col10alpha1 was transcribed in articular chondrocytes of the tibia, meniscal and patella-femoral cartilages of all ages, in normal and osteoarthritic mice. Transcripts were also present in ligament of some mature animals. Col11alpha2 followed a similar pattern of expression in CBA cartilages to Col2alpha1, being active in adult growth plate but generally inactive in adult articular cartilages. Young CBA and STR/ort mice expressed Col11alpha2 in articular cartilage and very strongly throughout the growth plate. Agc expression was detected in all articular cartilages at all ages in both strains. Interestingly, transcripts for all four genes were absent in tibial articular chondrocytes located close to osteoarthritic lesions in STR/ort mice, indicating that these cells are unable to synthesize matrix proteins. Adult STR/ort mice also showed evidence of tissue remodeling around the periphery of the knee joint. Cells in remodeling areas actively transcribed Col2alpha1, Col10alpha1, Col11alpha2 and Agc. CONCLUSION: It is unlikely that OA develops in STR/ort mice because of failure to express major proteins in joint tissue. However, once lesions develop in articular cartilage neighbouring chondrocytes fail to express genes encoding several matrix proteins.

Abnormal compartmentalization of cartilage matrix components in mice lacking collagen X: implications for function.

There are conflicting views on whether collagen X is a purely structural molecule, or regulates bone mineralization during endochondral ossification. Mutations in the human collagen alpha1 (X) gene (COL10A1) in Schmid metaphyseal chondrodysplasia (SMCD) suggest a supportive role. But mouse collagen alpha1 (X) gene (Col10a1) null mutants were previously reported to show no obvious phenotypic change. We have generated collagen X deficient mice, which shows that deficiency does have phenotypic consequences which partly resemble SMCD, such as abnormal trabecular bone architecture. In particular, the mutant mice develop coxa vara, a phenotypic change common in human SMCD. Other consequences of the mutation are reduction in thickness of growth plate resting zone and articular cartilage, altered bone content, and atypical distribution of matrix components within growth plate cartilage. We propose that collagen X plays a role in the normal distribution of matrix vesicles and proteoglycans within the growth plate matrix. Collagen X deficiency impacts on the supporting properties of the growth plate and the mineralization process, resulting in abnormal trabecular bone. This hypothesis would accommodate the previously conflicting views of the function of collagen X and of the molecular pathogenesis of SMCD.

Actions of a progestogen on human breast cancer cells: mechanisms of growth stimulation and inhibition.

Medroxyprogesterone acetate (MPA) reduced the proliferation of MCF-7 cells with a maximal response at 100 nM. A subline ('M' cells) resistant to this action remained responsive to antioestrogen but showed a poor response to dexamethasone (DEX). Experiments with mixtures of MPA and DEX showed that MPA acts on wild-type MCF-7 cells through a glucocorticoid as well as a progestogen mechanism. The growth of 'M' cells was stimulated by MPA at 100 nM or greater and the drug increased polyamine concentrations in 'wild-type' as well as 'M' cells. It is suggested that both progestogen and glucocorticoid receptors may mediate the effects of high-dose MPA therapy in breast cancer. The possible stimulation of the growth of some cell types by MPA requires further investigation.

Steroid hormone receptors in human salivary gland tumours.

Major salivary gland tumours were studied for the presence of hormone receptors for oestrogen and progesterone. Of the eight salivary gland tumours exhibiting varied histology, none showed high affinity receptors for oestrogen or progesterone. Salivary tissue from four patients with non-neoplastic salivary gland disease was also studied and found not to contain high affinity receptor sites. The absence of hormone receptors in these glands suggests that such tumours are not dependent on endocrine function.

Stability of the human nuclear oestrogen receptor: influence of temperature and ionic strength.

The measurement of oestrogen receptors in the nuclei of cells of human breast cancer is becoming increasingly important for patient management. However, the steroid-binding properties of the oestrogen nuclear receptor of human cells under different conditions of temperature and ionic strength have received little attention despite the relevance of the receptor for interpretation of assay data. This paper reports a study on the influence of temperature and ionic strength on the exchange rate of [3H]oestradiol from human breast and endometrial nuclear receptor. When the oestrogen nuclear receptor complex was bound to intact or sheared nuclei, the displacement of bound [3H]oestradiol into buffer containing excess unlabelled oestradiol increased with temperature but was significant over 24 h even at 4 degrees C for nuclei from both breast and endometrium. The use of protease inhibitors combined with relabelling of nuclear receptor after incubation confirmed that the observations at 4 degrees C represented exchange of hormone rather than degradation of the hormone-receptor complex. Degradation was seen at higher temperatures. Measurement of the on-rate of [3H]oestradiol onto nuclear receptor prefilled with unlabelled oestradiol showed that on-rate was also significant over 24 h at 4 degrees C. The displacement of oestradiol from salt-extracted, hydroxylapatite-precipitated receptor also increased with temperature although, in this case, no displacement could be detected until temperatures above 4 degrees C were reached. Only 40% of total oestrogen receptor detectable in intact human nuclei was solubilized by the standard method of salt extraction in 0.6 mol KCl/l. As the salt concentration was raised (0-0.6 mol KCl/l), an increase in the stripping of oestradiol from the hormone-receptor complex was observed. Such stripping of nuclear receptor during salt extraction would lead to a false impression of the proportion of 'empty' nuclear receptors. The results show that filled oestrogen nuclear receptor from human tumour tissue may be assayed by exchange at 4 degrees C over 24 h. This eliminates the protease degradation of receptor which occurs at higher assay temperatures.

Binding of methyltrienolone (R1881) to a progesterone receptor-like component of human prostatic cytosol.

The synthetic steroid methyltrienolone (R1881, 17beta-hydroxy-17alpha-methyl-estra-4,9,11-trien-3-one) binds with high affinity to protein in cytosols prepared from human hyperplastic prostate. R1881 also binds to the androgen receptor of rat prostate and the progesterone receptor of rabbit uterus. Other steroids compete with R1881 for unoccupied binding sites in the human prostatic cytosols in a manner similar to that observed with the rabbit uterine progesterone receptor, rather than the rat prostatic androgen receptor. The progesterone receptor-like binding sites are a feature of the prostatic stroma rather than the epithelium.

Biochemical investigations of separated epithelium and stroma from benign hyperplastic prostatic tissue.

Using arginase and hydroxyproline as biochemical markers, the yields and homogeneity of separated epithelial and stromal tissues from surgically removed benign hyperplastic prostate glands have been assessed. On the basis of these markers, about 30% of epithelial and 95% of stromal tissues were recovered. Dehydroepiandrosterone sulphate sulphatase activity was found predominantly in the epithelium, whereas testosterone 5alpha-reductase activity was predominantly in the stroma.

Prostatic distribution of sex hormone-binding globulin and cortisol-binding globulin in benign hyperplasia.

Sex hormone-binding globulin-(SHBG) and cortisol-binding globulin-(CBG) like proteins have been demonstrated in prostatic tissue surgically removed from patients with benign prostatic hyperplasia. These proteins are not easily removed by superfusion of tissue slices. Epithelial tissue was separated from stroma and found not to contain the SHBG- or CBG-like proteins. Substantial amounts of these proteins, however, remained associated with the stroma. It is suggested that they may be constituents of interstitial fluid in this tissue compartment. The possible significance of this in benign prostatic hyperplasia is discussed.