epidermal-growth-factor and Scleroderma--Systemic

Impaired wound healing and skin dehydration are the mainstay of systemic sclerosis (SSc) cutaneous manifestations. Aquaporin-3 (AQP3) has a pivotal role in skin hydration and wound healing. Epidermal growth factor receptor (EGFR) activation is impaired in SSc fibroblasts. It is unclear whether AQP3 downregulation or epidermal growth factor (EGF) signaling are the primary points of dysregulation in SSc patients.. Skin punch biopsies were obtained from 10 SSc patients and 10 healthy subjects. The mRNA and/or protein expression levels of AQP3, EGFR/p-EGFR, matrix metalloproteinase-1/2/9 (MMP-1/2/9), and tissue inhibitors of metalloproteinase-1 (TIMP1) at baseline and after EGF and transforming growth factor-β1 (TGF-β1) treatment was evaluated in extracted fibroblasts using real-time polymerase chain reaction and western blot analysis.. SSc fibroblasts expressed lower AQP3 and EGFR, compared with normal fibroblasts. Normal fibroblasts increased AQP3 expression in response to EGF whereas AQP3 expression had no change in EGF-treated-SSc fibroblasts. Likewise, EGFR was activated in response to EGF in the normal group but not SSc group. Baseline expression of MMP-1/2/9 and TIMP1 was not different between SSc and controls. EGF treatment did not result in alteration of any MMPs expression in either of the groups. Combination treatment resulted in a significant upregulation of MMP-1 in normal fibroblasts compared with SSc fibroblasts, while in SSc fibroblasts MMP-9 expression was upregulated in response to treatment with TGF-β1 only.. Downregulation of AQP3 expression in SSc fibroblasts may be related to reduced EGFR expression and activation. TGF-β1 (alone or in combination with EGF) only can upregulate AQP3 expression in SSc fibroblasts so, TGF-β1 affect MMP-1 and MMP-9 just in SSc fibroblasts.

Topics: Adult; Aquaporin 3; Cells, Cultured; Epidermal Growth Factor; ErbB Receptors; Female; Fibroblasts; Humans; Male; Middle Aged; Scleroderma, Systemic; Signal Transduction

Transforming growth factor beta receptors (TGFbetaRs) are known to be expressed at high levels in several fibrotic diseases, including systemic sclerosis. In the present study, we investigated the mechanism of up-regulation of TGFbetaR expression.. The levels of expression of TGFbetaR type II (TGFbetaRII) messenger RNA (mRNA), with or without stimulation by epidermal growth factor (EGF), were evaluated by Northern blot analysis, and the protein levels were determined by immunoblotting. The transcription activity of the TGFbetaRII gene was examined with luciferase assays using the -1670/+35 TGFbetaRII promoter luciferase construct.. EGF up-regulates the expression of TGFbetaRII mRNA and protein in human dermal fibroblasts. Actinomycin D, an RNA synthesis inhibitor, significantly blocked the EGF-mediated up-regulation of TGFbetaRII mRNA expression, whereas cycloheximide, a protein synthesis inhibitor, did not block this up-regulation. In addition, EGF treatment did not significantly affect the TGFbetaRII mRNA half-life. EGF-mediated induction of TGFbetaRII expression was inhibited by treatment of fibroblasts with the selective phosphoinositide 3-kinase (PI 3-kinase) inhibitors wortmannin or LY294002, and Akt inhibitor also blocked EGF-induced expression of TGFbetaRII. In addition, EGF induced TGFbetaRII promoter activity, and this induction was significantly blocked by wortmannin, LY294002, or Akt inhibitor. Cotransfection with a dominant-negative mutant of p85 (the regulatory component of PI 3-kinase) or Akt significantly reduced the induction of TGFbetaRII promoter activity by EGF. Moreover, a constitutive active form of p110 (a catalytic component of PI 3-kinase) induced TGFbetaRII promoter activity. In addition, scleroderma fibroblasts expressed increased levels of TGFbetaRII but did not show further up-regulation of TGFbetaRII expression by EGF.. These results indicate that EGF-mediated induction of TGFbetaRII expression occurs at the transcription level, does not require de novo protein synthesis, and involves the PI 3-kinase/Akt signaling pathway, and that abnormal activation of EGF-mediated signaling pathways, including PI 3-kinase or Akt, might play a role in the up-regulation of TGFbetaRII in scleroderma fibroblasts.

Topics: Androstadienes; Cells, Cultured; Chromones; Dactinomycin; Dermis; Dose-Response Relationship, Drug; Enzyme Inhibitors; Epidermal Growth Factor; Fibroblasts; Humans; Morpholines; Nucleic Acid Synthesis Inhibitors; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; RNA, Messenger; Scleroderma, Systemic; Signal Transduction; Transfection; Up-Regulation; Wortmannin

Abnormal growth regulation in lesional skin fibroblasts may be related to scleroderma pathogenesis. We report on the abnormal response of cultured fibroblasts derived from sclerotic lesions to various growth factors. We investigated the responses of skin fibroblasts (10 strains) and normal fibroblasts (9 strains) to the growth factors as PDGF, TGF-beta 1, EGF and basic FGF. Experiments were conducted during the proliferation and confluent stages. PDGF, EGF and basic FGF stimulated fibroblast growth during the proliferation and confluent stages, but the response of scleroderma fibroblasts was significantly lower than that of normal fibroblasts. TGF-beta 1 slightly stimulated confluent fibroblast growth and inhibited proliferating fibroblasts, and the response of scleroderma fibroblasts exceeded that of normal fibroblasts. The decreased response to growth-stimulating factors observed in scleroderma fibroblasts suggests that cultured fibroblasts derived from scleroderma lesions were already senescent because they have been activated by growth-stimulating factors and repeatedly divided in vivo. Thus, abnormal growth regulation of skin fibroblasts may be partially related to the pathogenesis of scleroderma.

Topics: Adolescent; Adult; Aged; Cell Division; Cells, Cultured; Child; Epidermal Growth Factor; Female; Fibroblast Growth Factor 2; Fibroblasts; Growth Substances; Humans; Male; Middle Aged; Platelet-Derived Growth Factor; Scleroderma, Systemic; Transforming Growth Factor beta

Epidermal growth factor receptor (EGF-R) of fibroblasts from 3 patients with scleroderma (progressive systemic sclerosis, PSS) was studied by radioiodinated-EGF binding assay. The binding was 60.9 +/- 4.0% of normal fibroblasts, and the Scatchard plots showed a decrease in the affinity for EGF, not in the number of EGF-R. PSS fibroblasts expressed higher levels (1.15-2.45-fold) of RNA for the v-erbB (EGF-R gene). All-transretinoic acid (retinoid) had little effect on EGF-R, v-erbB gene expression and the proliferation of PSS fibroblasts. These data concerning the abnormality in the EGF-R may all constitute a feature of PSS fibroblasts.

Topics: Cell Division; Epidermal Growth Factor; ErbB Receptors; Female; Fibroblasts; Gene Expression Regulation; Humans; Middle Aged; Oncogenes; Reference Values; Scleroderma, Systemic; Statistics as Topic; Stimulation, Chemical; Time Factors; Tretinoin

Transforming growth factor-beta (TGF-beta) has been found in all cells examined thus far, and has been shown to play an important role in inflammation and connective tissue formation. We now report that TGF-beta, alone or in combination with epidermal growth factor (EGF), led to a preferential increase in glycosaminoglycan synthesis by cultures of dermal fibroblasts from patients with progressive systemic sclerosis (PSS) when compared with normal fibroblasts (p less than 0.001). Transforming growth factor-beta increased collagen synthesis to the same extent in both PSS and normal fibroblasts, whereas EGF had no stimulatory activity on collagen synthesis. The addition of EGF to cultures incubated with TGF-beta led to a decrease in collagen synthesis compared with the effect seen with TGF-beta alone (p less than 0.02). These studies suggest that TGF-beta may play an important role in the accumulation of connective tissue seen in PSS and that the combined action of multiple growth factors may modulate the synthetic activity of human dermal fibroblasts.

Topics: Cells, Cultured; Collagen; Drug Combinations; Epidermal Growth Factor; Fibroblasts; Glycosaminoglycans; Humans; Peptides; Scleroderma, Systemic; Skin; Transforming Growth Factors

Purified type I topoisomerase from calf thymus as well as nuclear and cytoplasmic extracts from EGF-stimulated human and mouse fibroblasts in cell culture efficiently convert supercoiled plasmid DNA to the relaxed form. The purified IgG fraction from the sera of Japanese patients with the rheumatic disease scleroderma were shown to inhibit this relaxation activity. Thus, these patients likely produce autoantibodies to topoisomerase I. In addition, the human, bovine and murine enzymes share antigenic determinants recognized by the antisera.

Topics: Animals; Antibodies; Cattle; Cell Nucleus; Cells, Cultured; DNA Topoisomerases, Type I; Epidermal Growth Factor; Epitopes; Fibroblasts; Humans; Immunoglobulin G; Kinetics; Mice; Scleroderma, Systemic; Thymus Gland; Topoisomerase I Inhibitors

The circulating levels of hyaluronate were determined in 36 patients with scleroderma and in 36 control subjects matched for age and sex. The mean serum hyaluronate concentration in patients with progressive systemic sclerosis (n = 25) was 131 +/- 67 (SD) microgram/l and significantly greater (p less than 0.001) than that of the controls (mean level 49 +/- 21 (SD) microgram/l). Hyaluronate levels in patients with localised scleroderma (n = 4) were 141 +/- 47 (SEM) microgram/l and in patients with scleroderma-associated overlap syndromes (n = 7) 202 +/- 54 (SEM) microgram/l. The increase in serum hyaluronate probably reflected an enhanced synthesis or outflow of hyaluronate from the connective tissue, or both; it could not be explained by affection of the liver, which is the catabolic site of hyaluronate. The hyaluronate values were not related to certain serological indicators of inflammatory activity or to the extent of the skin lesions or the severity of internal organ manifestations. A positive correlation was noted between circulating platelet counts and hyaluronate levels (p less than 0.001). Plasma beta-thromboglobulin was measured in 15 of the patients with systemic sclerosis and found to correlate positively with platelet counts. Raised levels of beta-thromboglobulin were associated with the highest hyaluronate values. Platelet-derived growth factor, which stimulates connective tissue cells and is stored in the alpha-granules of platelets together with beta-thromboglobulin, was shown to enhance hyaluronate synthesis in fibroblast cultures. The results suggest an involvement in scleroderma of connective tissue activating substances released from platelets.

Topics: Cells, Cultured; Connective Tissue Diseases; Epidermal Growth Factor; Fibroblasts; Humans; Hyaluronic Acid; Middle Aged; Platelet Count; Platelet-Derived Growth Factor; Scleroderma, Systemic

To explore the mechanism of increased collagen synthesis by scleroderma skin fibroblasts in vitro, control and scleroderma fibroblasts were compared in confluent monolayer cultures growth-arrested by serum deprivation; responses to optimal mitogenic doses of platelet-derived growth factor, fibroblast growth factor, epidermal growth factor and nerve growth factor were compared. Platelet-derived growth factor had a selective mitogenic effect on control skin fibroblasts not observed with scleroderma skin fibroblasts. None of the factors studied had a selective effect on collagen synthesis independent of cell replication; scleroderma and control fibroblasts responded similarly. Therefore, the growth factors studied may not be involved in generating the activated scleroderma fibroblast directly; platelet-derived growth factor may play an indirect role in fibroblast replication in human fibrotic disorders.U

Topics: Cell Division; Cells, Cultured; Collagen; Epidermal Growth Factor; Fibroblast Growth Factors; Fibroblasts; Growth Substances; Humans; Nerve Growth Factors; Peptides; Platelet-Derived Growth Factor; Protein Biosynthesis; Scleroderma, Systemic