The Antifibrosis Effect Of Adrenomedullin In Human Lung Fibroblasts

Proliferation and collagen synthesis in fibroblasts are pathological features observedboth in pulmonary vascular structural remodeling (PVSR),lung fibrosis and the repair ofinjured lung tissue. Fibroblasts are collagen-secreting cells. An important function offibroblasts is to secrete extracellular matrix proteins in response to damage, which helps tothicken the vessel wall and form scars. Moreover, fibroblasts can produce angiotensin,basic fibroblast growth factor, transforming growth factor β1(TGF-β1) and various othercytokines to modulate cell growth and other activities. TGF-β1is an important metaboliccollagen regulatory cytokine. Prior studies have reported that TGF-β1may stimulatefibroblast proliferation and collagen production.Activation of Smad proteins is known to be the key signaling pathway of theprofibrotic effect of TGF-β1. TGF-β1induced rapid expression of p-Smad2/3in nuclei ofmyofibroblasts and TGF-β1upregulated the expression of type I collagen mRNA. Thesefindings suggest type I collagen expression is mediated by the TGF-β/Smad pathway.C-Myc is one of the most potent regulators of cell cycle progression in highereukaryotes. Down-regulation of c-Myc is a critical event for growthinhibition induced byTGF-β and is frequently impaired in cancer cells. TGF-β1can elevate the downregulatedc-Myc in cultured pterygial cells. The peak expressions of c-jun and c-myc were delayed inTGF-β1(1ng/ml)-added MT-9cells, At the transcriptional level, c-Myc downregulatesTGF-β1and TGF-β receptor2in mouse fibroblasts. TGF-β inhibits rat fibroblastsproliferation by suppression of c-Myc and induction of p15INK46, p21CIP1, or p27KIP.These results indicate that TGF-β1and c-myc mRNA expression were related.Adrenomedullin (AM) is a potent52-amino-acid vasodilator peptide that wasoriginally isolated from a human pheochromocytoma and is a multifunctional regulatory peptide. AM belongs to the calcitonin gene–related peptide (CGRP) family. AM acts as apeptide ligand that activates receptors on the cell surface. AM is produced and secreted byvarious types of cells, including fibroblasts, and AM exhibits important effects in thebiology of vascular cells, regulating vascular tone and promoting vasodilation. This peptidealso exhibits effects on cell growth and apoptosis. Cultured neonatal rat cardiac fibroblastsproduced and secreted AM, and AM may inhibit proliferation and protein synthesis in thesecells. These studies provide strong evidence for a regulatory function of AM. In fact, AMmay play an important role in modulating the growth of fibroblasts. However, themechanism by which AM mediates these functions has not been fully elucidated.There are few reports about c-myc, smad and adrenomedullin. It is reported that c-myctransactivates rat and human adrenomedullin genes and accelerates the degradation rate ofadrenomedullin mRNA. In human tubular epithelial cell line HK-2, AM at10(-8) M exertedno effect on TGF-β1-induced Smad2phosphorylation, but prevented the suppression of theinhibitory Smad6protein by TGF-β1and restored Smad2-Samd6complex formation. Theresults suggest that AM can attenuate TGF-β1-mediated renal tubulointerstitial extracellularmatrix (ECM) turnover via an antagonistic mechanism of inhibitory Smad in TGF-β1-elicited signaling.Aims In this study, we investigated AM and the AM receptor system in the humanfetal lung fibroblasts (HFLFs), human umbilical vein endothelial cells (HUVECs) andH1299cells. And we assessed the effects of AM on the cell proliferation, c-myc, COL1α1,COL3α1gene expession and Smad2/3phosphorylation in HFLFs. We also investigated theinfluence of AM on TGF-β1-induced cell proliferation, c-myc, COL1α1, COL3α1geneexpession.Methods Primary HFLFs culture were established by the outgrowth of fibroblasts.Cells were cultured in DMEM (high glucose) with10%fetal bovine Serum at37℃in ahumidified atmosphere with5%CO2. Cells were passaged every3days. The expression ofAM protein in fibroblasts was tested by immunohistochemistry analysis. The geneexpression of adrenomedullin and its receptors, adrenomedullin receptor (ADMR),calcitonin receptor–like receptor (CRLR), AM receptor chaperone receptor activity–modifying protein-1(RAMP1), RAMP2, and RAMP3were qualitatively measured byReverse transcriptase–polymerase chain reaction (RT-PCR) in the fibroblasts. The effects of AM and TGF-β1on the proliferation of fibroblasts were determined by themethanethiosulfonate (MTT) assay. After the addition of AM and TGF-β1, c-myc, α1(I)procollagen (COL1α1) and α1(Ⅲ) procollagen (COL3α1) gene expression of fibroblastswere determined by real-time quantitative PCR. Western blotting was used for themeasurement of p-Smad2/3protein assay in lung fibroblast.Results There were positive staining in HFLFs incubated with anti-adrenomedullin.AM and its receptors, ADMR, CRLR, RAMP1, RAMP2, and RAMP3gene expressioncould be detected in HFLF. AM, RAMP1and RAMP3gene expression could be detected inHUVE. AM, ADMR, CRLR, RAMP1and RAMP3gene expression could be detected inH1299cell. It suggested that adrenomedullin and its receptors were abundant expressed inHFLF. Cultured at24,48and72hours, the OD values of fibroblasts decreased aftertreatment with AM200nM. TGF-β15ng/ml stimulated fibroblasts proliferation and AM at200nM exerted inhibitory effect on cell proliferation induced by TGF-β1. At thetranscriptional level, TGF-β1induced expression of c-myc,COL1α1and COL3α1mRNAin HFLFs, and TGF-β1promoted p-Smad2/3protein of fibroblasts. After incubation withAM, c-myc and COL3α1mRNA expression and Smad2/3phosphorylation in fibroblastsdecreased. AM could suppress the expression of c-myc and COL3α1gene on the basic leveland the TGF-β1-stimulated.Conclusions HFLFs expressed AM and also expressed both receptors. AM inhibitedTGF-β1-induced cell proliferation and collagen gene expression in cultured fibroblasts. Ourresults indicate that AM may function as an antifibrosis factor that protects cells frompulmonary damage through its receptors and also mediated by the TGF-β/Smad and c-mycpathway.