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

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Abstract
BACKGROUND Nerve growth factor (NGF) immunoreactive proteins derived from human prostatic stromal cells (hPS) have been implicated in the paracrine regulation of prostate epithelial cell growth. However, mature NGFβ does not appear to be expressed by these cells. In order to determine whether NGF precursors are expressed by these cells, we investigated the potential processing and expression of precursor forms of NGF by human prostatic stromal cells, and examined the effects of NGF precursor moieties along with the other members of the neurotrophin family of gene products on soft agar colony formation of prostate epithelial cells. METHODS Specific antibodies to the peptide domains defined as N4 and L38, and the NGFβ moiety of prepro-NGF, were used in immunoblot assays to characterize the molecular weight forms of precursor NGF secreted by human prostatic stromal cells. The potential processing of NGF precursors with two enzymes, NGFγ and trypsin, was performed by incubation with stromal cell secretory protein containing precursor NGF. The selective effects of the N4, L38, and NGFβ peptide domains of precursor NGF, along with the remaining members of the neurotrophin family, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5), were examined 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 NGF precursors of 35 kDa and 27 kDa, along with the partially processed 22-kDa form of pro-NGF, whereas mature NGFβ was not observed. Treatment of precursor NGF with NGFγ and trypsin did not produce the large intermediate forms of pro-NGF, although these two enzymes did appear to cleave the N-terminal peptide from NGFβ. Of the N4, L38, and NGFβ peptide domains of precursor NGF, only NGFβ significantly stimulated the anchorage-independent growth of TSU-pr1 prostate epithelial cells in soft agar. The other members of the neurotrophin family of gene products had no effect on the anchorage-independent growth of prostate tumor cells. CONCLUSIONS Human prostate stromal cells secrete the 35-kDa and 27-kDa precursor forms of NGF arising from alternate start sites, and the partially processed 22-kDa form of pro-NGF. Whereas the N4, L38, and NGFβ peptide domains present within pro-NGF were previously shown to induce phosphorylation of the high-affinity NGF receptor, tropomyosin receptor kinase (Trk), only the NGFβ moiety was able to stimulate anchorage-independent growth of prostate tumor cells. Likewise, the other neurotrophin family members did not stimulate anchorage-independent growth of prostate tumor cells. Hence, it would appear that NGF may be the predominant neurotrophic growth factor for prostate growth, albeit via precursor forms of NGF, and that its effect appears to be selectively mediated via the NGFβ moiety of these NGF precursors. Prostate 41:39–48, 1999.