Evidence that furin is an authentic transforming growth factor-beta1-converting enzyme - PubMed (original) (raw)

Evidence that furin is an authentic transforming growth factor-beta1-converting enzyme

C M Dubois et al. Am J Pathol. 2001 Jan.

Abstract

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.

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Figures

Figure 1.

Figure 1.

A: Processing of TGF-β1 precursor by PCs in LoVo cells. LoVo cells were infected with vaccinia recombinants for both the TGF-β1 precursor (VV: TGF-β) or an unrelated control vaccinia recombinant (VV: POMC) and one of the PCs (PC1/PC3, VV: PC1; PC2, VV: PC2; PC5A, VV: PC5A; furin, VV: FUR, PACE-4, VV: PACE-4; PC5B, VV: PC5B; PC7, VV: PC7) at a multiplicity of infection of 5. Eighteen hours after infection, supernates or cell lysates were electrophoresed on 12% SDS-PAGE gels under reducing conditions. Immunoblots were performed with an anti-human LAP IgG (revealing an ∼55-kd proTGF-β1 and a 40-kd proregion forms) or PAN-specific anti-TGF-β antibodies (revealing an ∼12-kd mature TGF-β1 form). A representative experiment out of four performed is shown. B: Measure of TGF-β1 in cell supernatants or cell lysates. Eighteen hours after cell infection, LoVo cell supernates were collected, heat-activated (80°C, 5 minutes), and used to measure bioactive TGF-β1. A representative experiment out of four performed is shown.

Figure 2.

Figure 2.

A: Processing of TGF-β1 precursor by PCs in BSC-40 cells. Cells were infected with vaccinia recombinants for both the TGF-β1 precursor (VV: TGF-β) or an unrelated control vaccinia recombinant (VV: POMC) and one of the PCs (PC1/PC3, VV: PC1; PC2, VV: PC2; PC5A, VV: PC5A; furin, VV: FUR, PACE-4, VV: PACE-4; PC5B, VV: PC5B) at a multiplicity of infection of 5. Eighteen hours after infection, supernates were collected, concentrated, and electrophoresed on 12% SDS-PAGE gels under reducing conditions. Immunoblots were performed with an anti-human LAP IgG (revealing an ∼55-kd proTGF-β1 and an ∼40-kd proregion forms) or PAN-specific anti-TGF-β antibodies (revealing an ∼12-kd mature TGF-β1 form). A representative experiment out of three performed is shown. B: Measure of TGF-β1 in cell supernates. Eighteen hours after cell infection, cell supernates were collected, heat-activated (80°C, 5 minutes), and used to measure bioactive TGF-β1. A representative experiment out of three performed is shown.

Figure 3.

Figure 3.

A: Inhibition of TGF-β1 processing by α1-PDX. BSC-40 cells were infected with vaccinia recombinants for TGF-β1 precursor (VV: TGF-β), control vaccinia virus (VV: WT), furin-encoding vaccinia virus (VV: FUR) and α1-PDX-encoding vaccinia virus (VV: α1-PDX) at the indicated multiplicity of infection. Eighteen hours after cell infection, cell supernates were collected and electrophoresed on 12% SDS-PAGE gels under reducing conditions. Immunoblots were performed with an anti-human LAP, PAN-specific anti-TGF-β antibodies, furin-specific antisera (revealing an ∼95-kd form) and α1-antitrypsin-specific antisera (revealing an ∼55-kd α1-PDX form). B: Measure of TGF-β1 in cell supernates. Eighteen hours after cell infection, cell supernates were collected, heat-activated (80°C, 5 minutes), and used to quantitate bioactive TGF-β1 as described in Material and Methods. A representative experiment out of two performed is shown.

Figure 4.

Figure 4.

A: Furin and TGF-β1 mRNA expression in hematopoietic/immune cells and tissues. B: Furin and TGF-β1 mRNA expression in mice tissues. Northern blot analysis used total mRNA (5 μg/lane) and a rat TGF-β1 or a rat furin riboprobe. Ethidium bromide staining of 18S is shown as a control for mRNA integrity.

Figure 5.

Figure 5.

PC mRNA regulation by TG F-β1. The rat insulinoma cell line (Rin m5F) (A) or rat fibroblastic kidney cell line (NRK-49F) (B) were incubated for 7, 15, and 24 hours in the presence or absence of 5 ng of recombinant human TGF-β1. Total mRNAs (10 μg/lane) were probed with rat riboprobes specific for furin, PC1, PC5A/PC5B, PACE4, or GAPDH cDNA.

Figure 6.

Figure 6.

Co-regulation of furin and TGF-β1 mRNAs. NRK-49F cells were incubated for 4, 6, or 8 hours in the presence or absence of 10 μmol/L PMA or 5 ng/ml human recombinant TGF-β1. Total mRNAs (10 μg/lane) were probed with rat riboprobe specific for furin, TGF-β1, PC7, and a GAPDH cDNA.

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