Claire M. Dubois

Transforming Growth Factor β1 Induces Hypoxia-inducible Factor-1 Stabilization through Selective Inhibition of PHD2 Expression

The hypoxia-inducible transcription factor-1 (HIF-1) is central to a number of pathological processes through the induction of specific genes such as vascular endothelial growth factor (VEGF). Even though HIF-1 is highly regulated by cellular oxygen levels, other elements of the inflammatory and tumor microenvironment were shown to influence its activity under normal oxygen concentration. Among others, recent studies indicated that transforming growth factor (TGF) β increases the expression of the regulatory HIF-1α subunit, and induces HIF-1 DNA binding activity. Here, we demonstrate that TGFβ acts on HIF-1α accumulation and activity by increasing HIF-1α protein stability. In particular, we demonstrate that TGFβ markedly and specifically decreases both mRNA and protein levels of a HIF-1α-associated prolyl hydroxylase (PHD), PHD2, through the Smad signaling pathway. As a consequence, the degradation of HIF-1α was inhibited as determined by impaired degradation of a reporter protein containing the HIF-1α oxygen-dependent degradation domain encompassing the PHD-targeted prolines. Moreover, inhibition of the TGFβ1 converting enzyme, furin, resulted in increased PHD2 expression, and decreased basal HIF-1α and VEGF levels, suggesting that endogenous production of bioactive TGFβ1 efficiently regulates HIF-1-targeted genes. This was reinforced by results from HIF-1α knock-out or HIF-1α-inhibited cells that show impairment in VEGF production in response to TGFβ. This study reveals a novel mechanism by which a growth factor controls HIF-1 stability, and thereby drives the expression of specific genes, through the regulation of PHD2 levels.

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.

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.

Differential involvement of NF-κB and MAP kinase pathways in the generation of inflammatory cytokines by human neutrophils

The ability of human neutrophils to express a variety of genes encoding inflammatory mediators is well documented, and mounting evidence suggests that neutrophil‐derived cytokines and chemokines contribute to the recruitment of discrete leukocyte populations at inflammatory sites. Despite this, our understanding of the signaling intermediates governing the generation of inflammatory cytokines by neutrophils remains fragmentary. Here, we report that inhibitors of the p38 MAPK and MEK pathways substantially diminish the release of (and in the case of p38 inhibitors, the gene expression of) several inflammatory cytokines in neutrophils stimulated with LPS or TNF. In addition, various NF‐κB inhibitors were found to profoundly impede the inducible gene expression and release of inflammatory cytokines in these cells. The MAPK inhibitors did not affect NF‐κB activation; instead, the transcriptional effects of the p38 MAPK inhibitor appear to involve transcriptional factor IID. Conversely, the NF‐κB inhibitors failed to affect the activation of MAPKs. Finally, the MAPK inhibitors were found to prevent the activation a key component of the translational machinery, S6 ribosomal protein, in keeping with their post‐transcriptional impact on cytokine generation. To our knowledge, this constitutes the first demonstration that in neutrophils, the inducible expression of proinflammatory cytokines by physiological stimuli largely reflects the ability of the latter to activate NF‐κB and selected MAPK pathways. Our data also raise the possibility that NF‐κB or MAPK inhibitors could be useful in the treatment of inflammatory disorders in which neutrophils predominate.

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.

Inflammatory Cytokine Production by Human Neutrophils Involves C/EBP Transcription Factors

A growing number of neutrophil-derived cytokines have proven to be crucial to various inflammatory and immune processes in vivo. Whereas C/EBP (CCAAT/enhancer-binding protein) transcription factors are important for neutrophil differentiation from myeloid precursors, we report herein that they also regulate cytokine production in mature neutrophils. All known C/EBP proteins but C/EBPγ are expressed in neutrophils; most isoforms localize to the nucleus, except for C/EBPα, which is cytoplasmic. Neutrophil stimulation does not alter the overall levels, cellular distribution, or turnover of C/EBP proteins; it also does not further induce the constitutive DNA-binding activity detected in nuclear extracts, consisting of C/EBPβ and C/EBPε. However, nuclear C/EBPβ is rapidly phosphorylated upon cell stimulation, suggesting that it can activate cytokine promoters. Indeed, the transactivation of an IL-8 promoter-luciferase construct in a human neutrophil-like cell line was impaired when its C/EBP or NF-κB sites were mutated. Overexpression of a C/EBP repressor also impeded IL-8 promoter transactivation, as well as the generation of IL-8, Mip-1α, and Mip-1β in this cellular model, whereas TNF-α generation was mostly unaffected. Finally, overexpression of a C/EBPβ mutant (T235A) as well as chromatin immunoprecipitation assays unveiled an important role for this residue in cytokine induction. This is the first demonstration that C/EBP factors are important regulators of cytokine expression in human neutrophils.

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.

Autotaxin Promotes Cancer Invasion via the Lysophosphatidic Acid Receptor 4: Participation of the Cyclic AMP/EPAC/Rac1 Signaling Pathway in Invadopodia Formation

The ability of cancer cells to invade and metastasize is the major cause of death in cancer patients. Autotaxin (ATX) is a secreted lysophospholipase whose level of expression within tumors correlates strongly with their aggressiveness and invasiveness. ATX is the major enzyme involved in the production of lysophosphatidic acid (LPA), a phospholipid that is known to act mostly through its three first characterized receptors (LPA(1), LPA(2), and LPA(3)). Tumor cell invasion across tissue boundaries and metastasis are dependent on the capacity of invasive cancer cells to breach the basement membrane. This process can be initiated by the formation of the actin-rich cell protrusions, invadopodia. In this study, we show that ATX is implicated in the formation of invadopodia in various cancer cells types and this effect is dependent on the production of LPA. We further provide evidence that LPA(4) signaling in fibrosarcoma cells regulates invadopodia formation downstream of ATX, a process mediated through the activation of EPAC by cyclic AMP and subsequent Rac1 activation. Results using LPA(4) shRNA support the requirement of the LPA(4) receptor for cell invasion and in vivo metastasis formation. This work presents evidence that blocking the LPA receptor, LPA(4), in fibrosarcoma cells could provide an additional tool to improve the efficacy of treatment of metastasis in patients. Because LPA receptors and ATX are currently being targeted in preclinical trials, the current findings should stimulate future studies to evaluate the expression pattern and clinical outcome of LPA(4), together with other LPA receptors, in various cancer patients.

Hypoxia-Induced Invadopodia Formation Involves Activation of NHE-1 by the p90 Ribosomal S6 Kinase (p90RSK)

The hypoxic and acidic microenvironments in tumors are strongly associated with malignant progression and metastasis, and have thus become a central issue in tumor physiology and cancer treatment. Despite this, the molecular links between acidic pH- and hypoxia-mediated cell invasion/metastasis remain mostly unresolved. One of the mechanisms that tumor cells use for tissue invasion is the generation of invadopodia, which are actin-rich invasive plasma membrane protrusions that degrade the extracellular matrix. Here, we show that hypoxia stimulates the formation of invadopodia as well as the invasive ability of cancer cells. Inhibition or shRNA-based depletion of the Na+/H+ exchanger NHE-1, along with intracellular pH monitoring by live-cell imaging, revealed that invadopodia formation is associated with alterations in cellular pH homeostasis, an event that involves activation of the Na+/H+ exchange rate by NHE-1. Further characterization indicates that hypoxia triggered the activation of the p90 ribosomal S6 kinase (p90 RSK), which resulted in invadopodia formation and site-specific phosphorylation and activation of NHE-1. This study reveals an unsuspected role of p90RSK in tumor cell invasion and establishes p90RS kinase as a link between hypoxia and the acidic microenvironment of tumors.