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Molecular mechanisms underlying Ca2+-mediated motility of human pancreatic duct cells.

ABSTRACT: We recently reported that transforming growth factor-? (TGF-?) induces an increase in cytosolic Ca(2+) ([Ca(2+)](cyt)) in pancreatic cancer cells, but the mechanisms by which TGF-? mediates [Ca(2+)](cyt) homeostasis in these cells are currently unknown. Transient receptor potential (TRP) channels and Na(+)/Ca(2+) exchangers (NCX) are plasma membrane proteins that play prominent roles in controlling [Ca(2+)](cyt) homeostasis in normal mammalian cells, but little is known regarding their roles in the regulation of [Ca(2+)](cyt) in pancreatic cancer cells and pancreatic cancer development. Expression and function of NCX1 and TRPC1 proteins were characterized in BxPc3 pancreatic cancer cells. TGF-? induced both intracellular Ca(2+) release and extracellular Ca(2+) entry in these cells; however, 2-aminoethoxydiphenyl borate [2-APB; a blocker for both inositol 1,4,5-trisphosphate (IP(3)) receptor and TRPC], LaCl(3) (a selective TRPC blocker), or KB-R7943 (a selective inhibitor for the Ca(2+) entry mode of NCX) markedly inhibited the TGF-?-induced increase in [Ca(2+)](cyt). 2-APB or KB-R7943 treatment was able to dose-dependently reverse membrane translocation of PKC? induced by TGF-?. Transfection with small interfering RNA (siRNA) against NCX1 almost completely abolished NCX1 expression in BxPc3 cells and also inhibited PKC? serine phosphorylation induced by TGF-?. Knockdown of NCX1 or TRPC1 by specific siRNA transfection reversed TGF-?-induced pancreatic cancer cell motility. Therefore, TGF-? induces Ca(2+) entry likely via TRPC1 and NCX1 and raises [Ca(2+)](cyt) in pancreatic cancer cells, which is essential for PKC? activation and subsequent tumor cell invasion. Our data suggest that TRPC1 and NCX1 may be among the potential therapeutic targets for pancreatic cancer.

SUBMITTER: Dong H

PROVIDER: S-EPMC3006328 | biostudies-literature | 2010 Dec

REPOSITORIES: biostudies-literature

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Molecular mechanisms underlying Ca2+-mediated motility of human pancreatic duct cells.

Dong Hui H Shim Ki-Nam KN Li Jenny M J JM Estrema Christine C Ornelas Tiffany A TA Nguyen Flang F Liu Shanglei S Ramamoorthy Sonia L SL Ho Samuel S Carethers John M JM Chow Jimmy Y C JY

American journal of physiology. Cell physiology 20100922 6

We recently reported that transforming growth factor-β (TGF-β) induces an increase in cytosolic Ca(2+) ([Ca(2+)](cyt)) in pancreatic cancer cells, but the mechanisms by which TGF-β mediates [Ca(2+)](cyt) homeostasis in these cells are currently unknown. Transient receptor potential (TRP) channels and Na(+)/Ca(2+) exchangers (NCX) are plasma membrane proteins that play prominent roles in controlling [Ca(2+)](cyt) homeostasis in normal mammalian cells, but little is known regarding their roles in ...[more]

PMID: 20861471

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Project description:Aims/hypothesis: Duct cells isolated from adult human pancreas can be reprogrammed to express islet beta cell genes by adenoviral transduction of the developmental transcription factor neurogenin3 (Ngn3). In this study we aimed to fully characterize the extent of this reprogramming and intended to improve it. Methods: The extent of the Ngn3-mediated duct-to-endocrine cell reprogramming was measured employing genome wide mRNA profiling. By modulation of the Delta-Notch signaling or addition of pancreatic endocrine transcription factors Myt1, MafA and Pdx1 we intended to improve the reprogramming. Results: Ngn3 stimulates duct cells to express a focused set of genes that are abundant in islet endocrine cells and/or neural tissues. This neuro-endocrine shift, however, covers a minor fraction (5%) of the estimated genome-wide transcriptome difference between duct and islet endocrine cells. Interestingly, transduction of exogenous Ngn3 activates endogenous Ngn3 suggesting auto-activation of this gene. Furthermore, pancreatic endocrine reprogramming of human duct cells can be moderately enhanced by inhibition of Delta-Notch signaling as well as by co-expressing the transcription factor Myt1, but not MafA and Pdx1. Conclusions/interpretation: The results provide further insight into the plasticity of adult human duct cells and suggest measurable routes to enhance Ngn3-mediated in vitro reprogramming protocols for regenerative beta cell therapy in diabetes. We used Affymetrix HG133A and HG133B to get a comprehensive view on the reprogramming potential in vitro of human pancreatic duct cell cultures (n=3-4) at 3 and 14/20 days after ectopic adenoviral expression of murine neurogenin 3 as compared to GFP-expressing control vectors. The microarray analysis was performed on 3 independent samples that each contained RNA extracted from a pool of 3 independent donor pancreata. The total number of non-selected donor organs is 9. Transcripts were considered as differentially regulated by Ngn3 when 1.5 fold (LCB, unpaired P < 0.05) up- or down-regulated in AdGFP-Ngn3 versus AdGFP controls, at 3 and/or 14 dpi. Transcripts that showed differential expression between day 3 and day 14 in AdGFP-Ngn3 duct cells but not in AdGFP control cells, were also considered Ngn3-regulated

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Molecular mechanisms underlying Ca2+-mediated motility of human pancreatic duct cells.

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Molecular mechanisms underlying Ca2+-mediated motility of human pancreatic duct cells.

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