Nongenomic thyroid hormone signaling occurs through a plasma membrane-localized receptor - PubMed (original) (raw)

Nongenomic thyroid hormone signaling occurs through a plasma membrane-localized receptor

Hema Kalyanaraman et al. Sci Signal. 2014.

Abstract

Thyroid hormone (TH) is essential for vertebrate development and the homeostasis of most adult tissues, including bone. TH stimulates target gene expression through the nuclear thyroid receptors TRα and TRβ; however, TH also has rapid, transcription-independent (nongenomic) effects. We found a previously uncharacterized plasma membrane-bound receptor that was necessary and sufficient for nongenomic TH signaling in several cell types. We determined that this receptor is generated by translation initiation from an internal methionine of TRα, which produces a transcriptionally incompetent protein that is palmitoylated and associates with caveolin-containing plasma membrane domains. TH signaling through this receptor stimulated a pro-proliferative and pro-survival program by increasing the intracellular concentrations of calcium, nitric oxide (NO), and cyclic guanosine monophosphate (cGMP), which led to the sequential activation of protein kinase G II (PKGII), the tyrosine kinase Src, and extracellular signal-regulated kinase (ERK) and Akt signaling. Hypothyroid mice exhibited a cGMP-deficient state with impaired bone formation and increased apoptosis of osteocytes, which was rescued by a direct stimulator of guanylate cyclase. Our results link nongenomic TH signaling to a previously uncharacterized membrane-bound receptor, and identify NO synthase, guanylate cyclase, and PKGII as TH effectors that activate kinase cascades to regulate cell survival and proliferation.

Copyright © 2014, American Association for the Advancement of Science.

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Conflict of interest statement

Competing interests: The authors declare that they have no competing interests.

Figures

Fig. 1

Fig. 1. Nongenomic TH signaling requires a TRα isoform.

(A and B) Human POBs in hormone-deficient medium containing 0.1% FBS were incubated with (A) 1 nM T3 for the indicated times or (B) the indicated concentrations of T3, T4, or reverse T3 (rT3) for 10 min. Src, ERK, and Akt phosphorylation was assessed by Western blotting analysis with antibodies specific for Src (pTyr418), ERK½ (pTyr204), and Akt (pSer473). Equivalent loading was determined by analyzing blots for total Akt protein. Line graphs below the blots show means ± SEM of the fold-changes in pERK and pAkt abundances. Data are from three to five independent experiments. (C and D) POBs isolated from THRA f/f or THRB f/f mice were infected with control (LacZ-expressing) or CRE-expressing viruses. Forty-eight hours later, the cells were treated with vehicle or 1 nM T3 for 5 or 10 min. Phosphorylation of ERK and Akt was assessed as described for (A) and (B). The bar graphs show data for the 10-min time points, with vehicle-treated, control virus–infected cells assigned a value of 1. Data are means ± SEM from three independent experiments. **P < 0.01, ***P < 0.001 compared to vehicle-treated cells; ###P < 0.001 compared to T3-treated, control virus–infected cells. (E) MC3T3 cells were transfected with siRNAs specific for GFP or TRα and then were infected with viruses encoding LacZ, FLAG-tagged TRα1, or FLAG-tagged TRβ1. Cells were treated with vehicle or T3 for 10 min. Knockdown efficiency is shown in Fig. 2B and fig. S1G. Virus expression was assessed by Western blotting of cell lysates with an anti-FLAG antibody. Bar graph shows means ± SEM of the relative amounts of pERK and pAkt in the indicated samples. Data are from three independent experiments. ##P < 0.01, ###P < 0.001 compared to cells transfected with GFP-specific siRNA; **P < 0.01 for the indicated comparison.

Fig. 2

Fig. 2. Identification of previously uncharacterized TRα isoforms and of their function in nongenomic TH signaling.

(A) Diagram of the 410-amino-acid TRα1 protein showing the transcription activation domain (AD), DNA-binding (Bdg) domain, nuclear localization signal (NLS), and a C-terminal domain mediating T3=binding, receptor dimerization, and transcriptional activation. Putative translation initiation sites are indicated (M), as is the TH binding-incompetent TRΔα1 isoform transcribed from an intronic promoter. Met150 (M150) follows a flexible “hinge” region. Nucleotide sequences surrounding methionine codons are shown on the right and are conserved between mouse and human. (B) MC3T3 cells were transfected with siRNAs targeting GFP or TRα, and then were infected with virus encoding either LacZ or native (untagged) TRα1. Fourtyeight hours later, cell lysates were analyzed by Western blotting with antibodies against the C-terminus of TRα1 or CREB (loading control). Non-contiguous lanes from a single blot are shown and are separated by vertical lines. The asterisk indicates full-length TRα1; the blot is respresentative of three experiments. (C) MC3T3 cells were transfected with empty vector (E.V.) or the indicated amounts of plasmid encoding C-terminally FLAG-tagged TRα1 and then were analyzed by Western blotting with an anti-FLAG antibody. (D) MX3T3 cells were transfected with plasmids encoding the following constructs: full-length wild type TRα1 (WT); full-length receptor with isoleucine substituted for Met120/122 or Met150 (I120/122 or I150, respectively); full-length receptor with Met120, Met122, and Met150 mutated to isoleucines (Mut x3,); TRα1 from which codons 1 to 119 are deleted (Δ119); the Δ119 construct in which Met150 is mutated to isoleucine (Δ119/I150); TRα1 from which codons 1 to 149 were deleted (Δ149). Samples were analyzed by Western blotting with anti-FLAG antibody. Peptides translated from Met1, Met39, Met120/122, and Met150 are indicated. Note that the triple-FLAG tag causes proteins to migrate with a higher apparent molecular mass (see fig. S2D; data are representative of five experiments). (E and F) MC3T3 cells were transfected with siRNAs targeting GFP (first two lanes) or TRα (all other lanes), and then were infected with viruses encoding the TRα1 constructs described in (C). Δ119PH refers to a TH-binding–deficient mutant (P398H). Cells were treated with vehicle or 1 nM T3 treatment for 10 min and then were analyzed by Western blotting to determine ERK and Akt activation as described for Fig. 1, A and B. Western blots were analyzed with anti-FLAG antibody to determine the extent of viral infection. ns, nonspecific. Graphs show means ± SEM of the fold-increase in the abundances of pERK and pAkt from three independent experiments. Cells that were transfected with GFP-specific siRNA, infected with control virus, and treated with vehicle were assigned a value of 1. **P < 0.01, ***P < 0.001 compared to cells transfected with TRα-specific siRNA and infected with control virus; ##P < 0.05 compared to cells transfected with TRα-specific siRNA and infected with virus encoding WT TRα. (G) MC3T3 cells were cotransfected with a TRE-containing luciferase reporter plasmid and with empty vehicle or with plasmids encoding the indicated TRα1 constructs described in (C). Cells were then treated with vehicle or T3 for 24 hours. Data in the bar graph are means ± SEM of the relative luciferase activities in the indicated samples and are from five independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 for the indicated comparisons; #P < 0.05 compared to cells transfected with empty plasmid and treated with T3.

Fig. 3

Fig. 3. Membrane association of p30 TRα1 and co-localization with caveolin-1 and NOS3.

(A) Nuclear (Nuc), cytosolic (Cyto), and membrane (Mem) fractions of MC3T3 cells were analyzed by Western blotting with antibodies against the TRα1 C-terminus, CREB, or caveolin-1. Extracts of cells transfected with plasmid encoding non-tagged, WT TRα1 added to membranes (Mem+TRα1) served as a positive control. The asterisk indicates full-length TRα1. Analysis of concentrated nuclear extracts is shown in fig. S2A. (B) Solubilized membranes were incubated with T3-sepharose (T3) or control-sepharose (C), and bound proteins were analyzed by Western blotting with anti-TRα1 antibody. (10% of input is shown, representative of three experiments). (C) NIH-3T3 fibroblasts were transfected with siRNAs specific for GFP or TRα, and some cells were transfected with plasmid encoding nontagged TRα1-Δ149. Samples were analyzed by Western blotting as described for (A). (D) Murine POB membranes were fractionated over an Optiprep density gradient, and fractions of increasing density were analyzed by Western blotting with anti-TRα1 and anti-caveolin-1 antibodies (representative of two experiments). (E) Membranes from MC3T3 cells transfected with Myc-tagged TRα1-Δ149 were fractionated as described for (D) and were analyzed by Western blotting for the presence of the indicated proteins (representative of three experiments). (F) hPOBs were pre-treated with vehicle, 10 μM 2-bromo-palmitate (Br-Pal), or 10 mM methyl-β-cyclodextrin (MBCD) before being treated with 1 nM T3. The extent of ERK and Akt activation was assessed by Western blotting as described for Fig. 1A (representative of three experiments). (G) MC3T3 cells were transfected with siRNAs specific for GFP or TRα as well as with empty vector (E.V.) or with plasmids encoding the indicated TRα1 constructs. The cells were then treated with vehicle or T3 and analyzed by Western blotting as described for (F). Detection of Myc-tagged constructs is shown in the lower blot. Bar graph shows means ± SEM of the relative amounts of pERK and pAkt from four independent experiments. *P < 0.05, **P < 0.01 compared to cells transfected with TRα-specific siRNA and E.V; #P < 0.05, ##P < 0.01 compared to cells transfected with plasmid encoding TRα1-Δ149. (H) Cells were transfected with plasmids encoding Myc-tagged TRα1-Δ149 or TRα1-Δ149-A254/255. Plasma membranes (Mem) or whole-cell lysates (WCL) were prepared and analyzed by Western blotting with anti-Myc and anti-caveloin-1 antibodies (representative of three experiments). (I) MC3T3 cells transfected with plasmid encoding FLAG-tagged TRα1-Δ149 were incubated with mouse anti-FLAG and rabbit anti-caveolin-1 antibodies followed by a proximity ligation assay. The close proximity of p30 TRα1 and caveolin produced punctate red fluorescence. Cells were co-stained with FITC-labeled phalloidin and Hoechst 33342 (to stain the nucleus), and were analyzed by fluorescence microscopy. Scale bar: 2.5 μm. (J and K) Cells were transfected with (J) plasmid encoding FLAG-tagged TRα1-Δ149 or (K) plasmids encoding Myc-tagged TRα1-Δ149 and RFP-tagged NOS3. Cells were incubated with antibodies against the indicated proteins, DNA was stained with Hoechst, and the cells were analyzed by fluorescence microscopy. Scale bar: 2.5 μm; data are representative of three experiments.

Fig. 4

Fig. 4. The activation of Src, ERK, and Akt by TH requires NOS3 and membrane-bound PKGII.

(A) Diagram of the NO-cGMP signaling cascade, with enzyme inhibitors in red and activators in green. Src activation by SHP-1 or SHP-2 leads to ERK and Akt activation (14). (B to D) POBs isolated from (B and C) THRA f/f or THRB f/f mice were infected with LacZ- or CRE-expressing viruses. (C) In addition, cells were transfected with plasmids encoding the indicated TRα1 constructs. Forty-eight hours later, cells were treated with vehicle or 1 nM T3 for 10 min. (D) Human POBs were incubated for 30 min with 4 mM L-NAME (L-NA), 2 mM EGTA, or 10 μM LY294002 (LY) before being treated with vehicle or T3. Stable NO oxidation products (NOx represents nitrite and nitrate) were measured with the Griess reagent. Data are means ± SEM from three or four independent experiments. ***P < 0.001 compared to vehicle-treated cells, ###P < 0.001 compared to cells infected with control virus and treated with T3). (E) Fura-2AM–loaded hPOBs that were not pretreated or were pretreated with 2 mM EGTA were incubated with vehicle or 1 nM T3 at time zero. Intracellular Ca2+ mobilization was calculated from the 340:380 nm Fura-2 fluorescence ratio, and representative traces are shown. The bar graph shows means ± SEM of the peak Ca2+concentrations in the indicated numbers of cells. ATP served as a positive control. ***P < 0.001 compared to vehicle-treated cells; ###P < 0.001 compared to cells treated with T3 alone. (F and H) NOS3 f/f POBs were treated as described for (B). (F) Cells were then analyzed to measure NO production. (H) Cell lysates were analyzed by Western blotting as described for Fig. 1A with antibodies against the indicated proteins. Phosphorylated VASP was used as a positive control for PKG activation. Bar graphs show means ± SEM from three or four independent experiments. ***P < 0.001 compared to vehicle-treated cells; #P < 0.05, ###P < 0.001 compared to cells infected with control virus and treated with vehicle. Ns, not significant. (G) hPOBs were left untreated or were treated with LY294002 before being treated with vehicle or T3. The presence of phosphorylated NOS3 (pSer1177) and Akt (pSer473) was determined by Western blotting analysis, representative of three experiments. (I) hPOBs were pretreated with 4 mM L-NAME, 10 μM ODQ, or 50 μM Rp-CPT-PET-cGMPS (Rp) before being incubated with vehicle or 1 nM T3. Western blotting analysis was performed to determine the phosphorylation of Src, ERK, Akt, and VASP as described for (H). Bar graph shows means ± SEM from three to five independent experiments. ***P < 0.001 compared to vehicle-treated cells; ###P < 0.001 compared to cells treated with T3 alone. (J) hPOBs were treated with 1 nM T3, 10 μM PAPA-NONOate, 100 nM cinaciguat, or 100 μM 8-pCPT-cGMP for 10 min. Cells were then analyzed by Western blotting with antibodies against the indicated proteins, data are representative of three experiments. (K to M) MC3T3 cells were transfected with siRNAs specific for GFP, PKGI, or PKGII. (M) Cells were also infected with viruses encoding LacZ, WT PKGII, or mutant PKGII that cannot bind to the plasma membrane (G2A). Cells were treated with vehicle or T3 and were analyzed as described for (H). (K) The relative abundances of PKGI and PKGII mRNAs were quantified by RT-PCR analysis. (L and M) Cell lysates were analyzed by Western blotting with antibodies against the indicated proteins. Bar graphs in (L) and (M) show means ± SEM of the relative abundances of pERK and pAkt from three independent experiments. *P < 0.05, ***P < 0.001 compared to cells treated with GFP-specific siRNA in (K) and (L); **P < 0.01 compared to cells treated with GFP-specific siRNA and infected with control virus and ##P < 0.01 compared to cells expressing WT PKGII in (M).

Fig. 5

Fig. 5. Nongenomic TH signaling through the p30 TRα1–NO–cGMP pathway regulates cell proliferation and survival.

(A and B) Effect of nongenomic TH signaling on cell proliferation. (A) POBs isolated from THRA f/f and THRB f/f mice were infected with LacZ- or CRE-encoding adenoviruses. (B) MC3T3 cells were transfected with siRNAs targeting GFP or TRα and then were infected with adenoviruses encoding LacZ or the indicated TRa1 constructs, as described for Fig. 2. After 24 hours in hormone-free medium with 0.1% FBS, cells were treated with vehicle or 1 nM T3 for 30 min before being incubated with 8-Br-deoxyuridine (BrdU) in hormone-free medium for 18 hours. The percentages of cells that were labeled with BrdU were determined by immunofluorescence staining. (C) POBs from NOS3 f/f mice were infected with LacZ- or CRE-encoding adenoviruses. BrdU in S-phase nuclei was assessed as in B. DNA was counter-stained with Hoechst. (A to C) Data in bar graphs show means ± SEM of the percentages of BrdU-labeled cells from four independent experiments. ***P < 0.001 compared to vehicle-treated cells; ##P < 0.1, ###P < 0.001 compared to cells infected with control virus and treated with T3. (D) hPOBs were treated with 50μM Rp-CPT-PET-cGMPS (Rp), 10 μM U0126 (U), or 10 μM LY294002 (LY) for 1 hour before being incubated with vehicle or T3. BrdU uptake was determined as described for (A). Data are means ± SEM of the percentages of BrdU-labeled cells from four independent experiments. ***P < 0.001 compared to vehicle-treated cells; ###P < 0.001 compared to cells treated with T3 alone. (E to G) Effect of nongenomic TH signaling on cell death. (E) POBs from THRA f/f mice were infected with LacZ-encoding virus or with viruses expressing the indicated TRα constructs as described for (A). (F and G) NOS3 f/f mice were infected with LacZ- or CRE-expressing viruses as described for (A). (E to G) Cells were then subjected to serum-deprivation for 18 hours in the presence or absence of (E to G) 1 nM T3 or (F) 1 nM T3 or 100 nM cinaciguat (Cin). Cell death was assessed by (E and F) trypan blue uptake or (G) Western blotting analysis for cleaved caspase-3. Bar graphs show means ± SEM of the percentages of dead cells from three or four independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared to cells infected with LacZ-encoding virus and treated with T3; ##P < 0.01 for cells infected with LacZ-encoding virus compared to cells infected with TRα-encoding virus and treated with T3 (E) and for cinaciguat-treated compared to vehicle-treated cells in (F). (H) hPOBs were treated as described for (D), starved of serum, and analyzed as described for (E). Data are means ± SEM of the percentages of dead cells from four independent experiments. **P < 0.001 compared to vehicle-treated cells, #P < 0.05 compared to cells treated with T3 alone. (I) mPOBs were treated with vehicle or 1 nM T3 in the presence or absence of 4 mM L-NAME (L-NA) for 1 hour (left, undifferentiated cells) or 24 hours (right, differentiated cells). The relative abundances of the mRNAs for c-Fos, Fosl1, osteocalcin (Bglap2), osteopontin (Spp1), RANKL (Tnfsf11), and osteoprotegerin (Tnfrsf11b) were quantified by RT-PCR analysis and were normalized to that of HGPRT mRNA. The mean abundance of each transcript measured in vehicle-treated cells was assigned a value of one. Data are means ± SEM of the fold-increase in the indicated mRNA from three to six independent experiments. ***P < 0.001 for T3-treated cells compared to vehicle-treated cells; #P < 0.05, ###P < 0.001 compared to cells treated with T3 alone.

Fig. 6

Fig. 6. Hypothyroid mice are cGMP-deficient, but increasing their cGMP concentration improves bone formation and prevents osteocyte apoptosis.

Twelve week-old male C57BL6 mice were fed a control diet or an iodine-deficient diet containing 0.15% PTU for 4 weeks. During this time, mice received daily injections of either vehicle or cinaciguat (10 μg/kg). Calcein was injected 7 and 4 days before euthanasia. (A) Heart rate was measured 2 weeks after starting the diet, whereas serum T3 and T4 concentrations were measured at the time of euthanasia. Data are means ± SEM from seven mice for each condition. *P < 0.05, **P < 0.01, ***P < 0.001 compared to mice fed a control diet. (B) Serum cGMP concentrations were measured 2 hours after the last injection with cinaciguat. Data are means ± SEM from seven mice for each condition. *P < 0.05, ***P < 0.001 for the indicated comparisons. (C) Endosteal cortical bone surface and the area analyzed for trabecular bone are indicated by a black line on a trichrome-stained distal femur. (D) Femoral endosteal calcein labeling was assessed by fluorescence microscopy. Scale bar: 10 µm. (E) Single- and double-labeled surfaces were measured on fluorescence microscopy photographs, and were expressed as a percentage of the total endosteal bone surface assessed between 0.25 and 5 mm from the growth plate. ND, not detected. (F) Mineral apposition rate (MAR) and (G) bone formation rate (BFR) were calculated from the inter-label distances. Data in (E) to (G) are means ± SEM from six mice for each condition. *P < 0.05, **P < 0.01, ***P < 0.001 for the indicated comparisons. A minimal MAR value of 0.3 μm/day was assigned to the PTU group, which showed only single-labeled surfaces. (H and I) Serum concentrations of procollagen-1 N-terminal peptide (P1NP) and C-terminal telopeptide (CTX) for the indicated mice were measured by ELISA. (J and K) The numbers of osteoblasts (N.Ob, J) and osteoclasts (N.Oc, K) per mm of trabecular bone perimeter (B.Pm) were counted for the indicated mice. Data in (H) to (K) are means ± SEM from six mice for each condition. *P < 0.05, **P < 0.01, ***P < 0.001 for the indicated comparisons. (L) Apoptotic osteocytes were identified in cortical bone by TUNEL staining (black nuclei). Data are representative of four mice per group. (M) Bar graph shows means ± SEM of the percentages of TUNEL+ osteocytes from four mice for each group. (N) RNA was extracted from femurs from the indicated mice, and the relative abundances of osteocalcin (Bglap1), RANKL (Tnfsf11), and osteoprotegerin (Tnfrsf11b) mRNAs were quantified by RT-PCR analysis and normalized to that of GAPDH mRNA. Mean values of the control group were assigned a value of one. Data are means ± SEM from five or six mice for each condition. *P < 0.05, **P < 0.01, for the indicated comparisons.

Fig. 7

Fig. 7. Proposed mechanism of nongenomic TH signaling by p30 TRα1.

Binding of T3 to the plasma membrane–associated p30 TRα1 induces an increase in intracellular Ca2+ concentration, which leads to activation of the NO-cGMP-PKGII signaling cascade and the phosphorylation and activation of the SHP-1–SHP-2 phosphatase complex. This complex is bound, together with Src, to the cytoplasmic tail of the integrin β3 subunit (14). Src is activated by SHP-1–SHP-2 and initiates activation of MEK-ERK and PI3K-Akt signaling, which results in enhanced cell proliferation and survival. Lipid modifications important for the membrane-association of p30 TRα1, NOS3, and PKGII are in black.

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