Store-operated calcium entry (SOCE) is the main Ca 2؉ influx pathway involved in controlling proliferation of the human hepatoma cell lines Huh-7 and HepG2. However, the molecular nature of the calcium channels involved in this process remains unknown. Huh-7 and HepG2 cells express transient receptor potential canonical 1 (TRPC1) and TRPC6, as well as STIM1 and Orai1, and these 4 channels are the most likely candidates to account for the SOCE in these cells. We generated stable TRPC6-overexpressing or TRPC6-knockdown Huh-7 clones, in which we investigated correlations between the presence of the protein, the rate of cell proliferation, and SOCE amplitude. TRPC6-overexpressing Huh-7 cells proliferated 80% faster than did untransfected cells and their SOCE amplitude was 160% higher. By contrast, proliferation rate was 50% lower and SOCE amplitude 85% lower in TRPC6-knockdown clones than in untransfected cells. OAG (olyl acetyl glycerol)-induced calcium entry was similar in all cells, and small interfering RNA (siRNA) against TRPC1 had no effect on SOCE amplitude, highlighting the relationship among SOCE, TRPC6 and cell proliferation in Huh-7 cells. SOCE amplitude was reduced by STIM1 and Orai1 knockdowns, suggesting possible cooperation between these proteins and TRPC6 in these cells. Endothelial growth factor and hepatocyte growth factor increased TRPC6 expression and SOCE amplitude in Huh-7 cells, and cyclin D1 expression was decreased by STIM1, Orai1, and TRPC6 knockdowns. Conclusion: TRPC6 was very weakly expressed in isolated hepatocytes from healthy patients and expressed more strongly in tumoral samples from the liver of a cancer patient, strongly supporting a role for these calcium channels in liver oncogenesis. (HEPATOLOGY 2008;47:2068-2077 T he processes involved in the transformation of normal cells into tumorigenic cells and tumor progression are complex and only partially understood. 1,2 Many proteins in cancer cells are produced in larger or smaller amounts than in normal cells. Some of these proteins associated with cancer progression are involved in calcium homeostasis. Several types of cancer cells require an influx of calcium to proliferate, and it has been known for more than 30 years that the proliferation and growth of normal vertebrate cells depend on the physiological concentration of extracellular calcium. 3,4 Calcium influx is required at various stages of the cell cycle, 4 but little is known about the nature of the calcium channels involved in this process in nonexcitable cells such as liver cells. Two separate calcium entry pathways are simultaneously activated in liver cells. Phospholipase C stimulation results in InsP3 and diacylglycerol (DAG) generation, leading to capacitative or store-operated calcium entry (SOCE) because of a decrease in the calcium concentration of the endoplasmic reticulum (ER) lumen. A second type of calcium channel, activated independently of store depletion by the membrane-permeant analogue of DAG, 1-oleoy-2-acetyl-glycerol (OAG), 5 has also been described an...

Prostate smooth muscle cells predominantly express alpha1-adrenoceptors (alpha1-AR). alpha1-AR antagonists induce prostate smooth muscle relaxation and therefore they are useful therapeutic compounds for the treatment of benign prostatic hyperplasia symptoms. However, the Ca(2+) entry pathways associated with the activation of alpha1-AR in the prostate have yet to be elucidated. In many cell types, mammalian homologues of transient receptor potential (TRP) genes, first identified in Drosophila, encode TRPC (canonical TRP) proteins. They function as receptor-operated channels (ROCs) which are involved in various physiological processes such as contraction, proliferation, apoptosis, and differentiation. To date, the expression and function of TRPC channels have not been studied in prostate smooth muscle. In fura-2 loaded PS1 (a prostate smooth muscle cell line) which express endogenous alpha1A-ARs, alpha-agonists epinephrine (EPI), and phenylephrine (PHE) induced Ca(2+) influx which depended on the extracellular Ca(2+) and PLC activation but was independent of PKC activation. Thus, we have tested two membrane-permeable analogues of diacylglycerol (DAG), oleoyl-acyl-sn-glycerol (OAG) and 1,2-dioctanoyl-sn-glycerol (DOG). They initiated Ca(2+) influx whose properties were similar to those induced by the alpha-agonists. Sensitivity to 2-aminoethyl diphenylborate (2-APB), SKF-96365 and flufenamate implies that Ca(2+)-permeable channels mediated both alpha-agonist- and OAG-evoked Ca(2+) influx. Following the sarcoplasmic reticulum (SR) Ca(2+) store depletion by thapsigargin (Tg), a SERCA inhibitor, OAG and PHE were both still able to activate Ca(2+) influx. However, OAG failed to enhance Ca(2+) influx when added in the presence of an alpha-agonist. RT-PCR and Western blotting performed on PS1 cells revealed the presence of mRNAs and the corresponding TRPC3 and TRPC6 proteins. Experiments using an antisense strategy showed that both alpha-agonist- and OAG-induced Ca(2+) influx required TRPC3 and TRPC6, whereas the Tg-activated ("capacitative") Ca(2+) entry involved only TRPC3 encoded protein. It may be thus concluded that PS1 cells express TRPC3 and TRPC6 proteins which function as receptor- and store-operated Ca(2+) entry pathways.

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