The pathogenesis of fibrosis, especially involving post-translational modifications of collagen, is poorly understood. Lysyl hydroxylase 2 (long) (LH2 (long)) is thought to play a pivotal role in fibrosis by directing the collagen cross-link pattern. Here we show that LH2 (long) exerts a bimodal function on collagen synthesis in human dermal fibroblasts. Adenoviral-mediated overexpression of LH2 (long) resulted in a mRNA increase of collagen α1(I) but not of fibronectin and fibrillin-1. This was accompanied by a higher mRNA level of prolyl-4-hydroxylase but not of other ER proteins (Bip, Hsp47, LH1, LH3). The collagen mRNA increase led to an elevated collagen synthesis, which was higher in the fraction of extracellularly deposited, cell-associated collagen than in the medium. The cross-link pattern of cell-associated collagen showed an increase of the hydroxylysine-aldehyde-derived cross-link dihydroxylysinonorleucine and a decrease of the lysine-aldehyde-derived component hydroxylysinonorleucine. The helical lysyl hydroxylation of the procollagen molecule was unaltered. The increase of collagen synthesis in fibroblasts overexpressing LH2 (long) was independent from cross-linking as it was also observed in the presence of β-aminopropionitril, a cross-linking inhibitor. Together our data identify LH2 (long) as a bifunctional protein and underscores its potential role in the pathogenesis of fibrosis.

By ammonium sulphate precipitation, phosphocellulose and DEAE-Sephacel chromatography and Sephadex G-75 gel filtration, a factor was separated from rat liver cytosol which was shown to suppress the inhibitory effect of the steroid deoxycorticosterone (DOC) on the specific [3H]glucocorticoid binding to cytoplasmic receptors. By SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of factor-containing fractions its Mr was suggested to be about 40000. The possible role of this factor in the regulation of glucocorticoid receptor apparatus function is discussed.

The accumulation of free and protein-bound forms of [3H]corticosterone and [3H]dexamethasone by preparations of heart sarcolemma from adult male Wistar rats was studied. At physiological concentrations of glucocorticoids the sarcolemma preparations showed a considerably greater accumulation of the cytosol-bound form of glucocorticoids as compared to the free form. On the contrary, the formation of [3H]corticosterone-transcortine complex decreased the steroid uptake by the membranes under these conditions. The [3H]steroid-albumin complexes obtained by incubation of very high concentrations of [3H]glucocorticoids with pure albumin were retained by the membranes better than the free form of the hormones. The differences in the accumulation by membranes of steroid--cytosol and steroid--albumin complexes during temperature rise from 0 degrees to 20 degrees C were found. The increase in the uptake of steroid--cytosol complexes was evidently caused by thermal "activation" of the complexes, while the temperature influence on the steroid--albumin complexes accumulation by sarcolemma preparations was directly due to the uptake process. An addition of molybdate (blocker of steroid--receptor complexes activation) at a concentration of 100 mM completely eliminated the thermal effect on the steroid--cytosol complexes accumulation.

Accumulation of 3H-dexamethasone by the nuclei in a cell-free system was studied. The increase in temperature from 0 degrees to 20 degrees C and treatment of cytosol by KCl (0.4 M) or theophylline (10mM) significantly increased the absorption the bound hormone by the nuclear fraction. Activation of the steroid-protein complex induced by temperature and addition of theophylline did not change its size. The increase in the ionic strength decreased the Stokes radius from 53 A down to 39.5 A and the sedimentation coefficient value from 7S down to 4S. It is concluded that the heart tissue cytoplasm contains a glucocorticoid receptor.

Accumulation by rat heart cell nuclei in vitro of free and protein-bound [3H]corticosterone and [3H]dexamethasone was investigated. [3H]Glucocorticoid-protein complexes were prepared by incubation of labelled hormones with diluted blood serum and cytosol containing glucocorticoid cytoreceptor, transcortin-like protein and various amounts of transcortin. At concentrations of 2 × 10−9 to 25 × 10−9 m binding to cytoreceptor enhances nuclear accumulations of hormones. Uptake of [3H]corticosterone depends on the ratio of binding proteins present in cytosol. The higher the part of glucocorticoid bound to cytoreceptor the larger the amounts accumulated in nuclear fractions. At relatively high concentrations (5 × 10−8 m and higher) nuclei readily accumulate transcortin-bound and free forms of [3H]corticosterone and binding to transcortin evidently facilitates nuclear uptake. Studies with Triton X-100 and KCl extractions of incorporated [3H]glucocorticoids reveal heterogenity of the accumulation process. This process includes uptake of glucocorticoids by outer nuclear membrane as well as intranuclear accumulation. Binding to proteins facilitates in certain conditions uptake of hormones by nuclear membrane. 0.4 m KCl treatment of nuclei incubated with [3H]glucocorticoid-cytosol complex leads to extraction of essential amounts of intranuclear [3H]glucocorticoid fraction a part of which represents macromolecular complexes of hormones. KCl extracts from nuclei incubated with free of transcortin-bound steroid contain a comparatively low percentage of hormone, the greater part of which is present in free form.