In vivo functions of the proprotein convertase PC5/6 during mouse development: Gdf11 is a likely substrate

Abstract

The proprotein convertase PC5/6 cleaves protein precursors after basic amino acids and is essential for implantation in CD1/129/Sv/C57BL/6 mixed-background mice. Conditional inactivation of Pcsk5 in the epiblast but not in the extraembryonic tissue bypassed early embryonic lethality but resulted in death at birth. PC5/6-deficient embryos exhibited Gdf11-related phenotypes such as altered anteroposterior patterning with extra vertebrae and lack of tail and kidney agenesis. They also exhibited Gdf11-independent phenotypes, such as a smaller size, multiple hemorrhages, collapsed alveoli, and retarded ossification. In situ hybridization revealed overlapping PC5/6 and Gdf11 mRNA expression patterns. In vitro and ex vivo analyses showed that the selectivity of PC5/6 for Gdf11 essentially resides in the presence of a P1' Asn in the RSRR downward arrowN cleavage motif. This work identifies Gdf11 as a likely in vivo specific substrate of PC5/6 and opens the way to the identification of other key substrates of this convertase.

Fig. 1.

In situ hybridization analysis of PC5/6 expression in utero at E6.5. (A and B) Paraffin sections were hybridized with a ³⁵S-labeled cRNA PC5/6 an antisense (A) or sense (B) probe and were autoradiographed. (C and D) Sections were stained with cresyl violet and dipped in an autoradiography emulsion. (D) A higher magnification of the squared area in C showed a strong labeling on the large nuclei of the embryonic giant trophoblast cells (arrows). ep, epiblast.

Fig. 2.

Epiblast-specific KO of PC5/6 at E18.5. (A) Intercrossing of flox/flox mice with Δ1/+ mice harboring a heterozygote knockin of Cre at the Meox2 locus generates ≈25% of Δ1/flox and Cre positive embryos. All of the embryos that have a shortened or no tail were characterized by the only presence of Δ1 alleles at the PC5/6 locus and were Cre positive (cKO). Most of them exhibited abdominal herniations (red arrows). (B) In the smallest mutant embryos, subcutaneous hemorrhages were observed (white arrow). (C) In situ hybridization analysis of PC5/6 mRNA in cryosections of embryos at E18.5 using an antisense cRNA probe revealed a drastic diminution of the signal in cKO embryos, similar to the background obtained with a control sense probe (data not shown).

Fig. 3.

Skeletal defects in the PC5/6 mutant embryos at E18.5. (A) Note the smaller size, absence of tail, and retarded ossification of mandibles, limbs and vertebrae. (B) Up to four additional thoracic segments and (C) up to three additional vertebrosternal ribs were present in mutant embryos. Ribs were asymmetrically attached to the sternum that was incompletely fused. (D) Up to two additional lumbar segments (L7 and L8) were observed. Note the reduced ossification of vertebrae in the lumbar and sacral regions and the smaller size of the ilium bones.

Fig. 4.

Kidney, lung, and liver phenotypes of cKO embryos at E18.5. Paraffin sections were stained with hematoxylin/eosin. (A) Twelve of 13 embryos analyzed lacked both kidneys (ki). ad, adrenals; li, liver. (B) Note the collapsed alveoli and airways in the lung and (C) the multiple liver hemorrhages in mutant embryos.

Fig. 5.

Localization of PC5/6 and Gdf11 mRNA in WT embryos. Whole-mount in situ hybridization (Left) was performed at E9.5, whereas in situ hybridization on cryosections (Right) was carried out in embryos at E9.5 or E10.5 with cRNA PC5/6 and Gdf11 sense (data not shown) and antisense probes. de, diencephalon; ha, heart atrium; hv, heart ventricle; mb, mandibles; me, mesencephalon; psm, presomitic paraxial mesoderm; sca, spinal canal; sch, spinal chord; sfm, somite-forming paraxial mesoderm; tb, tail bud; te, telencephalon.

Fig. 6.

In vitro and ex vivo processing of mouse Gdf11. At the top are shown a schematic of the mGdf11 structure and the percentage in vitro cleavage of a 12-mer peptide encompassing the Gdf11 S1 site (WT) and its N297D mutant by purified soluble PC5/6A, furin, PC7, and PACE4. At the bottom are shown Western blot analyses using a C-terminal FLAG (FG) Ab of HEK293 media obtained 24 h after coexpression of recombinant WT mouse Gdf11 cDNA or its empty vector with the full-length forms of the indicated PCs (Left) or after coexpression of PC5/6A or PC5/6B with the Gdf11 cleavage site mutants R293A and R296A (Right). The migration positions of the uncleaved proGdf11 and its mature Gdf11 forms are emphasized.