The TRPC1 Channel Forms a PI3K/CaM Complex and Regulates Pancreatic Ductal Adenocarcinoma Cell Proliferation in a Ca2+-Independent Manner
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ABSTRACT:
SUBMITTER: Schnipper J
PROVIDER: S-EPMC9323718 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Transient receptor potential canonical (TRPC) channels are the most prominent nonselective cation channels involved in various diseases. However, the function, clinical significance, and molecular mechanism of TRPCs in colorectal cancer (CRC) progression remain unclear. In this study, we identified that TRPC1 was the major variant gene of the TRPC family in CRC patients. TRPC1 was upregulated in CRC tissues compared with adjacent normal tissues and high expression of TRPC1 was associated with more aggressive tumor progression and poor overall survival. TRPC1 knockdown inhibited cell proliferation, cell-cycle progression, invasion, and migration in vitro, as well as tumor growth in vivo; whereas TRPC1 overexpression promoted colorectal tumor growth and metastasis in vitro and in vivo. In addition, colorectal tumorigenesis was significantly attenuated in Trpc1-/- mice. Mechanistically, TRPC1 could enhance the interaction between calmodulin (CaM) and the PI3K p85 subunit by directly binding to CaM, which further activated the PI3K/AKT and its downstream signaling molecules implicated in cell cycle progression and epithelial-mesenchymal transition. Silencing of CaM attenuated the oncogenic effects of TRPC1. Taken together, these results provide evidence that TRPC1 plays a pivotal oncogenic role in colorectal tumorigenesis and tumor progression by activating CaM-mediated PI3K/AKT signaling axis. Targeting TRPC1 represents a novel and specific approach for CRC treatment.
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Project description:BACKGROUND: The Tic complex (Translocon at the inner envelope membrane of chloroplasts) mediates the translocation of nuclear encoded chloroplast proteins across the inner envelope membrane. Tic110 forms one prominent protein translocation channel. Additionally, Tic20, another subunit of the complex, was proposed to form a protein import channel - either together with or independent of Tic110. However, no experimental evidence for Tic20 channel activity has been provided so far. RESULTS: We performed a comprehensive biochemical and electrophysiological study to characterize Tic20 in more detail and to gain a deeper insight into its potential role in protein import into chloroplasts. Firstly, we compared transcript and protein levels of Tic20 and Tic110 in both Pisum sativum and Arabidopsis thaliana. We found the Tic20 protein to be generally less abundant, which was particularly pronounced in Arabidopsis. Secondly, we demonstrated that Tic20 forms a complex larger than 700 kilodalton in the inner envelope membrane, which is clearly separate from Tic110, migrating as a dimer at about 250 kilodalton. Thirdly, we defined the topology of Tic20 in the inner envelope, and found its N- and C-termini to be oriented towards the stromal side. Finally, we successfully reconstituted overexpressed and purified full-length Tic20 into liposomes. Using these Tic20-proteoliposomes, we could demonstrate for the first time that Tic20 can independently form a cation selective channel in vitro. CONCLUSIONS: The presented data provide first biochemical evidence to the notion that Tic20 can act as a channel protein within the chloroplast import translocon complex. However, the very low abundance of Tic20 in the inner envelope membranes indicates that it cannot form a major protein translocation channel. Furthermore, the independent complex formation of Tic20 and Tic110 argues against a joint channel formation. Thus, based on the observed channel activity of Tic20 in proteoliposomes, we speculate that the chloroplast inner envelope contains multiple (at least two) translocation channels: Tic110 as the general translocation pore, whereas Tic20 could be responsible for translocation of a special subset of proteins.
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Project description:Painful axotomy decreases K(ATP) channel current (IK(ATP)) in primary afferent neurons. Because cytosolic Ca(2+) signaling is depressed in injured dorsal root ganglia (DRG) neurons, we investigated whether Ca(2+)-calmodulin (CaM)-Ca(2+)/CaM-dependent kinase II (CaMKII) regulates IK(ATP) in large DRG neurons. Immunohistochemistry identified the presence of K(ATP) channel subunits SUR1, SUR2, and Kir6.2 but not Kir6.1, and pCaMKII in neurofilament 200-positive DRG somata. Single-channel recordings from cell-attached patches revealed that basal and evoked IK(ATP) by ionomycin, a Ca(2+) ionophore, is activated by CaMKII. In axotomized neurons from rats made hyperalgesic by spinal nerve ligation (SNL), basal K(ATP) channel activity was decreased, and sensitivity to ionomycin was abolished. Basal and Ca(2+)-evoked K(ATP) channel activity correlated inversely with the degree of hyperalgesia induced by SNL in the rats from which the neurons were isolated. Inhibition of IK(ATP) by glybenclamide, a selective K(ATP) channel inhibitor, depolarized resting membrane potential (RMP) recorded in perforated whole-cell patches and enhanced neurotransmitter release measured by amperometry. The selective K(ATP) channel opener diazoxide hyperpolarized the RMP and attenuated neurotransmitter release. Axotomized neurons from rats made hyperalgesic by SNL lost sensitivity to the myristoylated form of autocamtide-2-related inhibitory peptide (AIPm), a pseudosubstrate blocker of CaMKII, whereas axotomized neurons from SNL animals that failed to develop hyperalgesia showed normal IK(ATP) inhibition by AIPm. AIPm also depolarized RMP in control neurons via K(ATP) channel inhibition. Unitary current conductance and sensitivity of K(ATP) channels to cytosolic ATP and ligands were preserved even after painful nerve injury, thus providing opportunities for selective therapeutic targeting against neuropathic pain.
| S-EPMC2681318 | biostudies-literature
Project description:In contrast to other Classical Transient Receptor Potential TRPC channels the function of TRPC1 as an ion channel is a matter of debate, because it is often difficult to obtain substantial functional signals over background in response to over-expression of TRPC1 alone. Along these lines, heterologously expressed TRPC1 is poorly translocated to the plasma membrane as a homotetramer and may not function on its own physiologically, but may rather be an important linker and regulator protein in heteromeric TRPC channel tetramers. However, due to the lack of specific TRPC1 antibodies able to detect native TRPC1 channels in primary cells, identification of functional TRPC1 containing heteromeric TRPC channel complexes in the plasma membrane is still challenging. Moreover, an extended TRPC1 cDNA, which was recently discovered, may seriously question results obtained in heterologous expression systems transfected with shortened cDNA versions. Therefore, this review will focus on the current status of research on TRPC1 function obtained in primary cells and a TRPC1-deficient mouse model.
| S-EPMC4276908 | biostudies-literature
Project description:It has been clear that cancer-associated fibroblasts (CAFs) in the tumor microenvironment play an important role in pancreatic ductal adenocarcinoma (PDAC) progression. However, how CAFs relate to the patients' prognosis and the effects of chemoradiation therapy (CRT) has not been fully investigated. Tissue microarrays (TMAs) representing 167 resected PDACs without preoperative treatment were used for immunohistochemical studies (IHC) of palladin, ?-smooth muscle actin (SMA), and podoplanin. Correlations between the expression levels of these markers and clinicopathological findings were analyzed statistically. Whole sections of surgical specimens from PDACs with and without preoperative CRT, designated as the chemotherapy-first group (CF, n = 19) and the surgery-first group (SF, n = 21), respectively, were also analyzed by IHC. In TMAs, the disease-specific survival rate (DSS) at 5 years for all 167 cases was 23.1%. Seventy cases (41.9%) were positive for palladin and had significantly lower DSS (p = 0.0430). ?-SMA and podoplanin were positive in 167 cases (100%) and 131 cases (78.4%), respectively, and they were not significantly associated with DSS. On multivariable analysis, palladin expression was an independent poor prognostic factor (p = 0.0243, risk ratio 1.60). In the whole section study, palladin positivity was significantly lower (p = 0.0037) in the CF group (5/19) with a significantly better DSS (p = 0.0144) than in the SF group (16/22), suggesting that stromal palladin expression is a surrogate indicator of the treatment effect after chemoradiation therapy.
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Project description:SOCE (store-operated Ca2+ entry) is mediated via specific plasma membrane channels in response to ER (endoplasmic reticulum) Ca2+ store depletion. This route of Ca2+ entry is central to the dynamic interplay between Ca2+ and cAMP signalling in regulating the activity of Ca2+-sensitive adenylate cyclase isoforms (AC1, AC5, AC6 and AC8). Two proteins have been identified as key components of SOCE: STIM1 (stromal interaction molecule 1), which senses ER Ca2+ store content and translocates to the plasma membrane upon store depletion, where it then activates Orai1, the pore-forming component of the CRAC (Ca2+ release-activated Ca2+) channel. Previous studies reported that co-expression of STIM1 and Orai1 in HEK-293 (human embryonic kidney 293) cells enhances Ca2+-stimulated AC8 activity and that AC8 and Orai1 directly interact to enhance this regulation. Nonetheless, the additional involvement of TRPC (transient receptor potential canonical) channels in SOCE has also been proposed. In the present study, we evaluate the contribution of TRPC1 to SOCE-mediated regulation of Ca2+-sensitive ACs in HEK-293 cells stably expressing AC8 (HEK-AC8) and HSG (human submandibular gland) cells expressing an endogenous Ca2+-inhibited AC6. We demonstrate a role for TRPC1 as an integral component of SOCE, alongside STIM1 and Orai1, in regulating Ca2+ fluxes within AC microdomains and influencing cAMP production.
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Project description:Previous results link the mitochondrial potassium channel Kv1.3 (mitoKv1.3) to the regulation of apoptosis. By synthesizing new, mitochondria-targeted derivatives (PAPTP and PCARBTP) of PAP-1, a specific membrane-permeant Kv1.3 inhibitor, we have recently provided evidence that both drugs acting on mitoKv1.3 are able to induce apoptosis and reduce tumor growth in vivo without affecting healthy tissues and cells. In the present article, by exploiting these new drugs, we addressed the question whether mitoKv1.3 contributes to the regulation of cell proliferation as well. When used at low concentrations, which do not compromise cell survival, both drugs slightly increased the percentage of cells in S phase while decreased the population at G0/G1 stage of cells from two different pancreatic ductal adenocarcinoma lines. Our data suggest that the observed modulation is related to ROS levels within the cells, opening the way to link mitochondrial ion channel function to downstream, ROS-related signaling events that might be important for cell cycle progression.
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Project description:Pancreatic ductal adenocarcinoma (PDAC) stands as a highly aggressive and lethal cancer, characterized by a grim prognosis and scarce treatment alternatives. Within this context, TRPV6, a calcium-permeable channel, emerges as a noteworthy candidate due to its overexpression in various cancers, capable of influencing the cell behavior in different cancer entities. Nonetheless, the exact expression pattern and functional significance of TRPV6 in the context of PDAC remains enigmatic. This study scrutinizes the expression of TRPV6 in tissue specimens obtained from 46 PDAC patients across distinct stages and grades. We manipulated TRPV6 expression (knockdown, overexpression) in the human PDAC cell lines Panc-1 and Capan-1. Subsequently, we analyzed its impact on multiple facets, encompassing Ca2+ influx, proliferation, apoptosis, migration, chemoresistance, and tumor growth, both in vitro and in vivo. Notably, the data indicate a direct correlation between TRPV6 expression levels, tumor stage, and grade, establishing a link between TRPV6 and PDAC proliferation in tissue samples. Decreasing TRPV6 expression via knockdown hampered Ca2+ influx, resulting in diminished proliferation and viability in both cell lines, and cell cycle progression in Panc-1. The knockdown simultaneously led to an increase in apoptotic rates and increased the susceptibility of cells to 5-FU and gemcitabine treatments. Moreover, it accelerated migration and promoted collective movement among Panc-1 cells. Conversely, TRPV6 overexpression yielded opposing outcomes in terms of proliferation in Panc-1 and Capan-1, and the migration of Panc-1 cells. Intriguingly, both TRPV6 knockdown and overexpression diminished the process of tumor formation in vivo. This intricate interplay suggests that PDAC aggressiveness relies on a fine-tuned TRPV6 expression, raising its profile as a putative therapeutic target.
| S-EPMC10741494 | biostudies-literature
Project description:Skeletal muscle contraction is reputed not to depend on extracellular Ca2+. Indeed, stricto sensu, excitation-contraction coupling does not necessitate entry of Ca2+. However, we previously observed that, during sustained activity (repeated contractions), entry of Ca2+ is needed to maintain force production. In the present study, we evaluated the possible involvement of the canonical transient receptor potential (TRPC)1 ion channel in this entry of Ca2+ and investigated its possible role in muscle function. Patch-clamp experiments reveal the presence of a small-conductance channel (13 pS) that is completely lost in adult fibers from TRPC1(-/-) mice. The influx of Ca2+ through TRPC1 channels represents a minor part of the entry of Ca(2+) into muscle fibers at rest, and the activity of the channel is not store dependent. The lack of TRPC1 does not affect intracellular Ca2+ concentration ([Ca2+](i)) transients reached during a single isometric contraction. However, the involvement of TRPC1-related Ca2+ entry is clearly emphasized in muscle fatigue. Indeed, muscles from TRPC1(-/-) mice stimulated repeatedly progressively display lower [Ca2+](i) transients than those observed in TRPC1(+/+) fibers, and they also present an accentuated progressive loss of force. Interestingly, muscles from TRPC1(-/-) mice display a smaller fiber cross-sectional area, generate less force per cross-sectional area, and contain less myofibrillar proteins than their controls. They do not present other signs of myopathy. In agreement with in vitro experiments, TRPC1(-/-) mice present an important decrease of endurance of physical activity. We conclude that TRPC1 ion channels modulate the entry of Ca(2+) during repeated contractions and help muscles to maintain their force during sustained repeated contractions.
| S-EPMC2806157 | biostudies-literature
Project description:We evaluated currents induced by expression of human homologs of Orai together with STIM1 in human embryonic kidney cells. When co-expressed with STIM1, Orai1 induced a large inwardly rectifying Ca(2+)-selective current with Ca(2+)-induced slow inactivation. A point mutation of Orai1 (E106D) altered the ion selectivity of the induced Ca(2+) release-activated Ca(2+) (CRAC)-like current while retaining an inwardly rectifying I-V characteristic. Expression of the C-terminal portion of STIM1 with Orai1 was sufficient to generate CRAC current without store depletion. 2-APB activated a large relatively nonselective current in STIM1 and Orai3 co-expressing cells. 2-APB also induced Ca(2+) influx in Orai3-expressing cells without store depletion or co-expression of STIM1. The Orai3 current induced by 2-APB exhibited outward rectification and an inward component representing a mixed calcium and monovalent current. A pore mutant of Orai3 inhibited store-operated Ca(2+) entry and did not carry significant current in response to either store depletion or addition of 2-APB. Analysis of a series of Orai1-3 chimeras revealed the structural determinant responsible for 2-APB-induced current within the sequence from the second to third transmembrane segment of Orai3. The Orai3 current induced by 2-APB may reflect a store-independent mode of CRAC channel activation that opens a relatively nonselective cation pore.
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