RESUMO
Adrenomedullin (AM) is a regulatory peptide involved in cellular proliferation and protein synthesis. The authors investigated AM and the AM receptor system in the human fetal lung fibroblasts (HFLFs), and assessed whether AM can inhibit proliferation and collagen synthesis in HFLFs under hypoxia. Fibroblasts were exposed to hypoxia (2% O(2)) after the addition of AM. The effects of AM and transforming growth factor ß1 (TGF-ß1) on the proliferation of fibroblasts were determined by the methanethiosulfonate (MTS) assay. Total collagen synthesis was determined by [(3)H]proline incorporation. TGF-ß1 levels in the culture supernatant were measured by enzyme-linked immunosorbent assay (ELISA). The concentration of intracellular calciumion ([Ca(2+)](i)) in fibroblasts was detected with a laser scanning confocal microscope. AM, adrenomedullin receptor (ADMR), calcitonin receptor-like receptor (CRLR), AM receptor chaperone receptor activity-modifying protein-1 (RAMP1),RAMP2, and RAMP3 were detected in the HFLFs. The hypoxia-induced increases in cell proliferation, collagen synthesis, and TGF-ß1 production were inhibited by AM. AM also inhibited proliferation and collagen synthesis in fibroblasts induced by TGF-ß1. AM caused a decrease of the hypoxia-induced [Ca(2+)](i) in fibroblasts. This study suggests that AM is produced by HFLFs and AM may function as an antifibrosis factor that protects cells from hypoxic pulmonary damage through its receptors.
Assuntos
Adrenomedulina/metabolismo , Fibroblastos/metabolismo , Pulmão/metabolismo , Adrenomedulina/biossíntese , Adrenomedulina/genética , Adrenomedulina/farmacologia , Proteína Semelhante a Receptor de Calcitonina/metabolismo , Cálcio/metabolismo , Processos de Crescimento Celular/fisiologia , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/fisiologia , Colágeno/biossíntese , Colágeno/metabolismo , Fibroblastos/efeitos dos fármacos , Fibrose/genética , Fibrose/metabolismo , Humanos , Pulmão/efeitos dos fármacos , Proteínas Modificadoras da Atividade de Receptores/metabolismo , Receptores de Adrenomedulina/biossíntese , Receptores de Adrenomedulina/genética , Receptores de Adrenomedulina/metabolismo , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
OBJECTIVE: To observe the effects of adrenomedullin (AM) on transforming growth factor- ß1 (TGF-ß1) induced the expression of procollagen type 1 alpha 1 (Col1α1) and procollagen type 3 alpha 1 (Col3α1) as well as Smad2/3 phosphorylation in human fetal lung fibroblasts (HFLFs). METHODS: Primary HFLFs were cultured in vitro. After treated with TGF-ß1 and/or AM, the expression levels of Col1α1 and Col3α1 mRNA were determined by reverse transcription PCR (RT-PCR), and phospho-Smad2/3 (p-Smad2/3) protein levels in HFLFs were measured by Western blotting. RESULTS: TGF-ß1 increased the gene expression levels of Col1α1 and Col3α1, and promoted the phosphorylation levels of Smad2/3 protein in HFLFs. AM significantly reversed the expression level of Col3α1 mRNA, and inhibited p-Smad2/3 expression in HFLFs induced by TGF-ß1. CONCLUSION: AM could inhibit TGF-ß1-induced the procollagen expression in cultured HFLFs possibly through the Smad2/3 signaling pathway.
Assuntos
Adrenomedulina/farmacologia , Pró-Colágeno/genética , Transdução de Sinais/efeitos dos fármacos , Proteína Smad2/fisiologia , Proteína Smad3/fisiologia , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Células Cultivadas , Fibroblastos/metabolismo , Humanos , Pulmão/citologia , RNA Mensageiro/análise , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
The role of autophagy in cancer is controversial. Melatonin has been linked to several aspects of cancer progression and also to regulation of autophagy. Whether melatonin is involved in an autophagy-induced tumor suppressor mechanism or a cyto-protective mechanism is unknown. Therefore, we investigated the effects of melatonin on autophagy and its upstream regulator. We found that melatonin triggers an autophagic process by enhancing Beclin 1 expression and inducing a conversion of microtubule-associated protein 1 light chain 3(LC3)-I to LC3-II, the protein associated with the autophagosome membrane, in hepatoma H22 tumor-bearing mice. Moreover, melatonin inhibits the phosphorylation of the mammalian target of the rapamycin (mTOR) and Akt. Knockdown of Beclin 1 by either RNA interference or co-treatment with the autophagy inhibitor, 3-methyladenine(3-MA), significantly enhanced the melatonin-induced apoptosis in mouse hepatoma H22 cells. Our data provides the first evidence that melatonin induces protective autophagy that prevents mouse hepatoma H22 cells from undergoing apoptosis. A combination of melatonin with an autophagy inhibitor might be a useful therapeutic strategy for hepatocellular carcinoma.