Stephanie McMahon

Hypoxia-enhanced Expression of the Proprotein Convertase Furin Is Mediated by Hypoxia-inducible Factor-1 IMPACT ON THE BIOACTIVATION OF PROPROTEINS

Hypoxia is a common tumorigenesis enhancer, mostly owing to its impact on gene expression of many angiogenic and invasion-related mediators, some of which are natural substrates for the proprotein convertase furin. Analysis of furin promoters revealed the presence of putative binding sites for hypoxia-inducible factor-1 (HIF-1), a transcription complex that plays a pivotal role in cellular adaptation to hypoxia. In fact, we demonstrate herein that the levels of fur mRNA, encoding furin, are remarkably increased upon hypoxic challenge. Cotransfection of a HIF-1α dominant negative form in wild-type (WT) cells or transfection of a furin promoter-reporter gene in HIF-1-deficient cells indicated the requirement of HIF-1 for furin promoter activation by hypoxia. Direct HIF-1 action on the furin promoter was identified as a canonical hypoxia-responsive element site with enhancer capability. The hypoxic/HIF-1 regulation of furin correlated with an increased proteolytic activation of the substrates membrane-type 1 matrix metalloproteinase and transforming growth factor-β1. Our findings unveil a new facet of the physiological consequences of hypoxia/HIF-1, through enhanced furin-induced proteolytic processing/activation of proproteins known to be involved in tumorigenesis.

TACE/ADAM-17 maturation and activation of sheddase activity require proprotein convertase activity.

Proprotein convertases (PCs) have been proposed to play a role in tumor necrosis factor‐α converting enzyme (TACE) processing/activation. Using the furin‐deficient LoVo cells, as well as the furin‐proficient synoviocytes and HT1080 cells expressing the furin inhibitor α1‐PDX, we demonstrate that furin activity alone is not sufficient for effective maturation and activation of the TACE enzyme. Data from in vitro and in vivo cleavage assays indicate that PACE‐4, PC5/PC6, PC1 and PC2 can directly cleave the TACE protein and/or peptide. PC inhibition in macrophages reduced the release of soluble TNF‐α from transmembrane pro‐TNF‐α. We therefore conclude that furin, in addition to other candidate PCs, is involved in TACE maturation and activation.

Leukotriene D4‐induced, epithelial cell‐derived transforming growth factor β1 in human bronchial smooth muscle cell proliferation

Background Cysteinyl‐leukotrienes (cys‐LTs) orchestrate many pathognomonic features of asthma in animal models of allergic airway inflammation, including bronchial smooth muscle cell (BSMC) hyperplasia. However, because cys‐LTs alone do not induce mitogenesis in monocultures of human BSMC, the effect observed in vivo seemingly involves indirect mechanisms, which are still undefined.

Alternative pathway for the role of furin in tumor cell invasion process: Enhanced MMP-2 levels through bioactive TGFβ

The mammalian convertase furin plays a significant role in tumorigenesis and its overexpression was observed in a number of different cancer types. To date, however, few mechanisms of action have been described. Most of the information available concerns the invasion step and designates MT1-MMP, through the activation of MMP-2, as the bona fide substrate mediating furin activity. However, recent reports indicate furin-independent pathways for MT1-MMP activation. To gain further insights into the role of furin in the invasion process, we studied the in vitro invasive capacity of LoVo cells, a furin-deficient adenocarcinoma cell line transfected with wild-type furin. Furin complementation resulted in an increased cell invasiveness that correlated with their capacity to produce MMP-2. Chemical blockage of MMPs activity with BB-3103 or MMP-2-specific antibodies revealed that the increased invasive capacity of furin-complemented cells was mediated by MMP-2. Unexpectedly, furin complementation did not change the status of MT1-MMP expression or activation, but instead resulted in the production of mature and bioactive TGFbeta1. Western blot-analysis of TGFbeta1 fragmentation species indicated that TGFbeta maturation step required furin activity, whereas results from TGFbeta-inducible reporter assays in the presence of MMP inhibitors or exogenous MMP-2 suggested that the activation step was under MMP influence. In addition, blockage with TGFbeta neutralizing antibodies revealed that furin-induced invasiveness was mediated by endogenous production of TGFbeta. Taken together, our findings established the existence of a novel alternative/complementary pathway by which furin increases tumor cell invasion through an amplification/activation loop between MMP-2 and TGFbeta.

Leukotriene D4 Up-Regulates Furin Expression through CysLT1 Receptor Signaling

Leukotriene (LT)D(4) is suggested to play a role in airway remodeling, which is characterized by fibrogenesis and airway smooth muscle cell hyperplasia. In this study, we investigated the effects of LTD(4) on the expression of furin, a proprotein convertase involved in the maturation/activation of several substrates implicated in the remodeling processes. HEK293 cells stably transfected with the CysLT1 receptor were used to study the transcriptional regulation of furin by LTD(4). Stimulation of the cells with LTD(4) resulted in a time- and concentration-dependent induction of furin mRNA and protein expression. The study of furin gene (fur) promoters P1, P1A, and P1B revealed a selective transactivation of the P1 promoter by LTD(4). Mutations in the activator protein (AP)-1-binding element of the P1 promoter resulted in the partial loss of transactivation by LTD(4). Binding of AP-1 transcription factor to fur P1 promoter after stimulation with LTD(4) was demonstrated by electrophoretic mobility shift assay, and supershift assays indicated the formation of c-Jun/c-Fos complexes. LTD(4) induced the maturation of the furin substrates membrane-type 1 matrix metalloproteinase and transforming growth factor-beta1, which was inhibited by the furin inhibitor alpha1-PDX. Finally, LTD(4) induced furin gene expression in monocytic THP-1 cells, which was abrogated using a selective CysLT1 receptor antagonist and inhibitors of the mitogen-activated protein kinases MEK-1, p38, and JunK. Our data show for the first time that LTD(4), via the CysLT1 receptor, can transcriptionally activate furin production with consequent maturation of furin substrates relevant to airway remodeling. These findings suggest that CysLT1 is involved in remodeling processes through modulation of furin transcription.

Signalling Pathways Leading to Furin Expression in Cancer

With the continually increasing body of evidence implicating furin in health as well as in a large number of diseases, it has become important to identify the molecules involved in the signaling pathways leading to gene expression of this enzyme. Furin is dramatically overexpressed in cancer and various inflammatory conditions clearly pointing to the molecular mechanisms controlling its expression as a potential target for eventual rational therapeutic intervention. Currently, regulation of the most proximal furin promoter is beginning to be understood in the context of activation of certain signalling pathways involved in the growth and progression of neoplasms. In addition, several factors that modulate the furin-transcriptional unit are being elucidated at the level of cis-acting sequences. Strategies that take advantage of the signalling pathways that regulate furin expression for the treatment of cancer will be discussed