Furin, a predominant convertase of the cellular constitutive secretory pathway, is known to be involved in the maturation of a number of growth/differentiation factors, but the mechanisms governing ...its expression remain elusive. We have previously demonstrated that transforming growth factor (TGF) β1, through the activation of Smad transducers, regulates its own converting enzyme, furin, creating a unique activation/regulation loop of potential importance in a variety of cell fate and functions. Here we studied the involvement of the p42/p44 MAPK pathway in such regulation. Using HepG2 cells transfected withfur P1 LUC (luciferase) promoter construct, we observed that forced expression of a dominant negative mutant form of the small G protein p21ras (RasN17) inhibited TGFβ1-inducedfur gene transcription, suggesting the involvement of the p42/p44 MAPK cascade. In addition, TGFβ induced sustained activation/phosphorylation of endogenous p42/p44 MAPK. Further-more, the role of MAPK cascade in fur gene transcription was highlighted by the use of the MEK1/2 inhibitors, PD98059 or U0126, or co-expression of a p44 antisense construct that repressed the induction of fur promoter transactivation. Conversely, overexpression of a constitutively active form of MEK1 increased unstimulated, TGFβ1-stimulated, and Smad2-stimulated promoter P1 transactivation, and the universal Smad inhibitor, Smad7, inhibited this effect. Activation of Smad2 by MEK1 or TGFβ1 resulted in an enhanced nuclear localization of Smad2, which was inhibited upon blocking MEK1 activity. Our findings clearly show that the activation of the p42/p44 MAPK pathway is involved in fur gene expression and led us to propose a co-operative model whereby TGFβ1-induced receptor activation stimulates not only a Smad pathway but also a parallel p42/p44 MAPK pathway that targets Smad2 for an increased nuclear translocation and enhanced fur gene transactivation. Such an uncovered mechanism may be a key determinant for the regulation of furin in embryogenesis and growth-related physiopathological conditions.
The proprotein convertase furin participates in the maturation/bioactivation of a variety of proproteins involved in chondrogenesis events. These include parathyroid hormone-related peptide (PTHrP), ...an autocrine/paracrine factor that is crucial to both normal cartilage development and cartilage-related pathological processes. Despite the known importance of furin activity in the bioactivation of the polypeptides, the mechanisms that control furin regulation in chondrogenesis remain unknown. To gain insight into the molecular regulation of furin, we used the mouse prechondrogenic ATDC5 cell line, an established in vitro model of cartilage differentiation. Peak expression of both furin mRNA and furin PTHrP maturation was observed during chondrocyte nodule formation stage, an event that correlated with increased mRNA levels of Sox9, a potent high-mobility-group (HMG) box-containing transcription factor required for cartilage formation. Inhibition of furin activity led to a diminution in maturation of PTHrP, suggesting a relationship between Sox9-induced regulation of furin and chondrogenesis events. Transient transfection of Sox9 in nonchondrogenic cells resulted in a marked increase in furin mRNA and in the transactivation of the furin P1A promoter. Direct Sox9 action on the P1A promoter was narrowed down to a critical paired site with Sox9 binding capability in vitro and in vivo. Sox9 transactivation effect was inhibited by L-Sox5 and Sox-6, two Sox9 homologs also expressed in ATDC5 cells. Sox6 inhibitory effect was reduced when using Sox6-HMG-box mutants, indicating a repressive effect through direct HMG-box/DNA binding. Our work suggests a mechanism by which furin is regulated during chondrogenesis. It also adds to the complexity of Sox molecule interaction during gene regulation.
In 2009, Cheung, Fishman and Verma (2009) showed oncology to be under-represented in training program curricula. According to their results, oncology constitutes less than 10% of the content of ...university health programs (in medicine, nursing, and pharmacy) in more than 70% of 84 schools surveyed throughout Canada. This perceived disparity between the oncology curriculum dispensed and actual needs was again described in a similar study published in 2014 that was conducted with 159 educators and 518 residents in Canadian medical schools (Tam, Berry, Hsu, North, Neville, Chan, & Verma, 2014). To our knowledge, it continues to be true of nursing training programs despite cancer having become the leading cause of death in Canada: one in four Canadians will die of cancer, while two in five Canadians will develop cancer (Canadian Cancer Society Advisory Board, 2015). References
En 2009, Cheung, Fishman et Verma ont démontré que l'oncologie était sous-représentée dans les curriculums. Selon leurs résultats, moins de 10 % des contenus universitaires en santé (médecine, ...sciences infirmières, pharmacie) traitent d'oncologie, et ce, dans plus de 70 % des 84 écoles recensées au Canada. Une étude similaire parue en 2014, réalisée auprès de 159 éducateurs et 518 résidents au sein de facultés médicales canadiennes, décrit toujours cette perception d'inadéquation entre le curriculum d'oncologie enseigné et les besoins réels (Tam et al., 2014). À notre connaissance, cette situation prévaut toujours pour la formation en sciences infirmières, malgré que le cancer soit la première cause de décès au Canada. Ainsi, un Canadien sur quatre en mourra et deux Canadiens sur cinq en développeront un (Comité consultatif de la Société canadienne du cancer, 2015).
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.
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.
The convertase furin is involved in the maturation of key growth/aggregation mediators synthesized by the platelet producers, megakaryocytes, but the regulation of furin in these cells remains ...unknown. Computer-assisted search of the furin promoter sequence revealed multiple potential binding motifs for GATA-1, suggesting that furin is expressed and regulated in these cells. Using megakaryoblastic Dami cells, we observed that fur mRNA expression increased gradually on phorbol 12-myristate 13-acetate–induced differentiation, reaching maximum levels (8.3-fold increase) at 10 days. Transient transfections with P1, P1A, or P1B fur-LUC–promoter constructs revealed that in Dami cells, the P1 promoter is the strongest and the most sensitive to forced expression of GATA-1. Coexpression of GATA-1 and its comodulator, Friend of GATA-1 (FOG-1), resulted in a cooperative increase in P1 activity. Deletion analysis indicated that important GATA-1–regulated sequences are located in the most proximal region of the P1 promoter. Further analysis revealed 2 potential GATA-binding motifs at positions −66 and +62. Point mutation of each of the 2 motifs indicated that the intactness of the first GATA site is required for full basal and GATA-1–stimulated promoter activity. Finally, the inhibition of furin activity through gene transfer of the inhibitor α1-AT-PDX led to a block in maturation of the furin substrates transforming growth factor-β1 and platelet-derived growth factor. Taken together, these results indicate that the most proximal GATA element in the P1 promoter is needed forfur gene expression in megakaryoblastic cells. They also suggest that proper regulation of the fur gene in megakaryocytes has an impact on the activation of furin substrates involved in megakaryocyte maturation and platelet functions.
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.
Sequence analysis of the adhesion molecule E-cadherin had revealed a multibasic motif
4PArg-Gln-Lys-Arg
1P, 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.
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-1alpha 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-beta1. 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.
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