RESUMO
A novel human keratinocyte-derived autocrine factor (KAF) was purified from conditioned medium by using heparin affinity chromatography as the first step. Purified KAF stimulated the growth of normal human keratinocytes, mouse AKR-2B cells, and a mouse keratinocyte cell line (BALB/MK). Heparin sulfate inhibited KAF mitogenic activity on all cell types tested and inhibited the ability of KAF to compete with epidermal growth factor for cell surface binding. Interestingly, KAF stimulated the growth of BALB/MK cells at high cell density but failed to stimulate these cells at clonal density. Protein microsequencing of the first 20 NH2-terminal amino acid residues of purified KAF revealed identity to the NH2 terminus of human amphiregulin (AR). Northern (RNA) blot analysis with AR-specific cRNA demonstrated that human keratinocytes, as well as mammary epithelial cell cultures, expressed high levels of AR mRNA. In contrast, AR mRNA was not detected in normal human fibroblasts or melanocytes and was present at reduced levels in several mammary tumor cell lines. The mitogenic activity of purified AR was also shown to be inhibited by heparin sulfate, and an AR-specific enzyme-linked immunosorbent assay (ELISA) revealed that KAF and AR are antigenically related. We have previously shown that human keratinocytes can grow in an autocrine manner. Our present study demonstrates that one of the growth factors responsible for this autocrine growth (KAF) is similar or identical to AR and that KAF and AR bioactivity can be negatively regulated by heparin sulfate.
Assuntos
Glicoproteínas/genética , Substâncias de Crescimento/genética , Heparitina Sulfato/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular , Queratinócitos/fisiologia , Sequência de Aminoácidos , Anfirregulina , Animais , Sequência de Bases , Ligação Competitiva , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Replicação do DNA/efeitos dos fármacos , Família de Proteínas EGF , Fator de Crescimento Epidérmico/metabolismo , Glicoproteínas/isolamento & purificação , Glicoproteínas/farmacologia , Substâncias de Crescimento/isolamento & purificação , Substâncias de Crescimento/farmacologia , Humanos , Queratinócitos/efeitos dos fármacos , Masculino , Camundongos , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , RNA Mensageiro/genética , Homologia de Sequência do Ácido Nucleico , Pele/citologiaRESUMO
Endothelial cells most readily available and most frequently used in investigations of alloimmunity and cytokine expression and function are derived from human umbilical veins. It is unclear whether cells derived from fetal venous tissue are relevant to phenomena related to the adult allograft, especially in areas such as cardiac allograft vasculopathy, a chronic rejection process directed against the coronary arteries. Human aortic endothelial cells (HAECs) were compared to human umbilical vein endothelial cells (HUVECs) for their constitutive expression of poly (A)+ RNA coding for a group of cytokines known to stimulate smooth muscle cell proliferation, including acidic fibroblast growth factor, basic fibroblast growth factor, transforming growth factor alpha, transforming growth factor beta, platelet-derived growth factor A-chain, platelet-derived growth factor B-chain and amphiregulin. Poly (A)+ RNA coding for basic fibroblast growth factor and transforming factor-beta was consistently expressed by all nine isolates of HAECs, but platelet-derived growth factor A- and B-chain were expressed in only six of the nine isolates. In most cases this was related to the presence of transforming growth factor alpha expression. In contrast, HUVECs consistently expressed basic fibroblast growth factor, transforming growth factor-beta, and both platelet-derived growth factor chains. Transforming growth factor alpha expression was never seen in the HUVEC isolates. No endothelial cell isolate expressed mRNA coding for acidic fibroblast growth factor or amphiregulin. There appear to be differences between cytokine gene expression patterns by endothelial cells from different vascular beds.
Assuntos
Aorta/citologia , Endotélio Vascular/metabolismo , Regulação da Expressão Gênica , Substâncias de Crescimento/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular , Veias Umbilicais/citologia , Anfirregulina , Sequência de Bases , Células Cultivadas , Família de Proteínas EGF , Glicoproteínas/biossíntese , Glicoproteínas/genética , Substâncias de Crescimento/genética , Humanos , Recém-Nascido , Dados de Sequência Molecular , Poli A/biossíntese , Poli A/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genéticaRESUMO
We previously demonstrated that human keratinocyte cultures proliferate in the absence of polypeptide growth factors (autonomous growth) and that this autonomous growth is blocked by interaction of heparin with a human keratinocyte-derived autocrine factor (KAF) which we identified as amphiregulin (AR). In the present study, we demonstrate that sulfated polysaccharides other than heparin (low and high molecular weight dextran sulfates) also inhibit the AR-mediated autonomous proliferation of human keratinocytes. Furthermore, sulfated polysaccharides such as high and low molecular weight dextran sulfates, heparan sulfate and, to a lesser extent, chondroitin sulfates B and C were also shown to be inhibitors of human keratinocyte-derived AR (k-d AR)-stimulated DNA synthesis in quiescent murine AKR-2B cell cultures. Our results demonstrate that sulfation of polysaccharides is required for AR inhibitory activity, and that several sulfated polysaccharides (other than heparin) can act as inhibitors of AR-mediated autonomous proliferation in human epidermal keratinocytes and as inhibitors of k-d AR-mediated mitogenic activity in AKR-2B cells.