Investigation of the differential transport mechanism of cinnabar and mercury containing compounds

BackgroundCinnabar has a long history of uses in Chinese traditional medicines as an ingredient in various remedies. However, the detailed mechanism of cinnabar in medication remains unclear, and the toxicity of cinnabar has been a debate due to its containing mercury sulfide. This study was designed to investigate the differential transport mechanism of cinnabar and other Hg-containing compounds HgCl₂, MeHg and HgS, and to determine if organic anion transporters OAT1 and OAT3 were involved in the differential transport mechanism.Materials and methodsThe 293T cells were employed to investigate and compare the differential transport mechanism of cinnabar and HgCl₂, MeHg and HgS. Cells were incubated with a low dose (5 μM HgCl₂ and MeHg, 200 μM HgS and cinnabar), medium dose (10 μM HgCl₂ and MeHg, 400 μM HgS and cinnabar), and high dose (20 μM HgCl₂ and MeHg, 800 μM HgS and cinnabar) of HgCl₂, MeHg, HgS and cinnabar for 24 h. Following treatment, the cells were collected and the cell viability was determined by MTT assay. The intracellular mercury content was measured at 1, 4, and 24 h after treatment with 10 μM of the tested agents by an atomic fluorescence spectrophotometer. The effect of these tested agents on mitochondrial respiration was determined in a high-resolution oxygraphyat 24 h following treatment. Furthermore, the effect of modulation of expression of transporters OAT1 and OAT3 on the transport and cytotoxicity of the tested agents was evaluated. The up and down regulation of OAT1 and OAT3 were achieved by overexpression and siRNA transfection, respectively.ResultsCompared with HgCl₂ and MeHg, the cytotoxicity of cinnabar and HgS was lower, with cell viability at the high dose cinnabar and HgS being about 65%, while MeHg and HgCl₂ were 40% and 20%, respectively. The intracellular mercury accumulation was time-dependent. At 24 h the intracellular concentrations of HgCl₂ and MeHg were about 7 and 5 times higher, respectively, than that of cinnabar. No significant difference was found in the intracellular mercury content in cells treated with cinnabar compared to HgS. The knockdown and overexpression of the transporter OAT1 resulted in significant reduction and increase, respectively, in mercury accumulation in HgCl₂ -treated cells in relative to control cells, while no significant changes were observed in cells treated with cinnabar, MeHg, and HgS. In addition, the knockdown and overexpression of the transporter OAT3 caused significant reduction and increase, respectively, in mercury accumulation in both HgCl₂ and MeHg-treated cells in relative to control cells, while no significant changes were observed in cells treated with cinnabar and HgS. Furthermore, it was found that cells transfected with siOAT1 caused significant resistance to the cytotoxicity induced by HgCl₂, while no noticeable changes in cell viability were observed in cells treated with other tested agents. Additionally, cells transfected with OAT3 did not change cell sensitivity to cytotoxicity induced by all of the four tested agents.ConclusionThis study demonstrates that differential transport and accumulation of mercury in 293T cells exists among cinnabar and the three mercury-containing compounds HgCl₂, MeHg and HgS, leading to distinct sensitivity to mercury induced cytotoxicity. The kidney organic anion transporters OAT1 and OAT3 are partially involved in the regulation of the transport of HgCl₂ and MeHg, but not in the regulation of the transport of cinnabar.     

Lead alters intracellular protein signaling and suppresses pro-inflammatory activation in TLR4 and IFNR-stimulated murine RAW 264.7 cells,in vitro

Lead (Pb) is a persistent environmental pollutant that has a structure and charge similar to many ions, such as calcium, that are essential for normal cellular function. Pb may compete with calcium for protein binding sites and inhibit signaling pathways within the cell affecting many organ systems including the immune system. The aim of the current study was to assess whether the calcium/calmodulin pathway is a principal target of environmentally relevant Pb during pro-inflammatory activation in a RAW 264.7 macrophage cell line. RAW 264.7 cells were cultured with 5 μM Pb(NO₃)₂, LPS, rIFNγ, or LPS+rIFNγ for 12, 24, or 48 hr. Intracellular protein signaling and multiple functional endpoints were investigated to determine Pb-mediated effects on macrophage function. Western blot analysis revealed that Pb initially modulated nuclear localization of NFκB p65 and cytoplasmic phosphorylation of CaMKIV accompanied by increased phosphorylation of STAT1β at 24 hr. Macrophage proliferation was significantly decreased at 12 hr in the presence of Pb, while nitric oxide (NO) was significantly reduced at 12 and 24 hr. Cells cultured with Pb for 12, 24, or 48 hr exhibited altered cytokine levels after specific stimuli activation. Our findings are in agreement with previous reports suggesting that macrophage pro-inflammatory responses are significantly modulated by Pb. Further, Pb-induced phosphorylation of CaMKIV (pCaMKIV), observed in the present study, may be a contributing factor in metal-induced autophagy noted in our previous study with this same cell line.     

Cellular mechanisms and integrative timing of neuroendocrine control of GnRH secretion by kisspeptin

The hypothalamus integrates endogenous and exogenous inputs to control the pituitary–gonadal axis. The ultimate hypothalamic influence on reproductive activity is mediated through timely secretion of GnRH in the portal blood, which modulates the release of gonadotropins from the pituitary. In this context neurons expressing the RF-amide neuropeptide kisspeptin present required features to fulfill the role of the long sought-after hypothalamic integrative centre governing the stimulation of GnRH neurons. Here we focus on the intracellular signaling pathways triggered by kisspeptin through its cognate receptor KISS1R and on the potential role of proteins interacting with this receptor. We then review evidence implicating both kisspeptin and RFRP3 – another RF-amide neuropeptide – in the temporal orchestration of both the pre-ovulatory LH surge in female rodents and the organization of seasonal breeding in photoperiodic species.     

Feedback regulation mediated by Bcl-2 and DARPP-32 regulates inositol 1,4,5-trisphosphate receptor phosphorylation and promotes cell survival

Bcl-2 interacts with the inositol 1,4,5-trisphosphate receptor (InsP₃R) and thus prevents InsP₃-induced Ca²⁺ elevation that induces apoptosis. Here we report that Bcl-2 binds dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32), a protein kinase A (PKA)-activated and calcineurin (CaN)-deactivated inhibitor of protein phosphatase 1 (PP1). Bcl-2 docks DARPP-32 and CaN in a complex on the InsP₃R, creating a negative feedback loop that prevents exaggerated Ca²⁺ release by decreasing PKA-mediated InsP₃R phosphorylation. T-cell activation increases PKA activity, phosphorylating both the InsP₃R and DARPP-32. Phosphorylated DARPP-32 inhibits PP1, enhancing InsP₃R phosphorylation and Ca²⁺ release. Elevated Ca²⁺ activates CaN, which dephosphorylates DARPP-32 to dampen Ca²⁺ release by eliminating PP1 inhibition to enable it to dephosphorylate the InsP₃R. Knocking down either Bcl-2 or DARPP-32 abrogates this feedback mechanism, resulting in increased Ca²⁺ elevation and apoptosis. This feedback mechanism appears to be exploited by high levels of Bcl-2 in chronic lymphocytic leukemia cells, repressing B-cell receptor-induced Ca²⁺ elevation and apoptosis.Periodic elevations of intracellular Ca²⁺ serve as second messengers regulating mitochondrial metabolism, cell cycle entry, and cell survival (1, 2). Ca²⁺ signals are generated when inositol 1,4,5-trisphosphate receptors (InsP₃Rs) open in response to InsP₃, thus transferring Ca²⁺ from the endoplasmic reticulum into the cytoplasm and mitochondria. InsP₃R-mediated Ca²⁺ release is highly regulated, as excessive Ca²⁺ release causes cellular dysfunction and death (3). A number of proteins bind to InsP₃Rs and regulate channel opening, thus preventing excessive Ca²⁺ release (4). Among these is the antiapoptotic protein B-cell lymphoma-2 (Bcl-2) (5).The Bcl-2 homology domain 4 (BH4) domain of Bcl-2 mediates its interaction with InsP₃Rs (6, 7). This domain also binds the Ca²⁺/calmodulin-activated serine/threonine (Thr) protein phosphatase calcineurin (CaN) (8). The BH4 domain binds within the regulatory and coupling domain of the InsP₃R, located between the InsP₃ binding site near the N terminus and the channel domain near the C terminus (6). Protein kinase A (PKA) phosphorylates serine (Ser) 1589 and Ser1755 within this domain, increasing InsP₃-mediated channel opening and Ca²⁺ release (9). Bcl-2 is reported to regulate InsP₃R phosphorylation at Ser1755 and to thereby govern InsP₃R-mediated Ca²⁺ release in murine embryonic fibroblasts, although the mechanism is not yet identified (10).Bcl-2 plays an important role in regulating T-cell development and selection, processes that involve Ca²⁺ signaling and apoptosis regulation (11, 12). A 20-amino acid peptide (InsP₃R-derived peptide, or IDP) corresponding to the Bcl-2 binding site on the InsP₃R inhibits Bcl-2–InsP₃R interaction, thus eliminating Bcl-2’s control over InsP₃R-mediated Ca²⁺ elevation (6, 7). This peptide induces marked Ca²⁺ elevation and Ca²⁺-mediated apoptosis in primary chronic lymphocytic leukemia (CLL) cells and in B-cell lymphoma lines, indicating that Bcl-2–InsP₃R interaction contributes to the apoptosis-resistance characteristic of these lymphoid malignancies (13, 14).Here we report that Bcl-2 interacts with dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32), a potent inhibitor of protein phosphatase 1 (PP1) that is activated by PKA-mediated phosphorylation at Thr 34 and deactivated by CaN-mediated dephosphorylation at this site (15). Our findings indicate that Bcl-2 docks DARPP-32 and CaN on the InsP₃R, creating a negative feedback loop that responds to InsP₃R-mediated Ca²⁺ release by inhibiting InsP₃R phosphorylation at Ser1755, thereby preventing excessive Ca²⁺ elevation capable of inducing cell death. Our findings implicate this Bcl-2–CaN–DARPP-32 feedback mechanism in regulating Ser1755 phosphorylation and apoptosis in primary human CLL cells.     

GAP,an aequorin-based fluorescent indicator for imaging Ca2+ in organelles

Genetically encoded calcium indicators allow monitoring subcellular Ca²⁺ signals inside organelles. Most genetically encoded calcium indicators are fusions of endogenous calcium-binding proteins whose functionality in vivo may be perturbed by competition with cellular partners. We describe here a novel family of fluorescent Ca²⁺ sensors based on the fusion of two Aequorea victoria proteins, GFP and apo-aequorin (GAP). GAP exhibited a unique combination of features: dual-excitation ratiometric imaging, high dynamic range, good signal-to-noise ratio, insensitivity to pH and Mg²⁺, tunable Ca²⁺ affinity, uncomplicated calibration, and targetability to five distinct organelles. Moreover, transgenic mice for endoplasmic reticulum-targeted GAP exhibited a robust long-term expression that correlated well with its reproducible performance in various neural tissues. This biosensor fills a gap in the actual repertoire of Ca²⁺ indicators for organelles and becomes a valuable tool for in vivo Ca²⁺ imaging applications.Ca²⁺ is involved in the regulation of many intracellular processes that take place both in the cytosol and inside organelles (1–3). Therefore, accurate measurement of the calcium concentration ([Ca²⁺]) inside organelles is essential to discriminate discrete Ca²⁺ signals between the different compartments. Although synthetic Ca²⁺ indicators can be loaded into organelles, the signal has poor selectivity, as the dye is also present in the cytosol and must be carefully removed before measurements (4). The main advantage of Genetically Encoded Ca²⁺ Indicators (GECIs) is their ability to be targeted to specific intracellular locations. Both bioluminescent and fluorescent proteins have been successfully used to measure subcellular [Ca²⁺]. The photoprotein aequorin (5), purified from the jellyfish Aequorea victoria, was the first protein-based Ca²⁺ indicator, injected into cells in the early 1970s (6). After cloning of its cDNA (7), recombinant aequorin became the most frequently used probe to measure Ca²⁺ in organelles, including mitochondria (8), the endoplasmic reticulum (ER) (9), the nucleus (10), the Golgi apparatus (11), or secretory vesicles (12).Fluorescent GECIs achieve a better spatial resolution than bioluminescent sensors. They are generally composed of one or two fluorescent proteins, most of them variants of GFP, fused to a Ca²⁺-binding protein (13). Recently, a single EF-hand motif has been inserted in the GFP moiety to generate a Ca²⁺ fluorescent probe (14). Since the first cameleon based on FRET (15), the number of GECIs has exponentially increased, attempting optimization of critical features such as adequate expression, signal strength, or dynamic range. However, the in vivo use in mammals, one of the main applications of GECIs, has grown more slowly and has disclosed severe limitations (16, 17). Transgenic sensors usually showed a low expression, often resulting in its inactivation or reduced dynamic range. With the exception of troponin derivatives, most of the available GECIs, namely cameleons, camgaroos, pericams, or GCaMPs (circularly permutated EGFP-based Ca²⁺ sensors), are based on calmodulin, a highly regulated ubiquitous protein that binds a large number of targets (13). Although the interference with endogenous calmodulin has been reduced in the improved cameleons (18), the interaction with other cellular proteins cannot be ruled out. Thus, the loss of Ca²⁺ sensitivity observed in vivo may reflect the interaction of the probe with endogenous partners, which may disturb cellular functions.The jellyfish aequorin exhibits a number of advantages over mammalian EF-hand proteins. It is not toxic and appears not to interfere with other intracellular Ca²⁺-binding molecules, even when microinjected at high concentrations in mammalian cells. Moreover, the use of aequorin as a bioluminescence sensor has been extensively reported, ranging from subcellular Ca²⁺ measurements in many different cell types up to whole organisms, including transgenic animals (19–21).Here we describe a family of fluorescent Ca²⁺ sensors based on the fusion of two jellyfish proteins, GFP and apoaequorin. This Ca²⁺ probe shows a larger dynamic range compared with other GECIs and a robust photonic and thermal stability. It can be targeted to distinct compartments such as the nucleus, cytosol, or mitochondria, where it selectively and accurately monitors dynamic Ca²⁺ changes. In addition, we have generated a variant with a lower Ca²⁺ affinity suited for imaging Ca²⁺ changes in organelles with high resting [Ca²⁺] such as the ER or the Golgi apparatus. Finally, we demonstrate its in vivo applicability by generating transgenic mice where the Ca²⁺ biosensor maintained its in vitro features.     

Cortical control of adaptation and sensory relay mode in the thalamus

A major synaptic input to the thalamus originates from neurons in cortical layer 6 (L6); however, the function of this cortico–thalamic pathway during sensory processing is not well understood. In the mouse whisker system, we found that optogenetic stimulation of L6 in vivo results in a mixture of hyperpolarization and depolarization in the thalamic target neurons. The hyperpolarization was transient, and for longer L6 activation (>200 ms), thalamic neurons reached a depolarized resting membrane potential which affected key features of thalamic sensory processing. Most importantly, L6 stimulation reduced the adaptation of thalamic responses to repetitive whisker stimulation, thereby allowing thalamic neurons to relay higher frequencies of sensory input. Furthermore, L6 controlled the thalamic response mode by shifting thalamo–cortical transmission from bursting to single spiking. Analysis of intracellular sensory responses suggests that L6 impacts these thalamic properties by controlling the resting membrane potential and the availability of the transient calcium current I_T, a hallmark of thalamic excitability. In summary, L6 input to the thalamus can shape both the overall gain and the temporal dynamics of sensory responses that reach the cortex.Sensory signals en route to the cortex undergo profound signal transformations in the thalamus. One important thalamic transformation is sensory adaptation. Adaptation is a common characteristic of sensory systems in which neural output adjusts to the statistics and dynamics of past stimuli, thereby better encoding small stimulus changes across a wide range of scales despite the limited range of possible neural outputs (1–3). Thalamic sensory adaptation is characterized by a steep decrease in action potential (AP) activity during sustained sensory stimulation (4–7), decreasing the efficacy at which subsequent sensory stimuli are transmitted to the cortex.The widely reported duality of thalamic response mode is another key property of thalamic information processing which further affects how sensory input reaches the cortex. In burst mode, sensory inputs are relayed as short, rapid clusters of APs; in contrast, in tonic mode the same inputs are translated into single APs. Both tonic and burst modes have been described during anesthesia/sleep and wakefulness/behavior, with a pronounced shift toward the tonic mode during alertness (8–12).Although the exact information content of thalamic bursts is not yet clear, it has been suggested that bursting may signal novel stimuli to the cortex, whereas the tonic mode enables linear encoding of fine stimulus details, e.g., when an object is examined (13, 14). One issue hampering the interpretation of burst/tonic responses is that currently it is unknown if the cortex itself is involved in the rapid changes in firing modes seen in the awake and anesthetized animal (15, 16) and which mechanisms initiate these shifts in vivo.On the biophysical level, the response mode depends on the resting membrane potential (RMP), which controls the availability of the transient low-threshold calcium current (I_T) (17). Depolarization decreases the size of the I_T-mediated low-threshold calcium spike (LTS), and fewer burst spikes are fired (18). Similarly, RMP influences adaptation in that depolarization reduces the voltage distance to the AP threshold, thereby increasing the probability that smaller, depressed inputs will trigger APs (6). Thus, the dynamics of the RMP may govern several key properties of signal transformation in the thalamus, thereby providing a common mechanism for controlling thalamic adaptation and response mode.Although subcortical inputs have been shown to influence thalamic firing modes (7, 9), we investigated the impact of cortical activity on thalamic sensory processing. Cortico–thalamic projections from cortical layer 6 (L6) are a likely candidate for regulating thalamic sensory processing with high spatial and temporal precision, because these projections provide a major input to the thalamus and, as shown by McCormick et al. (19), depolarize and modulate firing of thalamic cells in vitro.However, because of the inability to study sensory signals in brain slices, the role of L6 on thalamic input/output properties during sensory processing is not clear. Here, in the ventro posteromedial nucleus (VPM) of the mouse whisker thalamus, we investigate how L6 impacts the transmission of whisker inputs to the cortex. Recent advances in cell-type–specific approaches to dissect specific circuits in vivo (20–22) allowed us to activate the L6–thalamic pathway specifically and determine its impact on thalamic sensory processing.We found that cortical L6 can change key properties of thalamic sensory processing by controlling the interaction of intrinsic membrane properties and sensory inputs. This mechanism enables the cortex to control the frequency-dependent adaptation and the gain of its own input.     

Alpha‐Tocopherol during lactation and after weaning alters the programming effect of prenatal high salt intake on cardiac and renal functions of adult male offspring

Maternal salt overload programs cardiovascular and renal alterations in the offspring. However, beneficial and harmful effects of high dose vitamin E supplementation have been described in humans and animals. We investigated the hypothesis as to whether cardiac and renal alterations can be programmed by gestational salt overload, and can become further modified during lactation and after weaning. Male Wistar rats were used, being the offspring of mothers that drank either tap water or 0.3 mol/L NaCl for 20 days before and during pregnancy. α‐Tocopherol (0.35 g/kg) was administered to mothers daily during lactation or to their offspring for 3 weeks post‐weaning. Systolic blood pressure (tcSBP) was measured in juvenile rats aged 210 days. The response of mean arterial pressure (MAP) and heart rate (HR) to intravenous infusion of angiotensin II (Ang II) was also examined. Left ventricle plasma membrane (PMCA) and sarcoplasmic reticulum Ca²⁺‐ATPase (SERCA) activities, and certain parameters of renal function, were measured. Maternal saline programmed for increased body mass and kidney mass/body mass ratio, increased tcSBP, increased mean arterial pressure and heart rate with anomalous response to infused Ang II. In the heart, saline increased PMCA and α‐Tocopherol per se increased PMCA/SERCA. In the kidney, the most remarkable result was the silent saline programming of Cr_Cl, which was sensitized for a sharp decrease after α‐Tocopherol. In conclusion, the combination of maternal saline overload and high α‐Tocopherol immediately after birth leads to simultaneous cardiovascular and renal alterations in the young offspring, like those encountered in type V cardiorenal syndrome.     

Cyst formation following disruption of intracellular calcium signaling

Mutations in polycystin 1 and 2 (PC1 and PC2) cause the common genetic kidney disorder autosomal dominant polycystic kidney disease (ADPKD). It is unknown how these mutations result in renal cysts, but dysregulation of calcium (Ca²⁺) signaling is a known consequence of PC2 mutations. PC2 functions as a Ca²⁺-activated Ca²⁺ channel of the endoplasmic reticulum. We hypothesize that Ca²⁺ signaling through PC2, or other intracellular Ca²⁺ channels such as the inositol 1,4,5-trisphosphate receptor (InsP3R), is necessary to maintain renal epithelial cell function and that disruption of the Ca²⁺ signaling leads to renal cyst development. The cell line LLC-PK1 has traditionally been used for studying PKD-causing mutations and Ca²⁺ signaling in 2D culture systems. We demonstrate that this cell line can be used in long-term (8 wk) 3D tissue culture systems. In 2D systems, knockdown of InsP3R results in decreased Ca²⁺ transient signals that are rescued by overexpression of PC2. In 3D systems, knockdown of either PC2 or InsP3R leads to cyst formation, but knockdown of InsP3R type 1 (InsP3R1) generated the largest cysts. InsP3R1 and InsP3R3 are differentially localized in both mouse and human kidney, suggesting that regional disruption of Ca²⁺ signaling contributes to cystogenesis. All cysts had intact cilia 2 wk after starting 3D culture, but the cells with InsP3R1 knockdown lost cilia as the cysts grew. Studies combining 2D and 3D cell culture systems will assist in understanding how mutations in PC2 that confer altered Ca²⁺ signaling lead to ADPKD cysts.The commonly occurring genetic kidney disorder, autosomal dominant polycystic kidney disease (ADPKD), is the result of mutations in polycystin 1 or 2 (PC1 or PC2). The progressive cyst formation within all segments of the nephron that defines the disorder leads to renal failure requiring treatment by dialysis and/or organ transplantation (1–3). Altered Ca²⁺ signaling is one of several pathways that have been implicated in the disease (4, 5). A major limitation toward elucidating the role of Ca²⁺ signaling in cyst formation has been the lack of easily manipulated, physiologically relevant experimental methodologies.In the past, ADPKD research has relied largely upon data from mouse models and cells maintained in 2D cell culture. Mouse models have played a significant role in understanding the biology of cyst formation but are unable to fully recapitulate the physiology of disease progression in humans due to the inherent differences between the species including life span, genetics, and environment. Two-dimensional cell culture has the ability to provide information on signaling pathways and response to therapies in a fast, high-throughput manner, but is incapable of replicating the inherent 3D nature of cyst formation. Advances in 3D tissue culture over the past 2 decades have improved the ability to model cyst development in vitro. However, previously published 3D tissue models of ADPKD have relied upon short-term culture of Madin-Darby canine kidney (MDCK) cells (6–12) or cells from patients (13–18) or PC1-null mice (19, 20; for review, see ref. 21). Recently, 3D tissues have been developed that incorporate mouse cells containing a shRNA-mediated knockdown of PC1 (9, 19). The benefits of this system include the use of a cell line, thus eliminating the need to isolate primary cells, and the use of cells with a stable genetic background.Ca²⁺ signaling underpins many cellular processes ranging from cell proliferation to cell death. Intracellular Ca²⁺ levels can be modified by opening of the inositol 1, 4, 5-trisphosphate receptor (InsP3R) or other intracellular Ca²⁺ release channels, including PC2. Over 99% of PC2 resides on the endoplasmic reticulum (22), where it is known to act as a modulator of the InsP3R and the ryanodine receptor (RyR) (23), with the remainder on the primary cilia. PC2 itself can function as a Ca²⁺-activated Ca²⁺ release channel (22, 24).Although it was demonstrated in 3D cultures that the knockdown of PC1 leads to cyst development (25), the effect of knocking down PC2 or other Ca²⁺-signaling proteins has not been shown. It has been hypothesized that the disruption of PC2, or the proteins that it interacts with, will result in cyst growth, as Ca²⁺ is a major signaling molecule (26, 27). Cells with decreased PC2 have been linked with decreased Ca²⁺ signaling (28), and overexpression of PC2 has been shown to act as an inhibitor of cell proliferation (29). Changing PC2 expression levels alters the uptake of Ca²⁺ into the endoplasmic reticulum, leading to liver cyst formation (30), but no direct link involving the release of Ca²⁺ from the endoplasmic reticulum has been implicated in renal cyst development. Similarly, changes in the expression of the InsP3R have been correlated with various disease conditions; for example, the InsP3R is upregulated in colorectal cancer (31), but downregulated in bile duct obstruction and cholestasis (32, 33).Here, we demonstrate that cyst formation can be followed for several weeks using a 3D culture system and that the disruption of intracellular Ca²⁺ signaling, through the knockdown of either InsP3R or PC2, leads to cyst development.