Activated hepatic stellate cells upregulate transcription of ecto-5′-nucleotidase/CD73 via specific SP1 and SMAD promoter elements

Abstract

sine is a potent modulator of liver fibrosis and inflammation. Adenosine has been shown to regulate such diverse activities as chemotaxis, contraction, and matrix production in hepatic stellate cells (HSC). Ecto-5=-nucleotidase/CD73 [EC 3.1.3.5] is the rate-limiting enzyme in adenosine production. Cd73-deficient mice are resistant to experimental liver fibrosis and have impaired adenosine generation. However, cell-specific expression and regulation of CD73 within the fibrotic liver have not been defined. In particular, prior evidence demonstrating that liver myofibroblasts, the cells believed to be responsible for matrix formation in the liver, express CD73 is lacking. Thus we tested the hypothesis that HSC and portal fibroblasts (PF), cells that undergo differentiation into liver myofibroblasts, express CD73 in a regulated fashion. We found that CD73 is weakly expressed in quiescent HSC and PF but is markedly upregulated at the transcriptional level in myofibroblastic HSC and PF. We furthermore found that CD73 protein and its functional activity are strongly increased in fibrous septa in rats subjected to experimental fibrosis. To determine the mechanism for the upregulation of Cd73 gene, we cloned the rat Cd73 promoter and then used serial truncation and site-directed mutagenesis to identify key regulatory elements. We identified two consensus SP1 motifs and one SMAD binding site, each of which was necessary for Cd73 gene upregulation. In conclusion, activated HSC upregulate Cd73 gene expression, via specific SP1 and SMAD promoter elements, after myofibroblastic differentiation. The ecto-5=-nucleotidase/CD73 enzyme is a novel cellular marker of activated liver myofibroblasts in vivo and in vitro and thus represents a promising molecular target for antifibrotic therapies in liver diseases.adenosine; hepatic fibrosis; liver myofibroblasts; portal fibroblast; purinergic signaling LIVER FIBROSIS WITH SUBSEQUENT cirrhosis is the most common cause of liver failure. This process results from an imbalance between synthesis and degradation of extracellular matrix (ECM) (25). Liver myofibroblasts are the primary cells responsible for the extensive ECM accumulation observed in liver fibrosis. These cells derive from a variety of sources including liver nonparenchymal cells, periportal and perivascular fibroblasts, and bone marrow-derived fibrocytes (37); however, the best characterized sources of liver myofibroblasts are hepatic stellate cells (HSC) and portal fibroblasts (PF) (16). As they differentiate, liver myofibroblasts become contractile, owing to production of ␣-smooth muscle actin (␣-SMA) and related proteins, and fibrogenic, owing to release of fibrillar collagens and other matrix proteins (16). Thus liver myofibroblasts represent excellent targets for antifibrotic therapies. Extracellular nucleosides (e.g., adenosine) and nucleotides (e.g., ATP) are potent signaling molecules that contribute to liver cellular homeostasis by regulating key physiological functions such as glucose metabolism, cholesterol transpor...

Expression of mediators of purinergic signaling in human liver cell lines

“…NTPDase8 has been identified as a hepatocyte canalicular marker [57]; however, expression was detected in HSC and Mz-ChA-1 cells as well. In contrast, CD73 is expressed in hepatocytes in normal liver; however, expression is dramatically redistributed to HSC [58] in the cirrhotic liver. Thus, there may be a disconnection between mRNA expression and protein expression in the cell lines we have examined.…”

An Elf2-like transcription factor acts as repressor of the mouse ecto-5′-nucleotidase gene expression in hepatic myofibroblasts

“…We have previously cloned and characterized the rat Nt5e promoter and shown that Nt5e gene expression is transcriptionally regulated in activated liver myofibroblasts during fibrosis in the rat species [15]. Here, we use a similar approach to identify Nt5e gene promoter in the mouse species and study the regulation of its transcriptional activity in activated mouse liver myofibroblasts.…”

“…In the healthy liver, Cd73 expression is mostly found in parenchyma, endothelium, and both quiescent hepatic stellate cells, and portal cells [14]. In the fibrosing liver, it is redistributed to, and transcriptionally regulated, the liver MF deriving from activated HSC and PF of murine and human origins [15][16][17][18]. For instance, Sp1/Smad transcription factors regulate Cd73 gene expression in rat HSC-derived liver MF [15].…”

“…In the fibrosing liver, it is redistributed to, and transcriptionally regulated, the liver MF deriving from activated HSC and PF of murine and human origins [15][16][17][18]. For instance, Sp1/Smad transcription factors regulate Cd73 gene expression in rat HSC-derived liver MF [15]. Moreover, other transcription factors that have functional relevance in the setting of chronic liver injury, such as Hif1α [19], Stat3 [20], and Tgf-β [21] have been shown to regulate Cd73 gene expression in other cell types.…”

Purinergic signalling in the liver in health and disease

“…Ecto-5′-nucleotidase (CD73) activity was found to be higher in the quiescent, rather than in the activated phenotype of a HSC line, suggesting that ecto-5′-nucleotidase-dependent adenosine production may play a role in the regulation of quiescent HSC functions [6]. However, it has been claimed recently that ecto-5′-nucleotidase (CD73) gene expression increased in both HSC and portal fibroblasts during myofibroblastic differentiation and represents a promising target for antifibrotic therapy [85].…”