Advanced Imaging Research Center, The University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, NE 4.210, Dallas, TX, 75390-8568, USA.
Department of Radiology, UT Southwestern Medical Center, Dallas, TX, 75390-8896, USA.
Mol Imaging Biol. 2021 Apr;23(2):230-240. doi: 10.1007/s11307-020-01557-x. Epub 2020 Nov 2.
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.
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.
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.
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.
我们之前通过 MRI 证实，高葡萄糖会刺激前列腺中锌离子的外排。据我们所知，这种现象以前尚未报道过，其机制尚不清楚。在这里，我们报告了一些初步观察结果，这些结果为我们提供了新的见解，了解在永生的人前列腺上皮细胞系 PNT1A 中葡萄糖刺激的锌分泌（GSZS）期间锌的处理。此外，我们鉴定了人良性前列腺增生（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。
使用细胞内荧光标记物 FluoZin-1-AM 评估 WT 和 mRNA 敲低 PNT1A 细胞系中 ZnT1 和 ZnT4 在锌稳态中的不同作用。此外，使用巴佛洛霉素 A1（Baf）破坏溶酶体并评估 GSZS 期间溶酶体储存的作用。使用细胞外锌响应荧光标记物 ZIMIR 评估暴露于高葡萄糖的 WT 和 ZnT1 mRNA 敲低细胞的动态锌外排。电子显微镜用于评估高葡萄糖刺激下的细胞内锌储存，并评估 Bafilomycin A1 如何影响锌转运。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）染色，这是一种细胞内锌响应荧光标记物，然后使用流式细胞术对细胞类型进行排序。
荧光研究表明，ZnT1 是前列腺上皮细胞中主要的锌外排转运体，通过 mRNA 敲低丧失 ZnT1 并结合溶酶体储存破坏导致细胞溶质锌增加近 4 倍。ZnT1 的敲低显着减少了 GSZS 期间的锌外排。电子显微镜（EM）显示，葡萄糖刺激显着增加了溶酶体中锌的储存；通过 Baf 或 ZnT4 mRNA 敲低破坏溶酶体增加多泡体（MVB）形成和细胞溶质锌水平。在人 BPH 组织中，只有腔上皮细胞含有大量的锌储存颗粒。
暴露于高葡萄糖的前列腺上皮细胞通过 ZnT1 通道诱导锌离子外排并通过 ZnT4 增加溶酶体储存来改变锌稳态。鉴于前列腺癌细胞经历了导致总锌水平降低的深刻代谢变化，了解葡萄糖暴露与前列腺中锌稳态之间的复杂相互作用可能为前列腺癌发生的发展提供新的见解。
