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. 2014 Mar;35(9):2868-77.
doi: 10.1016/j.biomaterials.2013.12.030. Epub 2014 Jan 4.

The Role of Mechanical Tension on Lipid Raft Dependent PDGF-induced TRPC6 Activation

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Free PMC article

The Role of Mechanical Tension on Lipid Raft Dependent PDGF-induced TRPC6 Activation

Lei Lei et al. Biomaterials. .
Free PMC article

Abstract

Canonical transient receptor potential channel 6 (TRPC6) can play an important role in governing how cells perceive the surrounding material environment and regulate Ca(2+) signaling. We have designed a TRPC6 reporter based on fluorescence resonance energy transfer (FRET) to visualize the TRPC6-mediated calcium entry and hence TRPC6 activity in live cells with high spatiotemporal resolutions. In mouse embryonic fibroblasts (MEFs), platelet-derived growth factor BB (PDGF) can activate the TRPC6 reporter, mediated by phospholipase C (PLC). This TRPC6 activation occurred mainly at lipid rafts regions of the plasma membrane because disruption of lipid raft/caveolae by methyl-β-cyclodextrin (MβCD) or the expression of dominant-negative caveolin-1 inhibited the TRPC6 activity. Culturing cells on soft materials or releasing the intracellular tension by ML-7 reduced this PDGF-induced activation of TRPC6 without affecting the PDGF-regulated Src or inositol 1,4,5-trisphosphate (IP3) receptor function, suggesting a specific role of mechanical tension in regulating TRPC6. We further showed that the release of intracellular tension had similar effect on the diffusion coefficients of TRPC6 and a raft marker, confirming a strong coupling between TRPC6 and lipid rafts. Therefore, our results suggest that the TRPC6 activation mainly occurs at lipid rafts, which is regulated by the mechanical cues of surrounding materials.

Keywords: Canonical transient receptor potential 6 (TRPC6); Fluorescence resonance energy transfer (FRET); Intracellular tension; Lipid rafts; Mechanical microenvironment.

Figures

Fig. 1
Fig. 1. The characterization of the TRPC6 FRET reporter
(A) The domain structure of the TRPC6 FRET reporter. This reporter includes a full length human TRPC6 with a cameleon Ca2+ reporter D3cpv at its C terminus. Cyan column and arrow presents ECFP and its emission; yellow column and arrow presents cpVenus and its emission, respectively. (B) and (D) FRET/CFP ratio images of HEK293T cells before (left) and after (right) 10 min stimulation by 100 μM OAG. HEK293T cells were transfected with (B) the wild type and mutant TRPC6 reporter or (D) the cameleon D3cpv reporter, wild type or mutant TRPC6 expression construct with D3cpv reporter as indicated. In all the panels, the color scale bars represent the FRET/CFP emission ratio, with cold and hot colors representing low and high activities of reporter, respectively. (C) and (E) Representative time courses of normalized FRET/CFP emission ratio of (C) TRPC6 reporter (black dots) and its mutant (white dots) or (E) D3cpv reporter (D3cpv plus empty vector, triangle; D3cpv plus wild type TRPC6, black dots; and D3cpv plus mutant TRPC6, white dots) before and after OAG stimulation, including cells from (B) or (D), respectively. 100 μM OAG was added at time 0 as indicated. The time courses represent the means±SD of each time point from multi-samples by setting the average FRET/CFP ratio of time points before stimulation to 1.0; “n” represents the cell number in each group. Size scale bar, 20 μm.
Fig. 2
Fig. 2. PDGF activates TRPC6 via the PLC pathway in MEFs
(A) and (B) Representative time courses of normalized FRET/CFP emission ratio of TRPC6 reporter (black dots) and its mutant (white dots) upon (A) 300 μM OAG or (B) 10 ng/ml PDGF stimulation. (C) Responses of TRPC6, KRas-Src or D3cpv reporter to 10 ng/ml PDGF stimulation in MEFs with (solid bars) or without (open bars) 2 μM U73122 pretreatment for 5 min. MEFs transfected with the TRPC6 reporter were pretreated with 1 μM TG for 1 hr before imaging to deplete the calcium storage ofintracellular organelles. MEFs transfected with D3cpv were maintained in Ca2+ free HBSS buffer during imaging to eliminate the calcium influx across the plasma membrane. (D) Responses of TRPC6 reporter to 300 μM OAG stimulation in MEFs with or without 2μM U73122 pretreatment for 5 min. The data represents the means±SD from multi-samples. “n” represents the cell number in each group. * indicates the significant difference between indicated groups (P < 0.01).
Fig. 3
Fig. 3. The activation of TRPC6 depends on lipid rafts and caveolae
(A) Responses of Lyn-D3cpv and KRas-D3cpv reporters upon 100 μM OAG or 10 nM ATP stimulation as indicated. Lyn-D3cpv or KRas-D3cpv reporter was transfected into HEK293T cells with (OAG stimulation) or without the co-transfection of wild type TRPC6 (ATP stimulation) as indicated. (B) and (D) Response of TRPC6 reporter to 100 μM OAG stimulation in HEK239T cells (B) with 10 μM MβCD pretreatment or (D) with the expression of dominate negative Caveolin-1 (Cav 1 S80E). MbCD was added into medium 1 hr before stimulation with OAG. (C) and (E) Responses of TRPC6, KRas-Src or D3cpv reporter to 10 ng/ml PDGF stimulation in MEFs (C) with 10 μM MβCD pretreatment or (E) with the expression of Cav1 S80E. MEFs transfected with TRPC6 reporter were pretreated with 1 μM TG for 1 hr before imaging. MEFs transfected with the D3cpv reporter were maintained in Ca2+ free HBSS buffer during imaging. (F) Confocal images of FITC-CTxB labeled ganglioside GM1 and TRPC6-mCherry in MEFs. “n” represents the cell number in each group. * indicates the significant difference between indicated groups (P < 0.01). Scale bar, 25 μm.
Fig. 4
Fig. 4. Substrate rigidity and intercellular tension affect TRPC6 activity
(A) and (B) Representative time courses of normalized FRET/CFP emission ratio of TRPC6 reporter inresponse to 65 dyn/cm2 shear stress stimulation (time 0) in HEK293T cells (A) or BAECs (B). (C) and (E) Response of TRPC6 reporter to 300 μM OAG stimulation in MEFs (C) cultured on rigid surface (glass) or soft substrate (gel with 600 pa stiffness) or (E) with 5 μM ML-7 pretreatment for 1hr. (D) and (F) Responses of TRPC6, KRas-Src, or D3cpv reporter to 10 ng/ml PDGF stimulation in MEFs (D) cultured on rigid surface (glass) or soft substrate (gel with 600 pa stiffness) or (F) with 5 μM ML-7 pretreatment for 1 hr. MEFs transfected with TRPC6 reporter were pretreated with 1 μM TG for 1 hr before imaging. MEFs transfected with D3cpv were maintained in Ca2+ free HBSS buffer during imaging. “n” represents the cell number in each group. * indicates the significant difference between indicated groups (P < 0.01).
Fig. 5
Fig. 5. Reduced intracellular tension decreases the diffusion coefficients of TRPC6 and ganglioside GM1
(A) The fluorescence intensity image of a cell expressing TRPC6-YFP before photobleaching (left), and at 0 and 2 min after photobleaching (middle and right), respectively. Yellow broken cycle highlights the region of interest for photobleaching. (B) Left: the concentration map after photobleaching (0 min), computed by normalizing the fluorescence intensity with the images before photobleaching. Right: the concentration map overlaid with a triangular mesh generated in the mobile region. (C) The time course of fluorescence recovery in the photobleached area as marked in (A). (D) The estimated diffusion coefficients of TRPC6 and ganglioside GM1 in MEFs transfected with TRPC6-YFP or stained with FITC-CTxB. MEFs were also treated with or without 5 μM ML-7 for 1 hr before FRAP assay. “n” represents the cell number in each group. * indicates the significant difference between indicated groups (P < 0.05). Scale bar, 20 μm.
Fig. 6
Fig. 6. A proposed signaling network depicting the PDGF induced TRPC6 activation at the plasma membrane
PDGF receptors dimerize upon binding to PDGF and cause the activation of receptor kinase activity. The activated PDGF receptors induce the activation of downstream Src, and PLC which generates IP3 and DAG. IP3 activates the IP3 receptor at ER while DAG can activate TRPC6 at the plasma membrane, specifically on the lipid rafts to induce the Ca2+ influx across the plasma membrane. Intercellular tension generated by the actomyosin contractility can affect the interaction between actin cytoskeleton and membrane lipid raft/caveolae microdomains to regulate the structure and function of lipid rafts, which eventually determines the TRPC6 response to PDGF-induced signaling.

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