GDF11 forms a bone morphogenetic protein 1-activated latent complex that can modulate nerve growth factor-induced differentiation of PC12 cells

Abstract

All transforming growth factor beta (TGF-beta) superfamily members are synthesized as precursors with prodomain sequences that are proteolytically removed by subtilisin-like proprotein convertases (SPCs). For most superfamily members, this is believed sufficient for activation. Exceptions are TGF-betas 1 to 3 and growth differentiation factor 8 (GDF8), also known as myostatin, which form noncovalent, latent complexes with their SPC-cleaved prodomains. Sequence similarities between TGF-betas 1 to 3, myostatin, and superfamily member GDF11, also known as bone morphogenetic protein 11 (BMP11), prompted us to examine whether GDF11 might be capable of forming a latent complex with its cleaved prodomain. Here we demonstrate that GDF11 forms a noncovalent latent complex with its SPC-cleaved prodomain and that this latent complex is activated via cleavage at a single specific site by members of the developmentally important BMP1/Tolloid family of metalloproteinases. Evidence is provided for a molecular model whereby formation and activation of this complex may play a general role in modulating neural differentiation. In particular, mutant GDF11 prodomains impervious to cleavage by BMP1/Tolloid proteinases are shown to be potent stimulators of neurodifferentiation, with potential for therapeutic applications.

FIG. 1.

Cleaved propeptide forms a noncovalent association with GDF11 and is cleaved by BMP1. (A) Schematic of the precursor molecule of GDF11. A small box on the NH₂ terminus represents the protein C epitope, while a vertical line represents the site at which propeptide and mature GDF11 sequences are separated by a recognition site for cleavage by furin-like proprotein convertases. (B) Electrophoretic patterns are shown on a Coomassie blue-stained 4-to-15% acrylamide gradient SDS-PAGE gel for GDF11 starting material (S.M.), affinity purified by binding to an anti-protein C column, and for the same material subsequent to overnight incubation in the absence (−) or presence (+) of BMP1. Approximate molecular masses (in kilodaltons) are shown (in parentheses) for the various bands. (C) Western blot analysis of GDF11 processing by BMP1 was performed, using an antibody directed against the protein C epitope on the NH₂ terminus on the GDF11 prodomain. SDS-PAGE separation of proteins prior to Western blotting was on a 4-to-15% gradient gel.

FIG. 2.

BMP1 cleavage site within the GDF11 prodomain. Shown is the alignment of the site at which BMP1 cleaves the GDF11 prodomain with sites at which BMP1 has previously been shown to cleave other substrates (11). Aspartate residues conserved at the P1′ positions of the various scissile bonds are in boldface, as are methionines and residues with aromatic side chains, noted as being NH₂ terminal to scissile bonds in the majority of previously identified substrates of BMP1-like proteinases.

FIG. 3.

BMP1 cleavage within prodomain sequences activates GDF11. Purified GDF11 latent complex starting material (GDF11 S.M.) is shown to have negligible levels of GDF11 signaling activity, similar to those of buffer containing no protein, and the same is true for GDF11 latent complex incubated in the absence of BMP1 (GDF11-BMP1), in an assay employing A204 cells transfected with the pGL3(CAGA)₁₂ reporter vector in which luciferase expression is under the control of multiple SMAD-responsive elements. In contrast, GDF11 latent complex incubated in the presence of BMP1 (GDF11 + BMP1) shows an almost sixfold induction of GDF11 signaling activity levels. GDF11 starting material in which the Asp in the P1′ position of the BMP1 cleavage site has been substituted for by Ala (GDF11 D120A S.M.) has no signaling activity above that of buffer, nor does the same material incubated either in the absence (GDF11 D120A-BMP1) or presence (GDF11+BMP1) of BMP1. As a control, BMP1 alone (BMP1) is shown to have no signaling activity in the assay. Numbers on the ordinate axis represent fold increases in signaling compared to buffer alone.

FIG. 4.

Inhibition of GDF11 signaling by addition of exogenous GDF11 prodomain-Fc fusion proteins. (A) A204 cells transfected with the pGL3(CAGA)₁₂ reporter vector were incubated with 10 ng/ml of active GDF11, GDF-8/myostatin, or activin and increasing amounts of either GDF11 prodomain-Fc fusion protein (Pro-Fc) or recombinant Fc domain (Fc), the latter as a negative control. (B) In the same type of assay, 10 ng/ml active GDF11 was incubated with increasing amounts of wild-type or mutant prodomain-Fc fusion protein, or recombinant Fc domain (as a negative control), with results demonstrating that GDF11 prodomain-Fc fusion protein bearing the D120A mutation inhibits active GDF11 as effectively as does the wild-type prodomain-Fc fusion protein. (C) In the same type of assay, preincubation of wild-type GDF11 prodomain-Fc fusion protein with BMP1 destroys its ability to inhibit signaling by active GDF11, whereas pre-incubation of prodomain-Fc fusion protein bearing the D120A substitution with BMP1 does not have a marked effect on its ability to inhibit active GDF11. Numbers on the ordinate axes in panels A to C represent fold increases in signaling compared to buffer alone.

FIG. 5.

MEFs employ BMP1/Tolloid-like proteinases to cleave propeptide sequences, thus activating GDF11. (A) Schematic of recombinant precursor GDF11 expressed by transfected MEFs in panels B and C. Small boxes on the NH₂ and COOH termini represent protein C and Flag epitope tags, respectively. A vertical line represents the site at which propeptide and mature GDF11 sequences are separated by a recognition site for cleavage by furin-like proprotein convertases. (B and C) Western blot assays using anti-protein C antibodies (B) or anti-Flag antibodies (C) were employed to monitor pro-GDF11 processing in transfected cultures of wild-type MEFs (WT) or MEFs derived from embryos doubly homozygous null for the Bmp1 and Tll1 genes (Bmp1 Tll1 null). (D and E) Conditioned media from wild-type (WT) or Bmp1 Tll1 doubly null MEFs (Bmp1 Tll1 null) transfected with empty vector, or from the same types of cells transfected with a pro-GDF-11 expression vector (WT-GDF11 and Bmp1 Tll1 null-GDF11), were tested for GDF11 signaling activity in the A204 cell pGL3(CAGA)₁₂ reporter gene assay. (D) Aliquots of 100, 200, and 500 μl were compared for each sample of conditioned medium from wild-type or doubly null MEF cells that had been transfected with the expression construct or with empty vector. Final volumes for the reporter gene assay were in each case 500 μl, with DMEM added as necessary. (E) Aliquots (200 μl) of samples of conditioned media were heat activated for 10 min at 80°C and then added to 300 μl DMEM prior to testing in the reporter gene assay. Numbers on the ordinate axis in panels D and E represent fold increases in signaling compared to buffer alone.

FIG. 6.

Active GDF11 is a potent, dose-dependent inhibitor, and GDF11 prodomain, especially if impervious to cleavage by BMP1-like proteinases, is a potentiator of NGF-induced PC12 cell differentiation. (A and B) PC12 cells were incubated in the presence of 50 ng/ml NGF plus 0, 2.5, 5, 10, or 20 ng/ml active GDF11 (A) or plus 5 ng/ml active GDF11 (B) in the presence of 250 ng/ml wild-type (Pro-Fc) or D120A-substituted (Pro D120A-Fc) GDF11 prodomain Fc fusion protein, or 80 ng/ml Fc domain (as a negative control). (C) Conditions for the experiment shown in panel C were the same as for panel B, except that exogenously added active GDF11 was not used. Percentages of PC12 cells undergoing differentiation to a neural-like phenotype under the various conditions were determined as described in Materials and Methods.

FIG. 7.

A highly specific inhibitor of BMP1-like proteinases potentiates the NGF-induced differentiation of PC12 cells. PC12 cells were incubated in the presence of 50 ng/ml NGF and 0, 1, 2, 5, or 10 μM BI-1 inhibitor.

FIG. 8.

Expression of endogenous GDF11 and BMP1, but not GDF8 or mTLL1, by PC12 cells. (A) RT-PCR was performed on the RNA from PC12 cells incubated in the presence (+) or absence (−) of 50 ng/ml NGF. (B) The lysates of PC12 cells incubated in the presence (+) or absence (−) of 50 ng/ml NGF were analyzed by Western blotting for the presence of endogenous BMP1. The same blot was reprobed with anti-α-tubulin antibody as a control for loading. (C) PC12 cells were mock transfected (−) or transfected with synthetic RNAi duplexes for GDF11 or for scrambled sequences. Cells were then treated with NGF and assayed for percent differentiation. (D) RT-PCR was performed on RNA from the PC12 cells of the experiment shown in panel C. PCR was for 25, 30, and 35 cycles for each sample.

FIG. 9.

Although they have opposite effects on differentiation, both NGF and active GDF11 induce PC12 cell cycle arrest and p27^Kip1 expression. (A and B) PC12 cells incubated in the presence (+) or absence (−) of 50 ng/ml NGF and/or 5 ng/ml active GDF11 were assayed for the percentage of cells incorporating BrdU (A), or were lysed and assayed by immunoblotting for levels of p27^Kip1 expression (B). Levels of α-tubulin were also determined by immunoblotting, as a control for loading. (C) PC12 cells incubated in the presence (+) or absence (−) of 50 ng/ml NGF and/or 250 ng/ml wild-type (Pro-Fc) or D120A-substituted (Pro D120A-Fc) GDF11 prodomain-Fc fusion protein, or 80 ng/ml Fc domain (as a negative control), were assayed for levels of p27^Kip1 expression.