ZnT-1 enhances the activity and surface expression of T-type calcium channels through activation of Ras-ERK signaling

Zinc transporter-1 (ZnT-1) is a putative zinc transporter that confers cellular resistance from zinc toxicity. In addition, ZnT-1 has important regulatory functions, including inhibition of L-type calcium channels and activation of Raf-1 kinase. Here we studied the effects of ZnT-1 on the expression and function of T-type calcium channels. In Xenopus oocytes expressing voltage-gated calcium channel (CaV) 3.1 or CaV3.2, ZnT-1 enhanced the low-threshold calcium currents (I(caT)) to 182 ± 15 and 167.95 ± 9.27% of control, respectively (P < 0.005 for both channels). As expected, ZnT-1 also enhanced ERK phosphorylation. Coexpression of ZnT-1 and nonactive Raf-1 blocked the ZnT-1-mediated ERK phosphorylation and abolished the ZnT-1-induced augmentation of I(caT). In mammalian cells (Chinese hamster ovary), coexpression of CaV3.1 and ZnT-1 increased the I(caT) to 166.37 ± 6.37% compared with cells expressing CaV3.1 alone (P < 0.01). Interestingly, surface expression measurements using biotinylation or total internal reflection fluorescence microscopy indicated marked ZnT-1-induced enhancement of CaV3.1 surface expression. The MEK inhibitor PD-98059 abolished the ZnT-1-induced augmentation of surface expression of CaV3.1. In cultured murine cardiomyocytes (HL-1 cells), transient exposure to zinc, leading to enhanced ZnT-1 expression, also enhanced the surface expression of endogenous CaV3.1 channels. Consistently, in these cells, endothelin-1, a potent activator of Ras-ERK signaling, enhanced the surface expression of CaV3.1 channels in a PD-98059-sensitive manner. Our findings indicate that ZnT-1 enhances the activity of CaV3.1 and CaV3.2 through activation of Ras-ERK signaling. The augmentation of CaV3.1 currents by Ras-ERK activation is associated with enhanced trafficking of the channel to the plasma membrane.     

Cloning and functional characterization of a mammalian zinc transporter that confers resistance to zinc.

A cDNA encoding a zinc transporter (ZnT-1) was isolated from a rat kidney cDNA expression library by complementation of a mutated, zinc-sensitive BHK cell line. This cDNA was used to isolate the homologous mouse ZnT-1 gene. The proteins predicted for these transporters contain six membrane-spanning domains, a large intracellular loop and a C-terminal tail. ZnT-1 is homologous to zinc and cobalt resistance genes of yeast. Immunocytochemistry with an antibody to a myc epitope added to the C-terminus of ZnT-1 revealed localization to the plasma membrane. Transformation of normal cells with a mutant ZnT-1 lacking the first membrane-spanning domain conferred zinc sensitivity on wild-type cells, suggesting that ZnT-1 functions as a multimer. Deletion of the first two membrane-spanning domains resulted in a non-functional molecule, whereas deletion of the C-terminal tail produced a toxic phenotype. Mutant cells have a slightly higher steady-state level of intracellular zinc and high basal expression of a zinc-dependent reporter gene compared with normal cells. Mutant cells have a lower turnover of 65Zn compared with normal cells or mutant cells transformed with ZnT-1. We propose that ZnT-1 transports zinc out of cells and that its absence accounts for the increased sensitivity of mutant cells to zinc toxicity.     

Cadmium and zinc induction of ZnT-1 mRNA in an established carp cell line

The cDNA of a zinc transporter-1 (ZnT-1) gene was cloned from an established cell line derived from common carp (Cyprinus carpio) using rapid amplification of cDNA ends (RACE). Using real-time quantitative PCR, we showed that both zinc (Zn) and cadmium (Cd) transiently upregulate ZnT-1 mRNA to comparable levels. The loosely bound cellular Zn pool, as estimated using the Zn-specific probe FluoZin-3, was increased threefold after exposure to 250 microM ZnCl(2). Correspondingly, the ZnT-1 mRNA level at 24 h was induced about fivefold, reflecting the need for more zinc export capacity. Total cellular zinc levels were not different from the controls after 72 h of exposure to 10, 50, or 250 microM ZnCl(2). A loss of total cellular Zn but little labile zinc changes were observed with up to 25 microM Cd. At 72 h, the total Zn was partially restored to the control levels, only 1 microM Cd allowed for a full recovery. Downregulation of ZnT-1 mRNA and partial loss of loosely bound Zn were observed with 50 microM Cd. Our results clearly show that although Zn and Cd can both regulate Zn export in EPC cells, the effects on the cellular Zn pools are quite different.     

负重爬梯与有氧跑台运动对糖尿病大鼠学习记忆能力的影响及其机制探讨

目的：研究负重爬梯与有氧跑台运动对糖尿病大鼠学习记忆能力的改善效果并探索其可能分子机制。方法：40只雄性大鼠,随机分为正常对照组（NC）、糖尿病对照组（DC）、糖尿病负重爬梯组（DL）和糖尿病有氧跑台组（DA）,以单次腹腔注射链脲佐菌素构建糖尿病大鼠模型。DL组在晚上进行负重爬梯训练,10次/组×3组/天,每次间歇2 min,6天/周×6周;DA组在同一时间进行20 m/min的跑台训练,30 min/d。于造模成功和运动干预结束后采用Morris水迷宫检测大鼠的学习记忆能力;第2次水迷宫测试结束后断颈处死大鼠,采用RT-QPCR法检测大鼠海马内脑源性神经营养因子（BDNF）、TRKB、CREB mRNA表达水平。结果：与NC组相比,DC组大鼠海马BDNF、CREB基因表达显著下降,学习记忆能力显著降低。与DC组相比,DL和DA组大鼠海马BDNF、CREB基因表达显著上调,学习能力显著提高;DL大鼠海马TrkB基因显著上调,大鼠空间记忆能力显著改善,而DA组大鼠海马TrkB基因无显著变化,大鼠空间记忆能力无改善,与DA组相比,DL组大鼠海马TRKB、CREB基因显著上调。结论：有氧跑台运动与负重爬梯运动介导BDNF/TrkB/CREB信号通路对糖尿病大鼠的学习能力均有促进作用,而负重爬梯运动对糖尿病大鼠记忆能力的改善优于有氧运动方式。     

Longitudinal changes in zinc transport kinetics, metallothionein and zinc transporter expression in a blood-brain barrier model in response to a moderately excessive zinc environment

A blood-brain barrier (BBB) model composed of porcine brain capillary endothelial cells (BCEC) was exposed to a moderately excessive zinc environment (50 micromol/L Zn) in cell culture, and longitudinal measurements were made of zinc transport kinetics, ZnT-1 (SLC30A1) expression and changes in the protein concentration of metallothionein (MT), ZnT-1, ZnT-2 (SLC30A2) and Zip1 (SLC39A1). Zinc release by cells of the BBB model significantly increased after 12-24 h of exposure, but decreased back to control levels after 48-96 h, as indicated by transport across the BBB from both the ablumenal (brain) and the lumenal (blood) directions. Expression of ZnT-1, the zinc export protein, increased by 169% within 12 h, but was no longer different from controls after 24 h. Likewise, ZnT-1 protein content increased transiently after 12 h of exposure, but returned to control levels by 24 h. Capacity for zinc uptake and retention increased from both the lumenal and the ablumenal directions within 12-24 h of exposure and remained elevated. MT and ZnT-2 were elevated within 12 h and remained elevated throughout the study. Zip1 was unchanged by the treatment. The BBB's response to a moderately high zinc environment was dynamic and involved multiple mechanisms. The initial response was to increase the cells' capacity to sequester zinc with additional MT and to increase zinc export with the ZnT-1 protein. But the longer-term strategy involved increasing ZnT-2 transporters, presumably to sequester zinc into intracellular vesicles as a mechanism to protect the brain and to maintain brain zinc homeostasis.     

Over-expression of Zip-13 mRNA in kidney and lung during dietary zinc deficiency in Wistar rats

Zinc is an essential nutrient for all organisms, which is involved in the function of numerous key enzymes in metabolism. Two gene families have been identified involved in zinc homeostasis. ZnT transporters reduce intracellular zinc while Zip transporters increase intracellular zinc. Previous studies in our laboratory have shown that Zip-1, ZnT-1, Zip-2 and LIV-1 mRNA are associated with zinc level in established human breast cancer in nude mice model. In this study, six zinc transporters: ZnT-1, ZnT-2, ZnT-4, Zip-1, Zip-8 and Zip-13 were chosen. We aim to determine the relation between zinc transporters and zinc level in kidney and lung of Wistar rats. Eighteen Wistar rats were randomly divided into three groups: normal group, zinc-deficiency group and pair-fed group. After 22 days, the rats were killed and organs samples were taken, then zinc transporters mRNA were detected by RT-PCR. Compared with the normal group, Zip-13 shows an up-regulation (P < 0.05) in zinc-deficiency group both in kidney and lung, and Zip-8 was significantly lower (P < 0.05) in zinc-deficiency group in kidney.     

Effects of hypoxia preconditioning on expression of metallothionein-1,2 and heme oxygenase-1 before and after kainic acid-induced seizures.

Global hypoxia preconditioning provides neuroprotection against a subsequent, normally damaging challenge. While the mechanistic pathways are unknown, changes in the expression of stress-related proteins are implicated. Hypoxia preconditioning attenuates the brain edema and neuropathology associated with kainic acid-induced status epilepticus in a protein synthesis-dependent manner when a kainic acid challenge is given up to one week post-preconditioning. Kainic acid initiates a glutamate-driven status epilepticus causing a Ca2+ and oxidative stress, resulting in injury to the piriform cortex and hippocampus. Stress-related gene expression [e.g. metallothioneins (MTs), heme oxygenase-1 (HO-1)] is enhanced during seizures in vulnerable brain areas, (e.g. piriform cortex). This study explores the effects of hypoxia preconditioning on expression of MT-1, MT-2 and HO-1 before and after kainic acid-induced seizures. Analysis of MT-1, MT-2 and HO-1 expression, through Western and Northern blotting, indicates that there is a variable pattern of induction and suppression of these two genes following hypoxia preconditioning alone as well as after kainic acid-induced seizures compared to non-preconditioned animals. These findings suggest that hypoxia preconditioning induces an adaptive response that prevents kainic acid seizure-associated neuropathology even when robust seizures occur. This may involve a variety of stress-related proteins, working in concert, each with their own individual expression profiles. Induction of this type of neuroprotection pharmacologically, or through preconditioning, will provide a better understanding of the stress response in brain.     

Zinc transporter mRNA expression in the RWPE-1 human prostate epithelial cell line

The human prostate gland undergoes a prominent alteration in Zn+2 homeostasis during the development of prostate cancer. The goal of the present study was to determine if the immortalized human prostate cell line (RWPE-1) could serve as a model system to study the role of zinc in prostate cancer. The study examined the expression of mRNA for 19 members of the zinc transporter gene family in normal prostate tissue, the prostate RWPE-1 cell line, and the LNCaP, DU-145 and PC-3 prostate cancer cell lines. The study demonstrated that the expression of the 19 zinc transporters was similar between the RWPE-1 cell line and the in situ prostate gland. Of the 19 zinc transporters, only 5 had levels that were different between the RWPE-1 cells and the tissue samples; all five being increased (ZnT-6, Zip-1, Zip-3A, Zip-10, and Zip-14). The response of the 19 transporters was also determined when the cell lines were exposed to 75 microM Zn+2 for 24 h. It was shown for the RWPE-1 cells that only 5 transporters responded to Zn+2 with mRNA for ZnT-1 and ZnT-2 being increased while mRNA for ZnT-7, Zip-7 and Zip-10 transporters were decreased. It was shown for the LNCaP, DU-145 and PC-3 cells that Zn+2 had no effect on the mRNA levels of all 19 transporters except for an induction of ZnT-1 in PC-3 cells. Overall, the study suggests that the RWPE-1 cells could be a valuable model for the study of the zinc transporter gene family in the prostate.     

A role for ZnT-1 in regulating cellular cation influx

The ZnTs are a growing family of proteins involved in lowering or sequestration of cellular zinc. Using fluorescent measurements of zinc transport we have addressed the mechanism of action of the most ubiquitously expressed member of this family, ZnT-1. This protein has been shown to lower levels of intracellular zinc though the mechanism has remained elusive. The rate of zinc efflux in HEK293 cells expressing ZnT-1 was not accelerated in comparison to control cells, suggesting that ZnT-1 may be involved in regulating influx rather than efflux of zinc. Co-expression of the L-type calcium channel, a major route for zinc influx, and ZnT-1 resulted in a 3-fold reduction in the rate of zinc influx in HEK293 and PC-12 cells, indicating that ZnT-1 modulates zinc permeation through this channel. Immunoblot analysis indicates that ZnT-1 expression does not modulate LTCC expression. Our findings therefore indicate that ZnT-1 modulates the permeation of cations through LTCC, thereby, regulating cation homeostasis through this pathway. Furthermore, ZnT-1 may play a role in cellular ion homeostasis and thereby confer protection against pathophysiological events linked to cellular Ca(2+) or Zn(2+) permeation and cell death.     

Neuroprotective Benefits of Aerobic Exercise and Organoselenium Dietary Supplementation in Hippocampus of Old Rats

The progressive decline of neurological functions, such as learning and memory, is an unavoidable consequence of aging. Our previous work suggested that the combination of physical exercise and a diet supplemented with diphenyl diselenide improves age-related memory decline in rats. The present study investigated the effects of physical exercise and a diet supplemented with diphenyl diselenide on the levels of proteins involved in the hippocampal neuroprotection to figure out the mechanisms related to the beneficial effects of this intervention in aged rats. Male Wistar rats (27 months old) were fed daily with standard chow supplemented with 1 ppm of diphenyl diselenide and subjected to swimming training with a workload (1% of body weight, 20 min/day) for 4 weeks. The hippocampus was dissected from the brain and used for the western blot and immunohistochemistry analyses. The results of this study demonstrate that the association of diphenyl diselenide-supplemented diet and swimming exercise increased the levels of proteins involved in neuroprotection and decreased the activation of those related to apoptosis and neuroinflammation in the hippocampus of old rats. This study suggests that physical exercise and a diet supplemented with (PhSe)₂ promoted neuroprotection in the hippocampus of aged rats.     

Effects of zinc on the induction of metallothionein isoforms in hippocampus in stress rats

Metallothioneins (MTs) are involved in the cellular metabolism of zinc and in cytoprotection against stress factors. Hippocampus plays a specific role in the body's response to stressors. The present study was conducted to evaluate the effects of zinc on the expression of metallothionein isoforms in the hippocampus of stress rats. The animal model of psychologic stress was developed by restraint for 4 weeks. Wistar rats were randomly assigned to 6 groups: control group, zinc-deficient group, zinc-supplemented group, and the corresponding 3 stress groups. Three separate diets of different zinc contents (1.73 ppm, 17.7 ppm, and 41.4 ppm, respectively) were used in this study. Compared with the control group, the stress groups had higher inductions of MTs and MT-1 and MT-3 mRNA in hippocampus. On the one hand, the expressions of MTs and their mRNAs in hippocampus were downregulated in the zinc-deficient group; however, their expressions were evidently enhanced in the stress zinc-deficient group. MT induction in the zinc-supplemented group was increased. Furthermore, the stress zinc-supplemented group had a more significant yield of MTs and their mRNAs. In addition, the levels of plasma cortisol, interleukin-6 (IL-6), IL-1, and nitric oxide (NO) were increased clearly in the zinc-deficient group and the stress groups. The results suggest that zinc deficiency may decrease and zinc supplementation may increase the expressions of MTs and their mRNAs in hippocampus; moreover, stress can increase their expressions dramatically. The impairment of stress on the body may be involved with the nutrition status of zinc, and zinc deficiency can lower the body's adaptability to stress.     

Modulation of the hippocampal protein machinery in voluntary and treadmill exercising rats

Information about protein expression studies in the brain of exercising and sedentary animals is limited. Cognitive functions change during exercise and the aim of this study was to investigate rat protein levels of the protein machinery in the hippocampus, the main cognitive brain area for spatial learning and memory, in exercising rats. Protein fluctuations may reflect functional variation during exercise. Male Sprague-Dawley rats, 23 months old, were used for the study: the first group consisted of sedentary rats, the second of rats undertaking voluntary exercise from 5 months to 23 months and the third undertaking involuntary exercise on a treadmill from 5 months to 23 months. Two-dimensional gel electrophoresis with subsequent mass spectrometrical identification assigning spots to proteins and determination of coomassie-densities was carried out. Heterogeneous nuclear ribonucleoprotein K, one protein variant of heat shock cognate 71 kDa protein and BAG family molecular chaperone regulator 5 showed differential protein levels in the three groups when a p-value of <0.005 was considered as statistically significant thus respecting multiple testing. The biological meaning of changed protein levels in hippocampus under different conditions of exercise is not known but warrants further investigation.     

Zinc homeostasis in the hippocampus of zinc-deficient young adult rats

On the basis of the evidence of the transient learning impairment of young adult rats fed a zinc-deficient diet for 4 weeks, zinc concentration in the hippocampus was examined in the zinc-deficient rats to understand the mechanism of brain dysfunction in zinc deficiency. Zinc concentration in the hippocampus, as well as that in other brain regions, was not decreased by 4-week zinc deprivation. When Timm's stain, with which histochemically reactive zinc in the presynaptic vesicles is detected, was compared between the control and zinc-deficient rats, the intensity of Timm's stain in the hippocampus was almost the same between them. In the hippocampus, zinc concentration in the synaptosomal fraction was not also decreased by 4-week zinc deprivation, whereas that in the crude nuclear fraction was significantly increased. These results suggest that zinc concentration in the presynaptic vesicles is not decreased in young adults rats by 4-week zinc deprivation. It is likely that zinc-requiring systems in the nucleus are more responsive to zinc deficiency than vesicular zinc. This responsiveness appears to be involved in the transient learning impairment.     

Crosstalk between L-type calcium channels and ZnT-1, a new player in rate-dependent cardiac electrical remodeling

Crosstalk between two membrane transport systems is an established mechanism underlying regulation. In this study, we investigated the interaction between ZnT-1, a putative plasma membrane zinc transporter, and L-type voltage-dependent calcium channels (LTCC). In the atrium of the myocardium decreased activity of the LTCC is a dominant feature of patients with atrial fibrillation. The trigger for this inhibition has been attributed to the rapid firing rates and consequent calcium overload in the atrial cardiomyocytes. However, the underlying mechanism of LTCC inhibition is still to be elucidated. Here, we showed that the expression of ZnT-1 inhibits the activity of L-type channels during electrical remodeling induced by rapid pacing. (i) Direct manipulations of ZnT-1 expression in cultured cardiomyocytes either by ZnT-1 overexpression or by ZnT-1 silencing with siRNA, decreased or enhanced, respectively, the barium influx through the LTCC. (ii) Co-expression of ZnT-1 with LTCC in Xenopus oocytes decreased whole cell barium current through LTCC. (iii) Rapid pacing of cultured cardiomyocytes (4 h, 100 ms cycle) increased ZnT-1 protein expression and inhibited the voltage-dependent divalent cation influx through the LTCC. Moreover, silencing ZnT-1 with siRNA prevented the rapid pacing induced inhibition of the LTCC (iv) Atrial pacing of anesthetized adult rats (4 h, 50 ms cycle) led to a significant increase in atrial ZnT-1 protein expression in parallel with the typical decrease of the refractory period in the atria. Taken together, these findings demonstrate that crosstalk between ZnT-1 and the L-type calcium channels may underlie atrial response to rapid pacing, suggesting that ZnT-1 is a significant participant in rate-dependent cardiac electrical remodeling.     

Zinc-regulating proteins, ZnT-1, and metallothionein I/II are present in different cell populations in the mouse testis.

Zinc ions play an important role in testis development and spermatogenesis. Thus, nutritional zinc deficiency leads to aberrant testicular development, reduced spermatogenesis, and male sterility. The precise actions of zinc in mediating these functions and the mechanisms by which zinc is itself regulated in the testis, however, have not been adequately elucidated. We have assessed the distribution of the zinc-regulating proteins ZnT-1 and metallothionein I/II (MT I/II) in the mouse seminiferous tubule. Co-labeling for ZnT-1 and MT I/II demonstrated unique patterns of distribution for these proteins, with ZnT-1 present in Sertoli cells in addition to luminal spermatozoa and MT I/II restricted to spermatocytes. These findings were confirmed by dual-label immunofluorescence for ZnT-1 and the Sertoli cell marker, vimentin, and by immunoelectron microscopy. The differential expression patterns of ZnT-1 and MTs support the hypothesis that ZnT-1 and MTs play different roles in the regulation of intracellular zinc in this organ. The specific expression of ZnT-1 in the Sertoli cells, moreover, is consistent with their role in maintaining a nurturing, closely regulated environment for spermatogenesis.     

ZnT-2, a mammalian protein that confers resistance to zinc by facilitating vesicular sequestration.

A cDNA encoding a second zinc transporter (ZnT-2) was isolated from a rat kidney cDNA expression library by complementation of a zinc-sensitive BHK cell line. The protein predicted from the open reading frame of ZnT-2 cDNA has 359 amino acids and initiates with a CTG codon. It resembles ZnT-1 (a plasma membrane protein that stimulates zinc efflux) in overall topology in that it has six membrane-spanning domains, a histidine-rich intracellular loop and a long C-terminal tail; however, the overall amino acid identity is only 26%. Unlike ZnT-1, which is in the plasma membrane and lowers cellular zinc by stimulating zinc efflux, ZnT-2 is localized on vesicles and allows the zinc-sensitive BHK cells to accumulate zinc to levels that are much higher than non-transformed cells can tolerate. Zinc was visualized within these vesicles with zinquin, a zinc-specific fluorescent probe. The intracellular compartment that accumulates zinc is acidic as revealed by staining with acridine orange or LysoTracker. Prolonged exposure of cells expressing ZnT-2 to zinc causes an accretion of intracellular vesicles. We suggest that ZnT-2 protects these cells from zinc toxicity by facilitating zinc transport into an endosomal/lysosomal compartment.     

Increased expression of zinc transporter ZIP4, ZIP11, ZnT1, and ZnT6 predicts poor prognosis in pancreatic cancer

IntroductionZinc homeostasis is regulated by SLC39A/ZIP, SLC30A/ZnT, and metallothionein (MT) families in human cells. Zinc dyshomeostasis may affect or be affected by the abnormal behavior of cancer cells. Although decreased serum zinc levels are observed in patients with pancreatic adenocarcinoma (PAAD), limited information is available regarding the expression pattern and prognostic roles of zinc homeostasis-related genes in PAAD.ObjectivesThe primary objective of this study was to explore the expression pattern and prognostic roles of zinc homeostasis-related genes in PAAD.MethodsThe expression pattern of 35 known zinc homeostasis-related genes in PAAD was systemically explored based on RNA-sequencing data from the Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) projects. The association between the expression levels of zinc homeostasis-related genes and survival of PAAD patients was evaluated using the Kaplan-Meier method and the log-rank test. Expressional correlation between zinc homeostasis-related genes with potential prognostic value in PAAD and normal pancreatic controls was evaluated using Pearson’s correlation analysis. Functional enrichment analyses were performed to elucidate possible mechanisms for the potential prognostic and therapeutic roles of these zinc homeostasis-related genes in PAAD. Effects of ZIP11, ZnT1, or ZnT6 knockdown on the proliferation and the migration of Capan-1 pancreatic cancer cells were assessed by the CCK-8 assay and the wound healing assay respectively.ResultsWe demonstrated that the expression levels of ZIP1, ZIP3, ZIP4, ZIP6, ZIP7, ZIP9, ZIP10, ZIP11, ZIP13, ZnT1, ZnT5, ZnT6, ZnT7, and ZnT9 were increased, whereas the expression levels of ZIP5, ZIP14, ZnT2, MT1 G, MT1H, and MT1X were decreased in PAAD tumors compared with normal pancreatic controls. Among these differentially-expressed genes related to zinc homeostasis, higher expression of ZIP4, ZIP11, ZnT1 or ZnT6 predicted poorer prognosis with the possible involvement of several cancer-related processes and pathways in PAAD patients. We further demonstrated that knockdown of ZIP11 attenuated Capan-1 cell proliferation with decreased activation of ERK1/2 pathway; knockdown of ZnT1 attenuated Capan-1 cell proliferation with decreased activation of ERK1/2, p38 MAPK, NF-kB, and mTOR pathways; knockdown of ZnT6 attenuated Capan-1 cell proliferation with decreased activation of ERK1/2, p38 MAPK, and NF-kB pathways.ConclusionsHigher expression of the zinc transporter ZIP4, ZIP11, ZnT1 or ZnT6 predicted poorer prognosis in patients with PAAD. These findings provide new clues for understanding the complex relationship between zinc homeostasis and pancreatic cancer.