Fig 2: miR-137-3p activates AMPKa through downregulating PDE4D. (a) Graphic representation of the miR-137-3p binding motifs within the 3'-UTR of PDE4D. (b) HEK293T cells were cotransfected the WT or TRU 3'-UTR of PDE4D with miR-137-3p agomir (50 nmol/L) for 48 h, and then, the cells were collected and the luciferase activity was determined using the Dual-Luciferase Reporter Assay System (n = 8). (c–e) Mice were fed with a HFD for 24 weeks to establish NAFLD and were also intraperitoneally injected with the miR-137-3p agomir, antagomir, or respective controls (100 mg/kg weekly) at the last 6 consecutive weeks. Relative PDE4D mRNA and protein levels in HFD mice treated with miR-137-3p agomir, antagomir, or their respective controls (n = 6). (f, g) The cAMP level and PKA activity in HFD mice treated with miR-137-3p agomir, antagomir, or their respective controls (n = 6). (h) Relative AC activity (n = 6). (i) Primary hepatocytes were transfected with siPDE4D or siRNA for 24 h, and relative PDE4D mRNA level was detected (n = 6). (j) Primary hepatocytes were preincubated with miR-137-3p antagomir (50 nmol/L) for 24 h and then stimulated with PO for 24 h. To knock down PDE4D, cells were pretransfected with siPDE4D or siRNA for 24 h before miR-137-3p antagomir treatment. AMPKa phosphorylation in miR-137-3p antagomir-treated primary hepatocytes with or without PDE4D silence (n = 6). (k) Mice were fed with a HFD for 24 weeks to establish NAFLD and were also intraperitoneally injected with the miR-137-3p agomir, antagomir, or respective controls (100 mg/kg weekly) at the last 6 consecutive weeks. To inhibit PDE4D, mice were intraperitoneally injected with 0.5 mg/kg rolipram or an equal volume of vehicle every other day for 8 consecutive weeks before the mice were sacrificed. Serum ALT and AST levels in miR-137-3p antagomir-treated HFD mice with or without PDE4D inhibition (n = 8). Data were expressed as the means ± standard deviations, and *p < 0.05 was considered significant. n.s. indicated no significance.

Fig 3: Pharmacologic PDE4D inhibition impairs mTORC1 signaling and pancreatic cancer cell growth.(A) High PDE4D levels in patients with pancreatic adenocarcinoma from GEPIA (74). *P < 0.05. (B) High PDE4D has low survival rate in patients with pancreatic adenocarcinoma (74). Log-rank test was performed for statistical analysis (A and B). (C) High PDE4D expression in patients with pancreatic ductal adenocarcinoma. Pancreatic ductal adenocarcinoma (PDAC) tissue sections paired with benign tissues. T, tumor; NLD, normal duct. Scale bar: 100 μm. Significance was assessed by 2-tailed Student’s t test. (D) PDE4D levels correlate with mTORC1 activity in pancreatic cancer cells. (E and F) PDE4D inhibition blocks mTORC1 activity. (E) MIA PaCa-2 cells were treated with roflumilast or GEBR-7b for 2 hours. mTORC1 activity was analyzed. (F) MIA PaCa-2 cells stably expressing shPDE4D have low mTORC1 activity. (G) PDE4D inhibition reduces pancreatic cancer cell size. Cell size was determined for shPDE4D MIA PaCa-2 cells, or MIA PaCa-2 cells treated with roflumilast or GEBR-7b for 48 hours. One-way ANOVA with Dunnett’s test was performed. P < 0.0001 for control vs. shPDE4D #1. P < 0.05 for control vs. shPDE4D #2 and control vs. GEBR-7b. P < 0.001 for control vs. roflumilast. (H) Pharmacological inhibition of PDE4D reduces proliferation of pancreatic cancer cells. MIA PaCa-2 cells with shPDE4D, or MIA PaCa-2 cells treated with roflumilast or GEBR-7b were counted. Data represent mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 by 2-way ANOVA with Dunnett’s multiple-comparison test. (I and J) Pharmacological inhibition of PDE4D reduces colony formation. (I) Colony formation assays in MIA PaCa-2 cells 2 weeks after seeding cells (1 × 103). (J) Colonies in I were counted using ImageJ for the quantification. Bar graph represents mean ± SD. ***P < 0.001, ****P < 0.0001 by 1-way ANOVA with Dunnett’s multiple-comparison test. Immunoblots probed for PDE4D, S6K1, 4EBP1, pCREB (Ser133) (measure of PKA activation), CREB, and β-actin are controls.

Fig 4: PDE4D promotes mTORC1 lysosomal localization.(A) Depletion of PDE4D inhibits amino acid–induced mTORC1 activation. HEK293A cells stably expressing shGFP (control) or shRNA targeting PDE4D (shPDE4D) were generated. Cells were starved in amino acid–free media for 2 hours, pretreated with or without forskolin (10 µM) for 1 hour, and then stimulated with amino acids for 1 hour. mTORC1 activity with pS6K1 (Thr389) was analyzed. S6K1, pCREB (Ser133) (measure of PKA activation), CREB, and ß-actin are controls. (B–D) Depletion of PDE4D blocks mTORC1 lysosomal localization. (B) HEK293A cell lines stably expressing shGFP (control) or shPDE4D were starved in amino acid–free media for 2 hours, pretreated with or without forskolin (10 µM) for 1 hour, and then stimulated with amino acids for 1 hour. Immunofluorescence experiments were performed with anti-mTOR (green) and -LAMP2 (lysosome marker, red) antibodies. Representative images were obtained under a Zeiss LSM 900 confocal microscope with 100× objective. (C) Staining intensity profiles across the 3.6-µm distance of the green (mTOR) and red (LAMP2) channels in the magnified pictures. (D) Ten immunofluorescence images per group were quantified using Squassh in ImageJ. The data represent mean ± SD. ****P < 0.0001 by 2-way ANOVA with Tukey’s test for multiple comparisons. (E) PDE4D localizes in the cytoplasm. Cells were fractionated after forskolin (10 µM) treatment for 1 hour and then analyzed by immunoblotting for PDE4D. LAMP2 (lysosome marker), lamin A/C (nuclear marker), and a-tubulin (cytoplasm marker) are controls. Arrow indicates PDE4D. (F) PDE4D does not localize to the lysosome. HEK293A were starved in amino acid–free media for 2 hours, pretreated with or without forskolin (10 µM) for 1 hour, and then stimulated with amino acids for 1 hour. Immunofluorescence experiments were performed with anti-PDE4D (green) and -LAMP2 (lysosome marker, red) antibodies. Representative images were obtained under an LSM 900 confocal microscope with 100× objective. AA, amino acid.

Fig 5: Targeting PDE4D suppresses in vivo tumor development of pancreatic cancer.(A and B) PDE4D inhibition suppresses tumor growth. MIA PaCa-2 cells stably expressing shGFP (control) or shPDE4D were subcutaneously injected into mice for tumor xenograft experiments. In addition, MIA PaCa-2 cells were subcutaneously injected into mice for tumor xenograft experiments and injected with either roflumilast (5 mg/kg) or GEBR-7b (0.3 mg/kg) intraperitoneally once every other day. Tumor growth in A was measured every week after injecting cancer cells into mice. Tumor weight in B was measured after resection of xenograft tumor from mice. Control n = 13, shPDE4D n = 12, roflumilast n = 13, and GEBR-7b n = 13. (C and D) PDE4D inhibition suppresses mTORC1 activity in pancreatic cancer. The xenograft tumor samples were assessed by immunoblotting (C) for mTORC1 activity via pS6K1. S6K1 and ß-actin are controls. The xenograft tumor samples were assessed by immunohistochemistry (D) for pS6 (Ser235/236). Quantification of immunohistochemical staining was obtained using ImageJ. Scale bars: 50 µm. Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by 2-way ANOVA with Dunnett’s multiple-comparison test (A) or 1-way ANOVA with Dunnett’s multiple-comparison test (B and C).