Purpose

We have previously demonstrated by MRI that high glucose stimulates efflux of zinc ions from the prostate. To our knowledge, this phenomena had not been reported previously and the mechanism remains unknown. Here, we report some initial observations that provide new insights into zinc processing during glucose-stimulated zinc secretion (GSZS) in the immortalized human prostate epithelial cell line, PNT1A. Additionally, we identified the subtypes of zinc-containing cells in human benign prostatic hyperplasia (BPH) tissue to further identify which cell types are likely responsible for zinc release in vivo.

Procedure

An intracellular fluorescence marker, FluoZin-1-AM, was used to assess the different roles of ZnT1 and ZnT4 in zinc homeostasis in wild type (WT) and mRNA knockdown PNT1A cell lines. Additionally, Bafilomycin A1 (Baf) was used to disrupt lysosomes and assess the role of lysosomal storage during GSZS. ZIMIR, an extracellular zinc-responsive fluorescent marker, was used to assess dynamic zinc efflux of WT and ZnT1 mRNA knockdown cells exposed to high glucose. Electron microscopy was used to assess intracellular zinc storage in response to high glucose and evaluate how Bafilomycin A1 affects zinc trafficking. BPH cells were harvested from transurtheral prostatectomy tissue and stained with fluorescent zinc granule indicator (ZIGIR), an intracellular zinc-responsive fluorescent marker, before being sorted for cell types using flow cytometry.

Results

Fluorescent studies demonstrate that ZnT1 is the major zinc efflux transporter in prostate epithelial cells and that loss of ZnT1 via mRNA knockdown combined with lysosomal storage disruption results in a nearly 4-fold increase in cytosolic zinc. Knockdown of ZnT1 dramatically reduces zinc efflux during GSZS. Electron microscopy (EM) reveals that glucose stimulation significantly increases lysosomal storage of zinc; disruption of lysosomes via Baf or ZnT4 mRNA knockdown increases multi-vesicular body (MVB) formation and cytosolic zinc levels. In human BPH tissue, only the luminal epithelial cells contained significant amounts of zinc storage granules.

Conclusions

Exposure of prostate epithelial cells to high glucose alters zinc homeostasis by inducing efflux of zinc ions via ZnT1 channels and increasing lysosomal storage via ZnT4. Given that prostate cancer cells undergo profound metabolic changes that result in reduced levels of total zinc, understanding the complex interplay between glucose exposure and zinc homeostasis in the prostate may provide new insights into the development of prostate carcinogenesis.

中文翻译：

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ZnT1 和 ZnT4 在前列腺上皮细胞葡萄糖刺激锌分泌中的作用

目的

我们之前通过 MRI 证明，高葡萄糖会刺激锌离子从前列腺流出。据我们所知，这种现象以前没有报道过，其机制仍然未知。在这里，我们报告了一些初步观察结果，这些观察结果为永生化人前列腺上皮细胞系 PNT1A 中葡萄糖刺激锌分泌 (GSZS) 过程中的锌加工提供了新的见解。此外，我们还确定了人类良性前列腺增生（BPH）组织中含锌细胞的亚型，以进一步确定哪些细胞类型可能负责体内锌的释放。

程序

使用细胞内荧光标记 FluoZin-1-AM 评估 ZnT1 和 ZnT4 在野生型 (WT) 和 mRNA 敲低 PNT1A 细胞系中锌稳态中的不同作用。此外，巴弗洛霉素 A1 (Baf) 用于破坏溶酶体并评估 GSZS 期间溶酶体储存的作用。ZIMIR 是一种细胞外锌响应荧光标记物，用于评估暴露于高葡萄糖的 WT 和 ZnT1 mRNA 敲低细胞的动态锌流出。使用电子显微镜评估细胞内锌储存对高葡萄糖的反应，并评估巴弗洛霉素 A1 如何影响锌运输。从经尿道前列腺切除术组织中收获 BPH 细胞，并用荧光锌颗粒指示剂 (ZIGIR)（一种细胞内锌响应荧光标记物）染色，然后使用流式细胞术分选细胞类型。

结果

荧光研究表明，ZnT1 是前列腺上皮细胞中主要的锌外流转运蛋白，通过mRNA 敲低结合溶酶体储存破坏而导致 ZnT1 损失，导致胞质锌增加近 4 倍。GSZS 期间 ZnT1 的敲低显着减少了锌的流出。电子显微镜（EM）显示葡萄糖刺激显着增加溶酶体中锌的储存；通过Baf 或 ZnT4 mRNA 敲低来破坏溶酶体会增加多囊泡体 (MVB) 的形成和胞质锌水平。在人类 BPH 组织中，只有管腔上皮细胞含有大量的锌储存颗粒。

结论

前列腺上皮细胞暴露于高葡萄糖会通过ZnT1 通道诱导锌离子流出并通过ZnT4增加溶酶体储存来改变锌稳态。鉴于前列腺癌细胞经历深刻的代谢变化，导致总锌水平降低，了解前列腺中葡萄糖暴露和锌稳态之间复杂的相互作用可能为前列腺癌发生的发展提供新的见解。