Three-dimensional collagen regulates collagen gene expression by a mechanism that requires serine/threonine kinases and is independent of mechanical contraction.

Integrin alpha1beta1, one of the cellular collagen receptors, can participate in the regulation of collagen accumulation by acting as a negative feedback regulator. The molecular mechanism behind this phenomenon has been unknown. We have plated cells inside three-dimensional collagen and analyzed a set of chemical inhibitors for various signal transduction pathways. Only two wide-spectrum serine/threonine kinase inhibitors, H-7 and iso-H-7 could prevent the down-regulation of alpha1(I) collagen mRNA levels in cells exposed to three-dimensional collagen. In monolayer iso-H-7 slightly down-regulated collagen gene expression, indicating that inside collagen it affected integrin signaling rather than having a direct stimulatory effect on collagen mRNA levels. The effect of iso-H-7 was not dependent on its ability to inhibit protein kinases A, C, or G. H-7 and iso-H-7 could also inhibit collagen gel contraction, but this mechanism was independent of collagen gene regulation. Three-dimensional collagen could also up-regulate the mRNA levels of several matrix metalloproteinases (MMPs) but H-7 and iso-H-7 had no effect on the regulation of MMP genes. Our data indicate that three-dimensional collagenous matrix regulates distinct cellular signaling pathways and that collagen gene regulation is independent of the other effects of the matrix.

Antibody against human alpha 1 beta 1 integrin inhibits HeLa cell adhesion to laminin and to type I, IV, and V collagens.

In HeLa cells beta 1 integrin forms heterodimers with alpha 1, alpha 2, alpha 3, alpha 5 and alpha 6 integrin subunits. Integrin alpha v beta 5 can also be detected. A monoclonal antibody SR-84 identified the alpha 1 integrin subunit in immunoprecipitation assays and inhibited alpha 1-related cell adhesion to different matrix proteins, laminin-1 and type I, IV, and V collagens, whereas its effect on adhesion to type II collagen was marginal. HeLa cells do not attach to type VI collagen. The presence of magnesium was essential for HeLa cell adhesion, whereas calcium alone was not sufficient and high concentrations of calcium even counteracted the effect of magnesium. Cell adhesion to type I collagen was sensitive to changes in pH, unlike cell adhesion to type IV collagen. We conclude that SR-84 is a valuable tool to study alpha 1 integrin-related functions, and that in HeLa cells alpha 1 beta 1 integrin is a magnesium-dependent receptor for type I, IV, and V collagens but not for type II and VI collagens.

In vitro assessment of the fibrogenicity of mineral dusts.

Relying on in vitro production of the macrophage fibrogenic factor, an attempt was made to quantitate the fibrogenicity of mineral particles. Having determined the optimal conditions by means of quartz, two series of experiments were conducted with respirable coal mine dusts; the first employed artificial mixtures of a mine dust, having a low natural quartz content, with various proportions of quartz, and the second native dusts from European mines. The fibrogenic responses in both series suggest that dust concentration is more important than its composition. Quartz and ash contents of native dusts bore no evident relation to fibrogenicity, high quartz and ash levels sometimes being associated with low collagen levels and vice versa. Comparison with other information disclosed disparities with epidemiology and different experimental assessments. Factors affecting the disarray may well include the neglected lipid response in vivo and individual susceptibility, aspects which do not appear beyond experimental assay.

Fibroblast RNA and macrophage proteins (including the fibrogenic factor) in experimental silicosis.

A hypothesis is presented for the action of silica-treated macrophages on protein synthesis in fibroblasts and also a method for the isolation of silica-attached materials in lung tissue. The increased protein synthesis in the fibroblasts is due, at least partly, to an increase in mRNA. Silica prevents the suppressing "macrophage effect" of macrophage-originated ribonuclease on fibroblasts. However, under certain conditions, collagen synthesis is stimulated by silica-treated macrophage preparations to such an extent that the effect cannot be explained by the inhibition of macrophage ribonuclease alone. We therefore postulate the existence of a fibrogenic factor, which is released by the macrophages. This factor has been demonstrated and can be purified from lung homogenate of SiO2-treated rats.

Macrophages and the synthesis of connective tissue components.

1. Macrophage homogenate, added to the incubation medium of granulation tissue slices, decreases the incorporation of labelled proline to collagen and other proteins, to a greater extent in the presence of certain prostaglandins and in oxygen atmosphere, but to a lesser degree if serotonin and cyclic AMP are added. 2. Rheumatoid synovial fluid, connective tissue active peptide (CTAP) of Castor and an extract from CCl4-damaged liver stimulate collagen synthesis in the presence of macrophages. 3. Preparations from non-treated macrophages stimulate the incorporation of glucosamine to acid mucopolysaccharides, mainly hyaluronate. 4. In a developing rat granuloma, the formation of collagen was stimulated by homogenized macrophages and by macrophage culture medium, but especially by whole macrophages in the presence of SiO2.

Effects of granulation-tissue extracts on collagen synthesis.

The effect of granulation-tissue extract on the synthesis of collagen in embryonic-chick tendon cells was found to depend on (i) the developmental phase of the granulation tissue, (ii) the concentration of the extract and (iii) the presence of diffusible components in it. The suppressing effect was maximal with concentrated extracts from 2--3-week granulation tissue (where the collagen synthesis is also maximal). The suppressing factor could be enriched by gel filtration chromatography. Cultured human synovial cells were affected like the embryonic-chick cells. When the sponge implant was imbibed with granulation-tissue extract in advance, the developing granuloma contained less collagen than the control. The stimulating effects with dilute extracts from granulation tissue were due to diffusible components. Blood serum and rheumatoid synovial fluid samples also stimulated collagen synthesis in granulation-tissue slices, less after dialysis.

Glycoproteins from experimental granulation tissue and their effects on collagen synthesis in embryonic chick tendon cells.

Buffer-soluble and pronase-liberated glycoproteins from experimental granulation tissue were fractionated by gel filtration and DEAE-cellulose chromatography. The age of the granuloma was reflected in the gel filtration pattern. Two glycoproteins were isolated, purified to homogeneity and analyzed for their carbohydrate and amino acid compositions. The collagen synthesis in embryonic chick tendon cells was measured in the presence of these fractions, which were found to be inhibiting even at 10(-6) M. These glycoproteins may be significant in the feedback regulation of the development of granulation tissue and fibrosis.