Human tryptase cleaves pro-nerve growth factor (pro-NGF): hints of local, mast cell-dependent regulation of NGF/pro-NGF action

Abstract

Several factors regulate nerve growth factor (NGF), which is formed from pro-NGF by intracellular and extracellular enzymatic cleavage. The close proximity between mast cells expressing the protease tryptase and NGF-producing smooth muscle-like peritubular cells in the testes of infertile patients led us to examine whether tryptase is among those factors. Human peritubular cells express functional tryptase receptors (PAR-2). Recombinant enzymatically active β-tryptase increased NGF levels in the culture medium of primary human peritubular cells, but the peptide agonist for PAR-2 (SLIGKV) did not. Neither tryptase nor the peptide increased NGF mRNA levels. To test whether the increase in NGF is due to enzymatic activity of tryptase acting on pro-NGF, supernatants of peritubular cells and synthetic pro-NGF were treated with tryptase. Results of Western blot studies indicate enzymatic cleavage of pro-NGF by active tryptase. Heat-inactivated tryptase or SLIGKV was not effective. Mass spectrometry analysis of in vitro cleavage products from recombinant tryptase and synthetic pro-NGF revealed multiple cleavage sites within the pro-NGF sequence. The results also indicate the generation of mature NGF and smaller NGF fragments as a result of tryptase action. Thus, tryptase-secreting mast cells in the vicinity of pro-NGF/NGF-secreting cells in any human tissue are likely able to alter the ratios of pro-NGF/NGF. As NGF and pro-NGF have different affinities for their receptors, this indicates a novel way by which mast cells, via tryptase, can modify the microenvironment in human tissues with regard to neurotrophin actions.

FIGURE 1.

Mast cells are abundant in infertile men. Mast cells are encountered in human testicular biopsies from men with impaired spermatogenesis (for details, see Ref. 33). They are identified by immunohistochemistry and contain immunoreactive tryptase (A). These cells accumulate in the peritubular compartment (arrows in A) and show the typical features of mast cells, including electron-dense vesicles at the ultrastructural level (B).

FIGURE 2.

NGF levels are increased by tryptase but not by SLIGKV. Using ELISA techniques, we detected that tryptase (100 and 1000 ng/ml) but not the agonist peptide SLIGKV (10 and 100 μm) statistically significantly augmented NGF levels after 24 h. Results are means (bars) plus S.E. are from n = 10 individual HTPC/-F cultures and were normalized to untreated controls. *, p < 0.05; **, p < 0.01.

FIGURE 3.

Tryptase and SLIGKV do not influence cell viability or NGF mRNA levels. A, both tryptase and SLIGKV did not alter cellular ATP levels (n = 7; means ± S.E.; RLU, relative luminescence units). B, semiquantitative RT-PCR (n = 5; top) and quantitative real time PCR (n = 4; bottom) showed that levels of NGF mRNA were not significantly affected by treatment with tryptase or the agonist peptide SLIGKV after 24 h. Bars are means + S.E.; n.s., not significant).

FIGURE 4.

NGF and its precursor pro-NGF in human peritubular cells and culture supernatant. A, immunocytochemistry of NGF and pro-NGF in cultured cells (n = 2). B, NGF (17 kDa) and pro-NGF (37 kDa) were detected in lysates of HTPC/-Fs (L; n = 12) and in the conditioned culture medium (S; n = 6) by Western blotting. Note that the NGF antibody recognizes both pro-NGF and NGF, whereas the pro-NGF antibody does not recognize NGF.

FIGURE 5.

Tryptase can cleave pro-NGF. A, a reduction in the levels of pro-NGF became obvious by Western blot detection of pro-NGF (n = 3) after treatment of the supernatant of HTPCs with or without 1000 ng/ml tryptase for 24 h. B, Western blot studies indicate a decrease of synthetic pro-NGF after treatment with tryptase (1000 ng/ml; 24 h; n = 8) but no decrease by SLIGKV (100 μm; n = 8) or heat-inactivated tryptase (1000 ng/ml; n = 7). Specificity of the pro-NGF antibody was shown by preadsorption (n = 2).

FIGURE 6.

Tryptase cleaves pro-NGF in vitro. A, cleavage sites of tryptase in the pro-NGF sequence (blue) and mature NGF sequence (black) as found by LC-MS/MS analyses in vitro using synthetic human pro-NGF and recombinant human tryptase. Arrows indicate cleavage sites. The cleavage site that is shared by tryptase, furin, and proconvertases is indicated by a red arrow. Note the multiple cleavage sites of the enzyme tryptase within the pro-NGF molecule and the three sites within the mature NGF region. B, MS/MS spectrum of peptide SSSHPIFHR. The N terminus of the peptide represents the N terminus of functional NGF. Annotation of characteristic y and b fragment ions was performed using Scaffold 2.04 software. C, MS/MS spectrum of peptide ALTMDGKQAAWR. The peptide represents an internal fragment of functional NGF. Annotation of characteristic y and b fragment ions was performed using Scaffold 2.04 software.