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PubMed1994

Production of monocyte chemoattractant protein-1 and macrophage inflammatory protein-1 alpha by inflammatory granuloma fibroblasts.

The formation of hepatic granulomas around persistently deposited Schistosoma mansoni eggs leads to parenchymal damage, ongoing fibrosis, and ultimate loss of liver function. In this study, the production of macrophage inflammatory protein-1 alpha (MIP-1) and monocyte chemoattractant protein-1 (MCP-1) by granuloma fibroblasts was examined to establish the potential contribution of intragranuloma fibroblasts to the maintenance of the chronic inflammation. Isolated fibroblasts from dispersed acute infection hepatic granulomas were grown in tissue culture for 3 to 4 weeks and used on the third or fourth passage. We initially surveyed fibroblasts for production of MIP-1 and MCP-1 by reverse transcription-polymerase chain reaction (RT-PCR) after stimulation with interleukin (IL)-1, tumor necrosis factor, interferon (IFN)-gamma, IL-4, or IL-10: cytokines found within the granuloma. These studies demonstrated constitutive expression of MCP-1 and differential up-regulation of MIP-1 on cytokine stimulation. Protein expression was then verified by immunohistochemical localization of MIP-1 and MCP-1 in paraformaldehyde-fixed fibroblasts and by direct quantitation of MIP-1 and MCP-1 in culture supernatants by specific ELISAs. These studies demonstrated constitutive expression of MCP-1 in unstimulated and cytokine-stimulated granuloma fibroblasts. In contrast, IL-1 (0.1 to 2.5 ng/ml), IFN-gamma (10 micrograms/ml), and IL-10 (2.5 to 10 ng/ml) were able to induce the significant production of MIP-1 by the granuloma fibroblasts. Interestingly, normal noninflammatory fibroblasts from uninfected mice showed no significant production of MIP-1 or MCP-1 in response to these cytokines. These results suggest that granuloma fibroblasts may be phenotypically altered compared with normal fibroblasts and have a significant role in leukocyte recruitment, granuloma growth, and maintenance of the egg-induced lesion.

Animals↗
PubMed1993

Thrombin stimulates fibroblast chemotaxis and replication.

The serine protease alpha-thrombin, a product of the circulating zymogen prothrombin, plays multiple roles in homeostasis and coagulation. During blood clotting, it is present within fibrin matrices and is likely to be presented to local cell populations. It is known to be a fibroblast mitogen, but its effects on fibroblast recruitment have not been assessed. Here we compared the effect of human alpha-thrombin on chemotaxis and proliferation of human and rat skin fibroblasts and assessed the mechanism of these actions. Fibroblast chemotaxis was assayed using a 48-well Boyden chamber and replication assessed by a spectrophotometric method, based upon the uptake and subsequent elution of methylene blue by fibroblasts. Two fibroblast cell lines were used; fetal rat skin (FR) and newborn human foreskin (HS68). Human alpha-thrombin stimulated FR fibroblast chemotaxis over a wide range of doses (10(-12) M to 10(-7) M). Maximal migration was seen at 10(-10) M; 39 +/- 2.5 cells/high power field (h.p.f.) compared with 19 +/- 3 cells/h.p.f. for media control. In the same assay platelet-derived growth factor, a well characterized fibroblast chemoattractant, caused a maximal stimulation of 44 +/- 5 cells/h.p.f. at a concentration of 3 x 10(-9) M. A similar stimulation was observed with HS68 fibroblasts, although for this cell line maximal chemotaxis (190 +/- 12.5% of control) was seen at 10(-8) M thrombin. Fibroblast replication was optimal at 1.25 x 10(-9) M thrombin (134 +/- 4 and 127 +/- 5% of control for FR and HS68, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Ehrlich ascites tumour cell spreading is induced upon contact with immobilized isolated fibroblast plasma membranes and fibronectin.

Ehrlich ascites tumour (EAT) cells normally growing in suspension, adhere and spread upon contact with fibroblasts. Changes in EAT cell morphology and adhesiveness induced in contact with purified fibroblast plasma membranes, fibroblast extracellular matrix and fibronectin were investigated. EAT cells adhered readily to glass covered with fibroblast extracellular matrix or fibronectin, but they did not spread. Immobilization of fibroblast plasma membranes or fibronectin on nitrocellulose restored EAT cell ability to spread. Tumour cells spread on immobilized plasma membranes or fibronectin maintained a "fried egg" morphology, while these spread on living fibroblasts were rather elongated. EAT cells preincubated with anti-fibronectin serum lost their ability to adhere to any of the tested substrata: living and fixed fibroblasts, fibroblast extracellular matrix, nitrocellulose with immobilized fibroblast plasma membranes or fibronectin. When anti-fibronectin serum was applied only to these substrata and it was washed out before EAT cell plating, EAT cell adhesion and spreading were not significantly changed. Extraction of extracellular matrix components from immobilized fibroblast plasma membranes did not influence EAT adhesion and spreading. The results are discussed with reference to the specific role of fibronectin in the growth of EAT cells as a solid tumour or suspension.

Animals↗
PubMed1995

Dual role of fibroblasts in tumor cell growth.

Vast experience with cultivating biopsies from human tumors indicates that in most cases the admixture of fibroblasts has a negative effect on growth of tumor cells. Only rarely is observed help provided by the fibroblasts. It has also long been known that fibroblasts can inhibit by contact themselves and also produce growth factor(s) promoting cell proliferation. We have used three permanent squamous cell carcinoma lines and fibroblasts derived from biopsies of trachea to study this paradox. The inhibitory activity of fibroblasts is contact-dependent in a cell membrane-bound factor, which is trypsin sensitive. We prepared microsomal fractions (MF) from aged (more than 40 passages) fibroblasts and followed their effect on proliferation of the tumor cell lines in MTT assay. MF from all three fibroblast lines inhibited the tumor cells. Most regularly was this phenomenon observed with line UM-SCC 22B. Not all tumor cells are immortal. On the contrary, a large portion of them undergoes terminal differentiation. With the line UM-SCC 22B we tested the possibility that MF from fibroblasts can increase the portion of tumor cells which will senescence after few mitoses. The immortal cells were defined as cells capable in 8 or 13 days of forming colonies of more than 50 or 200 cells. The senescent cells were defined as cells not capable of producing within the same period of time colonies of more than 12 or 30 cells. The MF was able to increase the number of small colonies, i.e. the number of senescent tumor cells. The fibroblasts of the same passage level were releasing soluble growth factor(s) promoting growth of cells. Fibroblasts can apparently simultaneously inhibit growth by contact and release factor(s) promoting growth of cells. The final outcome is a result of the balance between these two forces. This balance is regulated by many intrinsic and extrinsic forces.

Carcinoma, Squamous Cell↗
PubMed1997

Repair phenotype in corneal fibroblasts is controlled by an interleukin-1 alpha autocrine feedback loop.

PURPOSE: To explore the role of autocrine interleukin-1 alpha (IL-1 alpha) as a central regulator of the repair phenotype in corneal fibroblasts. METHODS: Disruption of the actin cytoskeleton with cytochalasin B (CB), which mimics changes in shape that occur in repair tissues, was used to stimulate repair gene expression in early-passage fibroblasts. Changes in expression of IL-1 alpha, IL-8, collagenase, and ENA-78 were determined by Northern blot analysis, radioimmunoassay, and an enzyme-amplified sensitivity immunoassay (EASIA). Expression of repair genes was also examined in repair fibroblasts, isolated from healing, penetrating keratectomy wounds in rabbits. RESULTS: Blocking IL-1 alpha activity prevented both constitutive and stimulated increases in synthesis of IL-8 and collagenase in early-passage cultures of corneal fibroblasts, demonstrating the role of IL-1 alpha as a necessary intermediate for expression of these genes. Evidence is also presented that the IL-1 alpha autocrine controls expression of an IL-8 related factor, ENA-78. Unlike early-passage fibroblasts, fibroblasts freshly isolated from the uninjured cornea did not express IL-1 alpha. However, fibroblasts freshly isolated from remodeling corneal repair tissue 3 weeks after injury were found to express substantial levels of IL-1 alpha, regulated through an autocrine feedback loop. Neutralization experiments demonstrated that the IL-1 alpha autocrine is largely responsible for controlling both collagenase and IL-8 synthesis in repair fibroblasts, as it is in early-passage fibroblasts. CONCLUSIONS: These findings provide evidence that activation of an autocrine IL-1 alpha feedback loop is an important mechanism by which fibroblasts adopt a repair phenotype during remodeling of the cornea.

Animals↗
PubMed1997

Fibroblasts as sentinel cells. Synthesis of chemokines and regulation of inflammation.

In this commentary, we have suggested that the fibroblast should be considered a sentinel cell. This concept is based on the fibroblast's ability to function both as a structural element and as a vital immunoregulatory cell. In some tissues, these capabilities may be ascribable to subsets of fibroblasts, rather than to some of the general fibroblast populations. The pioneering research of Xia et al, as well as that of others, highlights the need to explore the importance of fibroblasts as playing critical roles in disease. Emerging concepts regarding tissue-specific fibroblasts and fibroblast heterogeneity need to be considered in studies of their biosynthetic capabilities. Of special importance is the recent insight that the NF kappa B/RelB family of transcription proteins have apparently different regulatory roles in fibroblasts and hematopoietic cells. Therefore, with regard to therapeutic strategies targeting molecules such as RelB, caution should be exercised as their interruption may have very different consequences in macrophages compared with fibroblasts. For example, inhibition of RelB in macrophages may well prevent enhanced chemokine expression, whereas in fibroblasts, a critical governor for preventing chemokine expression would be lost. Overall, this could lead to exacerbation of inflammation rather than to an attenuation of the process.

Animals↗
PubMed1997

Introduction of exogenous class II trans-activator in MHC class II-deficient ABI fibroblasts results in incomplete rescue of MHC class II antigen expression.

Previously, we have shown that fibroblasts established from type III bare lymphocyte syndrome patient ABI are characterized by the absence of MHC class II gene expression and a strongly reduced amount of MHC class I transcripts. Complementation analysis has suggested that the gene defective in these ABI fibroblasts is different from that encoding the class II trans-activator (CIITA), which has been attributed an essential role in both constitutive and inducible expression of MHC class II genes. In the present study it is shown by reverse transcriptase-PCR analysis that the amount of CIITA transcripts in ABI fibroblasts is greatly reduced compared with that in fibroblasts derived from a healthy individual. Transient cotransfection of a construct in which CIITA is under the control of a constitutive promoter with an HLA-DRA promoter-luciferase reporter plasmid resulted in enhanced luciferase expression in ABI fibroblasts. Furthermore, ABI fibroblasts stably transfected with CIITA re-express functional HLA-DR Ags, but do not express HLA-DQ and DP Ags at the cell surface. Comparison of these data with those obtained for normal fibroblasts and fibroblasts defective for CIITA indicate that the gene defect and the resulting lack of MHC class II expression in ABI fibroblasts can only partly be corrected by the introduction of CIITA. Furthermore, DNase I hypersensitivity analysis of ABI fibroblasts has revealed a closed chromatin structure in the promoter region of the MHC class II DRA gene. However, CIITA transfection resulted in an open DNA configuration, which suggests a role for CIITA in provoking changes in the chromatin structure of the DRA gene.

Cell Line, Transformed↗
PubMed1996

Modulation of prolidase activity during in vitro aging of human skin fibroblasts the role of extracellular matrix collagen.

The mechanism related to the decrease in the number of fibroblasts in the dermis during aging and the decrease in the biosynthetic capacity of existing fibroblasts to produce collagen is not known. Since prolidase [E. C. 3. 4. 13. 9] has been found to play an important role in the recycling of proline for collagen synthesis and cell growth, we performed comparative studies on prolidase activity and extracellular collagen content in in vitro aged human skin fibroblasts. We found that during aging of human skin fibroblasts there is a correlated decrease of prolidase activity and extracellular collagen content. The correlation is bifunctional. Decrease in prolidase activity contributes to decrease of extracellular collagen content, and removal of extracellular collagen form cultured fibroblasts contributes to a decrease of fibroblast prolidase activity. Using plates coated with different ECM proteins, it was found that extracellular type I collagen in cultured fibroblasts was the most potent activator of intracellular prolidase activity. Our results demonstrate that during in vitro aging of human skin fibroblasts, the prolidase activity of fibroblasts decreases. This phenomenon may be related to the reduced number of fibroblasts and decrease of extracellular collagen content in in vitro aging of the cells.

Cell Division↗
PubMed1998

Respiratory activity and growth of human skin derma fibroblasts.

A study has been made on the speed of growth and respiratory activity of fibroblast cultures from control derma, cheloid (hypertrophic) scar and stabilized scar taken from human skin. The speed of growth and the efficiency of plaque formation of fibroblasts from cheloid scar were greater in comparison with those of fibroblasts from stabilized scar and were stimulated by the addition to the culture medium of the exudate from post-traumatic ulcer. Measurement of the contents of cytochromes showed a decrease in the content of cytochromes b562 and c + c1 in the fibroblast culture from both cheloid and stabilized scar as compared to the fibroblast culture from control derma. Cytochrome aa3 content did not show significant difference among the three types of fibroblast cultures. The respiratory activities supported by pyruvate plus malate, succinate or ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine did not show, however, significant difference among the three fibroblast cultures. These observations show that the speed of growth of skin fibroblasts does not depend on the overall respiratory capacity. The exudate stimulated the activity of cytochrome c oxidase in fibroblasts from control derma, and cheloid scar. This effect and the accompanying stimulation of fibroblast growth might be correlated with the balance of oxygen free radicals.

Adult↗
PubMed2003

Keratinocyte growth factor produced by gastric fibroblasts specifically stimulates proliferation of cancer cells from scirrhous gastric carcinoma.

It has been previously reported (M. Yashiro et al., Jpn. J. Cancer Res., 84: 883-886, 1994) that a growth factor secreted by human gastric fibroblasts stimulated proliferation of human scirrhous gastric carcinoma cells in vitro, suggesting a similar paracrine action in the gastric submucosa. The present study established the identity of the growth factor as keratinocyte growth factor (KGF). Increase in numbers and incorporation of [(3)H]thymidine in scirrhous gastric carcinoma cell lines (OCUM-2M and OCUM-11) in response to culture medium from a gastric fibroblast line (NF-8 and NF-21) were duplicated by substitution of KGF and inhibited by addition of anti-KGF antibody. Effects were specific for scirrhous carcinoma cells in distinction to well-differentiated gastric carcinoma cell lines. Fibroblasts, especially gastric fibroblasts, expressed KGF mRNA, whereas gastric cancer cells did not. Conversely, scirrhous gastric cancer cells expressed more KGF receptor mRNA than well-differentiated gastric adenocarcinoma cell, whereas gastric fibroblasts did not express this mRNA. ELISA detected high concentrations of KGF in medium from gastric fibroblasts, much lower concentration in medium from other fibroblasts, and no KGF in medium from gastric cancer cells. Western analysis indicated that KGF in gastric fibroblasts lysates had a molecular weight of M(r) 19,000, within the range suggested in our previous report. Thus, gastric fibroblasts secretion of KGF is likely to underline the remarkable proliferation of scirrhous gastric cancer cells in a paracrine manner.

Adenocarcinoma, Scirrhous↗
PubMed1993

Hepatocyte growth factor, keratinocyte growth factor, their receptors, fibroblast growth factor receptor-2, and the cells of the cornea.

PURPOSE: The purpose of this study was to determine whether messenger RNA coding for hepatocyte growth factor (HGF), HGF receptor (MET), keratinocyte growth factor (KGF), KGF receptor, and fibroblast growth factor (FGF) receptor-2 were produced in primary cultures of human corneal epithelial, stromal fibroblast, and endothelial cells, as well as ex vivo corneal epithelium, endothelial cells transfected with the SV40 large T antigen, and control embryonic lung fibroblasts. The effects of exogenous HGF and KGF, compared to epidermal growth factor, on the proliferation of first passage corneal cells were also examined. METHODS: Polymerase chain reaction was used to amplify complementary DNA for each modulator from each cell type. Hot blotting was used to demonstrate the specificity of amplification products. Proliferation of first passage corneal epithelial, stromal fibroblast, and endothelial cells in response to varying concentrations of HGF, KGF, and epidermal growth factor was measured. RESULTS: Specific amplification products for messenger RNA coding for each modulator were detected in each corneal cell type, although very low levels of HGF and KGF messenger RNA appeared to be present in corneal epithelial cells relative to stromal fibroblasts and corneal endothelial cells. Amplification products that may have been derived from alternative transcripts were detected for several of the modulators. HGF and KGF stimulated proliferation in a dose-response manner in first passage corneal epithelial and endothelial cells, but not stromal fibroblast cells. CONCLUSIONS: Human corneal epithelial, stromal fibroblasts, and endothelial cells produce messenger RNA coding for HGF and KGF, although low levels appear to be present in the epithelial cells. All three major cell types of the cornea produce messenger RNA coding for HGF receptor, KGF receptor, and FGF receptor-2. The proliferation of human corneal epithelial and endothelial cells, but not stromal fibroblasts, was stimulated by exogenous HGF and KGF. HGF and KGF likely have intracrine, autocrine, and/or paracrine functions in the cornea. Exogenous HGF and KGF may be useful in corneal preservation and for regulating corneal wound healing.

Adolescent↗
PubMed1986

Connective tissue components in synovial fibroblast cultures exposed to interleukin 1 and prostaglandin E2.

Long-term synovial fibroblast cultures were exposed to interleukin 1 (IL-1) or prostaglandin E2 (PGE2). The normally spindle-shaped fibroblasts changed to stellate-shaped cells, resembling the HLA-DR-positive, collagenase-producing cells which are normally seen only in primary cultures from enzyme-digested rheumatoid synovial tissue. However, the IL-1- or PGE2-induced fibroblasts were not HLA-DR-positive. This suggests that these cell populations represent originally different cell lines or that the expression of HLA-DR antigens is not induced by the agents used. For further characterization of these stellate cells, the location of fibronectin and type I collagen was studied by specific antibodies and the pericellular coat around fibroblasts was visualized by the erythrocyte exclusion method. Both IL-1 and PGE2 treatments destroyed the intercellular fibronectin network. Type I collagen was detected as intracellular granules. The stellate fibroblasts were usually full of these granules in contrast to intact fibroblasts in which the number of collagen fluorescence granules varied greatly. The pericellular coat known to be formed mainly by hyaluronic acid was similar around spindle and stellate-shaped fibroblasts. Rheumatoid arthritis-derived fibroblasts did not differ from their non-rheumatoid counterparts in any of the experiments. The effect of IL-1 and PGE2 on fibroblasts simulates the interaction between mononuclear cells and fibroblasts in synovial stroma and also potentially the interactions between different cell types in synovial lining.

Arthritis, Rheumatoid↗
PubMed2000

Simultaneous analysis of [3H]-thymidine uptake and alpha 1(I) procollagen mRNA expression in systemic sclerosis skin fibroblasts--an in situ hybridization study.

Heterogeneity of DNA synthesis and collagen synthesis has been reported in skin fibroblasts from systemic sclerosis (SSc) patients. The uptake of [3H]-thymidine and expression of alpha 1(I) procollagen mRNA by cultured skin fibroblasts from four normal controls and four SSc patients was analyzed simultaneously. The grains overlying the cytoplasm representing alpha 1(I) procollagen mRNA and overlying the nucleus representing [3H]-thymidine uptake were counted using computer-aided image analysis. The results were analyzed statistically. Procollagen mRNA expression by SSc fibroblasts was significantly greater than by control fibroblasts (P < 0.01). The distribution curve of [3H]-thymidine uptake showed two peaks representing low- and high-uptake cells. Significantly more SSc fibroblasts than control fibroblasts showed high [3H]-thymidine uptake (P < 0.05). The number of SSc fibroblasts expressing low amounts of alpha 1(I) procollagen mRNA was significantly lower than the number of control fibroblasts (P < 0.05). [3H]-thymidine uptake by SSc fibroblasts expressing high amounts of alpha 1(I) procollagen mRNA was significantly lower than by those expressing low amounts (P < 0.05). These results indicate that elevated DNA synthesis and elevated collagen mRNA synthesis in SSc skin fibroblasts are due to different clones with high DNA-synthesizing ability and high collagen-producing ability.

Aged↗
PubMed2005

Transforming growth factor beta stimulates rheumatoid synovial fibroblasts via the type II receptor.

Transforming growth factor (TGF)-beta regulates the function of fibroblasts, and has been shown to have a role in the pathogenesis of rheumatoid arthritis (RA) because several studies have demonstrated the presence of TGF-beta in the synovial tissue and synovial fluids of RA patients. In this study, we examined the expression of TGF-beta receptors in synovial fibroblasts of patients with RA and demonstrated the significance in functional responses of synovial fibroblasts to TGF-beta in this disorder. Transforming growth factor beta1 stimulated the expression of connective tissue growth factor (CTGF) in fibroblasts of patients with RA more than in those of patients with osteoarthritis (OA). Transforming growth factor beta1 induced the chemotactic migration of RA synovial fibroblasts and inhibited their proliferation significantly more than OA synovial fibroblasts. Both RA and OA synovial fibroblasts expressed detectable amounts of TGF-beta receptor type II mRNA, but the expression was higher in RA patients than in OA patients, as assessed by reverse transcriptase-polymerase chain reaction. There was no significant difference in the expression of TGF-beta receptor type I or type III in synovial fibroblasts between RA and OA patients. These results indicate that synovial fibroblasts of RA patients express the increased TGF-beta receptor type II, which is associated with altered responses to TGF-beta observed in CTGF expression, chemotaxis, and proliferation of RA synovial fibroblasts, and may have an important role in the pathogenesis of RA.

Journal Article↗
PubMed1993

Suppression of in vitro proliferative scar fibroblast contraction by interferon alfa-2b.

Increased fibroblast activity and collagen production have been observed frequently in proliferative scars. Previous studies have demonstrated that interferons suppress collagen production by means of normal, keloid, and hypertrophic scar-derived fibroblasts. The fibroblast-populated collagen lattice is an in vitro model used to study fibroblast function. We used fibroblast-populated collagen lattices to evaluate the effect of interferon on fibroblasts harvested from normal human skin, human keloid, and hypertrophic scar tissues. Human recombinant interferon alfa-2b (1000 IU/ml) was added to the culture media. The collagen gel, prepared from rat tail tendon bundles, was overlaid with 5 x 10(4) fibroblast cells. Keloid fibroblast-populated collagen lattices showed the highest contraction. Contraction in all the groups appeared suppressed by interferon alfa-2b during the first 72 hours of study (p < 0.05). The reduction in fibroblast-populated collagen lattice contraction by interferon alfa-2b was similar among the groups. The contractile properties of fibroblasts taken from normal human skin, keloids, and hypertrophic scars in this in vitro study were suppressed by interferon alfa-2b. This suggested that interferon alfa-2b may be beneficial for the treatment of proliferative scars.

Journal Article↗
PubMedDate not supplied

Effects of transforming growth factor-beta1 on extracellular matrix gene expression by human fibroblasts from a laryngeal stenotic lesion.

Transforming growth factor-beta1 appears to play important roles in normal wound healing by increasing synthesis of extracellular matrix components. However, the role of transforming growth factor-beta1 in the production of excessive scar tissue by fibroblasts from stenotic lesions of the larynx has not been evaluated. We examined the effect of transforming growth factor-beta1 on the steady-state messenger RNA levels of elastin, alpha2(l) procollagen, and lysyl oxidase (the enzyme that cross-links both of these structural proteins) in cell cultures of diploid human fibroblasts established from fetal skin, newborn foreskin, and an adult laryngeal stenotic lesion. Time-course and dose-response experiments demonstrated that treatment with 500 pmol/L transforming growth factor-beta1 for 20 hours induced maximal levels of mRNA for elastin (7- to 59-fold) and alpha2(l) procollagen (1.7- to 2.4-fold) in all three cultures of fibroblasts. Transforming growth factor-beta1 also increased levels of lysyl oxidase mRNA in fibroblasts cultured from newborn foreskin (2.4-fold) and a stenotic lesion (10-fold) but had minimal effects on the fibroblasts cultured from fetal skin (1.1-fold), which constitutively expressed high levels of lysyl oxidase mRNA. Furthermore, the fibroblast culture established from a laryngeal stenotic lesion responded with the highest fold-induction for all three mRNAs. Inhibition of mRNA synthesis by actinomycin D showed that transcription was required for transforming growth factor-beta1 induction of elastin, alpha2(l) procollagen, and lysyl oxidase mRNA in all three cultures of fibroblasts. Inhibition of protein synthesis by cycloheximide showed that translation was required for maximal induction by transforming growth factor-beta1 of elastin mRNA but had no observable effect on alpha2(l) procollagen mRNA in all three cultures of fibroblasts. In addition, translation was required for maximal induction of the lysyl oxidase mRNA by transforming growth factor-beta1 in the fibroblasts cultured from a stenotic lesion but not for fibroblast cultures established from fetal and adult skin. These results show that transforming growth factor-beta1 coordinately increases mRNA levels for the structural extracellular matrix proteins collagen and elastin, as well as for the cross-linking enzyme, lysyl oxidase. These data also support the hypothesis that transforming growth factor-beta1 may contribute to the formation of laryngeal stenotic lesions.

Journal Article↗
PubMed2001

Fibroblast growth factor 10 induces proliferation and differentiation of human primary cultured keratinocytes.

Fibroblast growth factor 10 is a novel member of the fibroblast growth factor family, which is involved in morphogenesis and epithelial proliferation. It is highly homologous to the keratinocyte growth factor (or fibroblast growth factor 7), a key mediator of keratinocyte growth and differentiation. Both fibroblast growth factor 10 and keratinocyte growth factor bind with high affinity to the tyrosine kinase keratinocyte growth factor receptor. Here we analyzed the effect of fibroblast growth factor 10 on primary cultures of human keratinocytes, grown in chemically defined medium, and we compared the proliferative and differentiative cell responses to fibroblast growth factor 10 with those induced by keratinocyte growth factor and epidermal growth factor. Cell counting, 5-bromo-2'-deoxyuridine incorporation, and western blot analysis showed that fibroblast growth factor 10, similarly to keratinocyte growth factor, not only is a potent mitogen for human keratinocytes, but also promotes the expression of both early differentiation markers K1 and K10 and late differentiation marker filaggrin in response to the Ca2+ signal, and seems to sustain the proliferative activity in suprabasal stratified cells. Immunoprecipitation/western blot analysis revealed that fibroblast growth factor 10, similarly to keratinocyte growth factor, is able to induce tyrosine phosphorylation of keratinocyte growth factor receptor and of cellular substrates such as PLCgamma.

Antigens, Differentiation↗
PubMed1993

Keratinocyte growth factor and hepatocyte growth factor/scatter factor are heparin-binding growth factors for alveolar type II cells in fibroblast-conditioned medium.

Epithelial-mesenchymal interactions mediate aspects of normal lung growth and development and are important in the restoration of normal alveolar architecture after lung injury. To determine if fibroblasts are a source of soluble growth factors for alveolar type II cells, we investigated the effect of fibroblast-conditioned medium (CM) on alveolar type II cell DNA synthesis. Serum-free CM from confluent adult human lung fibroblasts was concentrated fivefold by lyophilization. Type II cells were isolated from adult rats by elastase dissociation and incubated with [3H]thymidine and varying dilutions of concentrated CM and serum from day 1 to 3 of culture. Stimulation of type II cell DNA synthesis by fibroblast-CM was maximal after 48 h of conditioning and required the presence of serum. The activity of the CM was eliminated by boiling and by treatment with trypsin, pepsin, or dithiothreitol and was additive with saturating concentrations of acidic fibroblast growth factor, epidermal growth factor, and insulin. The growth factor activity bound to heparin-Sepharose and was eluted with 0.6 and 1.0 M NaCl. Neutralizing antibody studies demonstrated that the primary mitogens isolated in the 0.6 and 1.0 M NaCl fractions were keratinocyte growth factor (KGF, fibroblast growth factor 7) and hepatocyte growth factor/scatter factor (HGF/SF), respectively. HGF/SF was demonstrated in the crude CM and KGF was detected in the 0.6 M NaCl eluent by immunoblotting. Northern blot analysis confirmed that the lung fibroblasts expressed both KGF and HGF/SF transcripts. Human recombinant KGF and HGF/SF induced a concentration- and serum-dependent increase in rat alveolar type II cell DNA synthesis. We conclude that adult human lung fibroblasts produce at least two soluble heparin-binding growth factors, KGF and HGF/SF, which promote DNA synthesis and proliferation of rat alveolar type II cells in primary culture. KGF and HGF/SF may be important stimuli for alveolar type II cell proliferation during lung growth and after lung injury.

Animals↗