Proteolytic processing of the transforming growth factor beta precursor (pro-TGF beta) is an essential step in the formation of the biologically active TGF beta homodimeric protein (TGF beta). The 361-amino-acid precursor pro-TGF beta 1 has within its primary structure the R-H-R-R processing signal found in many constitutively secreted precursor proteins and potentially recognized by members of the mammalian convertase family of endoproteases. To determine whether cleavage of pro-TGF beta 1 can be achieved by the furin convertase in vitro, purified precursor was incubated in the presence of a truncated/secreted form of the enzyme. Immunoblots showed that the 55-kDa pro-TGF beta 1 was converted into the 44 and 12.5 kDa bands corresponding to the pro-region and the mature monomer, respectively. Treatment of pro-TGF beta 1 with furin resulted in a 5-fold increase in the production of biologically active TGF beta 1. Furthermore, when expressed in the furin-deficient LoVo cells, no processing of pro-TGF beta 1 was observed. In contrast, efficient processing was observed when pro-TGF beta was coexpressed with the furin convertase. Collectively, these results provide evidence that in our experimental systems the TGF beta 1 precursor is efficiently and correctly processed by human furin thus permitting release of the biologically active peptide.

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.

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) ␤1, 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 with fur P1 LUC (luciferase) promoter construct, we observed that forced expression of a dominant negative mutant form of the small G protein p21 ras (RasN17) inhibited TGF␤1-induced fur gene transcription, suggesting the involvement of the p42/p44 MAPK cascade. In addition, TGF␤ 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, TGF␤1-stimulated, and Smad2-stimulated promoter P1 transactivation, and the universal Smad inhibitor, Smad7, inhibited this effect. Activation of Smad2 by MEK1 or TGF␤1 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 TGF␤1-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.

TGF ␤ 1 is known for its potent and diverse biological effects, including immune regulation, and cell growth and differentiation. We have recently shown that TGF ␤ 1 precursor is processed by human furin COOH-terminal to the R-H-R-R 278 cleavage site to generate authentic mature TGF ␤ 1. In the present study, we demonstrate that steady-state furin mRNA levels are increased in rat synovial cells by 2 and 20 ng/ml TGF ␤ 1. Stimulation with TGF ␤ 1 results in a significant increase in furin mRNA levels, starting at 3 h with the peak effect observed at 12 h (2.5-fold increase Ϯ 0.4). TGF ␤ 1 did not increase furin mRNA stability, and treatment of synovial cells with actinomycin D, before TGF ␤ 1 addition prevented the increase in fur gene expression, suggesting that the observed regulation occurs at the level of gene transcription. Treatment of synovial and NRK-49F fibroblastic cells with exogenous TGF ␤ 1 (5 ng/ml) or TGF ␤ 2 (10 ng/ml) translates into an increase in pro-TGF ␤ 1 processing as evidenced by the appearance of a 40-kD immunoreactive band corresponding to the TGF ␤ 1 NH 2 -terminal pro-region. Furin processing activity stimulated by TGF ␤ 2 correlates with significant increase in extracellular mature and heat-activable TGF ␤ 1 as determined by an isoform-specific ELISA assay. Taken together, these results demonstrate for the first time that TGF ␤ 1 upregulates gene expression of its own converting enzyme, and that this expression is translated into augmented processing of the TGF ␤ 1 precursor form. Such adaptive responsiveness of the TGF ␤ 1 convertase may represent an important aspect of TGF ␤ 1 bioavailibility in TGF ␤ 1-related processes and pathological conditions. ( J.

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 TGFbeta1 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 TGFbeta1 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 TGFbeta1-induced luciferase activity indicating the participation of endogenous Smads. Increased levels of TGFbeta1-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 TGFbeta1-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.

Glatiramer acetate is a disease-modifying drug approved for the treatment of relapsing-remitting multiple sclerosis. Since its discovery almost four decades ago, and in particular since the observation of its beneficial clinical effects in the late 1980s and early 1990s, numerous data have been generated and contribute pieces of a puzzle to help explain the mechanism of action of glatiramer acetate. Two major themes have emerged, namely (i) the induction of glatiramer acetate-reactive TH2 immunoregulatory cells, and (ii) the stimulation of neurotrophin secretion in the central nervous system that may promote neuronal repair.

We have developed a method of induction of airway eosinophilia and neutrophilia in guinea pigs by intravenous injection of various types of Sephadex beads. In the first series of experiments, we have shown that G-50 Sephadex beads (Superfine, 24 mg/kg in conscious animals) induced a large accumulation of inflammatory cells in alveolar walls. The bronchoalveolar lavage (BAL) fluid from animals treated with this dose of Sephadex beads contained about 85 x 10(6) cells as compared to 20 x 10(6) cells in control animals. The eosinophils corresponded to 41% of the BAL cell population as assessed with Wright-Giemsa staining. However, in the BAL fluid from these bead-treated animals, a significant increase of monocytes, lymphocytes, and neutrophils was also observed. We have also tested the potency of G-75, G-100, and G-200 Sephadex beads (Superfine) to induce eosinophilia in guinea pig. Nonlethal intravenous doses of G-75 (14.27 mg/kg), G-100 (8.0 mg/kg), and G-200 (10.71 mg/kg) Sephadex beads were selected and induced variable levels of airway eosinophilia and neutrophilia in conscious guinea pigs. The percentage of eosinophil recovered in the BAL fluid corresponded to 35, 61, and 44% of total cells for G-75, G-100, and G-200, respectively. The neutrophils corresponded to 24, 2, and 12% of the total BAL cells for G-75, G-100, and G-200, respectively. Since the size of the beads did not seem to correlate with the intensity of airway eosinophilia and neutrophilia, the effect of lower doses of the G-50 Sephadex beads (9.86-0.43 mg/kg) on the inflammatory cell infiltration was also tested. Results showed that there was a correlation between the neutrophil number and the number of beads (r = 0.996), whereas the number of eosinophils was less directly correlated to the bead number (r = 0.812). The alveolar eosinophils were purified from BAL fluid by centrifugation on a continuous Percoll gradient (65%) to separate eosinophils from neutrophils. Normodense eosinophils (density 1.087-1.100 g/ml) obtained from Sephadex-treated animals were found at the bottom of the continuous Percoll gradient (25 x 10(6); 98% purity). These highly purified eosinophils released thromboxane A2 (TxA2) following stimulation with 2 microM ionophore A23187. The method of accumulation and purification of guinea pig alveolar eosinophils is simple, rapid, and provides a large number of pure normodense cells for further biological studies.(ABSTRACT TRUNCATED AT 400 WORDS)

Kurloff cells may represent a major component of NK cell activity in the guinea pig. We have pursued to characterize the mechanism of their action. Using murine target cells, we found Kurloff cell cytotoxicity to be selective for the NK-sensitive YAC-1 target cell, with minimal activity against the NK-resistant P815 target cell. In the presence of PHA, but not ConA, cytotoxicity was markedly augmented against both YAC-1 and P815. While effector-target conjugate formation was observed with YAC-1 cells but not P815 cells in control cultures, it was augmented with both target cell types in cultures with PHA. Pretreatment alone with PHA was ineffective, however. NK cell activity of Kurloff cells was dependent on extracellular Ca++ and entry of Ca++ into the effector cells, as demonstrated by abrogation of cytotoxicity when extracellular Ca++ was chelated with EDTA or EGTA, or following treatment with the Ca++ channel blockers verapamil and diltiazem. Furthermore, inhibition of PKC by H7 resulted in significant reduction of Kurloff cell-mediated NK activity, while pretreatment of effector cells with the PKC activator TPA enhanced NK activity. Kurloff cells could also be stimulated to produce serine esterases by contact with target cells or treatment with phorbol ester and ionophore. Finally, a majority of Kurloff cells, identified by the monoclonal antibody 14D1, reacted with the human NK cell marker CD56. Taken together, these data suggest that Kurloff cells have NK-like characteristics and activity, with target cell selectivity, and that their lytic mechanisms involve influx of extracellular Ca++, PKC activation and serine esterase production.