Regulation of the human cathepsin E gene by the constitutive androstane receptor

Abstract

Cathepsin E (CTSE) is an aspartic protease that has been linked to antigen processing and innate immunity. Elevated levels of CTSE expression have also been associated with several forms of cancer, including carcinomas exhibiting highly invasive character. In this study, we performed DNA microarray experiments, together with quantitative reverse transcriptase PCR analyses and enzymatic activity determinations to identify human CTSE as a novel target gene for regulation by the constitutive androstane receptor (CAR), a nuclear receptor activated by the liver tumor promoting agent, phenobarbital. In particular, two motifs within the 5′-flanking region of the human CTSE gene were identified as direct sites of interaction with CAR/RXRα heterodimers, a direct repeat-3 site at position −766 and a direct repeat-4 site at position −1407. Thus, these studies demonstrate CAR-mediated regulation of CTSE within primary hepatocyte cultures from several individual donors and suggest that elevated CTSE activity may play a functional role in the etiology of hepatocarcinogenesis. KeywordsCathepsin E; Constitutive androstane receptor; Hepatocytes; Primary hepatocytes; Liver; Human; Hepatocytes Cathepsin E (CTSE) 1 is an intracellular aspartic protease that hydrolyzes various biologically active peptides [1]. Unlike related proteases, CTSE is localized outside of the lysosomal system and likely functions in extralysosomal proteolysis [2]. Although its exact biological functions remain elusive, several physiological roles of CTSE have been reported, including roles in immune defense and antigen processing [3][4][5]. In this respect, CTSEdeficient mice exhibit markedly increased susceptibility to both gram positive and gram negative bacterial infection, likely due to impaired regulation of bacterial elimination [5]. Given the apparent association of enhanced CTSE expression with hepatocellular carcinoma, we hypothesize that liver tumor promoting agents may function to enhance CTSE expression levels in liver and that increased proteolytic activity may underlie certain mechanistic aspects of the tumor promotion process. In the studies reported here, we identify CTSE expression in human hepatoctyes. In particular, microarray expression profiling analyses indicated that exposures to the classical liver tumor promoter, phenobarbital (PB), resulted in increased CTSE transcript levels within primary hepatocyte cultures from several individual donors, a result that was confirmed by quantitative RT-PCR experiments. Further, we demonstrate that the constitutive androstane receptor (CAR) acts as a transcriptional regulator of the human CTSE gene and that these events lead to PB-inducible increases in functional hepatic CTSE enzymatic activity. Materials and methods ChemicalsPhenobarbital sodium salt was obtained from UWMC Drug Services (Seattle, WA). Clotrimazole, meclizine and dimethyl sulfoxide (DMSO) were purchased from Sigma (St. Louis, MO). CITCO was purchased from BIOMOL Research Laboratories (Plymouth Meeting, PA). Cell cultureCO...

Emerging roles of cathepsin E in host defense mechanisms

“…Recent evidence has demonstrated that the A variety of intra-and extracellular proteases, including aspartic proteinases and cysteine cathepsins, are highly up-regulated in certain types of cancers (69)(70)(71)(72)(73)(74)(75)(76)(77)(78)(79)(80)(81)(82)(83)(84). In addition, there is a change in the localization of the endolysosomal proteases to extracellular spaces.…”

“…The proteolytic activity of extracellular aspartic and cysteine cathepsins has long been associated with many types and stages of cancer (73)(74)(75)(76)(77)(81)(82)(83)(84)(85)(86)(87). Cathepsin E is also known to be up-regulated and secreted in several forms of cancer and thus suggests their clinical utility as a potential biomarker for these cancers (69,(71)(72)(73)(74)(75)(76)(77)(78)(79)(80)88). However, the clinical significance of the increased cathepsin E expression in carcinogenesis has been controversial.…”

The human constitutive androstane receptor promotes the differentiation and maturation of hepatic-like cells

“…Subsequent to their activation, both CAR and the pregnane X receptor (PXR) function as transcriptional regulators of genes participating in hepatic biotransformation and drug transport (Chang and Waxman, 2006), affecting the dispositional fate of many drugs (Wei et al, 2000), chemical carcinogens (Xie et al, 2003) as well as endogenous substances such as steroids (Xie et al, 2003), heme and bilirubin (Xie et al, 2003), thyroid hormone (Maglich et al, 2004), and cholesterol/bile acids (Guo et al, 2003; Stedman et al, 2005). CAR’s function has also emerged as an important regulator of lipid and energy metabolism (Wada et al, 2009) and as a modulator of genes involved in a diverse array of physiological processes that include cell growth and differentiation (Page et al, 2007; Liu et al, 2009; Baskin-Bey et al, 2006; Blanco-Bose et al, 2008). …”