Nerve growth factor and proprotein convertases furin and PC7 in transected sciatic nerves and in nerve segments cultured in conditioned media: Their presence in Schwann cells, macrophages, and smooth muscle cells

Abstract

Synthesis of proteins such as nerve growth factor (NGF) is induced after nerve lesion. The NGF precursor (pro‐NGF) requires a posttranslational processing by proprotein convertases to become active. In this report, we re‐examine the localization of NGF protein and mRNA in injured nerve and show that the candidate pro‐NGF convertases furin and PC7 colocalize with NGF in non‐neuronal cells in nerve. By Northern blot analysis, 1.5‐kb and 1.3‐kb NGF mRNAs were shown to be increased in distal and immediately proximal nerve segments on days 1, 4, and 14 after lesion; by Western blot analysis, NGF proteins of high molecular weight were detected after injury. In vivo, two phases of NGF immunopositivity were observed, in macrophages and perivascular cells shortly after lesion and in endoneurial cells on day 1 and 4. To identify the cells containing NGF, nerve segments were incubated in serum‐containing medium with or without conditioning by white blood cells isolated from the circulation. Both hybridization and immunoreactivity signals for NGF were elevated after incubation of nerve segments for 4 hours in conditioned media, so that cells with NGF immunoreactivity could be identified by antibodies to specific cell markers. In these nerve fragments, Schwann cells, perivascular smooth muscle cells, and macrophages contained NGF immunoreactivity. The concentration of furin and PC7 mRNA also increased in lesioned nerves. By immunocytochemical investigation of nerve explants, furin and PC7 were detected in endoneurial cells, macrophages and perivascular cells and were colocalized with NGF. These in vitro and in vivo findings suggest that both furin and PC7 are associated with NGF in several cell types of the sciatic nerve and, hence, may be implicated in intracellular processing of pro‐NGF. J. Comp. Neurol. 403:471–485, 1999. © 1999 Wiley‐Liss, Inc.

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

“…Twelve-micrometer-thick cryosections of embryos at E9.5, E10.5, or E18.5 were fixed in 4% formaldehyde at 4°C for 1 h. For PC5/6 labeling in utero, E6.5 uteri from pregnant females were fixed in 4% formaldehyde overnight at 4°C, embedded in paraffin, and cut into 5-m sections. After deparaffination and rehydratation, these or the above cryosections were hybridized with mouse PC5/6 antisense and control sense cRNA probe as described (9,41). To generate a mGdf11 cRNA probe, a cDNA segment covering the sequence encoding amino acids 67-404 was amplified by using sense 5Ј-GCAGCACAGCCGCGAGCTG and antisense 5Ј-GATACCGGTGGAG-CAGCCACATCGATCCAC oligonucleotides and subcloned in pDrive (Qiagen).…”

Molecular cloning and embryonic expression of zebrafish PCSK5 co‐orthologues: Functional assessment during lateral line development

“…Cryosections (12 mm thick) were prepared from embryonic day-19 (E19) mice (whole body sections), fixed in 4% formaldehyde, and hybridized as previously described (Marcinkiewicz et al, 1999) with mouse sense (negative control, not shown) and antisense cRNA probes. The latter (807 bases) correspond to the mouse PC5 coding region for residues 80 to 348 and were synthesized using 35 S-UTP and 35 S-CTP (>1,000 Ci/mmol; Amersham Biosciences, Piscataway, NJ).…”

The Proprotein Convertase PCSK9 Induces the Degradation of Low Density Lipoprotein Receptor (LDLR) and Its Closest Family Members VLDLR and ApoER2

“…The cells were then incubated for 30 min with 1% bovine serum albumin (Fraction V, Sigma) containing 0.1% Triton X-100, followed by overnight incubation at 4°C with rabbit polyconal antibodies Ab:PCSK9 (1,250 Ci/mmol; Amersham Biosciences), to obtain high specific activities of ϳ1000 Ci/mmol. Eight to 10-m whole mouse cryosections obtained at day 1 after birth (P1) were fixed for 1 h in 4% formaldehyde and hybridized overnight at 55°C as described (31). For autoradiography, the sections were dipped in photographic emulsion (NTB-2, Kodak), exposed for 6 -12 days, developed in D19 solution (Kodak), and stained with hematoxylin and eosin.…”