The Zinc Sensing Receptor, a Link Between Zinc and Cell Signaling

Abstract

Zinc is essential for cell growth. For many years it has been used to treat various epithelial disorders, ranging from wound healing to diarrhea and ulcerative colon disease. The physiological/molecular mechanisms linking zinc and cell growth, however, are not well understood. In recent years, Zn²⁺ has emerged as an important signaling molecule, activating intracellular pathways and regulating cell fate. We have functionally identified an extracellular zinc sensing receptor, called zinc sensing receptor (ZnR), that is specifically activated by extracellular Zn²⁺ at physiological concentrations. The putative ZnR is pharmacologically coupled to a Gq-protein which triggers release of Ca²⁺ from intracellular stores via the Inositol 1,4,5-trisphosphate (IP₃) pathway. This, in turn results in downstream signaling via the MAP and phosphatydilinositol 3-kinase (PI3 kinase) pathways that are linked to cell proliferation. In some cell types, e.g., colonocytes, ZnR activity also upregulates Na⁺/H⁺ exchange, mediated by Na⁺/H⁺ exchanger isoform 1 (NHE1), which is involved in cellular ion homeostasis in addition to cell proliferation. Our overall hypothesis, as discussed below, is that a ZnR, found in organs where dynamic zinc homeostasis is observed, enables extracellular Zn²⁺ to trigger intracellular signaling pathways regulating key cell functions. These include cell proliferation and survival, vectorial ion transport and hormone secretion. Finally, we suggest that ZnR activity found in colonocytes is well positioned to attenuate erosion of the epithelial lining of the colon, thereby preventing or ameliorating diarrhea, but, by signaling through the same pathways, a ZnR may enhance tumor progression in neoplastic disease.

Introduction

The zinc gradient across the plasma membrane may reach six orders of magnitude, as cytoplasmic Zn²⁺ is tightly buffered by a variety of mechanisms (1). Specific binding sites for Zn²⁺ are present on numerous proteins, including Zn²⁺ fingers on transcription factors, that bind it with high affinity, and metallothioneins, to which the Zn²⁺ is more loosely bound. Dynamic changes in extracellular Zn²⁺ occur upon its release from cells in organs such as pancreas, brain, and salivary gland (2–4), while changes in intracellular Zn²⁺ may result from oxidative stress (5,6). Such dynamic changes in Zn²⁺ gradients and the availability of specific Zn²+ binding domains suggest that Zn²⁺ ions, once considered merely structural elements, are, in fact, important signaling molecules that influence many aspects of cell physiology.

The signaling effects of Zn²⁺ may be mediated by intracellular or extracellular Zn²⁺ ions. It is generally accepted that an increase in free intracellular Zn²⁺ is associated with cell death. For example, release of intracellular Zn²⁺, triggered by formation of reactive oxygen species (ROS) or by nitrosilation, induces proapoptotic molecules, e.g., p38, and activation of K⁺- channels leading to cell death (7,8). Chelation of intracellular Zn²⁺ then, using a high affinity Zn²⁺ chelator, could interfere with this process. Chelation of intracellular Zn²⁺, however, may remove zinc from intracellular metalloproteins, resulting in protein synthesis-dependent, caspase-3 mediated apoptosis (9).

To protect cells from the consequences of a decrease in cellular zinc, changes in plasma Zn²⁺ often precede the reduction of this ion within cells. Symptoms of zinc deficiency, therefore, particularly attenuation of cell proliferation, are observed long before changes in intracellular zinc are observed (10). Thus, sensing changes in extracellular Zn²⁺, and activation of signaling to regulate cell processes, before key components (e.g., zinc finger proteins) are affected, is essential. This Zn²⁺-sensing function might also provide protection against a sudden rise in intracellular Zn²⁺ in tissues in which Zn²⁺ is released during normal activity, e.g., in endocrine and exocrine glands or at glutamatergic synapses in the mammalian forebrain.

Extracellular Zn²⁺ indirectly activates cell signaling, by interacting with major membrane transporters and ion channels, most notably, the dopamine transporter, NMDA, glycine, GABA, and purinergic receptors (11–15). A high affinity binding site has also been identified recently on the store-operated channel (SOC) (16).

A growing body of evidence suggests that extracellular Zn²⁺ also activates signal transduction pathways that induce cell proliferation and survival. Thus, Zn²⁺ upregulates the PI3 kinase pathway, leading to activation of AKT in fibroblasts (17). Zinc also has been shown to induce transactivation of the epidermal growth factor receptor (EGFR) by Src in airway epithelial cells (18–20). In colonocytes, extracellular Zn²⁺ triggered the activation of ERK1/2 (extracellular-signal regulated kinase), which, in turn, was accompanied by induction of p21(CiP/WAF1) and cyclin D1 (21). Evidence of a direct mechanism linking extracellular Zn²⁺ to cellular signaling, however, has been lacking. We hypothesized that such a mechanism exists and subsequently described the interaction of extracellular Zn²⁺ with a specific target, an extracellular Zn²⁺ sensing receptor, that mediates Zn²⁺-dependent intracellular signaling (22).

Identification of a Putative Zn²⁺ Sensing Receptor

Because of the synergistic effect of Zn²⁺ and Ca²⁺ on cell growth, and the general role of Ca²⁺ in regulating many of the signaling pathways known to be activated by zinc, we first asked if Zn²⁺-dependent signaling may be mediated by changes in intracellular Ca²⁺. Extracellular Zn²⁺ in colonocytes, keratinocytes, and salivary gland cells, was subsequently shown to trigger a rise in intracellular Ca²⁺ released from thapsigargin-sensitive stores (22,23) (Table 1). Inhibitors of Gαq and the phospholipase C, (PLC), attenuated this Zn²⁺-dependent Ca²⁺ rise, indicating that it is mediated by activation of a Gαq-coupled receptor (22,24). However, PLC could also be activated by a mechanism that involves, among others, receptor tyrosine kinases (23). To determine the possible role of growth factor receptor in mediating the Zn²⁺-dependent Ca²⁺ response cells were treated with the general inhibitor of tyrosine kinases, genistein (50 µM), prior to application of Zn²⁺. The Zn²⁺ dependent Ca²⁺ rise was not attenuated by the inhibitor indicating it is not mediated by receptor tyrosine kinases (Figure 1A). This activity proved highly specific to extracellular Zn²⁺ as it was not triggered by other heavy metal ions tested, e.g. Mn²⁺, Cu²⁺, and Fe²⁺ (22). This is in agreement with previous works showing that cation sensing receptors are not activated by Zn²⁺ (25). Interestingly, a putative Cd²⁺-sensing receptor was described in fibroblasts (26,27), Zn²⁺ did not activate the Ca²⁺ release mediated via the IP₃ pathway in these cells, similar to our results (Table 1). Although Zn²⁺ did not activate this putative metal receptor, it acted as a competitive inhibitor to Cd²⁺. Finally, the Zn²⁺-dependent Ca²⁺ rise is distinct from the activity of the Ca²⁺ sensing receptor (CaR) (28), as overexpression of CaR did not produce an increase in Ca²⁺ following application of extracellular Zn²⁺. Thus, our experiments revealed a Gq-coupled, Zn²⁺ sensing receptor, ZnR, linking changes in extracellular Zn²⁺ and downstream signal transduction pathways (Figure 1B, 22). Although the molecular identity of the Zn²⁺ sensing receptor has not yet been determined, our preliminary data indicate that ZnR activity is mediated by hetero-oligomerization of two members of the venous-fly trap subgroup of G-protein coupled receptors, which form a functional zinc sensing receptor. Such heterodimerization, plays a role in the versatility of the taste sensing receptors, it remains to be elucidated if it also plays a role in cation sensing.

Table 1 :Summary of currently known cells which exhibit Zn²⁺ -dependent Ca²⁺ signaling via the putative ZnR pathway.

Figure 1

A. The Zn²⁺ -dependent Ca²⁺ rise triggered in HaCaT, keratinocytic cell line, is insensitive to the general tyrosine kinase inhibitor genistein. B. A model for ZnR signaling mediating Zn²⁺ -dependent cell growth and survival. Our results indicate that a ZnR is the major link between changes in extracellular Zn²⁺ and physiological cell function, such as: secretion, proliferation and survival. Desensitization, induced by Zn²⁺, largely attenuates the Zn²⁺ -dependent signaling.

Figure 2

Extracellular Zn²⁺ -signaling, activated by the putative ZnR, reduces butyrate induced cell death. Colonocytic culture, HT29 cells, was treated with butyrate (30 mM, 24 h) and cells were imaged using brightfield microscopy. Massive loss of cells is observed (left picture) following the butyrate treatment. Application of Zn²⁺ for ten minutes prior to the butyrate treatment reduced the loss of cells in the culture (right picture).

Extracellular Zn²⁺-Dependent Signaling in the Colon

In colonocytes, the putative ZnR mediates Zn²⁺-dependent activation of the MAP kinase and the PI3 kinase pathways (29). Desensitization of the ZnR by Zn²+, is followed by inhibition of the Zn²⁺-dependent Ca²⁺ rise as well as by phosphorylation of ERK1/2, indicating that the ZnR is a principal link between extracellular Zn²⁺ and ERK1/2. Activation of the ZnR signaling pathway also upregulates the Na⁺/H⁺ exchanger isoform 1 (NHE1) and enhances the recovery from acidic pH (29). Once thought to merely regulate pHi, this Na⁺/H⁺ exchanger is now believed to be involved in a much broader range of activities, particularly in regulation of cell proliferation and apoptosis (30–32). Butyrate, a short chain fatty acid produced by bacterial fermentation in the colon, induces apoptosis of colonocytes and has been implicated in ulcerative colon diseases (33–35). Interestingly, it has been demonstrated that zinc confers protection in ulcerative colitis, though the mechanism is not known (36). Upregulation of NHE1 by ZnR activity and subsequent anti-apoptotic and proliferative signaling may explain how zinc helps prevent ulcerative colitis. We have shown that application of extracellular Zn²⁺ prior to acidification with butyrate significantly enhances recovery of the colonocytes from the acidification produced by butyrate. Furthermore, short exposure of colonocytes to concentrations of Zn²⁺ sufficient to activate but not desensitize the ZnR, or to induce changes in intracellular Zn²⁺, attenuated butyrate-induced cell death (Hershfinkel, et al., In preparation and Figure 2). That this effect was not mediated by regulation of pH_i is shown by the fact that inhibition of Na⁺/H⁺ exchange, using the NHE1 inhibitor, cariporide, did not alter colon cell survival. This indicates that an extracellular Zn²⁺-dependnet mechanism can reduce the acid load by activating the NHE1, but must activate an additional pathway to mediate Zn²⁺-dependent cell survival. One candidate is the pro-survival glycoprotein, clusterin (CLU) (also known as apolipoprotein J) (37). Initial experiments have shown that activation of the ZnR signaling pathway leads to enhancement of clusterin expression. The level of expression, moreover, was enhanced further by application of zinc and butyrate, suggesting a synergistic mechanism. Considering the strong pro-survival effects of clusterin described in the colon (37,8), it is tempting to speculate that such a mechanism may underlie the protective effect of zinc in preventing butyrate-induced cell death. This may further suggest a role for ZnR in the etiology of colon cancer.

A Paracrine Role for the Putative ZnR

Paracrine effects of signaling molecules trigger intracellular signaling pathways in neighboring cells that do not express a specific receptor. We posit that paracrine effects are of particular importance for amplifying signaling mediated by endogenous zinc, especially in the skin where zinc is involved in wound healing via an unknown mechanism. Yet, zinc is released only in minute amounts following the infliction of a wound or other epithelial damage (39). The release of intracellular Ca²⁺, such as that induced by the pathway triggered by ZnR activity, is a principle mediator of paracrine responses, and has been shown to induce the release of important agonists such as ATP (40–12). In addition, it has been demonstrated previously that Zn²⁺ regulates ATP-sensitive purinergic receptor stimulation in a Ca²⁺-dependent manner (43).

Zinc is packaged in secretory granules of many exocrine glands, such as the salivary glands, and the pancreas (1). Although a specific role for salivary Zn²⁺ is unknown, loss of taste and salivary secretion dysfunctions have been linked to zinc deficiency, suggesting that this ion plays an important role in salivary secretion (44,45). We have observed that Zn²⁺, acting via the ZnR pathway, mediates release of intracellular Ca²⁺ in the HSY, ductal salivary gland cell line (parotid origin) (24). The endogenous release of ATP and, thereby, activation of the P2Y purinergic, receptor is important for regulation of the ionic content, volume and pH of the salivary fluid (24). We therefore studied the role of extracellular Zn²⁺ in regulating ATP secretion in co-cultures of HSY and vascular smooth muscle cells that do not express a functional ZnR. Activation of the salivary-ZnR in HSY cells was followed by a rise in Ca²⁺ in the vascular cells that was inhibited by the ATP scavenger, apyrase. Our results, therefore, indicate that a salivary-ZnR also acts paracrinically, i.e., by enhancing secretion of ATP, thereby linking zinc and the ZnR activity to key signaling pathways involved in salivary secretion (24). Clearly this model may encompass other secreted molecules, and may, thereby, extend the ability of ZnR to activate intracellular signaling. It may be particularly relevant with regard to the role of ZnR in wound healing. Although zinc is well known to enhance wound healing, we have identified ZnR-dependent signaling on keratinocytes and not on the fibroblasts that play such an important role in this process (22). A paracrine effect could explain how keratinocytic ZnR is initially activated and subsequently induces ATP secretion. The secreted ATP then serves to trigger crosstalk between the keratinocytes and fibroblasts, mediated by the purinergic P2Y receptor. In fact, we observed that producing a scratch in a keratinocytic monolayer, triggers a Zn²⁺-dependent Ca²⁺ rise (Sharir et al., In preparation), this may then initiate a signaling cascade leading to proliferation of fibroblasts as well.

Extracellular Zn²⁺ Dependent Signaling in the Prostate

Robust changes in extracellular Zn²⁺ are observed in the prostate during tumorigenesis (46,47). In the normal, non-neoplastic prostate, Zn²⁺ is found at very high, i.e., millimolar concentrations. It is involved in metabolic functions that result in secretion of citrate which binds the Zn²⁺ (48,49). In prostate cancer, a tenfold decrease in Zn²⁺ and citrate is observed (50). Yet, cellular signaling, activated by such changes in Zn²⁺, that affect cell growth and survival, have not been described in the prostate. We have shown that an androgen-independent prostate cancer cell line, PC-3, mediates Zn²⁺-dependent intracellular Ca²⁺ signals similar to the putative ZnR (Dubi et al., In preparation). The characteristic intracellular Ca²⁺ release mediated by such ZnR, and the subsequent activation of MAP and PI3 kinases, may enhance prostate cell proliferation and survival. Using crystal violet staining we show that application of Zn²⁺ (for a short ten minutes daily), that activates the ZnR-dependent intracellular signaling but does not significantly change intracellu-lar Zn²⁺, enhances cell proliferation (Figure 3). The profound reduction in Zn²⁺ during prostate cancer, however, may suggest that ZnR activity, inducing cell growth will be attenuated in prostate cancer. This apparent paradox may be reconciled by our demonstration that the epithelial ZnR is completely desensitized by high Zn²⁺ concentrations such as found in the normal prostate (29). We propose, therefore, that the ZnR activity is quiescent in the non-neoplastic prostate. The reduced Zn²⁺ concentrations occurring in the neoplastic prostate are not inducing desensitization of the ZnR, and thus Zn²⁺-dependent signaling may be activated leading to cell growth. During prostate cancer, ZnR signaling can be triggered by Zn²⁺ which is released from cells following the tissue destruction triggered by the invasion of the tumor to enhance cell proliferation and survival. Further in vivo studies using Zn²⁺ chelation should clarify the novel role of Zn²⁺-dependent signaling activation in prostate cancer progression.

Figure 3

Extracellular Zn²⁺ -signaling, activated by the putative ZnR, enhanced cell growth of prostate cancer cells. Prostate cancer cell line, PC-3, was treated with Zn²⁺ -containing (100 µM, ten minutes) or Zn²⁺ -free Ringer’s solution daily, for five days. Cultures were then stained with crystal violet. Enhanced cell growth is shown in the cells treated with Zn²⁺ (right picture) compared with the control cells (left picture).

Conclusions

Cell signaling triggered by the putative ZnR, and its profound role in mediating Zn²⁺-dependent signaling, supports our hypothesis that such a receptor is a major link between extracellular zinc and its physiological functions such as secretion, or cell proliferation, and survival (see Figure 1). The ZnR, as a putative member of the G-protein coupled receptor (GPCR) family, which represents two percent of the human genome but is a target for about 50 percent of current pharmaceutical compounds, is particularly attractive as a target candidate for therapeutic interventions (51). At the very least, regulation of ZnR activation is likely to be of significant importance in wound healing, diarrhea, and salivary dysfunction. In each of these conditions, zinc is already used therapeutically, though development of more potent ligands to activate such a ZnR may prove beneficial. The simplicity of Zn²⁺ as a ligand holds out the promise that effective zinc mimetics can be developed. Inhibiting the function of a ZnR, either by reducing ligand availability or by means of its desensitization, also may provide a novel and efficacious strategy for attenuating tumor progression. The availability of effective in vivo zinc chelators and carriers, e.g., clioquinol, may, moreover, suggest an effective approach toward this goal.