Biosynthesis and secretion of procollagenase by rabbit synovial fibroblasts. Inhibition of procollagenase secretion by monensin and evidence for glycosylation of procollagenase.

Monolayer cultures of rabbit synovial fibroblasts stimulated with phorbol myristate acetate to produce large amounts of collagenase (EC 3.4.24.7) were used to study the biosynthesis and secretion of this enzyme. [3H]Leucine was added to cell cultures for pulse-chase and continuous-labelling experiments. The labelled procollagenase synthesized was identified by immunoprecipitation followed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and fluorography. The amounts of intracellular and extracellular proenzyme were quantified by measuring radioactivity incorporated into the proteins. procollagenase was synthesized as doublet proteins of Mr 57 000 and Mr 61 000. Immunoprecipitable proenzyme proteins were first detected in culture medium 35 min after [3H]leucine was added to the cells. Monensin treatment of the cells inhibited procollagenase secretion and led to intracellular accumulation of the proenzyme. Cells treated with tunicamycin produced only the 57 000-Mr form, indicating that in rabbit synovial cells the 61 000-Mr form was post-translationally modified by addition of oligosaccharides to asparagine residues. The ratios of glycosylated to unglycosylated forms in cell lysates and in culture medium were 0.22:1 and 0.07:1 respectively.     

Tumor necrosis factor inhibits collagen and fibronectin synthesis in human dermal fibroblasts

Tumor necrosis factor (TNF) caused inhibition of collagen production by confluent cultures of human dermal fibroblasts in a dose-dependent manner. Concomitant increase of prostaglandin E2 release was observed as a result of TNF-induced cell activation. However, a blockade of the cyclooxygenase pathway of arachidonate metabolism by indomethacin did not abrogate the inhibitory effect of TNF on collagen synthesis, suggesting that this effect could be independent of prostaglandin metabolism. Gel electrophoresis of the newly synthesized macromolecules from the culture media showed that both type I and type III collagens as well as fibronectin were affected by the inhibition. Electrophoresis of cell layer-associated proteins demonstrated that the reduction in amounts of collagen and fibronectin in the medium did not result from an intracellular accumulation of these macromolecules. Production of procollagens was reduced in parallel to that of collagens, suggesting that the effect of TNF is exerted before the processing steps of procollagens. These results clearly show that TNF could play a role in modulation of matrix deposition by fibroblasts during inflammatory processes.     

Relaxin modulates proinflammatory cytokine secretion from human decidual macrophages

Relaxin (RLN) is a systemic hormone from the corpus luteum, and its levels remain low during normal human gestation. Indeed, elevation of circulating RLN has long been associated with preterm birth, for which there has been no physiological explanation. Recent studies have shown that RLN suppresses endotoxin-induced cytokine secretion from THP-1 monocytic cells by acting on the glucocorticoid receptor (GR), but its effects on primary macrophages are unknown. Therefore, in the present study, we examined the effects of RLN on cytokine secretion from primary decidual macrophages (DMs) obtained at term before labor. Unlike THP-1 cells, RLN had no effects on the cytokine responses induced by either lipopolysaccharide (LPS) or interleukin (IL) 1B, mimicking infection-induced or sterile inflammation, respectively. However, RLN alone for 4 h significantly decreased (P < 0.05) colony-stimulating factor 2 (CSF2; also known as granulocyte-macrophage colony-stimulating factor) and IL8 but for 24 h significantly increased IL6 (P < 0.01). We show that DMs express both the RLN receptor (RXFP1) and the GR. RLN suppression of CSF2 and IL8 was sensitive to the GR-antagonist mifepristone (RU-486). However, RLN activation of RXFP1 induced a dose-dependent cAMP response, which when mimicked by forskolin also caused significantly increased (P < 0.05) secretion of IL6. Thus, RLN may be anti-inflammatory in DMs via activation of the GR but proinflammatory via activation of RXFP1 and cAMP. In summary, we have shown that RLN targeting DMs may modulate proinflammatory cytokine secretion at the maternal-fetal interface and contribute to the localized inflammatory response associated with parturition in women.     

Functional diversity of lysyl hydroxylase 2 in collagen synthesis of human dermal fibroblasts

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.     

The contraction of collagen matrices by dermal fibroblasts

Floating collagen gel cultures containing human foreskin fibroblasts have been observed to undergo a rapid contraction process. The initial rate of contraction (i.e., within the first 2 hr) was observed to be a linear function of cell number within the concentration range of 10(5)-10(6) cells/gel. Observation of thick, deresined sections of such contracting gels in the SEM, as well as observation of thin sections in the TEM, suggest that the fibroblasts exert a tension upon the surrounding collagen fibers. These observations further indicate that the fibroblasts migrate from the interior regions of the gel matrix and eventually form a monolayer of cells encapsulating the contracted collagen disc. These observations are discussed in terms of the possible mechanisms involved in gel contraction.     

Interleukin-4 stimulates collagen synthesis by normal and scleroderma fibroblasts in dermal equivalents.

Interleukin-4 (IL-4) is one of the products of T-lymphocytes and mast cells, inflammatory cells which accumulate in connective tissues at early stages of fibrosis. We tested the effects of IL-4 on human fibroblasts from normal and scleroderma skin seeded in three dimensional collagen lattices ("dermal equivalents"). IL-4 (10 and 100 U/ml) stimulated collagen synthesis in a dose-dependent manner. No significant alteration of lattice retraction and cell proliferation was observed. At the concentration 100 U/ml, Il-4 was approximately twice more efficient on collagen synthesis than Transforming Growth Factor beta (10 ng/ml). IL-4 secretion in connective tissues might be an important factor for the development of fibrotic processes.     

Microgravity and hypergravity effects on collagen biosynthesis of human dermal fibroblasts

Astronauts experiencing long periods of space flight suffer from severe loss of bone tissue, particularly in those bones that carry the body weight under normal gravity. It is assumed that the lack of mechanical load decreases connective tissue biosynthesis in bone-forming cells. To test this assumption, quantitative and qualitative aspects of collagen synthesis under microgravity, normal gravity, and hypergravity conditions were investigated by incubating human fibroblast cultures with [³H]-proline for 4, 7, 10, and 20 h during the Spacelab D2-mission in 1993. Quantitative analysis revealed an increase of collagen synthesis under microgravity conditions, being up to 143% higher than in 1 g controls. In contrast, hypergravity samples showed a decrease in collagen synthesis with increasing g, being at the 13% level at 10 g. The relative proportion of collagen in total synthesized protein showed a slight decrease with increasing g. The secretion of collagen by the cells, proline hydroxylation of individual collagen -chains, and the relative proportions of synthesized collagens I, III, and V were not affected under any of the applied conditions.Our research was supported financially by Dara GmbH Bonn (grant. no. 01QV 8866), the Deutsche Forschungsgemeinschaft (SFB A1/367) and BMFT grant. no. 01 KM 9303/8.     

Stimulation of procollagenase synthesis in human rheumatoid synovial fibroblasts by mononuclear cell factor/interleukin 1

In order to define mechanisms regulating the synthesis of procollagenase in human rheumatoid synovial fibroblasts, the proteins synthesized by cultured cells were labeled with [35S]methionine. Labeled medium proteins were analyzed by SDS-PAGE directly and after immunocomplexing with a specific antibody to human fibroblast collagenase. Labeling of both the predominant form of the enzyme (Mr approximately 55 000) as well as a minor species (Mr approximately 61 000) was increased following incubation with the monokine, mononuclear cell factor/interleukin 1. The approximately 61 kDa form of the procollagenase appears to be a glycosylated form of the approximately 55 kDa precursor based on binding to Con A-Sepharose and decrease in the approximately 61 kDa form after culture in the presence of tunicamycin. Thus, mononuclear cell factor, homologous with interleukin 1, partially purified from monocyte conditioned medium increased incorporation of [35S]methionine into several medium proteins, including those complexed by the anticollagenase antibody. In the presence of mononuclear cell factor/interleukin 1, labeling of the procollagenase was increased 12-14-fold over control cultures incubated with medium alone. Therefore, one of the mechanisms involved in increase of collagenase activity in the medium of cultured synovial fibroblasts in the presence of mononuclear cell factor/interleukin 1 is a stimulation of enzyme protein synthesis.     

A heterocyclic molecule kartogenin induces collagen synthesis of human dermal fibroblasts by activating the smad4/smad5 pathway

Declined production of collagen by fibroblasts is one of the major causes of aging appearance. However, only few of compounds found in cosmetic products are able to directly increase collagen synthesis. A novel small heterocyclic compound called kartogenin (KGN) was found to stimulate collagen synthesis of mesenchymal stem cells (MSCs). So, we hypothesized and tested that if KGN could be applied to stimulate the collagen synthesis of fibroblasts.     

Hyaluronan affects protein and collagen synthesis by in vitro human skin fibroblasts

Given the importance of hyaluronan (HA) for the homeostasis of connective tissues during embryogenesis and aging and its role in tissue repair, the aim of the present study was to examine the effect of exogenous HA on the synthesis of total protein, collagen and HA by in vitro human dermal fibroblasts. With differences between different cell strains, HA, at concentrations between 0.5 and 1 microM, induced a significant decrease in total protein synthesised and secreted into the medium compared to controls (P < 0.05), and particularly in collagen (-40%; P < 0.05). The ratios between collagen types I and III and between collagen types V and I were normal. Pulse and chase experiments showed that protein degradation was normal. The presence of exogenous HA did not affect HA synthesis. Data strongly indicate that a relatively high concentration of HA in the extracellular space, such as during development and in the first phases of tissue repair, would partially limit the deposition of the extracellular matrix, and of collagen in particular. This would suggest a role for HA in delaying tissue differentiation during embryogenesis and in preventing fibrosis and scar formation in fetus and in the early phases of wound healing.     

Effect of strain on human dermal fibroblasts in a three-dimensional collagen sponge

Our aim was to design a simple compression system and investigate the influence of mechanical stress on skin-like structures. Many mechanical compression studies have employed intricate culture systems, so the relationship between extracellular matrix material and the response of skin cells to mechanical stress remains unknown. Our approach uses only glass vials, 6-well plates and standard laboratory equipment. We examined the influence of mechanical stress on human skin fibroblasts embedded within a collagen sponge. The results show that mechanical compression increases MMP-1 and MMP-2 release by the cells into the the cell culture. Our results suggest that pressure on the skin may affect extracellular matrix degradation through some as yet unidentified pathways and that IL-6 mRNA expression may be involved in this effect. Using our approach, the effects of static mechanical stress on protein expression by cells in the culture medium and in sponges can be easily examined, and therefore this system will be useful for further analyses of skin responses to mechanical stress.     

Synthesis and release of procollagenase by cultured fibroblasts.

An inactive collagenase was harvested from both serum-free and serum-supplemented fibroblast monolayer cultures in periods of active collagen synthesis. The latent collagenase did not hydrolyze collagen and did not bind the potent collagenase inhibitor alpha2-macroglobulin. Activation with trypsin imparted to the enzyme the ability to hydrolyze collagen at neutral pH in a typical manner and to form an inhibited complex with alpha2-macroglobulin. The molecular weights, determined by calibrated gel filtration, were 78,000 and 60,000 for the latent and active enzymes, respectively. The data indicate that collagenase is released from the cells in inactive form, as a zymogen.     

Aging-related changes and topology of adhesion responses sensitive to cycloheximide on collagen substrata by human dermal fibroblasts

Human dermal fibroblasts (both papillary and reticular) were tested during in vivo or in vitro aging for their responses on collagen and/or fibronectin (FN) substrata, as well as on topologically mixed substrata. Cycloheximide treatments were used to evaluate whether receptors to these matrix molecules, mediating F-actin reorganization into stress fibers, are altered during aging processes. Late-passage (but not mid-passage) papillary and reticular cells from both an elderly male and a newborn infant spread effectively on collagen +/- FN but failed to generate stress fibers after lengthy pretreatment of cells with cycloheximide. In contrast, treatment with cycloheximide only when cells were reattaching was not inhibitory. None of the treatments had any effect on stress fiber formation of cells on FN-only substrata, demonstrating that drug sensitivity was specific for collagen responses. The inhibition could be reversed by rinsing cycloheximide out of cultures and could be prevented by prior growth of cells in ascorbate-supplemented medium to stimulate production/maturation of collagen and possibly collagen-specific receptors. Adjacent regions of coverslips were adsorbed with collagen and a proteolytic fragment of plasma FN lacking the collagen-binding domain but retaining other binding domains; cells bridging the interface were of special interest. When fragment F155 containing both the RGDS cell-binding and the heparin II-binding domains was tested in this paradigm, cells generated stress fibers continuous from the collagen-facing side into the F155-facing side of the same cell, consistent with the compatability of both collagen and FN receptors in generating the same stress fiber. However, F110 lacking the heparin II domain was incapable of facilitating stress fiber formation; fibers formed effectively on the collagen side and terminated abruptly at the collagen:F110 interface. These experiments demonstrate stringent regulation of where stress fibers begin, span, and terminate in the cytoplasm by matrix receptors at the cell's undersurface and establish that there are alterations of collagen-specific receptors as a consequence of in vitro aging, but not of in vivo aging, in both papillary and reticular human dermal fibroblasts.     

Tissue form of type VII collagen from human skin and dermal fibroblasts in culture

The triple-helical domain of type VII collagen was isolated from human placental membranes by mild digestion with pepsin, and polyclonal antibodies were raised in rabbits against this protein. After affinity purification the antibodies specifically recognized type VII collagen in both the triple-helical and the unfolded state. They also reacted with the fragments P1 and P2, derived from the triple-helical domain by further proteolysis with pepsin, but did not crossreact with other biochemical components of the dermal connective tissue. In skin the presence of a fragment of type VII collagen, similar to that isolated from placenta, was demonstrated by SDS-PAGE and immunoblotting. Type VII collagen represented less than 0.001% of the total collagen extracted by pepsin digestion from newborn or adult skin. The tissue form of type VII collagen was obtained from dermis after artificial epidermolysis with strongly denaturing buffers under conditions reducing disulfide bonds. The protein was identified by immunoblotting with the antibodies. The molecule was composed of three polypeptides with an apparent molecular mass of about 250 kDa, each. Similar large-molecular-mass chains could be identified by immunoblotting in extracts of human fibroblasts in culture.     

Collagenolytic cleavage products of collagen type I as chemoattractants for human dermal fibroblasts

The chemoattractive properties of collagen in native (triple-helical) and denatured (random coil) conformation were compared in a Boyden chamber type assay to those of collagen fragments derived from cleavage with mammalian or bacterial collagenase using human embryonic dermal fibroblasts as target cells. Chemotaxis to native collagen required low collagen concentrations because fibril formation at high concentrations and at physiological pH and ionic strength prevented chemoattractiveness. Chemotaxis of denatured collagen was comparable to that of native collagen in solution. Cleavage of native collagen with mammalian collagenase increased, digestion with bacterial collagenase abolished its chemotactic activity. It is thought that these data may reflect the in vivo situation during inflammation and wound repair.     

Relaxin regulates fibrillin 2, but not fibrillin 1, mRNA and protein expression by human dermal fibroblasts and murine fetal skin

Relaxin modulates connective tissue remodeling by altering matrix molecule expression. We have found that relaxin specifically inhibits a microfibril component, fibrillin 2 (FBN2), without affecting fibrillin 1 (FBN1). Human dermal fibroblasts (HDFs) grown or stimulated to overexpress fibrillin expression were used to show that relaxin specifically down-regulated FBN2 mRNA and protein levels. Continuous exposure of HDFs to relaxin (30ng/ml) significantly (P<0.05) decreased fibrillin 2 protein (40%) while FBN1 protein expression was unchanged. Our in vitro studies were confirmed using relaxin null mice whereby the absence of relaxin was associated with increased FBN2 mRNA and protein in fetal skin from pregnant relaxin knockout mice. The regulation of FBN2 expression may be associated with functional changes in elastic tissues during development and growth.