Delsite R, Djakiew D
Department of Cell Biology, Georgetown University Medical Center, Washington, DC, USA.
Prostate. 1999 Sep 15;41(1):39-48. doi: 10.1002/(sici)1097-0045(19990915)41:1<39::aid-pros6>3.0.co;2-e.
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 NGFbeta 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.
Specific antibodies to the peptide domains defined as N4 and L38, and the NGFbeta 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, NGFgamma and trypsin, was performed by incubation with stromal cell secretory protein containing precursor NGF. The selective effects of the N4, L38, and NGFbeta 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.
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 NGFbeta was not observed. Treatment of precursor NGF with NGFgamma 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 NGFbeta. Of the N4, L38, and NGFbeta peptide domains of precursor NGF, only NGFbeta 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.
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 NGFbeta peptide domains present within pro-NGF were previously shown to induce phosphorylation of the high-affinity NGF receptor, tropomyosin receptor kinase (Trk), only the NGFbeta 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 NGFbeta moiety of these NGF precursors.
源自人前列腺基质细胞（hPS）的神经生长因子（NGF）免疫反应性蛋白参与前列腺上皮细胞生长的旁分泌调节。然而，这些细胞似乎不表达成熟的NGFβ。为了确定这些细胞是否表达NGF前体，我们研究了人前列腺基质细胞对NGF前体形式的潜在加工和表达，并检测了NGF前体部分以及神经营养因子家族其他基因产物对前列腺上皮细胞软琼脂集落形成的影响。
使用针对定义为N4和L38的肽结构域以及前体NGF的NGFβ部分的特异性抗体，通过免疫印迹分析来鉴定人前列腺基质细胞分泌的前体NGF的分子量形式。用两种酶NGFγ和胰蛋白酶对NGF前体进行潜在加工，方法是将其与含有前体NGF的基质细胞分泌蛋白一起孵育。在软琼脂集落形成试验中，检测前体NGF的N4、L38和NGFβ肽结构域以及神经营养因子家族的其他成员脑源性神经营养因子（BDNF）、神经营养因子-3（NT-3）和神经营养因子-4/5（NT-4/5）刺激前列腺肿瘤上皮细胞生长的能力。
对基质细胞分泌蛋白进行免疫印迹分析鉴定出35 kDa和27 kDa的NGF前体，以及部分加工的22 kDa前体NGF形式，但未观察到成熟的NGFβ。用NGFγ和胰蛋白酶处理前体NGF并未产生大的中间形式的前体NGF，尽管这两种酶似乎确实从前体NGFβ上切割下了N端肽。在前体NGF的N4、L38和NGFβ肽结构域中，只有NGFβ能显著刺激TSU-pr1前列腺上皮细胞在软琼脂中的非锚定依赖性生长。神经营养因子家族的其他基因产物对前列腺肿瘤细胞非锚定依赖性生长没有影响。
人前列腺基质细胞分泌源自不同起始位点产生的35 kDa和27 kDa前体形式的NGF，以及部分加工的22 kDa前体NGF形式。虽然先前已证明前体NGF中存在的N4、L38和NGFβ肽结构域可诱导高亲和力NGF受体原肌球蛋白受体激酶（Trk）磷酸化，但只有NGFβ部分能够刺激前列腺肿瘤细胞的非锚定依赖性生长。同样，其他神经营养因子家族成员也不刺激前列腺肿瘤细胞的非锚定依赖性生长。因此，似乎NGF可能是前列腺生长的主要神经营养生长因子，尽管是通过NGF的前体形式，并且其作用似乎是通过这些NGF前体的NGFβ部分选择性介导的。
