Francine Grondin

Evidence that Furin Is an Authentic Transforming Growth Factor-β1-Converting Enzyme

Transforming growth factor (TGF)-beta1 plays an essential role in cell growth and differentiation. It is also considered as a gatekeeper of immune homeostasis with gene disruption leading to autoimmune and inflammatory diseases. TGF-beta1 is produced as an inactive precursor polypeptide that can be efficiently secreted but correct proteolytic cleavage is an essential step for its activation. Assessment of the cleavage site has revealed a unique R-H-R-R sequence reminiscent of proprotein convertase (PC) recognition motifs and has previously demonstrated that this PC-like cleavage site is correctly cleaved by furin, a member of the PC family. Here we report that among PC members, furin more closely satisfies the requirements needed to fulfill the role of a genuine TGF-beta1 convertase. Even though six members of the PC family have the ability to cleave TGF-beta1, ectopic expression of alpha(1)-antitrypsin Portland (alpha(1)-AT-PDX), a potent furin inhibitor, blocked 80% of TGF-beta1 processing mediated by endogenous enzymes as demonstrated in an in vitro digestion assay. Genetic complementation of a furin-deficient LoVo cell line with the wild-type gene restores the production of mature and bioactivable TGF-beta1. Moreover, both furin and TGF-beta are coordinately expressed and regulated in vitro and in vivo in the hematopoietic and immune system, an important tissue target. These results demonstrate for the first time that furin is an authentic and adaptive TGF-beta1-converting enzyme whereas other members of the PC family might substitute or supplement furin activity. Our study advances our comprehension of the complexity of the TGF-beta system and should facilitate the development of therapeutically useful TGF-beta inhibitors.

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.

Hypoxia-inducible Factor Mediates Hypoxic and Tumor Necrosis Factor α-induced Increases in Tumor Necrosis Factor-α Converting Enzyme/ADAM17 Expression by Synovial Cells

Chronic hypoxia and inflammatory cytokines are hallmarks of inflammatory joint diseases like rheumatoid arthritis (RA), suggesting a link between this microenvironment and central pathological events. Because TACE/ADAM17 is the predominant protease catalyzing the release of tumor necrosis factor α (TNFα), a cytokine that triggers a cascade of events leading to RA, we examined the regulation of this metalloprotease in response to hypoxia and TNFα itself. We report that low oxygen concentrations and TNFα enhance TACE mRNA levels in synovial cells through direct binding of hypoxia-inducible factor-1 (HIF-1) to the 5′ promoter region. This is associated with elevated TACE activity as shown by the increase in TNFα shedding rate. By the use of HIF-1-deficient cells and by obliterating NF-κB activation, it was determined that the hypoxic TACE response is mediated by HIF-1 signaling, whereas the regulation by TNFα also requires NF-κB activation. As a support for the in vivo relevance of the HIF-1 axis for TACE regulation, immunohistological analysis of TACE and HIF-1 expression in RA synovium indicates that TACE is up-regulated in both fibroblast- and macrophage-like synovial cells where it localizes with elevated expression of both HIF-1 and TNFα. These findings suggest a mechanism by which TACE is increased in RA-affected joints. They also provide novel mechanistic clues on the influence of the hypoxic and inflammatory microenvironment on joint diseases.

Proprotein cleavage of E-cadherin by furin in baculovirus over-expression system: potential role of other convertases in mammalian cells.

Sequence analysis of the adhesion molecule E‐cadherin had revealed a multibasic motif [4PArg‐Gln‐Lys‐Arg1P], reminiscent of the minimal cleavage signal for furin, the prototype of the proprotein convertase family, and/or other members sharing similar sequence specificity. Mutation of this site was sufficient to abolish processing of E‐cadherin in fibroblasts reinforcing the possibility that proprotein convertases are involved in the maturation of this adhesion molecule. Here we demonstrate that even though furin can efficiently and specifically cleave proE‐cadherin in a baculovirus‐based co‐expression system, the furin‐deficient LoVo cells were found to process endogenous E‐cadherin as efficiently as normal cell lines. This suggests, for the first time, that E‐cadherin is not only a substrate for furin but for other mammalian convertases sharing similar sequence specificity.

Involvement of Smads in TGFβ1-induced furin (fur) transcription

Furin is recognized as being one of the main convertases of the cellular constitutive secretion pathway but the mechanisms regulating its expression are still unknown. We have previously demonstrated that TGFβ1 up‐regulates its own converting enzyme, furin, creating a novel activation/regulation cycle of potential importance in a variety of physiological and pathophysiological conditions. The fur (fes upstream region) gene is regulated via three alternative promoters; P1, P1A, and P1B. To gain insight into the molecular mechanism(s) underlying this up‐regulation, we performed transient cell transfections with P1, P1A, and P1B promoter luciferase constructs. Transfection experiments in HepG2 cells revealed that fur P1 promoter is the strongest and the most sensitive to TGFβ1 stimulation (5 ng/ml) (3.2‐fold vs. 2.4‐fold for P1A and 2.1‐fold for P1B). Cotransfection with either a dominant negative mutant form of Smad2 [Smad2(3SA)] or a known Smad inhibitor [Smad7] inhibit constitutive and TGFβ1‐induced luciferase activity indicating the participation of endogenous Smads. Increased levels of TGFβ1‐induced transcriptional activation of the P1 promoter by overexpression of Smad2 and/or Smad4 is greatly reduced in the presence of Smad2(3SA) and completely inhibited by Smad7, suggesting the participation of endogenous Smad2/Smad4 complexes. Furthermore, the fork‐head activin signal transducer (FAST‐1), known to interact with Smad2/Smad4 complexes, is a potent stimulator of TGFβ1‐induced transactivation of the fur P1 promoter. Five prime‐deletion analysis of this promoter identified the proximal region (between positions −8734 and −7925), as the nucleotide stretch that carries most of the transcriptional activation of fur P1 promoter by Smad2. Overall, the present data demonstrate that Smad2 and Smad4 possibly in complex with FAST‐1 or other DNA binding partners participate in the constitutive and inducible transactivation of the fur P1 promoter. This represents the first detailed study of the transcriptional regulation of the fur gene.

Enhanced TGFβ1 maturation in high five cells coinfected with recombinant baculovirus encoding the convertase furin/pace: Improved technology for the production of recombinant proproteins in insect cells

One important limitation of the widely used insect baculovirus overexpression system is its inefficiency to properly process heterologous proteins which are initially biosynthesized as larger inactive precursor proteins. One example is transforming growth factor beta 1 (TGFbeta1), a 25-kDa homodimeric protein with pleiotropic functions. As many growth factors, the inactive TGFbeta1 precursor molecule needs to be proteolytically cleaved C-terminal to a basic sequence to yield the mature and active homodimer. In insect cells, a large proportion of overexpressed TGFbeta1 was found in an inactive precursor form suggesting that the levels of endogenous convertases are limiting for the production of mature and bioactive TGFbeta1 in this system. We have demonstrated that furin, a member of a novel family of mammalian prohormone convertases (PCs) can efficiently process TGFbeta1 precursor resulting in the production of the mature and active growth factor. Taking advantage of this observation, we have developed an improved overproduction system for TGFbeta1 by coexpressing prohTGFbeta1 and human furin convertase in High Five cells. Using this system, the production of mature active TGFbeta1 increased in a dose-dependent fashion reaching up to 7. 8-fold the amount obtained with the growth factor only. Thus, eliminating the rate-limiting step in recombinant TGFbeta1 production maximizes its processing efficiency and the yield of the mature active growth factor. Such simple and efficient technology could be useful for large scale production of other proproteins which undergo similar maturation processes and share furin recognition sequences at the junction between the proregion and the mature polypeptide.

The serpin proteinase inhibitor 8: an endogenous furin inhibitor released from human platelets.

The cornerstone of hemostasis is the ability of the organism to limit the enzymatic processes involved, thereby avoiding thrombosis. For this, anticoagulant systems in place involve serpins, such as PAI-1 and antithrombin III, which bind to their targeted serine proteases and limit their period of activity. We have previously identified the serine protease furin as a platelet-derived enzyme with an intrinsic role in platelet functions. We now report that furin enzymatic activity decreased rapidly following platelet activation, corresponding with the increase in formation of a high 180 M(r) SDS-stable complex composed of furin and the PI8 serpin. PI8 is shown to be a platelet-derived constituent, synthesized by megakaryocytes and stored in platelets prior to its release. Immunoprecipitation and purification of the PI8-furin complex confirmed their direct interaction and indicates that one of the roles of PI8 is to inhibit furin enzymatic activity. Furthermore, our findings demonstrate the inhibitory capacity of exogenous PI8 in platelet aggregation assays. The finding that PI8 is released by platelets and controls functional responses suggests a role for this serpin in platelet-regulated pathophysiological responses.