Cilia have high cAMP levels that are inhibited by Sonic Hedgehog-regulated calcium dynamics

Abstract

Protein kinase A (PKA) phosphorylates Gli proteins, acting as a negative regulator of the Hedgehog pathway. PKA was recently detected within the cilium, and PKA activity specifically in cilia regulates Gli processing. Using a cilia-targeted genetically encoded sensor, we found significant basal PKA activity. Using another targeted sensor, we measured basal ciliary cAMP that is fivefold higher than whole-cell cAMP. The elevated basal ciliary cAMP level is a result of adenylyl cyclase 5 and 6 activity that depends on ciliary phosphatidylinositol (3,4,5)-trisphosphate (PIP₃), not stimulatory G protein (Gα_s), signaling. Sonic Hedgehog (SHH) reduces ciliary cAMP levels, inhibits ciliary PKA activity, and increases Gli1. Remarkably, SHH regulation of ciliary cAMP and downstream signals is not dependent on inhibitory G protein (Gα_i/o) signaling but rather Ca²⁺ entry through a Gd³⁺-sensitive channel. Therefore, PIP₃ sustains high basal cAMP that maintains PKA activity in cilia and Gli repression. SHH activates Gli by inhibiting cAMP through a G protein-independent mechanism that requires extracellular Ca²⁺ entry.

Keywords: Hedgehog; PIP3; PKA; cAMP; cilia.

Fig. 1.

(A) Images of mouse embryonic fibroblasts expressing the PKA sensor AKAR in the whole cell or cilia (5HT6-AKAR4). The dotted lines represent the regions of interest that were measured for the whole cell or cilia. The solid green line outlines the whole cell of the cell expressing the cilia-targeted PKA sensor. (B) FRET measured in cells that had not formed cilia is similar for AKAR4 and 5HT6-AKAR4, indicating that the 5HT6 tag does not alter AKAR4 response. When measured in cilia, higher FRET ratios are detected, indicating high PKA activity in cilia compared with the rest of the cell (*P < 0.05, compared with the whole cell; n = 3 experiments). ns, not significant. Data are presented as mean ± SEM.

Fig. 2.

(A) Cartoon depiction of mCherry-cADDis and cilia-targeted 5HT6-mCherry-cADDis. (B) Colocalization of 5HT6-cADDis and immunostained Arl13b in MEFs that are also DAPI-stained. The red channel is pixel-shifted. (C) Green and red fluorescence from whole cells (WC) expressing mCherry-cADDis and cilia-targeted mCherry-cADDis. (D) Response of mCherry-cADDis to AC activation by 1, 10, and 100 μM L-858051 concentrations. The red fluorescence from mCherry remains constant whereas the green fluorescence from cADDis diminishes with AC stimulation. (E) Summary fluorescence change in cADDis alone (black), mCherry in mCherry-cADDis (red), and cADDis in mCherry-cADDis (green) in response to various L-858051 concentrations. The reduction of green fluorescence in cADDis (n = 9 cells) and mCherry-cADDis (n = 8 cells) is similar. mCherry intensity does not change in response to AC stimulation. (F) Quantitation of mCherry/cADDis intensity reflects relative cAMP levels (n = 8 cells). (G) Ciliary cADDis fluorescence diminishes in response to a 5-min stimulation of the 5HT6 receptor using 100 nM 2-methyl-5-hydroxytryptamine (2-M-5HT) as well as direct AC activation by application of 100 μM L-858051 for 5 min. mCherry fluorescence remains constant throughout. (H) Receptor or AC-stimulated cAMP levels in cilia as described in G, measured using the mCherry/cADDis ratio, normalized to basal ciliary cAMP levels (*P < 0.05; n = 7 cilia). Data are presented as mean ± SEM.

Fig. S1.

(A) Forskolin (100 μM) and the water-soluble analog L-858051 (100 μM) stimulate similar cAMP production measured using mCherry-cADDis. (B) Whole-cell mCherry/cADDis ratios are similar in MEFs expressing the mCherry-cADDis sensor or the 5HT6-mCherry-cADDis sensor in nonciliated cells. (C) Three different strategies to target mCherry-cADDis to the cilia, using the 5HT6 receptor, SST3R, or Arl13b. Regardless of the targeting method, ciliary cAMP is similar, indicating that the targeting protein does not affect cAMP levels. Data are presented as mean ± SEM.

Fig. 3.

(A) Images of 5HT6-mCherry-cADDis–expressing cells with a cilium and in nonciliated cells (whole cell). The dotted lines represent the region of interest measured. The solid green line outlines the whole cell of the cell expressing the cilia-targeted 5HT6-mCherry-cADDis. (B) Dose response to the cell-permeable cAMP analog 8-Br-2′-O-Me-cAMP-AM. Cells expressing 5HT6-mCherry-cADDis were incubated with the cyclase inhibitor MDL-12330A to inhibit cAMP production, and then the sensor intensity was measured after addition of the cell-permeable cAMP analog (n = 3; 6 to 10 ROIs per point for each experiment). The mCherry/cADDis ratios were plotted versus [cAMP analog] to produce the dose response. (C) The ratio of mCherry to cADDis is higher in cilia compared with the whole cell, demonstrating that cilia have higher basal cAMP levels compared with the whole cell (*P < 0.05; n = 3 experiments). (D) Ciliary cAMP concentrations are five times higher than whole-cell levels, determined by interpolation of ratios on a standard curve (*P < 0.05; n = 3; 6 to 10 ROIs per experiment). Data are presented as mean ± SEM.

Fig. S2.

(A) mCherry/cADDis ratio is higher in the cilia of IMCD3 cells (*P < 0.05). (B) Dose response of mCherry-cADDis to the cAMP analog in IMCD3 cells. (C) Interpolated cAMP values of ciliary and cytosolic cAMP values replicate those found in MEFs. Whole-cell cAMP, 1.01 μM; ciliary cAMP, 4.30 μM (*P < 0.05). Data are presented as mean ± SEM.

Fig. 4.

(A) Direct inhibition of AC using 100 μM MDL-12330A for 30 min reduces ciliary cAMP to whole-cell levels (*P < 0.05; n = 3 experiments) but does not affect whole-cell cAMP levels. (B) Inhibition of PDE using 100 μM IBMX overnight increases ciliary cAMP but not whole-cell cAMP levels (*P < 0.05; n = 3 experiments). (C) Western blot using an anti-AC5/6 antibody showing effective knockdown using siRNA to AC5 and AC6; mortalin is used as a loading control. The bar graph shows relative reduction in AC6 mRNA (*P < 0.05). (D) Knockdown of AC5/6-reduced ciliary cAMP measured using the mCherry/cADDis ratio (*P < 0.05; n = 3 experiments; 15 to 40 ROIs per experiment). (E) Western blot showing effective siRNA knockdown of Gα_s in MEFs; mortalin is used as a loading control. The bar graph is a quantitation of three independent knockdown experiments. (F) Knockdown of Gα_s does not reduce ciliary cAMP measured using the mCherry/cADDis ratio (n = 3 experiments; 7 to 14 ROIs per experiment). (G) Summary FRET/CFP data from cells expressing the PIP₃ sensor InPAkt or 5HT6-InPAkt in cilia (*P < 0.05; n = 3 experiments) or the whole cell (n = 3 experiments) show that cilia have higher PIP₃ levels. (H) Inhibition of PI3K using 10 nM LY294002 overnight reduces basal cilia cAMP levels (*P < 0.05; n = 3 experiments). (I) Expression of the lipid phosphatase PTEN in cilia reduces cAMP levels (*P < 0.05; n = 3 experiments). Data are presented as mean ± SEM.

Fig. S3.

AC5 and AC6 were both detected in RT-PCR and qPCR (not shown); however, AC6 was the prominent subtype.

Fig. S4.

(A) Knockdown of Gα_s prevents stimulation of β2AR with isoproterenol (1 μM) but not direct stimulation of adenylate cyclase with L-858051 (100 μM). (B) Time courses of live-cell fluorescence changes in cilia show cAMP increases in response to stimulation of 5HT6R with 2-methyl-5-hydroxytryptamine (100 nM), a Gα_s-coupled receptor, and direct AC stimulation by L-858051 (100 μM). Both receptor and direct AC-stimulated cAMP production are inhibited by SHH. Gd³⁺ effectively inhibits the effect of SHH on cAMP production. (C) cAMP production with 3 μM L-858051 is inhibited by stimulation of the transfected Gα_i/o-coupled NPY2 receptor with PYY (3-36) (100 μM) in the presence of boiled PTX (250 ng/mL) but not PTX (250 ng/mL). Data are presented as mean ± SEM.

Fig. 5.

SHH effects on Ca²⁺, cAMP, PKA, and Gli. (A) Overnight application of 100 μM Gd³⁺ inhibits SHH (10 nM)-mediated increase in ciliary Ca²⁺ (*P < 0.05; n = 3 experiments; 7 to 12 ROIs each). (B) SHH reduces ciliary cAMP; Gd³⁺ prevents the effect of SHH (*P < 0.05; n = 3 experiments). (C) SHH inhibits PKA activity in cilia measured using the PKA sensor AKAR4. Gd³⁺ prevents SHH inhibition of PKA (*P < 0.05; n = 3 experiments). (D) In MEFs, SHH reduces Gli3R and increases Gli3-FL and Gli1; Gd³⁺ inhibits SHH effects. Western blot is representative of three similar experiments. Mortalin is used as a loading control. (E) Immunofluorescence to detect localization of transfected Gli2-HA in cilia after various treatments. Arl13b immunofluorescence (green) is used to demarcate cilia; nuclei are stained using DAPI. Gli2 localizes to cilia after SHH treatment. The SHH effect on Gli2 localization is inhibited by Gd³⁺. Inhibition of PI3K leads to localization of Gli2 to cilia in the absence of SHH. White arrows indicate colocalization. (F) Summary data for ciliary localization of Gli2-HA after various treatments. The data are from two independent experiments. A total of 42 to 55 cilia were counted for the four conditions to determine the percentages. (G) In MEFs treated with PTX (250 ng/mL; overnight), SHH maintains its ability to reduce ciliary cAMP whereas Gd³⁺ still prevents the SHH effect (*P < 0.05; n = 3 experiments). (H) SHH increase of Gli1 expression is also maintained after PTX treatment but was prevented by Gd³⁺ (*P < 0.05). Data are presented as mean ± SEM.

Fig. S5.

(A) Activators of the SHH pathway SHH, SAG, and 20S significantly increase ciliary calcium levels and reduce ciliary cAMP levels compared with untreated ciliary levels (*P < 0.05). Effects on calcium and cAMP can be blocked by Gd³⁺ (^#P < 0.05). (B) The Trp channel blocker ruthenium red reverses HH agonist-mediated cAMP decrease by SHH, SAG, and 20S (*P < 0.05). Data are presented as mean ± SEM.

Fig. S6.

PKD2 but not PKD2L1 was detected by RT-PCR in MEFs and IMCD3 cells. We used several other primer sets and still could not detect PKD2L1 in either cell type (not shown). Using a qPCR probe to PKD2L1, we did not detect significant mRNA expression in MEFs or IMCD3 cells (n = 2). We used three other qPCR probes to PKD2L1 and still failed to detect significant mRNA in IMCD3 cells. PKD2 was detected in both cell types. Calculating ΔCT as (CT_PKD2 − CT_GAPDH), in IMCD3 cells: ΔCT, 2.9; in MEFs: ΔCT, 4.2.

Fig. 6.

Model of ciliary HH signaling. (Left) Under basal conditions, PIP₃ (depicted in red on the inner leaflet of the membrane) maintains AC5/6 activity in the cilia, resulting in high cAMP concentration. Relative to the whole cell, high Ca²⁺ is present in the cilia. Basal cAMP activates PKA, shifting the Gli balance in favor of GliR. (Middle) After SHH stimulation, Smoothened translocates to the ciliary membrane, and channel activity and Ca²⁺ levels are increased (12) that inhibit AC5/6. The resulting reduction in cAMP reduces PKA activity, shifting the balance to GliA. (Right) Gadolinium blocks Ca²⁺ entry through channels, inhibiting the effects of SHH.