A cAMP signalosome in primary cilia drives gene expression and kidney cyst formation (2022)
Figure 7

Figure 7: An increase in ciliary cAMP levels is transduced by PKA-dependent phosphorylation of CREB in the cilium.

(A) Wild-type (WT), cilia-bPAC, or cyto-bPAC (magenta) mIMCD-3 cells were stimulated by light (for 1 h prior to fixation, 465 nm, 38.8 µW/cm²), and treated with DMSO (control) or 50 µM Ciliobrevin-D, a dynein-inhibitor. Cells were labeled with DAPI (blue) to label the DNA, an ARL13B antibody (white) to label cilia, and a phospho-specific (Ser133) CREB antibody (pCREB, green). Arrows indicate the direction and the length of the shift of the respective fluorescence channel. The box indicates the position of the magnified view shown on the right of each panel.

B) Quantification of the ciliary pCREB signal in cilia-bPAC and cyto-bPAC cells. Data are shown as mean ± S.D., n > 3. Each data point represents an independent experiment and corresponds to the median of > 73 cilia (biological replicates); p values for an unpaired, two-sided Student's t-test with Welch's correction are indicated.

(C) mIMCD-3 Ift27-/- cells with or without stable cilia-bPAC expression (magenta) were stimulated with light (for 1 h prior to fixation, 465 nm, 38.8 µW/cm²). Cells were labeled with DAPI (blue) to label the DNA, an ARL13B antibody (white) to label cilia, and a phospho-specific (Ser133) CREB antibody (pCREB, green). Arrows indicate the direction and the length of the shift of the respective fluorescence channel. The box indicates the position of the magnified view shown on the right of each panel.

(D) Quantification of the ciliary pCREB signal. Data are shown as mean ± S.D., n > 3. Each data point represents the median of > 90 cilia from the same experiment; p values are indicated for an unpaired, two-sided Student's t-test with Welch's correction.

(E) Quantitative RT-PCR analysis comparing Ptgs2 expression in light-stimulated control (+) or Ift27-/- mIMCD-3 cells, both expressing cilia-bPAC. Data are shown as mean ± S.D., p values were calculated by a two-sided, unpaired Student's t-test. Data points show individual experiments.

(F) Quantification of the ciliary pCREB signal in mIMCD-3 cells, expressing the PKA-inhibiting peptide cilia-PKI and cilia-bPAC or cilia-bPAC only, in the dark and after light stimulation (for 1 h prior to fixation, 465 nm, 38.8 µW/cm²). For each cell line, the ciliary pCREB signal of light-stimulated cells was normalized to the median pCREB signal measured for cells in the dark. Data are shown as violin plots, bars indicate median and interquartile range, n = 7 (-:1242 cilia (biological replicates), +:482 cilia (biological replicates)); p value for a Kolmogorov Smirnov test is indicated.

(G) Scheme indicating the model for ciliary cAMP signaling controlling CREB-dependent gene expression.

Tables

Table 1

Table 1: Gene symbols of differentially expressed genes. Genes represent data shown in Fig. 6, grouped according to the Venn Diagram (Fig. 6B). Detailed table including log2-foldchanges and adjusted p-values are shown in Appendix Table S4.

Table 2

Table 2: Results from a reactome analysis of the genes differentially expressed in cilia-bPAC cells after light stimulation for 1 h. Only reactomes covering at least four of the differentially expressed genes are shown.

Table 3: Enriched transcription factor binding sites in differentially expressed genes triggered by cilia-bPAC vs cyto-bPAC stimulation. Binding sites unraveled and p-values reported using the pcaGoPromoter package (Hansen et al., 2012). No p value indicated for p > 0.05.

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