NELL2 Modulates Cell Proliferation and Apoptosis via ERK Pathway in the 1 Development of Benign Prostatic Hyperplasia

31 Benign prostatic hyperplasia (BPH) is a quite common illness but its etiology and 32 mechanism remain unclear. Neural epidermal growth factor-like like 2 (NELL2) plays 33 multifunctional roles in neural cell growth and is strongly linked to the urinary tract 34 disease. Current study aims to determine the expression, functional activities and 35 underlying mechanism of NELL2 in BPH. Human prostate cell lines and tissues from 36 normal human and BPH patients were utilized. Immunohistochemical staining, 37 immunofluorescent staining, RT-PCR and Western-blotting were performed. We 38 further generated cell models with NELL2 silenced or overexpressed. Subsequently, 39 proliferation, cycle, and apoptosis of prostate cells were determined by CCK-8 assay 40 and flow cytometry analysis. The epithelial-mesenchymal transition (EMT) and 41 fibrosis process were also analyzed. Our study revealed that NELL2 was upregulated 42 in BPH samples and localized in the stroma and the epithelium compartments of 43 human prostate tissues. NELL2 deficiency induced a mitochondrial-dependent cell 44 apoptosis, and inhibited cell proliferation via phosphorylating ERK1/2 activation. 45 Additionally, suppression of ERK1/2 with U0126 incubation could significantly 46 reverse NELL2 deficiency triggered cell apoptosis. Consistently, overexpression of 47 NELL2 promoted cell proliferation and inhibited cell apoptosis. However, NELL2 48 interference was observed no effect on EMT and fibrosis process. Our novel data 49 demonstrated that upregulation of NELL2 in the enlarged prostate could contribute to 50 the development of BPH through enhancing cell proliferation and inhibited a 51 mitochondrial-dependent cell apoptosis via the ERK pathway. The NELL2-ERK 52 system might represent an important target to facilitate the development of future 53 therapeutic approaches in BPH. 56 57


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Downregulation of NELL2 resulted in cell apoptosis and ERK pathway 303 activation. 304 To better understand the underlying mechanism of this cell survival-inhibiting 305 effect, cell cycle and cell apoptosis was further detected via flow cytometry analysis.

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Compared with the control cells, downregulation of NELL2 had no effect on cell 307 cycle promotion (Fig 3A) but triggered a significant increase of the apoptotic BPH-1 308 and WPMY-1 cells (Fig. 3C). The alteration of proteins involved in cell cycle and cell cascade, was also significantly increased (Fig 3D, E). Moreover, we found that 313 Cytochrome c was also upregulated, suggesting that NELL2 deficiency could trigger 314 prostatic cell apoptosis through a mitochondrial-dependent manner (Fig 3D, E). The  Downregulation of NELL2 had no effect on EMT and fibrosis process. 331 It was reported that NELL2 promoted neural differentiation through increasing 332 N-cadherin expression(19). As a calcium-dependent glycoprotein, N-cadherin was 333 critical for a variety of pathophysiological mechanisms, including EMT in BPH(38). blotting analysis (Fig. 5B). The CCK-8 assay indicated that NELL2 promoted cell 347 growth at 48 and 72h (Fig. 5C). The flow cytometry analysis also revealed that 348 NELL2 inhibited the apoptotic BPH-1 and WPMY-1 cells (Fig. 5D). Furthermore, triggered a significant increase of the apoptotic cells (Fig. 6B). Furthermore, NELL2 362 overexpression promoted cell growth ( Fig 6C) and inhibit cell apoptosis (Fig 6D) in 363 RWPE-1 cells.

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Our novel data demonstrated that NELL2 was localized in the stroma and the 366 epithelium compartments of human prostate tissues and upregulated in hyperplastic 367 prostate tissues. We also showed that NELL2 deficiency could induce a 368 mitochondrial-dependent cell apoptosis, and inhibit cell proliferation via 369 phosphorylating ERK1/2 activation in vitro (Fig 7). Our study suggested that NELL2  Therefore, it is of great necessity for our subsequent study to determine that NELL2 385 proteins can either be expressed solely in the epithelia or in both epithelia and stroma. 386 We demonstrated that NELL2 protein was localized in both epithelia and stroma of 387 human hyperplastic prostate, which was not entirely consistent with a previous report

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There is indirect evidence that all these three MAPK cascades might be implicated in  (data not shown). It will be interesting to investigate in the future.

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Collectively, our current study reveals that NELL2 protein is localized in both the Moreover, this process is completely dependent on activation of the ERK pathway.

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Our data suggests that the NELL2-ERK system could play vital roles in the 524 development of BPH and it might be rediscovered as new therapeutic targets for BPH.

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In the future, genetic mice with conditional knockout NELL2 gene in prostate could 526 be an intriguing model to explore the in vivo function of NELL2.

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Data availability statement 529 The data used to support the findings of this study are available from the 530 corresponding author upon reasonable request.     Clinical Science. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version is available at https://doi.org/10.1042/CS20210476