Characterization of nerve growth factor precursor protein expression by human prostate stromal cells: A role in selective neurotrophin stimulation ofprostate epithelial cell growth

BACKGROUND. Nerve growth factor (NGF) immunoreactive proteins derived from human prostatic stromal cells (hPS) have been implicated in the paracrine r egulation of prostate epithelial cell growth. However, mature NGF beta does not appear to be expressed by these cells. In order to determine whether N GF precursors are expressed by these cells, we investigated the potential p rocessing and expression of precursor forms of NGF by human prostatic strom al cells, and examined the effects of NGF precursor moieties along with the other members of the neurotrophin family of gene products on soft agar col ony formation of prostate epithelial cells. METHODS. Specific antibodies to the peptide domains defined as N4 and L38, and the NGF beta moiety of prepro-NGF, were used in immunoblot assays to ch aracterize the molecular weight forms of precursor NGF secreted by human pr ostatic stromal cells. The potential processing of NGF precursors with two enzymes, NGF gamma and trypsin, was performed by incubation with stromal ce ll secretory protein containing precursor NGF. The selective effects of the N4, L38, and NGF beta peptide domains of precursor NGF, along with the rem aining members of the neurotrophin family, brain-derived neurotrophic facto r (BDNF), neurotrophin3 (NT-3), and neurotrophin-4/5 (NT-4/5), were examine d for their ability to stimulate growth of prostate tumor epithelial cells in an assay of soft agar colony formation. RESULTS. Immunoblot analysis of stromal cell secretory protein identified N GF precursors of 35 kDa and 27 kDa, along with the partially processed 22-k Da form of pro-NGF, whereas mature NGF beta was not observed. Treatment of precursor NGF with NGF gamma and trypsin did not produce the large intermed iate forms of pro-NGF, although these two enzymes did appear to cleave the N-terminal peptide from NGF beta. Of the N4, L38, and NGF beta peptide doma ins of precursor NGF, only NGF beta significantly stimulated the anchorage- independent growth of TSU-pr1 prostate epithelial cells in soft agar. The o ther members of the neurotrophin family of gene products had no effect on t he anchorage-independent growth of prostate tumor cells. CONCLUSIONS. Human prostate stromal cells secrete the 35-kDa and 27-kDa pre cursor forms of NGF arising from alternate start sites, and the partially p rocessed 22-kDa form of pro-NGF. Whereas the N4, L38, and NGF beta peptide domains present within pro-NGF were previously shown to induce phosphorylat ion of the high-affinity NGF receptor, tropomyosin receptor kinase (Trk), o nly the NGF beta moiety was able to stimulate anchorage-independent growth of prostate tumor cells. Likewise, the other neurotrophin family members di d not stimulate anchorage-independent growth of prostate tumor cells. Hence , it Mould arrear that NGF may be the predominant neurotrophic growth facto r for prostate growth, albeit via precursor forms of NGF, and that its effe ct appears to be selectively mediated via the NGF beta moiety of these NGF precursors. Prostate 41:39-48, 1999. (C) 1999 Wiley-Liss, Inc.