Cell death prevention, mitogen-activated protein kinase stimulation, and increased sulfatide concentrations in Schwann cells and oligodendrocytes by prosaposin and prosaptides

Abstract

Prosaposin, the precursor of saposins A, B, C, and D, was recently identified as a neurotrophic factor. Herein prosaposin was found to increase sulfatide concentrations in primary and transformed Schwann cells (iSC) and oligodendrocytes (differentiated CG4 cells). Of the four mature saposins, only saposin C was found to increase sulfatide concentrations in these cell types. A similar result was obtained by using peptides (prosaptides) encompassing the neurotrophic sequence located in the saposin C domain. Dose-response curves demonstrated maximal enhancement by saposin C and prosaptides at low nanomolar concentrations (5-10 nM). The increase in sulfatide concentration by a 14-mer prosaptide, TX14(A), in CG4 oligodendrocytes was about 3-fold greater than in primary Schwann cells. A mutant prosaptide with a single amino acid replacement of Asn --> Asp was inactive. Prosaptides did not induce cell proliferation of primary Schwann cells, iSC cells, or CG4 oligodendrocytes but nanomolar concentrations of prosaptides prevented cell death of iSC cells and CG4 oligodendrocytes. Immunoblot analysis demonstrated that phosphorylation of both mitogen-activated protein kinase p-42 and p-44 isoforms were enhanced 3- to 5-fold after 5 min of treatment with prosaptides at concentrations of 1-5 nM. These findings suggest that prosaposin and prosaptides bind to a receptor that initiates signal transduction to promote myelin lipid synthesis and prolong cell survival in both Schwann cells and oligodendrocytes. Prosaposin may function as a myelinotrophic factor in vivo during development and repair of myelinated nerves explaining the deficiency of myelin observed in prosaposin-deficient mice and humans.

Figure 1

Enhancement of sulfatide content in primary Schwann cells by prosaposin and saposin A–D. Primary Schwann cells were incubated in medium containing prosaposin (250 ng/ml) or individual saposins (50 ng/ml) for 48 h, and sulfatide concentrations were determined.

Figure 2

Effect of prosaptides on sulfatide content in primary Schwann cells. (a) Effect of the 14-mer prosaptide, TX14(A). The cells were cultured in medium containing TX14(A) at concentrations from 0 to 50 ng/ml for 48 h and duplicate values were averaged. (b) Effects of 769P and 769M and an antiserum raised against 769P. Sulfatide concentrations were determined.

Figure 3

Effect of TX14(A) on sulfatide content in CG4 oligodendrocyte cells. (a) The cells were cultured for 48 h in medium containing TX14(A) at concentrations from 0 to 20 ng/ml. Control lane contains 5 ng of sulfatide. (b) Graphical representation of results shown in a.

Figure 4

Effect of TX14(A) on growth of primary Schwann cells. (a) Cells were incubated for 48 h in DMEM with 10% fetal calf serum and bovien pituitary extract (0–84 μg/ml) in the absence (open square) or presence (solid circle) of 50 μM forskolin. (b) Cells were incubated for 48 h in DMEM with 10% fetal calf serum and 0–50 μM forskolin (open squares) or 0–50 nM TX14(A) in the absence (solid circle) or presence (solid square) of 50 μM forskolin.

Figure 5

Prevention of cell death in iSC Schwann cells by TX14(A). The 14-mer prosaptide was added at 1 or 5 nM. After 48 h of culture, trypan blue-positive cells were scored.

Figure 6

Phosphorylation of MAPK in primary Schwann and iSC cells after stimulation with TX14(A). (a Upper) Western blot of MAPK phosphorylation in primary Schwann cell lysates with a polyclonal antibody that recognizes phosphorylated MAPK p42 and p44. (Lower) Western blot of MAPK phosphorylation in primary Schwann cell lysates with a polyclonal antibody that recognizes unphosphorylated MAPK p42 and p44. Lanes: 1, control; 2, TX14(A) at 1 nM; 3, TX14(A) at 5 nM. (b) Ratio of phosphorylated MAPK to unphosphorylated MAPK in primary Schwann cells by densitometric analysis. (c) Ratio of phosphorylated MAPK to unphosphorylated MAPK in iSC Schwann cells by densitometric analysis.