Capacitative calcium entry and transient receptor potential canonical 6 expression control human hepatoma cell proliferation

Abstract

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...

The role of TRPC6 in HGF-induced cell proliferation of human prostate cancer DU145 and PC3 cells

“…These observations fit well with TRPC6 mediating the HGF-induced inflow of extracellular Ca 2+ . TRPC6 channels function as store-operated calcium channels that account for the sustained calcium inflow triggered by the IP 3 -evoked depletion of intracellular Ca 2+ stores [9]. Furthermore, TRPC6, as a receptoroperated Ca 2+ channel in primary hPCE cells, is also directly activated by DAG [11,12].…”

“…It is not clear how TRPC6 is activated under basal conditions in our cultures. It is possible that growth factors, such as VEGF [19] and EGF [9], in the culture medium during basal conditions activate TRPC6. Inconsistent with our results, some research has indicated that overexpressing wild-type TRPC6 in some types of cancer cells does not enhance cell proliferation [10,27].…”

“…Through a phospholipase C (PLC)-dependent mechanism, they can be activated by G-protein coupled receptors or receptor tyrosine kinases [7]. Recently, it has been shown that TRPC6 contributes to the proliferation of some types of cancer cells [8][9][10]. In addition, in the primary human prostate cancer epithelial (hPCE) cells and LNCaP cells, agonist-mediated stimulation of α 1 -adrenergic receptors requires the coupled activation of TRPC6 channels and nuclear factors of activated T cells to promote cell proliferation [11,12].…”

“…HGF induces epithelial tubular cell proliferation, migration, scattering and tubulogenesis through KCNA1, TRPC6 and NHE1 [16]. In addition, El Boustany et al [9] found that endothelial growth factor (EGF) and HGF increase TRPC6 levels and induce a large increase in store-operated calcium entry amplitude in Huh-7 cells. However, the mechanisms by which HGF induces this calcium entry are not yet understood.…”

A Reciprocal Shift in Transient Receptor Potential Channel 1 (TRPC1) and Stromal Interaction Molecule 2 (STIM2) Contributes to Ca2+ Remodeling and Cancer Hallmarks in Colorectal Carcinoma Cells

“…cell migration and invasion (10,14). STIM1 and ORAI1 underlie I CRAC and regulate glioblastoma cell proliferation, apoptosis, and invasion (15,16) and are involved in neuroblastoma proliferation as well (17).…”