Selective inhibition of collagen synthesis by the Ca2+ ionophore A23187 in cultured human fibroblasts

The question of whether the Ca2+ ionophore A23187 affects collagen production relative to total protein synthesis or has possible effects on collagen degradation was investigated. Cultured normal human fibroblasts were incubated with radioactive proline, and the radioactivity of collagenase-sensitive and -resistant proteins was used to calculate the rates of protein production. The net production of collagen relative to total proteins was inhibited by A23187 in a dose-related manner, and 50% inhibition of basal collagen production was achieved with 0.6 microM A23187. There was a 70% decrease in the absolute rate of collagen production in the presence of 0.6 microM A23187 which represented a 4-fold greater inhibition of collagen production than of noncollagen protein production. The major mechanism for the decreased net production of collagen was decreased synthesis, rather than increased degradation. Ca2+ mobilization induced by cholecystokinin octapeptide was also associated with selective inhibition of collagen production in normal human fibroblasts. These studies establish that the Ca2+ ionophore A23187 induces a selective decrease in collagen polypeptide synthesis by normal human fibroblasts and suggest a modulatory role of Ca2+ on collagen metabolism.     

G protein-coupled receptor kinase-2 is a novel regulator of collagen synthesis in adult human cardiac fibroblasts

Cardiac fibroblasts (CF) make up 60-70% of the total cell number in the heart and play a critical role in regulating normal myocardial function and in adverse remodeling following myocardial infarction and the transition to heart failure. Recent studies have shown that increased intracellular cAMP can inhibit CF transformation and collagen synthesis in adult rat CF; however, mechanisms by which cAMP production is regulated in CF have not been elucidated. We investigated the potential role of G protein-coupled receptor kinase-2 (GRK2) in modulating collagen synthesis by adult human CF isolated from normal and failing left ventricles. Baseline collagen synthesis was elevated in failing CF and was not inhibited by β-agonist stimulation in contrast to normal controls. β-adrenergic receptor (β-AR) signaling was markedly uncoupled in the failing CF, and expression and activity of GRK2 were increased 3-fold. Overexpression of GRK2 in normal CF recapitulated a heart failure phenotype with minimal inhibition of collagen synthesis following β-agonist stimulation. In contrast, knockdown of GRK2 expression in normal CF enhanced cAMP production and led to greater β-agonist-mediated inhibition of basal and TGFβ-stimulated collagen synthesis versus control. Inhibition of GRK2 activity in failing CF by expression of the GRK2 inhibitor, GRK2ct, or siRNA-mediated knockdown restored β-agonist-stimulated inhibition of collagen synthesis and decreased collagen synthesis in response to TGFβ stimulation. GRK2 appears to play a significant role in regulating collagen synthesis in adult human CF, and increased activity of this kinase may be an important mechanism of maladaptive ventricular remodeling as mediated by cardiac fibroblasts.     

Transforming growth factor beta (TGF beta) causes a persistent increase in steady-state amounts of type I and type III collagen and fibronectin mRNAs in normal human dermal fibroblasts.

It has been previously shown that transforming growth factor beta (TGF beta) is capable of stimulating fibroblast collagen and fibronectin biosynthesis. The purpose of this study was to examine the mechanisms involved in TGF beta stimulation of fibroblast biosynthetic activity. Our results indicate that TGF beta causes a marked enhancement of the production of types I and III collagens and fibronectin by cultured normal human dermal fibroblasts. The rate of collagen production by fibroblasts exposed to TGF beta was 2-3-fold greater than that of control cells. These effects were associated with a 2-3-fold increase in the steady-state amounts of types I and III collagen mRNAs and a 5-8-fold increase in the amounts of fibronectin mRNAs as determined by dot-blot hybridization with specific cloned cDNA probes. In addition, the increased production of collagen and fibronectin and the increased amounts of their corresponding mRNAs remained elevated for at least 72 h after removal of TGF beta. These findings suggest that TGF beta may play a major role in the normal regulation of extracellular matrix production in vivo and may contribute to the development of pathological states of fibrosis.     

Effects of interleukin-18 on cardiac fibroblast function and gene expression

Fibroblasts are the primary cell type responsible for synthesis and remodeling of the extracellular matrix in the heart. A number of factors including growth factors, hormones and mechanical forces have been identified that modulate the production of extracellular matrix by cardiac fibroblasts. Inflammatory mediators including pro-inflammatory cytokines and chemokines also impact fibrosis of the heart. Recent studies have illustrated that interleukin-18 promotes a pro-fibrotic response in cardiac fibroblasts; however the effects of this cytokine on other aspects of fibroblast function have not been examined. While fibroblasts have long been known for their role in production and remodeling of the extracellular matrix, other functions of these cells are only now beginning to be appreciated. We hypothesize that exposure to interleukin-18 will stimulate other aspects of fibroblast behavior important in myocardial remodeling including proliferation, migration and collagen reorganization. Fibroblasts were isolated from adult male rat hearts and bioassays performed to determine the effects of interleukin-18 on fibroblast function. Treatment of fibroblasts with interleukin-18 (1-100ng/ml) resulted in increased production of extracellular matrix components and remodeling or contraction of three-dimensional collagen scaffolds by these cells. Furthermore, exposure to interleukin-18 stimulated fibroblast migration and proliferation. Treatment of heart fibroblasts with interleukin-18 resulted in the rapid activation of the c-Jun N-terminal kinase (JNK) and phosphoinositide 3-kinase (PI3-kinase) pathways. Studies with pharmacological inhibitors illustrated that activation of these pathways is critical to interleukin-18 mediated alterations in fibroblast function. These studies illustrate that interleukin-18 plays a role in modulation of cardiac fibroblast function and may be an important component of the inflammation-fibrosis cascade during pathological myocardial remodeling.     

L-ascorbic acid 2-phosphate stimulates collagen accumulation, cell proliferation, and formation of a three-dimensional tissuelike substance by skin fibroblasts

Proliferation of human skin fibroblasts was stimulated significantly by the presence of L-ascorbic acid 2-phosphate (Asc 2-P). The presence of Asc 2-P (0.1-1.0 mM) in the culture medium for 3 weeks enhanced the relative rate of collagen synthesis to total protein synthesis 2-fold as well as cell growth 4-fold. Coexistence of L-azetidine 2-carboxylic acid (AzC), an inhibitor of collagen synthesis, attenuated both effects of Asc 2-P in a dose-dependent manner. Supplementation of the medium with Asc 2-P also accelerated procollagen processing to collagen and deposition of collagen in the cell layer. Among the acidic glycosaminoglycans (GAG), another major component of extracellular matrix (ECM), deposition of sulfated forms was increased by the additive. Electron microscopic observations showed multilayered, rough endoplasmic reticulum-rich cells surrounded by dense ECM. These results indicate that Asc 2-P is useful in culture systems as a long-acting vitamin C derivative and also that it promotes reorganization of a three-dimensional tissuelike substance from skin fibroblasts in culture by stimulating collagen accumulation in the fibroblasts.     

Modulation of fibroblast functions by interleukin 1: increased steady- state accumulation of type I procollagen messenger RNAs and stimulation of other functions but not chemotaxis by human recombinant interleukin 1 alpha and beta

Interleukin-1 (IL-1) is synthesized by and released from macrophages in response to a variety of stimuli and appears to play an essential role in virtually all inflammatory conditions. In tissues of mesenchymal origin (e.g., cartilage, muscle, bone, and soft connective tissue) IL-1 induces changes characteristic of both destructive as well as reparative phenomena. Previous studies with natural IL-1 of varying degrees of purity have suggested that it is capable of modulating a number of biological activities of fibroblasts. We have compared the effects of purified human recombinant (hr) IL-1 alpha and beta on several fibroblast functions. The parameters studied include cell proliferation, chemotaxis, and production of collagen, collagenase, tissue inhibitor of metalloproteinase (TIMP), and prostaglandin (PG) E2. We observed that hrIL-1s stimulate the synthesis and accumulation of type I procollagen chains. Intracellular degradation of collagen is not altered by the hrIL-1s. Both IL-1s were observed to increase the steady-state levels of pro alpha 1(I) and pro alpha 2(I) mRNAs, indicating that they exert control of type I procollagen gene expression at the pretranslational level. We found that both hrIL-1 alpha and beta stimulate synthesis of TIMP, collagenase, PGE2, and growth of fibroblasts in vitro but are not chemotactic for fibroblasts. Although hrIl-1 alpha and beta both are able to stimulate production of PGE2 by fibroblasts, inhibition of prostaglandin synthesis by indomethacin has no measurable effect on the ability of the IL-1s to stimulate cell growth or production of collagen and collagenase. Each of the IL-1s stimulated proliferation and collagen production by fibroblasts to a similar degree, however hrIL-1 beta was found to be less potent than hrIL-1 alpha in stimulating PGE2 production. These observations support the notion that IL-1 alpha and beta may both modulate the degradation of collagen at sites of tissue injury by virtue of their ability to stimulate collagenase and PGE2 production by fibroblasts. Furthermore, IL-1 alpha and beta might also direct reparative functions of fibroblasts by stimulating their proliferation and synthesis of collagen and TIMP.     

Interleukin-2 inhibits 3T3 fibroblast proliferation

In order to clarify the role played by interleukin-2 (IL-2) in the regulation of fibroblast function, we investigated the effect of rat IL-2 and human recombinant IL-2 on 3T3 fibroblast proliferation and collagen synthesis. Fibroblasts were incubated with various concentrations of IL-2 for different periods of time. IL-2 was found to decrease in time- and dose-dependent manner the proliferation of 3T3 fibroblasts. This effect correlated with ability of IL-2 to enhance PGE₂ production by 3T3 fibroblasts. When 3T3 fibroblasts were cocultured with rat peritoneal mast cells (MC), the growth-inhibiting effect of IL-2 was significantly less pronounced. Treatment of the cultures with IL-2 had no effect on collagen production by both 3T3 fibroblasts and fibroblasts cocultured with MC. In conclusion, in this study we provide evidence that IL-2, the key cytokine in T-cell growth and differentiation, can affect fibroblast functions.     

Promotion of collagen production by human fibroblasts with gastric cancer cells in vitro

To elucidate the histogenesis of gastric scirrhous cancer, the promotion of collagen production by normal human skin fibroblasts (HSF-1) with human gastric cancer cells (KATO-III, MKN-45 and MKN-28) was investigated by direct coculture and parabiotic culture. Argyrophilic collagenous fibers were demonstrated among fibroblasts on both direct cocultures and parabiotic cultures of the fibroblasts with gastric cancer cells. Microscopic examination showed that these fibers appeared earlier and were more abundant and thicker in direct cocultures and parabiotic cultures than in single cultures of fibroblasts. Gastric cancer cells in single or parabiotic culture did not form argyrophilic fibers. For quantitative proof of the promotion of collagen production by fibroblasts with gastric cancer cells, hydroxyproline produced by fibroblasts was measured. Much higher fibroblast hydroxyproline values were obtained in parabiotic cultures with gastric cancer cell lines than in single cultures of HSF-1. Moreover, the rate of collagen synthesis by HSF-1 was much higher than that of any gastric cancer cell line tested. These results demonstrate that gastric cancer cells enhance collagen production by fibroblasts in vitro. This finding suggests that they may produce a factor promoting fibroblast collagen synthesis and that this may contribute to the formation of stromal collagen in human gastric scirrhous cancer.     

Collagen synthesis in human fibroblasts: Effects of ascorbic acid and regulation by hydrocortisone

The effects of hydrocortisone and ascorbic acid on collagen and noncollagen protein synthesis, and on growth were examined in fibroblasts derived from normal human dermis. When the medium was supplemented with 0.28 mM ascorbic acid, the apparent rate of collagen production increased 2--3 fold over the culture cycle. Ascorbic acid also caused a small increase in the apparent rate of synthesis of noncollagen protein and an elevation in growth rate and maximum cell density. Growth was not required for the increase in collagen production since addition of ascorbate to confluent cultures induced a similar increase. Hydrocortisone (1.5 μM) blocked the ascorbate-related increase in collagen production during growth and in confluent cultures. The hormone simultaneously increased the apparent rate of noncollagen protein production and maximum cell density, suggesting that the effect on collagen synthesis was specific. Inhibition of collagen production by hydrocortisone was observed only in the presence of ascorbate, while the increase in growth and noncollagen protein production occurred in the presence and absence of the vitamin.     

BRS-3 activation transforms the effect of human bronchial epithelial cells from PGE2 mediated inhibition to TGF-beta1 dependent promotion on proliferation and collagen synthesis of lung fibroblasts

Airway re-modelling in asthma usually results in an irreversible weakness of pulmonary ventilation, however, its initiating or controlling mechanism remains unclear. In this study, we hypothesize that signal communication between airway epithelial cells and sub-mucosal fibroblast cells may play an important role in the maintenance of structure homeostasis in a physiologic condition and in initiation of airway remodelling in a stressed condition. To test the hypothesis, a co-cultured system of human bronchial epithelial cells (BEC) and human lung fibroblasts (HLF) were designed to observe the effects of BEC, in the normal state or in a BRS-3 activated state, on the proliferation and collagen synthesis of HLF. The results showed that the proliferation activities of both BEC and HLF inhibited each other under the normal state. BRS-3-activated BEC can transform the reciprocal inhibition into promoting effects. The secretion of TGF-beta1 increased and the synthesis of PGE2 decreased from BRS-3-activated BEC, which were correlated with the proliferation and collagen synthesis of HLF. The proliferation activities of HLF were weakened by co-culture with TGF-beta1 antisense oligonucleotides (ASO) treated BEC. It was concluded that, in the normal state, BEC inhibits the activities of fibroblasts through release of PGE2 to maintain the airway homeostasis; however when stressed, for example by BRS-3 activation, BEC promote the activities of fibroblasts mediated by TGF-beta1, thereby facilitating the airway re-modelling.     

Modulation of collagen production following bleomycin-induced pulmonary fibrosis in hamsters. Presence of a factor in lung that increases fibroblast prostaglandin E2 and cAMP and suppresses fibroblast proliferation and collagen production

To elucidate mechanisms involved in the regulation of lung collagen content we studied hamsters with bleomycin-induced pulmonary fibrosis. Lung collagen in this model is increased as the result of greatly increased lung collagen synthesis rates. However, collagen synthesis rates are subsequently restored to normal. Hamster lung explants from both normal and bleomycin-exposed hamsters were cultured, and the effects of explant conditioned medium (CM) on lung fibroblast (IMR-90) proliferation and collagen production in vitro were determined. Lung explant CM increased fibroblast prostaglandin (PG)E2 production and intracellular cAMP, and decreased both fibroblast proliferation and collagen production in a dose-dependent manner. Greater activity was observed with lung explant CM from bleomycin-exposed lungs. Incubation of fibroblasts with indomethacin prior to addition of CM blocked CM-mediated changes in PGE2 and cAMP and inhibited changes in fibroblast proliferation and collagen production. Exogenous PGE2 or dibutyryl cAMP also suppressed fibroblast proliferation and collagen production. The suppressive activity in lung-conditioned medium is nondialyzable, has an apparent molecular weight of 15,000-20,000 by gel filtration, and is heat-stable. It is not species-restricted since CM from hamster lung affected human and hamster lung fibroblasts similarly. Activity is present preformed in lung and bronchoalveolar lavage fluid, although bronchoalveolar macrophages produce a nondialyzable factor in culture which suppresses fibroblast proliferation. The suppressive activity identified in fibrotic lung may represent a means for limiting collagen accumulation following tumor injury.     

Transforming growth factor-beta 1 in adipose derived stem cells conditioned medium is a dominant paracrine mediator determines hyaluronic acid and collagen expression profile

Conditioned medium from adipose derived stem cells (ADSC-CM) stimulates both collagen synthesis and migration of fibroblasts, and accelerates wound healing in vivo. Recently, the production and secretion of growth factors has been identified as an essential function of adipose-derived stem cells (ADSCs). However, the main soluble factor of ADSC-CM which mediates paracrine effects and its underlying mechanism has not been elucidated yet. In this study, we considered transforming growth factor-beta1 (TGF-β1) as a strong candidate for paracrine effect of ADSC-CM and investigated collagen synthesis and hyaluronic acid synthase (HAS) expression. After ADSC-CM addition, collagen type I, type III, HAS and hyaluronic acid (HA) expressions on human dermal fibroblasts (HDFs) were evaluated. Furthermore, to clarify effects of TGF-β1 as a paracrine mediator, TGF-β1 antibody and external supplementary TGF-β1 were treated to HDFs. Collagens type I, type III, HAS-1 and HAS-2 mRNA expressions of HDFs were greatly increased by ADSC-CM treatment, however there was no change in TGF-β1 antibody treated HDFs compared with non-treated control. These results strongly demonstrate that TGF-β1 plays an important role as a paracrine mediator of ECM synthesis. The fact that TGF-β1 contained in ADSC-CM not only accelerates collagen deposition but also increase hyaluronic acid synthesis of HDFs through HAS-1 and HAS-2 expression was also elucidated in this study. Therefore, ADSC-CM shows promise for the treatment of cutaneous wounds and accelerates granulation formation during healing process.     

Modulation of human dermal fibroblast extracellular matrix metabolism by the lymphokine leukoregulin

The effect of leukoregulin, a 50-kD lymphokine with unique antitumor properties, was studied in vitro on several fibroblast functions. Leukoregulin did not inhibit fibroblast proliferation, as measured by cell enumeration and [3H]thymidine incorporation, and had no cytotoxic effect in terms of increased membrane permeability detected by trypan blue exclusion, two of the major leukoregulin actions on tumor cells. Leukoregulin induced a dose-dependent decrease in collagen synthesis, demonstrated by decreased [3H]proline incorporation into collagenase-digestible protein, as early as 6 h after the addition of the lymphokine to human fibroblasts. Leukoregulin inhibited the synthesis of both type I and type III collagen, as measured by SDS-PAGE and by specific radioimmunoassay. Neutralizing antibodies to interleukin-1 alpha, interleukin-1 beta, tumor necrosis factor-alpha, and interferon-gamma failed to alter the effect of leukoregulin on collagen synthesis, attesting that leukoregulin action was not due to contamination by these cytokines. Inhibition of collagen synthesis occurred concomitantly with increased secretion of prostaglandin E2 and a transient rise in intracellular cyclic AMP content, peaking at 6 h. However, blocking prostaglandin synthesis with indomethacin did not counteract inhibition of collagen synthesis by leukoregulin, demonstrating independence of this action of leukoregulin from cyclooxygenase metabolites. Leukoregulin also stimulated glycosaminoglycan production in a dose-dependent manner, affecting the synthesis of hyaluronic acid as the major fibroblast-derived extracellular glycosaminoglycan. In addition, secretion of neutral proteases (collagenase, elastase, caseinase) was increased. These observations indicate that leukoregulin is able to regulate synthesis of molecules critical to the deposition of the extracellular matrix by nontransformed nonmalignant fibroblasts.     

Regulation of Collagen Synthesis in Human Dermal Fibroblasts in Contracted Collagen Gels by Ascorbic Acid, Growth Factors, and Inhibitors of Lipid Peroxidation

Ascorbic acid has been shown to stimulate collagen synthesis in monolayer cultures of human dermal fibroblasts. In the present studies, we examined whether the presence of a collagen matrix influences this response of dermal fibroblasts to ascorbic acid. Fibroblasts and collagen were mixed and allowed to gel and contract for 6 days to form a matrix prior to determining the concentration and time dependence for ascorbic acid to affect collagen synthesis by fibroblasts within the matrix. Collagen synthesis was stimulated at levels at or above 10 μM ascorbic acid and was maximal after 2 days of treatment. This concentration and time dependence is similar to that of cells grown in monolayer cultures. The effects of transforming growth factor-β (TGF-β) and fibroblast growth factor (FGF) were also examined in this model. TGF-β increased and FGF inhibited collagen synthesis in the gels, as has been shown for cells in monolayer cultures. The effects of potential inhibitors of lipid peroxidation induced by ascorbic acid were also examined in these matrices and compared to previous results obtained in monolayer cultures. Propyl gallate, cobalt chloride, α,α-dipyridyl, and α-tocopherol inhibited the ascorbic acid-mediated stimulation of collagen synthesis while mannitol had no effect. Natural retinoids inhibited total protein synthesis without the specific effect on collagen synthesis that was seen in monolayer cultures. These results indicate that ascorbic acid stimulates collagen synthesis in fibroblasts grown in a collagen matrix in a manner similar to that found in monolayer cultures. In contracting collagen gels, however, the magnitude of the effect is less and retinoids do not specifically inhibit collagen synthesis.     

Interleukin 1 increases collagen type IV production by murine mammary epithelial cells

The effects of human interleukin 1 (IL 1) on collagen type IV production by normal mouse mammary epithelial cells were examined. Human IL 1 was derived from the culture media of peripheral blood monocytes or placental cells that were stimulated with silica. Although crude culture media of silica-stimulated monocytes or placental cells had no enhancing activity for type IV collagen production, IL 1-containing fractions obtained by Sephacryl S-200 gel chromatography and isoelectrofocusing from such media possessed considerable activity. To confirm the effects of IL 1 on collagen production, human monocyte-derived IL 1 was highly purified by sequential isoelectrofocusing, anion-exchange (AX 300), high-performance liquid chromatography (HPLC), and HPLC gel filtration (TSK 3000). The same HPLC gel filtration fractions contained both an activity that stimulated collagen synthesis by mammary cells and thymocyte growth-promoting activity. These activities of IL 1 differed from a number of other factors, such as epidermal growth factor and another factor produced by placental cells that stimulated type IV collagen production but not thymocyte proliferation. In fact, IL 1 induced 100-fold less collagen type IV production by mammary epithelial cells than was needed to induce thymocyte proliferation. Our data suggest that IL 1-like molecules, which reportedly are produced by many tissue cell types, may therefore play a role in promoting a basement membrane formation at stromal-epithelial boundaries.