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Homer1 mediates CaSR-dependent activation of mTOR complex 2 and initiates a novel pathway for AKT-dependent β-catenin stabilization in osteoblasts - PubMed

  • ️Tue Jan 01 2019

Homer1 mediates CaSR-dependent activation of mTOR complex 2 and initiates a novel pathway for AKT-dependent β-catenin stabilization in osteoblasts

Mark S Rybchyn et al. J Biol Chem. 2019.

Abstract

The calcium-sensing receptor (CaSR) is critical for skeletal development, but its mechanism of action in osteoblasts is not well-characterized. In the central nervous system (CNS), Homer scaffolding proteins form signaling complexes with two CaSR-related members of the G protein-coupled receptor (GPCR) family C, metabotropic glutamate receptor 1 (mGluR1) and mGluR5. Here, we show that CaSR and Homer1 are co-expressed in mineralized mouse bone and also co-localize in primary human osteoblasts. Co-immunoprecipitation experiments confirmed that Homer1 associates with CaSR in primary human osteoblasts. The CaSR-Homer1 protein complex, whose formation was increased in response to extracellular Ca2+, was bound to mechanistic target of rapamycin (mTOR) complex 2 (mTORC2), a protein kinase that phosphorylates and activates AKT Ser/Thr kinase (AKT) at Ser473 siRNA-based gene-silencing assays with primary osteoblasts revealed that both CaSR and Homer1 are required for extracellular Ca2+-stimulated AKT phosphorylation and thereby inhibit apoptosis and promote AKT-dependent β-catenin stabilization and cellular differentiation. To confirm the role of the CaSR-Homer1 complex in AKT initiation, we show that in HEK-293 cells, co-transfection with both Homer1c and CaSR, but neither with Homer1c nor CaSR alone, establishes sensitivity of AKT-Ser473 phosphorylation to increases in extracellular Ca2+ concentrations. These findings indicate that Homer1 mediates CaSR-dependent AKT activation via mTORC2 and thereby stabilizes β-catenin in osteoblasts.

Keywords: Akt PKB; G protein-coupled receptor (GPCR); Homer1; bone formation; calcium-sensing receptor (CaSR); cell signaling; family C G-protein–coupled receptor; mTOR complex (mTORC); osteoblast; scaffold protein; β-catenin.

© 2019 Rybchyn et al.

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.

We propose Homer1 may link CaSR to mTORC2 in osteoblasts to promote AKT-dependent β-catenin stabilization. A proposal of this study is that Homer-1 may link CaSR to mTORC2 activation upstream of AKT (indicated by ?). AKT phosphorylation is known to occur via two discrete mechanisms: PI3K-dependent phosphorylation at Thr308, and mTORC2-dependent phosphorylation at Ser473. AKT subsequently inhibits GSK3 and directly phosphorylates β-catenin to promote β-catenin stabilization and allowing subsequent nuclear translocation leading to cellular differentiation. AKT also suppresses apoptosis and promotes growth in the cell. Kinase target of Torin1 is mTOR. PM = plasma membrane.

Figure 2.
Figure 2.

CaSR and Homer1 were necessary for mTORC2-dependent dual phosphorylation of AKT in response to elevated extracellular Ca2+ in osteoblasts. A, phosphorylations of AKT at Thr308/Ser473 from same osteoblasts by Western blotting in response to Ca2+o shift 1–2 m

m

over 1 h. B, Western blotting of AKT phosphorylated at Ser473/Thr308 and mTOR phosphorylated at Ser2448 in response to Ca2+o shift 1–2 m

m

for 15 min in the presence of Torin1 (pretreatment 5 min). Concentration is shown as fold times the half-maximal inhibitory concentration (IC50). IC50 Torin1 ∼2 n

m

. C and E, Western blotting of phosphorylations of AKT at Thr308/Ser473, in response to Ca2+o shift 1–2 m

m

for 15 min after CaSR (siCaSR) (C) or Homer1 (siHomer1) (E) protein levels reduced (+) compared with control transfected cells (−). D and F, densitometry of triplicate blots shown for silenced CaSR (C) and Homer1 (E), respectively. #, solid, vertical black lines on the presented Western blotting indicate where lanes have been spliced from the same Western blotting for presentation purposes. The data shown in C–F are also presented in Fig. 4 as part of the broader signaling cascade investigated in the study. *, p < 0.05; **, p < 0.01.

Figure 3.
Figure 3.

CaSR and Homer1 were co-expressed in mineralized bone, co-localized in vitro, and co-immunoprecipitated with mTOR and the mTORC2-specific protein Rictor. A, deconvolution microscopy images of the mineralized area of the long bones of WT (top row) or CaSR−/− (bottom row) mice stained for CaSR (green) or Homer1 (red). Nuclei (blue) stained with DAPI. ×63 objective. Scale, 50 μm. B, confocal microscopy images of a monolayer culture of primary human osteoblasts taken with ×10 objective (top row, scale bar, 10 μm) or a single osteoblast taken under oil with ×63 objective (bottom row, scale bar, 2 μm) stained for CaSR (green) or Homer (red). Nuclei (blue) were stained with DAPI. C, human osteoblasts were exposed to Ca2+o shift 1–3.5 m

m

for 2 min and then lysed with a nonionic detergent. Soluble protein was immunoprecipitated with anti-Homer1 (H) or an isotype control (Iso). Detection of Homer1 (panel i), as a control between Ca2+o treatments, CaSR (panel ii), mTOR (panel iii), Rictor (panel iv), and Raptor (panel v) by Western blotting from the immunoprecipitated fraction. Total lysate is Western blotting of nonimmunoprecipitated osteoblast lysate; # is nonglycosylated CaSR. D, densitometry from triplicate blots, where bands were detected, shown in C for Homer1 (panel i), CaSR (panel ii), mTOR (panel iii), and Rictor (panel iv). ns, no significance; *, p < 0.05; ***, p < 0.001.

Figure 4.
Figure 4.

CaSR and Homer1 modulated Ca2+-dependent activation of the AKT pathway in human osteoblasts. A–D, phosphorylation of AKT at Ser473/Thr308, GSK3α–Ser21, GSK3β–Ser9, β-catenin–Ser552, mTOR–Ser2448, and β-actin (loading control) from human osteoblasts by Western blotting. A, osteoblasts exposed to various Ca2+o concentrations for 15 min. B–D, panel i, osteoblasts were exposed to Ca2+o shift from 1–2 m

m

for 15 min after silencing: B, CaSR (siCaSR); C, Homer1 (siHom1); D, Gα12 (siGα12) (+), and the lysates were then processed for Western blotting detection of various proteins and phosphoproteins as shown. The impact of siRNA on the silenced gene is shown at the bottom of each column in each of B (panel i), C (panel i), and D (panel i), where expression is shown as “silenced gene.” For each transfection, the effect of a nondirected control siRNA sequence (siCTRL) is shown (−). B and C, panel ii, densitometry results from triplicate blots shown B (panel i), C (panel i), and D (panel i). *, p < 0.05; **, p < 0.01, ns, not significantly different. # Solid, vertical black lines on the presented Western blotting indicate where lanes have been spliced from the same Western blotting for presentation purposes. The AKT, CaSR, and Homer1 data shown in B and C are also shown in Fig. 2, which focuses independently on AKT.

Figure 5.
Figure 5.

CaSR and Homer1-modulated Ca2+-dependent β-catenin nuclear translocation in human osteoblasts. A, nuclear β-catenin (β-cat) is shown in response to panel i, Ca2+o shift from 1 to 2 m

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over a 60-min period, or panels ii and iii, a 30-min Ca2+o shift 1 to 2 m

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with reduction in CaSR protein level (siCaSR; panel ii) or reduction in Homer1 protein level (siHomer1; panel iii) by siRNA. The nuclear (nuc) protein laminB1 (lamB1) was used as a loading control for the nuclear fraction. Panels iv and v, densitometry from triplicate blots for silenced CaSR and Homer1. Panel vi, OPG protein levels, corrected for total cell protein, measured by whole-cell ELISA in cells that were incubated for 24 h in 2 m

m

Ca2+o ± AKT kinase inhibitor AKT-XI (20 μ

m

). B, Western blotting of OPG that was chemically extracted from marrow flushed mouse long bones from WT (CaSR+/+), heterozygous (CaSR+/−), or CaSR knockout (CaSR−/−). β-Actin loading control. *, p < 0.05; **, p < 0.01.

Figure 6.
Figure 6.

CaSR and Homer1-modulated Ca2+-dependent phosphorylation of AKT–Ser473 and GSK3β–Ser9 in MG63 osteosarcoma cells. A, Western blotting showing level of AKT–Ser473/total AKT (pan), GSK3β–Ser9/total GSK3β (pan), and α-tubulin (loading control) in response to various Ca2+o concentrations for 15 min in MG63 osteosarcoma cells. Prior to Ca2+o treatment, cells were transfected with siRNA directed at Homer1 (siHom), CaSR (siCaR), or a nondirected sequence (siCTRL) for 48 h. Densitometry values (dens) were calculated by dividing the detected level of phosphorylated protein by the level of total protein (ImageJ), and normalizing this number to the ratio obtained from the 1 m

m

Ca2+o lane for each silenced condition. B, Western blotting showing expression level of Homer1 and CaSR in response to each directed siRNA (+) compared with cells transfected with a nondirected sequence (−) in MG63 cells. # Solid, vertical black lines on the presented Western blotting (B, panel, siCaR blots only) indicate where lanes have been spliced from the same Western blotting for presentation purposes. The experiment was independently repeated with similar results.

Figure 7.
Figure 7.

Ca2+-dependent stimulation of alkaline phosphatase activity and rescue from oxidative stress–induced apoptosis depend on Homer1 and CaSR. ALP activity in response to various Ca2+ concentrations for 3 days in absence of siRNA (A), presence of siRNA to CaSR (siCaSR) (B), and presence of siRNA to Homer1 (siHomer1) (C). For each transfection, the effect of a nondirected control siRNA sequence (siCTRL) is shown. ALP corrected for total cellular protein (t.c.p). D and E, caspase-3 activity in osteoblasts treated with Ca2+ (D) at indicated concentrations for 5 h then 125 μ

m

H2O2 for 2.5 h. E and F, osteoblasts transfected with siRNA to reduce CaSR (siCaSR) (E) or Homer1 (siHomer1) (F) or a nondirected control sequence (siCTRL) and then treated with 2 m

m

Ca2+ or vehicle (1 m

m

Ca2+) for 5 h then 125 μ

m

H2O2 for 2.5 h. G and H, caspase-3 in osteoblasts treated with 2 m

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Ca2+ and Torin1 (IC50 ∼2 n

m

) (G) or wortmannin (IC50∼1 n

m

) (H) for 5 h and then 125 μ

m

H2O2 for 2.5 h. Caspase-3 corrected for t.c.p. ns, not significant, *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with vehicle (Veh).

Figure 8.
Figure 8.

Co-transfection of HEK-293 cells with CaSR and Homer1c established AKT–Ser473 sensitivity to extracellular Ca2+. A, Western blotting of HEK-293 cells transiently transfected with Homer1c (HEK-Homer1c) or nontransfected cells (NT) as a control, showing Homer1c protein levels at various time points post-transfection. 48 h post-transfection was chosen as the time point for subsequent signaling analyses B–D, Western blotting of AKT–Ser473 phosphorylation levels in response to 0.1–5 m

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extracellular Ca2+ in HEK-CaSR (B), HEK-293 cells (C) transiently transfected with Homer1c, or HEK-CaSR cells (D) transiently transfected with Homer1c. ns, not significantly different; **, p < 0.01; ***, p < 0.001 compared with 0.1 m

m

Ca2+o; ##, p < 0.01 compared with 0.5 m

m

Ca2+o; $, p < 0.05 compared with 1 m

m

Ca2+o.

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