Background
Cadmium is ranked 7
th in the “Top 20 Hazardous Substances Priority List “ by the Agency for Toxic Substance and Disease Registry and the U.S. Environmental Protection Agency [
1]. Individuals at the highest risk for cadmium-related disease include cigarette smokers, those on a steady diet rich in high fiber foods or contaminated shellfish, women having low body-iron stores, and malnourished populations [
2‐
5]. In acute doses, Cd
+2 has been shown to cause damage to the central nervous system, lung, bone, gastrointestinal tract, liver, ovary, testis, placenta, and the developing embryo [
6,
7]. Chronic exposure to low amounts of Cd
+2 has been shown to cause renal proximal tubular metabolic acidosis and osteomalacia (renal Fanconi syndrome) [
8]. The elimination of Cd
+2 from the body is very slow and thus accumulates as a total body burden, predominantly in the kidney, with age. Cadmium is also classified as a human “Category 1” carcinogen due to its strong correlation with lung cancer [
5,
9‐
11]. Association of Cd
+2 with cancers of other organs have also been suggested, but the data are currently inconclusive. This laboratory has been interested in the possible association of Cd
+2 with the development and progression of human urothelial cancer. There is an extremely strong association of human bladder cancer with the consumption of cigarettes and tobacco, with some reports suggesting a two- to four-fold increased risk and that 50% of the bladder cancers in men would not occur in the absence of cigarette smoking [
12,
13]. The number of cigarettes smoked, degree of inhalation, type of tobacco, use of filters, and smoking cessation have all been shown to have specific relationships with the development of bladder cancer [
14]. Cigarette smoke is by far one of the greatest sources of Cd
+2 exposure with each cigarette containing between 1–2 μg of Cd
+2 and 40-60% of the Cd
+2 in inhaled smoke enters the systemic circulation [
7,
13‐
17]. The high level of Cd
+2 accumulation in individuals who smoke cigarettes, along with the strong association of bladder cancer and smoking, is a major factor indirectly implicating Cd
+2 in the development of urothelial cancer. There are also several epidemiological studies which have implicated Cd
+2 in the development of bladder cancer [
5,
9,
18,
19].
This laboratory has developed a model of Cd
+2 and As
+3 induced human urothelial cancer through the direct malignant transformation of the UROtsa cell line, a cell line retaining characteristics of human urothelium [
20,
21]. This laboratory has also been interested in As
+3, a bladder carcinogen highly implicated in the incidence of bladder cancers due to human exposure in drinking water [
22‐
24], and have developed an
in vitro bladder carcinogenesis model for As
+3 similar to that of Cd
+2. The As
+3-transformed cells serve as an interesting control to that transformed by Cd
+2, due to the divergent chemical properties of As
+3. The laboratory has subsequently isolated and characterized 6 additional Cd
+2 transformed cell lines and 5 additional As
+3 transformed cell lines [
25‐
27]. These cell lines were all shown to retain morphological characteristics consistent with human urothelial cancer and to display phenotypic differences characteristic of tumor heterogeneity. The histology of subcutaneous tumor transplants produced by these transformed isolates displayed histological features of human urothelial carcinoma with areas of squamous differentiation. This observation is important since urothelial carcinoma is the most prominent type of bladder cancer in western countries and accounts for over 95% of all cases and is 5
th in overall occurrence [
28]. To the authors’ knowledge, there has been no examination of the mechanism by which Cd
+2 might enter the urothelial cell in order to elicit cell transformation. Recent studies have shown a relationship between a specific allelic difference in the mouse
Slc39a8 gene encoding the ZIP8 transporter and the specific phenotypes of Cd-induced testicular necrosis and acute renal failure [
29,
30]. Subsequent studies have shown that the ZIP8 transporter, which is utilized by Cd
+2 for transport can also transport one or more essential divalent cation(s) that are critical to cellular function [
31]. In cell culture studies, manganese (Mn) was shown to be the best inhibitor of ZIP8-mediated Cd
+2 uptake; possessing a low Km of 2.2 μM. These studies show that ZIP8 is a Cd
+2 or Mn
+2/HCO
3- symporter, but a role for the transport of Zn
+2 cannot be ruled out. ZIP8 has been localized to the apical surface of two cell types; between the blood and vascular endothelial cells of the testis [
29,
30], and between the glomerular filtrate and renal proximal tubule cells [
30]. ZIP8 has also been shown to exist in glycosylated and non-glycosylated forms [
30,
31] and can alter their localization as a function of extracellular Zn
+2 concentration [
32]. The role of ZIP transporters in cadmium damage to the testis and kidney has been the subject of a recent review [
33]. The finding that the ZIP8 transporter can transport Cd
+2 into several cell types suggested that this transporter might also be operative in the urothelial cell. The first goal of the present study was to determine the expression and localization of ZIP8 in HPT cells since the
in situ expression of ZIP8 has previously been shown for this cell type. The second goal was to determine if ZIP8 was expressed in normal human urothelium and if expression was altered in human urothelial cancer. The final goal of the study was to determine ZIP8 expression and localization in human urothelial cells transformed by Cd
+2 and As
+3.
Discussion
The first goal of the present study was to determine the expression and localization of ZIP8 in HPT cells. These cells were chosen for analysis since the
in situ expression of ZIP8 has previously been shown for this cell type along with an association of ZIP8 with Cd-induced damage to the proximal tubule [
29‐
33]. In addition, the renal MDCK cell line, which retains the property of vectorial active transport, has been used to characterize the localization and expression of ZIP8 [
30‐
32]. An analysis of the expression of ZIP8 in the HPT cells largely confirmed what has been found in previous studies employing the MDCK cell line [
30‐
32]. The HPT cells were shown to express two forms of the ZIP8 protein, one at approximately 49 kDa and the other at approximately 80 kDa. The 49 kDa band identified by the ZIP8 antibody is in agreement with the molecular weight expected for the non-glycosylated ZIP8 protein as derived from the sequence available from the NCBI database. The human 49 kDa band identified as ZIP8 in the HPT cells is also in general agreement with that obtained for MDCK cells transfected with the mouse ZIP8 sequence [
31]. The approximate 80 kDa band found in extracts of the HPT cells is assumed to be the glycosylated form of the ZIP8 protein. This is based on the molecular weight and association with the membrane fraction of the HPT cell extracts, findings similar to that found for the MDCK cells transfected with the mouse ZIP8 sequence [
31]. The ZIP8 protein was also localized to the endoplasmic reticulum and apical cell surface of the HPT cells, an identical localization to that found for the ZIP8-transfected MDCK cells [
31]. The finding that there were occasional profiles of HPT cells with paranuclear staining of ZIP8 defines a difference in ZIP8 localization compared to the MDCK cells. There are also findings in other organs and cell types, such as lung and breast epithelium, that show different localizations of ZIP8 and higher molecular weights for the glycosylated form of ZIP8 [
34,
35]. The significance of glycosylated and non-glycosylated forms is interpreted to reflect processing of the ZIP8 protein for deployment to the plasma membrane. Glycosylation occurs on asparagines (N-linked) initially in the ER. There are no consensus O-linked glycosylation sites in the protein. Final processing of N-linked glycosyl groups on proteins occurs in the Goli apparatus [
36,
37]. Confocal localization suggests that the protein has considerable ER distribution, and this would be consistent with the 49 kDa form. It is possible that when the initial glycosylation begins, the N-linked glycosyl groups are not enough to significantly increase the molecular weight of the protein and/or to retard the mobility of the protein on a Western blot. The 43 kDa band, corresponding to isoform C which has the first 67 amino acids missing, part of which is the signal peptide, and is predicted not to be transported into the lumen of the ER for glycosylation.
The results of an analysis of ZIP8 in protein extracts of human renal tissue were similar to that of the HPT cells, showing both a 49 kDa and 80 kDa band that was reactive with the ZIP8 antibody. There was also an additional, approximately 43 kDa band in the human renal tissue extract that was reactive with the ZIP8 antibody. This band could be a degradation product due to the processing interval between surgical removal and tissue procurement or it could be an additional isoform of the ZIP8 protein that has a predicted molecular weight 43.1 kDa by NCBI and identified as ZIP8 isoform 2 on the Swiss-Prot database. This 43.1 kDa band was also found in extracts of normal urothelial tissue. The 43 kDa band was not found in extracts of HPT cells or the parental UROtsa cell line. Previous studies have shown ZIP8 to be expressed only in the proximal tubules of the kidney in mice [
30]. However, immuno-staining of ZIP8 on archival specimens of human kidney showed ZIP8 to be present in both proximal and distal tubule cells and in some stromal elements in normal urothelium, presenting the possibility of isoform 2 being present in other tubule segments and/or stromal cell types. This discrepancy between mice and human expression patterns could be due to specie specific differences. Overall, the results in the HPT cells regarding the expression of ZIP8 were largely those expected from previous studies. This is important due to the implication of ZIP8 in enhanced cadmium-induced renal proximal tubular damage in mice [
30]. The HPT cells have been used as a model for the study of Cd-induced toxicity in the past [
38‐
41] and the current observation that they have basal expression of ZIP8 should provide the research community with an effective
in vitro model to further elucidate the role of ZIP8 in Cd-induced proximal tubule renal damage.
The second goal of the present study was to determine if ZIP8 was expressed in normal human urothelium and if expression was altered in human urothelial cancer. The results demonstrated that ZIP8 was expressed in the normal urothelium. Immunostaining showed that ZIP8 was expressed in the urothelial cells of all 5 independent specimens of normal urothelium. However, the expression of ZIP8, while uniform within each specimen, was highly variable among the five samples, with staining for ZIP8 varying from very weak to strong in intensity. Immunostaining also showed ZIP8 to have a paranuclear localization in addition to punctate staining within the cytoplasm. Western analysis of ZIP8 expression in 5 independent specimens of normal urothelium showed the presence of the 49 kDa band, but not the higher molecular weight band associated with the glycosylated form of the ZIP8 protein. The corresponding analysis of ZIP8 expression in the UROtsa cell line is of interest regarding the variability of expression and the paranuclear localization of ZIP8 in the normal urothelium. First, the level of expression of the ZIP8 protein in the UROtsa cell line was shown to be dependent on the time following replenishment of the growth medium, with expression being elevated significantly following feeding of the cells with fresh growth medium, followed by a rapid reduction in expression within 36 hrs of the addition of fresh growth medium. It has also been shown that the availability of Zn
+2 can influence the trafficking of the ZIP8 protein to the apical cell surface in MDCK cells [
32]. One can speculate that the variability of expression of ZIP8 demonstrated among the independent specimens of normal urothelium may reflect differences in the nutritional status of the patient from which the samples originate. The possibility that ZIP8 expression can vary with nutritional status would render interpreting differences in expression between levels in tissues and fluids from normal versus disease states very difficult. Second, the ZIP8 protein was also localized to the paranuclear region of the parental UROtsa cells, a finding in agreement with the paranuclear localization of the ZIP8 protein in the five patient samples of normal urothelium. The association of ZIP8 protein with the cell nucleus could indicate a possible involvement in providing Zn
+2 to Zn-requiring transcription factors. The present findings appear to be the first description of ZIP8 expression and localization in human urothelium.
As part of the above goal it was also proposed to determine if ZIP8 expression was altered in human urothelial cancer. As noted above, the wide variability of expression of ZIP8 in normal urothelium renders comparison very difficult between the normal and malignant urothelial cells. The most striking finding from the analysis was that out of the 14 cases of low and high grade urothelial cancer examined for ZIP8 expression, there was one high grade, invasive urothelial cancer that showed no expression of the ZIP8 protein. An additional difference in the expression of ZIP8 among the urothelial cancers was that there was no paranuclear localization of ZIP8 in any of the high grade urothelial cancers that showed positive staining for ZIP8. Otherwise, expression in the remaining high and low grade cancers displayed a wide variability of ZIP8 expression similar to that noted in specimens of normal urothelium. The fact that the one urothelial cancer that did not express the ZIP8 protein was in the high grade invasive group could be important since the loss of ZIP8 could be associated with tumor progression. This would need to be confirmed on a much larger sample set of urothelial tumors having data on patient outcome.
The final goal of the study was to determine ZIP8 expression and localization in human urothelial cells transformed by Cd+2 and As+3. The results showed that both Cd+2 and As+3 transformed UROtsa cells and their tumor transplants expressed higher levels of ZIP8 mRNA and protein compared to the parental cell line. There was no notable difference in the expression of ZIP8 between the Cd+2 and As+3 transformed cell lines, ruling out a Cd+2 specific alteration in ZIP8 expression that might be associated with the development of urothelial cancer. One difference noted between the As+3 and Cd+2 transformed cell lines compared to the parental UROtsa cell line was that the transformed lines consistently expressed the 80 kDa band of ZIP8 associated with the glycosylated form of the protein. The parental UROtsa cell line displayed the 80 kDa band only transiently after the cells were fed fresh growth medium. The transformed cell lines also showed some localization of ZIP8 to the cell membrane, but the majority was localized to the cytoplasm and paranuclear region of the cells. Again, these differences are difficult to interpret due to the time dependence of ZIP8 expression with growth medium replenishment in the parental UROtsa cell line. Similar to that found for the archival specimens of high grade urothelial cancer, the tumor transplants generated from the As+3 and Cd+2 transformed cells showed no evidence of paranuclear staining for ZIP8.
It remains to be elucidated why or how ZIP8 is overexpressed in metal transformed cells. It was unexpected that As
+3-transformed cells also over-express this transporter. As
+3 is not expected to be transported by ZIP8 due to the divergent properties of these metals. As
+3 exists as a trihydroxylated, neutral species known as arsenous acid As(OH)
3 with the pK for the donation of the first hydrogen being greater at pH 9.0 [
42], and is thought to be transported via the aquaporin transporters [
43,
44]. It is well known that global gene expression patterns are considerably altered during metal carcinogenesis, and that alterations in epigenetic regulation have been appreciated to play a fundamental role [
45]. Epigenetic alterations leading to the overexpession or silencing of specific loci have been correlated to methylation/demethylation of CpG islands and post-translational modifications of histone tails within the promoters of altered genes. Specific explanations for why particular loci are silenced or conducive to overexpression by long-term exposure and/or transformation by metals still remain to be determined, although in a few specific cases, alteration in the expression or activity of methylases and demethylases have be identified. Epigenetics alterations are thus suspected in the case of ZIP8 overexpression in Cd
+2- and As
+3-transformed cells. It is tempting to speculate that specific metal transport pathways may be involved.
Competing interests
None of the authors’ have competing interests.
Authors’ contributions
AA: Postdoctoral fellow. RT-PCR and western analysis of ZIP8 expression. TB: Undergraduate research summer student who validated the initial finding of elevated ZIP8 mRNA expression from microarray data. SG: Supervised the postdoctoral fellow in western analysis and assisted with data analysis. MAS & ZDZ: Procurement of human and animal tissue, mouse autopsy, tissue fixation, and evaluation of immunostaining and histology. JD: Supervised, trained, and assisted postdoctoral fellow with immunofluorescent localization. DAS: Worked on the manuscript with postdoctoral fellow, designed study, final data integration. SS: Wrote the manuscript and trained and supervised postdoctoral fellow in cell culture and RT-PCR analysis. All authors read and approved the final manuscript.