Zinc and manganese homeostasis closely interact in mammalian cells.

Understanding the homeostatic interactions among essential trace metals is important for explaining their roles in cellular systems. Recent studies in vertebrates suggest that cellular Mn metabolism is related to Zn metabolism in multifarious cellular processes. However, the underlying mechanism remains unclear. In this study, we examined the changes in the expression of proteins involved in cellular Zn and/or Mn homeostatic control and measured the Mn as well as Zn contents and Zn enzyme activities to elucidate the effects of Mn and Zn homeostasis on each other. Mn treatment decreased the expression of the Zn homeostatic proteins metallothionein (MT) and ZNT1 and reduced Zn enzyme activities, which were attributed to the decreased Zn content. Moreover, loss of Mn efflux transport protein decreased MT and ZNT1 expression and Zn enzyme activity without changing extracellular Mn content. This reduction was not observed when supplementing with the same Cu concentrations and in cells lacking Cu efflux proteins. Furthermore, cellular Zn homeostasis was oppositely regulated in cells expressing Zn and Mn importer ZIP8, depending on whether Zn or Mn concentration was elevated in the extracellular milieu. Our results provide novel insights into the intricate interactions between Mn and Zn homeostasis in mammalian cells and facilitate our understanding of the physiopathology of Mn, which may lead to the development of treatment strategies for Mn-related diseases in the future.

Reduced Mn uptake of pleiotropic ZIP8 SNP is caused by its loss of Mn-responsive accumulation on the cell-surface.

Zrt/Irt-like protein 8 (ZIP8), which is a zinc transporter, plays a pivotal role as a manganese transporter. Recent studies have shown that a ZIP8 SNP (rs13107325 C→T, A391T) is associated with multiple diseases, likely by causing systemic Mn deficiency. However, the underlying molecular mechanisms remain unclear. We attempted to address this issue in cell-based experiments using Madin-Darby canine kidney cells stably expressing ZIP8 WT or the A391T SNP mutant under the control of the Tet-regulatable promoter. We showed that the A391T mutant lost the property of Mn-responsive accumulation on the cell surface, which was observed in WT ZIP8. We also showed that the loss of Mn-responsive accumulation of A391T mutant was associated with its reduced Mn uptake, compared to WT ZIP8, in the Mn uptake assay using the radioisotope 54Mn. Our results potentially explain how the ZIP8 A391T substitution is associated with disease pathogenesis caused by Mn deficiency.

Sophisticated expression responses of ZNT1 and MT in response to changes in the expression of ZIPs.

The zinc homeostatic proteins Zn transporter 1 (ZNT1) and metallothionein (MT) function in dampening increases in cytosolic zinc concentrations. Conversely, the expression of ZNT1 and MT is expected to be suppressed during decreases in cytosolic zinc concentrations. Thus, ZNT1/MT homeostatic responses are considered to be essential for maintaining cellular zinc homeostasis because cellular zinc concentrations are readily altered by changes in the expression of several Zrt-/Irt-like proteins (ZIPs) under both physiological and pathological conditions. However, this notion remains to be tested experimentally. Here, we investigated the aforementioned homeostatic process by analyzing ZNT1 and MT protein expression in response to ZIP expression. Overexpression of cell-surface-localized ZIPs, such as ZIP4 and ZIP5, increased the cellular zinc content, which caused an increase in the expression of cell-surface ZNT1 and cytosolic MT in the absence of zinc supplementation in the culture medium. By contrast, elimination of the overexpressed ZIP4 and ZIP5 resulted in decreased expression of ZNT1 but not MT, which suggests that differential regulation of ZNT1 and MT expression at the protein level underlies the homeostatic responses necessary for zinc metabolism under certain conditions. Moreover, increased expression of apically localized ZIP4 facilitated basolateral ZNT1 expression in polarized cells, which indicates that such a coordinated expression mechanism is crucial for vectorial transcellular transport. Our results provide novel insights into the physiological maintenance of cellular zinc homeostasis in response to alterations in cytosolic zinc concentrations caused by changes in the expression of ZIPs.