26585

GREM1

Ingestion of aristolochic acid (AA) is associated with the development of aristolochic acid nephropathy (AAN), which is characterized by progressive tubulointerstitial fibrosis, chronic renal failure and urothelial cancer. Our previous study showed that bone morphogenetic protein-7 (BMP-7) could attenuate AA-induced epithelial-to-mesenchymal transition (EMT) in human proximal tubule epithelial cells (PTEC). However, how gremlin (a BMP-7 antagonist) antagonizes the BMP-7 action in PTEC remained unsolved. The aim of the current study was to investigate the role of gremlin in AA-induced EMT in PTEC (HK-2 cells). HK-2 cells were treated with AA (10 mumol/L) for periods up to 72 h. Cell viability was determined by tetrazolium dye (MTT) assay. Morphological changes were assessed by phase-contrast microscopy. Markers of EMT, including E-cadherin and alpha-smooth muscle actin (alpha-SMA) were detected by indirect immunofluorescence stains. The BMP-7 and gremlin mRNA and protein expression in HK-2 cells were analyzed by quantitative real-time PCR (real-time RT-PCR) and western blotting after exposure to AA. The level of phosphorylated Smad1/5/8, a marker of BMP-7 activity, was also determined by western blot analysis. Cells were transfected with gremlin siRNA to determine the effects of gremlin knockdown on markers of EMT following treatment with AA. Our results indicated that AA-induced EMT was associated with acquisition of fibroblast-like cell shape, loss of E-cadherin, and increases of alpha-SMA and collagen type I. Interestingly, exposure of HK-2 cells to 10 mumol/L AA increased the mRNA and protein expression of gremlin in HK-2 cells. This increase was in parallel with a decrease in BMP-7 expression and a down-regulation of phosphorylated Smad1/5/8 protein levels. Moreover, transfection with siRNA to gremlin was able to recover BMP-7 signaling activity, and attenuate EMT-associated phenotypic changes induced by AA. Together, these observations strongly suggest that gremlin plays a critical role in the modulation of reno-protective action of BMP, and that inhibition of gremlin will be a promising means of developmenting novel treatments for AAN.CI - Copyright (c) 2012 Elsevier Ireland Ltd. All rights reserved.

Transforming growth factor (TGF)-beta2, gremlin and connective tissue growth factor (CTGF) are known to play important roles in the induction of epithelial mesenchymal transition (EMT) and extracellular matrix (ECM) synthesis. However, the complex functional relationship among gremlin, CTGF and TGF-beta2 in the induction of EMT and ECM synthesis in human lens epithelial cells (HLECs) has not been reported. In this study, we found that TGF-beta2, CTGF and gremlin can individually induce the expression of alpha-smooth muscle actin (alpha-SMA), fibronectin (Fn), collagen type I (COL-I), Smad2 and Smad3 in HLECs. Blockade of CTGF and gremlin effectively inhibited TGF-beta2-induced expression of alpha-SMA, Fn, COL-I, Smad2, and Smad3 in HLECs. Furthermore blockade of Smad2 and Smad3 effectively inhibited CTGF and gremlin induced expression of alpha-SMA, Fn, COL-I in HLECs. In conclusion, TGF-beta2, CTGF and gremlin are all involved in EMT and ECM synthesis via activation of Smad signaling pathway in HLECs. Specifically silencing CTGF and gremlin can effectively block the TGF-beta2-induced EMT, ECM synthesis due to failure in activation of Smad signaling pathway in HLECs.CI - Copyright (c) 2014 Elsevier Inc. All rights reserved.

Gremlin is a developmental gene upregulated in human chronic kidney disease and in renal cells in response to transforming growth factor-beta (TGF-beta). epithelial mesenchymal transition (EMT) is one process involved in renal fibrosis. In tubular epithelial cells we have recently described that Gremlin induces EMT and acts as a downstream TGF-beta mediator. Our aim was to investigate whether Gremlin participates in EMT by the regulation of the Smad pathway. Stimulation of human tubular epithelial cells (HK2) with Gremlin caused an early activation of the Smad signaling pathway (Smad 2/3 phosphorylation, nuclear translocation, and Smad-dependent gene transcription). The blockade of TGF-beta, by a neutralizing antibody against active TGF-beta, did not modify Gremlin-induced early Smad activation. These data show that Gremlin directly, by a TGF-beta independent process, activates the Smad pathway. In tubular epithelial cells long-term incubation with Gremlin increased TGF-beta production and caused a sustained Smad activation and a phenotype conversion into myofibroblasts-like cells. Smad 7 overexpression, which blocks Smad 2/3 activation, diminished EMT changes observed in Gremlin-transfected tubuloepithelial cells. TGF-beta neutralization also diminished Gremlin-induced EMT changes. In conclusion, we propose that Gremlin could participate in renal fibrosis by inducing EMT in tubular epithelial cells through activation of Smad pathway and induction of TGF-beta.

Idiopathic pulmonary fibrosis (IPF) is characterized by activation and injury of epithelial cells, the accumulation of connective tissue and changes in the inflammatory microenvironment. The bone morphogenetic protein (BMP) inhibitor protein gremlin-1 is associated with the progression of fibrosis both in human and mouse lung. We generated a transgenic mouse model expressing gremlin-1 in type II lung epithelial cells using the surfactant protein C (SPC) promoter and the Cre-LoxP system. Gremlin-1 protein expression was detected specifically in the lung after birth and did not result in any signs of respiratory insufficiency. Exposure to silicon dioxide resulted in reduced amounts of lymphocyte aggregates in transgenic lungs while no alteration in the fibrotic response was observed. Microarray gene expression profiling and analyses of bronchoalveolar lavage fluid cytokines indicated a reduced lymphocytic response and a downregulation of interferon-induced gene program. Consistent with reduced Th1 response, there was a downregulation of the mRNA and protein expression of the anti-fibrotic chemokine CXCL10, which has been linked to IPF. In human IPF patient samples we also established a strong negative correlation in the mRNA expression levels of gremlin-1 and CXCL10. Our results suggest that in addition to regulation of epithelial-mesenchymal crosstalk during tissue injury, gremlin-1 modulates inflammatory cell recruitment and anti-fibrotic chemokine production in the lung.

Backgroud/Aims: mesenchymal stromal cells (MSCs) are a major component of the tumor microenvironment (TME). Several studies focusing on tumor-derived MSCs have demonstrated that they exhibit a strong ability to promote the tumor epithelial-mesenchymal transition (EMT). However, the factors mediating these effects are poorly understood. METHODS: Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and immunohistochemistry assays were used to detect the expression of Gremlin1 (GREM1) in human esophageal squamous cell carcinoma (ESCC) tissues. ShRNA silencing, flow cytometry, cell counting kit (CCK8) assay, invasion assay, western blot were used to detect the effect of GREM1 in ECa109, TE-1 cell lines and xenograft tumor models. RESULTS: In the current study, we found that the GREM1 was overexpressed in human ESCC tissues. The conditioned medium from mesenchymal stromal cells (MSCs-CM) enhanced the malignancy of xenograft esophageal tumors in vivo, as well as the cell proliferation, viability and invasion of the esophageal carcinoma cell lines ECa109 and TE-1 in vitro. Furthermore, the shRNA silencing of GREM1 in MSCs (shGREM1-MSCs) reversed the increased malignancy of the esophageal tumor in vivo, while the conditioned medium from shGREM1-MSCs (shGREM1-MSCs-CM) affected the cell cycle and cell invasion in vitro. These processes were accompanied by the EMT in the ECa109 and TE-1 cell lines with an alteration in the expression levels of mesenchymal and epithelial markers. Furthermore, the TGF-beta/BMP (transforming growth factor-beta/bone morphogenetic protein) signaling pathway participated in the shGREM1-MSCs-CM-induced anti-tumor effect on enhanced esophageal malignancy induced by MSCs-CM treatment. CONCLUSIONS: Taken together, our study suggested that GREM1 delivered by MSCs promoted EMT in ESCC in vitro and in vivo, which is partly through TGF-beta/BMP signaling pathway. The results provide experimental evidence to a potential therapeutic target in the treatment of esophageal cancer.CI - (c) 2018 The Author(s). Published by S. Karger AG, Basel.

Sonic hedgehog (SHH) signaling is an important promotor of desmoplasia, a critical feature in pancreatic cancer stromal reactions involving the activation of pancreatic stellate cells (PSCs). Gremlin 1 is widely overexpressed in cancer-associated stromal cells, including activated PSCs. In embryonic development, SHH is a potent regulator of Gremlin 1 through an interaction network. This subtle mechanism in the cancer microenvironment remains to be fully elucidated. The present study investigated the association between Gremlin 1 and SHH, and the effect of Gremlin 1 in pancreatic cancer. The expression of Gremlin 1 in different specimens was measured using immunohistochemistry. The correlations among clinicopathological features and levels of Gremlin 1 were evaluated. Primary human PSCs and pancreatic cancer cell lines were exposed to SHH, cyclopamine, GLI family zinc finger-1 (Gli-1) small interfering RNA (siRNA), and Gremlin 1 siRNA to examine their associations and effects using an MTT assay, reverse transcription-quantitative polymerase chain reaction analysis, western blot analysis, and migration or invasion assays. The results revealed the overexpression of Gremlin 1 in pancreatic cancer tissues, mainly in the stroma. The levels of Gremlin 1 were significantly correlated with survival rate and pT status. In addition, following activation of the PSCs, the expression levels of Gremlin 1 increased substantially. SHH acts as a potent promoter of the expression of Gremlin 1, and cyclopamine and Gli-1 siRNA modulated this effect. In a screen of pancreatic cancer cell lines, AsPC-1 and BxPC-3 cells expressed high levels of Gremlin 1, but only AsPC-1 cells exhibited a high expression level of SHH. The results of the indirect co-culture experiment suggested that paracrine SHH from the AsPC-1 cells induced the expression of Gremlin 1 in the PSCs. Furthermore, Gremlin 1 siRNA negatively regulated the proliferation and migration of PSCs, and the proliferation, invasion and epithelial-mesenchymal transition of AsPC-1 and BxPC-3 cells. Based on the data from the present study, it was concluded that an abnormal expression level of Gremlin 1 in pancreatic cancer was induced by SHH signaling, and that the overexpression of Gremlin 1 enabled pancreatic cancer progression.