Recent studies indicate that the transient receptor potential canonical 6 (TRPC6) channel is highly expressed in several types of cancer cells. However, it remains unclear whether TRPC6 contributes to the malignancy of human non-small cell lung cancer (NSCLC). We used a human NSCLC A549 cell line as a model and found that pharmacological blockade or molecular knockdown of TRPC6 channel inhibited A549 cell proliferation by arresting cell cycle at the S-G2M phase and caused a significant portion of cells detached and rounded-up, but did not induce any types of cell death. Western blot and cell cycle analysis show that the detached round cells at the S-G2M phase expressed more TRPC6 than the still attached polygon cells at the G1 phase. Patch-clamp data also show that TRPC whole-cell currents in the detached cells were significantly higher than in the still attached cells. Inhibition of Ca2+-permeable TRPC6 channels significantly reduced intracellular Ca2+ in A549 cells. Interestingly, either blockade or knockdown of TRPC6 strongly reduced the invasion of this NSCLC cell line and decreased the expression of an adherent protein, fibronectin, and a tight junction protein, zonula occluden protein-1 (ZO-1). These data suggest that TRPC6-mediated elevation of intracellular Ca2+ stimulates NSCLC cell proliferation by promoting cell cycle progression and that inhibition of TRPC6 attenuates cell proliferation and invasion. Therefore, further in vivo studies may lead to a consideration of using a specific TRPC6 blocker as a complement to treat NSCLC.
Regulation of the epithelial sodium channel (ENaC), which regulates fluid homeostasis and blood pressure, is complex and remains incompletely understood. The TIP peptide, a mimic of the lectin-like domain of TNF, activates ENaC by binding to glycosylated residues in the extracellular loop of ENaC-α as well as to a hitherto uncharacterized internal site. Molecular docking studies suggested three residues, Val 567 , Glu 568 , and Glu 571 , located at the interface between the second transmembrane and C-terminal domains of ENaC-α, as a critical site for binding of the TIP peptide. We generated Ala replacement mutants in this region of ENaC-α and examined its interaction with TIP peptide (3M, V567A/E568A/E571A; 2M, V567A/E568A; and 1M, E571A). 3M and 2M ENaC-α but not 1M ENaC-α, displayed significantly reduced binding capacity to TIP peptide and to TNF. When overexpressed in H441 cells,3M mutant ENaC-α formed functional channels with similar gating and density characteristics as the WT subunit and efficiently associated with the β and γ subunits in the plasma membrane. We subsequently assayed for increased open probability time and membrane expression, both of which define ENaC activity, following addition of TIP peptide. TIP peptide increased open probability time in H441 cells overexpressing wild type and 1M ENaC-α channels, but not 3M or 2M ENaC-α channels. On the other hand, TIP peptide-mediated reduction in ENaC ubiquitination was similar in cells overexpressing either WT or 3M ENaC-α subunits. In summary, this study has identified a novel site in ENaC-α that is crucial for activation of the open probability of the channel, but not membrane expression, by the lectin-like domain of TNF.
Clinical evidence suggests that statins reduce cancer incidence and mortality. However, there is lack of in vitro data to show the mechanism by which statins can reduce the malignancies of cancer cells. We used a human B lymphoma Daudi cells as a model and found that lovastatin inhibited, whereas exogenous cholesterol (Cho) stimulated, proliferation cell cycle progression in control Daudi cells, but not in the cells when transient receptor potential canonical 6 (TRPC6) channel was knocked down. Lovastatin decreased, whereas Cho increased, the levels of intracellular reactive oxygen species (ROS) respectively by decreasing or increasing the expression of p47-phox and gp91-phox (NOX2). Reducing intracellular ROS with either a mimetic superoxide dismutase (TEMPOL) or an NADPH oxidase inhibitor (apocynin) inhibited cell proliferation, particularly in Cho-treated cells. The effects of TEMPOL or apocynin were mimicked by inhibition of TRPC6 with SKF-96365. Lovastatin decreased TRPC6 expression and activity via a Cho-dependent mechanism, whereas Cho increased TRPC6 expression and activity via an ROS-dependent mechanism. Consistent with the fact that TRPC6 is a Ca 2+ -permeable channel, lovastatin decreased, but Cho increased, intracellular Ca 2+ also via ROS. These data suggest that lovastatin inhibits malignant B cell proliferation by reducing membrane Cho, intracellular ROS, TRPC6 expression and activity, and intracellular Ca 2+ .
The renal epithelial sodium channel (ENaC) provides regulated sodium transport in the distal nephron. The effects of intracellular calcium ([Ca2+]i) on this channel are only beginning to be elucidated. It appears from previous studies that the [Ca2+]i increases downstream of ATP administration may have a polarized effect on ENaC, where apical application of ATP and the subsequent [Ca2+]i increase have an inhibitory effect on the channel, whereas basolateral ATP and [Ca2+]i have a stimulatory effect. We asked whether this polarized effect of ATP is, in fact, reflective of a polarized effect of increased [Ca2+]i on ENaC and what underlying mechanism is responsible. We began by performing patch clamp experiments in which ENaC activity was measured during apical or basolateral application of ionomycin to increase [Ca2+]i near the apical or basolateral membrane, respectively. We found that ENaC does indeed respond to increased [Ca2+]i in a polarized fashion, with apical increases being inhibitory and basolateral increases stimulating channel activity. In other epithelial cell types, mitochondria sequester [Ca2+]i, creating [Ca2+]i signaling microdomains within the cell that are dependent on mitochondrial localization. We found that mitochondria localize in bands just beneath the apical and basolateral membranes in two different cortical collecting duct principal cell lines and in cortical collecting duct principal cells in mouse kidney tissue. We found that inhibiting mitochondrial [Ca2+]i uptake destroyed the polarized response of ENaC to [Ca2+]i. Overall, our data suggest that ENaC is regulated by [Ca2+]i in a polarized fashion and that this polarization is maintained by mitochondrial [Ca2+]i sequestration.
Adrenomedullin (ADM) is a vasodilator that causes natriuresis and diuresis. However, the direct effect of ADM on osmotic water permeability in the rat inner medullary collecting duct (IMCD) has not been tested. We investigated whether ADM and its ADM receptor components (CRLR, RAMP2, and 3) are expressed in rat inner medulla (IM) and whether ADM regulates osmotic water permeability in isolated perfused rat IMCDs. The mRNAs of ADM, CRLR, and RAMP2 and 3 were detected in rat IM. Abundant protein of CRLR and RAMP3 were also seen but RAMP2 protein level was extremely low. Adding ADM (100 nM) to the bath significantly decreased osmotic water permeability. ADM significantly decreased aquaporin-2 (AQP2) phosphorylation at Serine 256 (pS256) and increased it at Serine 261 (pS261). ADM significantly increased cAMP levels in IM. However, inhibition of cAMP by SQ22536 further decreased ADM-attenuated osmotic water permeability. Stimulation of cAMP by roflumilast increased ADM-attenuated osmotic water permeability. Previous studies show that ADM also stimulates phospholipase C (PLC) pathways including protein kinase C (PKC) and cGMP. We tested whether PLC pathways regulate ADM-attenuated osmotic water permeability. Blockade of either PLC by U73122 or PKC by rottlerin significantly augmented the ADM-attenuated osmotic water permeability and promoted pS256-AQP2 but did change pS261-AQP2. Inhibition of cGMP by L-NAME did not change AQP2 phosphorylation. In conclusion, ADM primarily binds to the CRLR-RAMP3 receptor to initiate signaling pathways in the IM. ADM reduced water reabsorption through a PLC-pathway involving PKC. ADM-attenuated water reabsorption may be related to decreased trafficking of AQP2 to the plasma membrane. cAMP is not involved in ADM-attenuated osmotic water permeability.
Our recent studies indicate that hydrogen peroxide (H 2 O 2 ) only at high concentrations can cause oxidative stress in renal epithelial cells and induce apoptosis of podocytes. Consistently, the present study shows that H 2 O 2 , even at 1mM, failed to induce intracellular oxidative stress and apoptosis of the podocytes due to efficient activity of catalase, an enzyme which degrades H 2 O 2 to produce water and oxygen (O 2 ). However, H 2 O 2 acted as a source of O 2 to allow acute ethanol to induce superoxide production and cause apoptosis of the podocytes. In contrast, acute ethanol alone did not elevate intracellular superoxide, even though it stimulates expression and translocation of p47phox to the plasma membrane. Inhibition of catalase abolished not only O 2 production from H 2 O 2 degradation, but also NOX2-dependent superoxide production in the podocytes challenged by both H 2 O 2 and acute ethanol. In parallel, acute ethanol in the presence of H 2 O 2 , but neither ethanol nor H 2 O 2 alone, stimulated transient receptor potential canonical 6 (TRPC6) channels and caused TRPC6-dependent elevation of intracellular Ca 2+ . These data suggest that exogenous H 2 O 2 does not induce oxidative stress due to rapid degradation to produce O 2 in the podocytes, but the oxygenated podocytes become sensitive to acute ethanol challenge and undergo apoptosis via a TRPC6-dependent elevation of intracellular Ca 2+ . Since cultured podocytes are considered in hypoxic conditions, H 2 O 2 may be used as a source of O 2 to establish an ischemia-reperfusion model in some type of cultured cells in which H 2 O 2 does not directly induce intracellular oxidative stress.
Hypertrophy is an adaptive response that enables organs to appropriately meet increased functional demands. Previously, we reported that calcineurin (Cn) is required for glomerular and whole kidney hypertrophy in diabetic rodents (Gooch, J. L., Barnes, J. L., Garcia, S., and Abboud, H. E. (2003). Calcineurin is activated in diabetes and is required for glomerular hypertrophy and ECM accumulation. Am. J. Physiol. Renal Physiol. 284, F144–F154; Reddy, R. N., Knotts, T. L., Roberts, B. R., Molkentin, J. D., Price, S. R., and Gooch, J. L. (2011). Calcineurin Aβ is required for hypertrophy but not matrix expansion in the diabetic kidney. J. Cell Mol. Med. 15, 414–422). Because studies have also implicated the reactive oxygen species-generating enzymes NADPH oxidases (Nox) in diabetic kidney responses, we tested the hypothesis that Nox and Cn cooperate in a common signaling pathway. First, we examined the role of the two main isoforms of Cn in hypertrophic signaling. Using primary kidney cells lacking a catalytic subunit of Cn (CnAα−/− or CnAβ−/−), we found that high glucose selectively activates CnAβ, whereas CnAα is constitutively active. Furthermore, CnAβ but not CnAα mediates hypertrophy. Next, we found that chronic reactive oxygen species generation in response to high glucose is attenuated in CnAβ−/− cells, suggesting that Cn is upstream of Nox. Consistent with this, loss of CnAβ reduces basal expression and blocks high glucose induction of Nox2 and Nox4. Inhibition of nuclear factor of activated T cells (NFAT), a CnAβ-regulated transcription factor, decreases Nox2 and Nox4 expression, whereas NFAT overexpression increases Nox2 and Nox4, indicating that the CnAβ/NFAT pathway modulates Nox. These data reveal that the CnAβ/NFAT pathway regulates Nox and plays an important role in high glucose-mediated hypertrophic responses in the kidney.
The thiazide-sensitive sodium chloride cotransporter (NCC) and the epithelial sodium channel (ENaC) are two of the most important determinants of salt balance and thus systemic blood pressure. Abnormalities in either result in profound changes in blood pressure. There is one segment of the nephron where these two sodium transporters are coexpressed, the second part of the distal convoluted tubule. This is a key part of the aldosterone-sensitive distal nephron, the final regulator of salt handling in the kidney. Aldosterone is the key hormonal regulator for both of these proteins. Despite these shared regulators and coexpression in a key nephron segment, associations between these proteins have not been investigated. After confirming apical localization of these proteins, we demonstrated the presence of functional transport proteins and native association by blue native PAGE. Extensive coimmunoprecipitation experiments demonstrated a consistent interaction of NCC with α-And γ-ENaC. Mammalian two-hybrid studies demonstrated direct binding of NCC to ENaC subunits. Fluorescence resonance energy transfer and immunogold EM studies confirmed that these transport proteins are within appropriate proximity for direct binding. Additionally, we demonstrate that there are functional consequences of this interaction, with inhibition of NCC affecting the function of ENaC. This novel finding of an association between ENaC and NCC could alter our understanding of salt transport in the distal tubule.