Korean J Physiol Pharmacol.  2022 Nov;26(6):479-499. 10.4196/kjpp.2022.26.6.479.

Cell-cell contacts via N-cadherin induce a regulatory renin secretory phenotype in As4.1 cells

• Chang JW
• Kim S
• Lee EY
• Leem CH
• Kim SH
• Park CS

• KMID: 2534524
• DOI: http://doi.org/10.4196/kjpp.2022.26.6.479

Abstract

The lack of a clonal renin-secreting cell line has greatly hindered the investigation of the regulatory mechanisms of renin secretion at the cellular, biochemical, and molecular levels. In the present study, we investigated whether it was possible to induce phenotypic switching of the renin-expressing clonal cell line As4.1 from constitutive inactive renin secretion to regulated active renin secretion. When grown to postconfluence for at least two days in media containing fetal bovine serum or insulin-like growth factor-1, the formation of cell-cell contacts via N-cadherin triggered downstream cellular signaling cascades and activated smooth muscle-specific genes, culminating in phenotypic switching to a regulated active renin secretion phenotype, including responding to the key stimuli of active renin secretion. With the use of phenotype-switched As4.1 cells, we provide the first evidence that active renin secretion via exocytosis is regulated by phosphorylation/dephosphorylation of the 20 kDa myosin light chain. The molecular mechanism of phenotypic switching in As4.1 cells described here could serve as a working model for full phenotypic modulation of other secretory cell lines with incomplete phenotypes.

Keyword

MRTF-A; Myosin light chain; Myosin light-chain kinase; Myosin light chain phosohatase-1; N-cadherin; Phenotype switching

Figure

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  Fig. 1 Growth curve and cadherin expression before (lane 1) and after confluence (lane 2). (A) Growth curve. Cells were plated (3 × 103 cells/well) onto 24-well plates (day 0), cultured for 10 days and counted on a hemocytometer every 2–3 days (n = 3). (B) Phase contrast microscopy of ~70% and 100% confluent cells (×200). Scale bar: 60 μm. (C) Expression of cadherins. Protein (40 μg) obtained from cell lysates of cells grown to ~70% confluence (lane 1) or from cells on day 2 postconfluence (lane 2) were resolved by 7% acrylamide SDS-PAGE, followed by Western blotting with antibodies against epithelial cadherin (E-cad), placental cadherin (P-Cad), or neural cadherin (N-Cad). The numbers on the left indicate the molecular masses of standard proteins in kilo Daltons (kDa). The density of the bands was compared by unpaired Student’s tests (n = 3 for each group). β-actin was used as the protein loading control for each gel. NS, no significant difference. (D) N-cad expression at ~70% confluence (upper panel) and 100% confluence (lower panel). Cell nuclei were stained with propidium iodide (DAPI). Scale bar: 10 μm.

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  Fig. 2 BILA 2157 BS inhibits renin activity. Incubation media from postconfluent cells with a renin activity of 268.3 ± 51 ng ANG I/ml/h (relative value 1.0) was incubated with varying concentrations of BILA 2157 BS for 1 h. Active renin activity was determined by radioimmunoassay for ANG I. Each data point represents the mean ± SD of three samples. ANG I, angiotensin I.

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  Fig. 3 Effects of Ca2+, calmidazolium, ML-7, isoproterenol and forskolin on active renin secretion in postconfluent As4.1 cells. Cells on day 2 postconfluence were incubated in Ca2+-free DMEM containing 1 mM EGTA (–Ca2+ DMEM) (A, left panel) and then in 2 mM Ca2+-containing DMEM and 10% FBS (+Ca2+ DMEM) successively for 1 h each or in the reverse order (A, right panel, p < 0.001, n = 6). In the next series of experiments, cells were incubated in +Ca2+ DMEM for 1 h (control) and then another 1 h in +Ca2+ DMEM + 10 % FBS containing either calmidazolium (3 × 10−5 M) (B, n = 6), ML-7 (6 × 10−5M) (C, n = 6), isoproterenol (10−8–10−5 M) (D, n = 6) or forskolin (3 × 10−5 M) plus IBMX (10−4 M) (E, n = 6). The rate of active renin secretion was determined by radioimmunoassay for ANG I in (A) and (B) or by ELISA in (C), (D), and (E). ANG I, angiotensin I; DMEM, Dulbecco’s modified eagle medium; FBS, fetal bovine serum. Samples within groups were compared using paired Student’s t-tests and between groups using unpaired Student’s t-tests (A–C, E) or ANOVA (D), *p < 0.001 vs. control, except between samples at the two highest concentrations, which were compared by unpaired Student’s t-tests. †p < 0.001.

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  Fig. 4 Expression of N-cadherin, MRTF-A, sm MHC, nm MHC and PP1β changes to As4.1 cells before and after confluence. Cell lysates from cells grown to 70%–80% confluence in the presence of 10% FBS (A, lane 1), cells at day 2 postconfluence in the continuous presence of 10% FBS (A, lane 2) or in the absence of 10% FBS for two days after achieving 100% confluence (A, lane 3). After the aspiration of the incubation media, cells were lysed and centrifuged. Supernatant protein (60 μg) was resolved by SDS-PAGE on 7% or 15% acrylamide slab gels followed by Western blotting for N-cad, MRTF-A, sm MHC, nm MHC, and PP1β and then analyzed by densitometry. In the case of MRTF-A, a nuclear fraction was prepared, and 100 μg was resolved (B, upper panel). p-values were obtained by ANOVA. To assess nuclear localization of MRTF-A, cells were plated onto coverslips and fixed and permeabilized as described in the Methods section. Cells were then immunostained with primary MRTF-A antibody (1:100) and then with FITC-labelled goat anti-mouse secondary antibody to MRTF-A (1:1,000). Nuclei were stained with propidium iodide (DAPI) (B, lower panel). The density of preconfluent cells was arbitrarily set to 1. Fluorescence was analyzed by an image analyzer. Lane 1, preconfluent cells; lane 2, postconfluent cells in the presence of 10% FBS. p-values were obtained by unpaired Student’s t-tests (n = 4). Scale bar: 10 μM. (C) Postconfluent cells were incubated in DMEM + 10% FBS with or without CCG-1423 (5 × 10−6 M), an inhibitor of nuclear translocation of MRTF-A, for 1 h and then with ML-7 (6 × 10−5 M) for another 1 h. Secreted renin was measured by ELISA (C, n = 5). MRTF-A, myocardin related transcription factor-A; sm MHC, smooth muscle myosin heavy chain; nm MHC, nonmuscle myosin heavy chain; PP1β, protein phosphatase 1β; FBS, fetal bovine serum. NS, no significant difference. p-values within groups were obtained by paired Student’s t-tests, and those between groups were obtained by ANOVA.

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  Fig. 5 Effects of MRTF-A knockout on phenotypic changes. Cells at ~70% confluence were transfected with control plasmids (A, lane 1) or the MRTF-A gene was knocked out using MRTF-A HDR CRISPR-associated 9 (Cas9) (A, lane 2) for three days. On day 2 postconfluence, the expression of MRTF-A, N-cad, sm MHC, nm MHC, and PP1β in the supernatant fraction (80 μg) was determined as described in the legend of . p-values were obtained by unpaired Student’s t-tests. (B) Control and knockout cells were incubated before and after stimulation with ML-7 (6 × 10−5 M) for 1 h each, and secreted active renin was measured by ELISA (B, n = 4). MRTF-A, myocardin related transcription factor-A; HDR, homology directed repair; CRISPR, clustered regularly interspaced short palindromic repeats; sm MHC, smooth muscle myosin heavy chain; nm MHC, nonmuscle myosin heavy chain; PP1β, protein phosphatase 1β. NS, no significant difference. p-values were obtained either by paired Student’s t-tests (within groups) or by ANOVA (between groups).

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  Fig. 6 Effects of SRF knockout on phenotypic changes. Cells were transfected with control plasmids (A, lane 1) or SRF HDR CRISPR/Cas9 plasmids (A, lane 2) as described in the legend of . Then, the expression of each protein was determined in the supernatant fraction (80 μg). Both control and knockout cells were incubated with ML-7 (6 × 10−5 M) before and after stimulation for 1 h each, and secreted active renin was measured by ELISA (B, n = 5). SRF, serum response factor; HDR, homology directed repair; CRISPR, clustered regularly interspaced short palindromic repeats. p-values were obtained either by paired Student’s t-tests (within groups) or by ANOVA (between groups).

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  Fig. 7 Effects of N-cad expression on pMLC20 and PP1β. Cells were cultured to 100% confluence and maintained for two more days in the presence of 10% FBS (lane 1) or in the presence of both FBS and IGF-1 antibody (10 μg/ml) (lane 2). Proteins in the supernatant (40 μg) were resolved by Western blotting as described above (n = 4). pMLC20, phosphorylated 20 kDa myosin light chain; PP1β, protein phosphatase 1β; FBS, fetal bovine serum; IGF-I, insulin-like growth factor-I. p-values were obtained by unpaired Student’s t-tests.

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  Fig. 8 Effects of MYLK knockdown on phenotypic changes. Cells were transfected with control or siRNA of MYLK plasmids as described in the legend of . Expression of MYLK and the cytosolic pMLC20 level (A) were determined using supernatant protein (60 μg). p-values were obtained by unpaired Student’s t-tests (n = 3). Active renin secretion from control and knockdown cells was determined by incubating cells in DMEM + 10% FBS for 1 h (B, n =6). pMLC20, phosphorylated 20 kDa myosin light chain; DMEM, Dulbecco’s modified eagle medium; FBS, fetal bovine serum. p-values by unpaired Student’s t-tests.

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  Fig. 9 Effects of PP1β knockdown on phenotypic changes. Cells were transfected with control (lane 1) or siRNA of PP1β plasmids as described in the legend for . The expression of PP1β (A, upper panel) and cytosolic pMLC20 level (A, lower panel) were determined using supernatant protein (60 μg). p-values were obtained by unpaired Student’s t-tests (n = 4). Active renin secretion was determined before and after stimulation by ML-7 (6 × 10−5 M) for 1 h each, and secreted renin was measured by ELISA (B). PP1β, protein phosphatase 1β; pMLC20, phosphorylated 20 kDa myosin light chain. p-values were obtained either by paired Student’s t-tests (within groups) or by ANOVA (between groups) (n = 6).

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  Fig. 10 Effects of ML-7 and calyculin A on the phosphorylation of MLC20. On day 2, postconfluent cells were incubated in DMEM + 10% FBS (control) in the presence of ML-7 (6 × 10−5 M; ML-7) and in the presence of both calyculin A (2 × 10−7 M) and ML-7 (6 × 10−5 M; ML-7 + Caly) for 1 h. The level of pMLC20 was determined using supernatant protein (80 μg). MLC20, 20 kDa myosin light chain; DMEM, Dulbecco’s modified eagle medium; FBS, fetal bovine serum. p-values were obtained by ANOVA (n = 5).

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  Fig. 11 Imaging of exocytotic discharge of renin and neutral red by ML-7. Cells on postconfluent day 2 were plated on cover slips were permeabilized and stained with antibody to renin. Many cytoplasmic stained granules are seen (A, control). When cells were pretreated with ML-7 (6 × 10−5 M) for 2 min and stained with renin antibody, no granules were observed (ML-7). Scale bar: 10 μm. Cells were incubated with 100 μM neutral red for 3 h, washed with fresh DMEM, and photographed with a light microscope. Numerous neutral red stained pink granules are seen (B). N denotes nucleus. Scale bar: 3.33 μm. (C, D) When neutral red-loaded granules were viewed at the isosbestic absorbent light at 470 nm, the granules observed were black. White granules are likely discharged granules prior to imaging. When cells were perfused with ML-7 (6 × 10−5 M), #1, #2, and #3 neutral red-loaded granules were discharged over 200 msec. (D) The time-lapsed movie shows the discharge of neutral red-loaded secretory granules (Supplementary Video). DMEM, Dulbecco’s modified eagle medium.

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  Fig. 12 Schematic summary of the transcriptional cascades regulating expression of proteins associated with renin secretory phenotypes in As4.1 cells. N-cad expression at the plasma membrane upon cell-cell contact triggers MRTF-A-SRF-CArG box, which activates transcription of smooth muscle-specific genes encoding contractile proteins in association with the regulatory phenotype of active renin secretion (left side). Without N-cad expression, β-catenin (signaling cascade in the middle) and growth factors (signaling cascade of right side) trigger a signaling cascade of growth factors of cell proliferation. MRTF-A, myocardin related transcription factor-A; SRF, serum response factor; MLC20, 20 kDa myosin light chain; PP1β, protein phosphatase 1β; FBS, fetal bovine serum; IGF-I, insulin-like growth factor-I; sm MHC, smooth muscle myosin heavy chain; nm MHC, nonmuscle myosin heavy chain.

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