Cyclic GMP-dependent protein kinase II inhibits cell proliferation, Sox9 expression and Akt phosphorylation in human glioma cell lines

Abstract

Earlier we used a glioma model to identify loci in the mouse genome, which were repeatedly targeted by platelet-derived growth factor (PDGF)-containing Moloney murine leukemia viruses. The gene Prkg2, encoding cyclic guanosine monophosphate (cGMP)-dependent protein kinase II, cGKII, was tagged by retroviral insertions in two brain tumors. The insertions were both situated upstream of the kinase domain and suggested creating a truncated form of the cGKII protein. We transfected different human glioma cell lines with Prkg2 and found an overall reduction in colony formation and cell proliferation compared with controls transfected with truncated Prkg2 (lacking the kinase domain) or empty vector. All glioma cells transfected with the cGKII phosphorylate vasodilator-stimulated phosphoprotein, VASP, after cGMP analog treatment. Glioma cell lines positive for the Sox9 transcription factor showed reduced Sox9 expression when Prkg2 was stably transfected. When cGKII was activated by cGMP analog treatment, Sox9 was phosphorylated, Sox9 protein expression was suppressed and the glioma cell lines displayed loss of cell adhesion, inhibition of Akt phosphorylation and G1 arrest. Sox9 repression by siRNA was similarly shown to reduce glioma cell proliferation. Expression analysis of stem and glial lineage cell markers also suggests that cGKII induces differentiation of glioma cell lines. These findings describe an anti-proliferative role of cGKII in human glioma biology and would further explain the retroviral tagging of the cGKII gene during brain tumor formation in PDGF-induced tumors.

Figure 1

(a) Schematic picture of the two proviral integrations of Moloney murine leukemia virus (MMLV)/platelet-derived growth factor (PDGF) in Prkg2 on mouse chromosome 5. Major structural domains of the protein are presented. Data were obtained from National Center for Biotechnology Information, Mouse 37 assembly and ENSEMBL Release 51, 2008. (b) Transiently transfected Cos-1 cells (after 72 h) overexpressing an empty pcDNA vector (lane 1), full-length cGKII (lane 2, ~85 kDa) or truncated ΔcGKII (lane 3, ~35 kDa). (c) Colony-forming assay showing a number of colonies formed in four different glioma cell lines (U-1242MG, U-343MG, U-2987MG and U-Cl2:6MG) transfected with Prkg2, ΔPrkg2 or empty pcDNA (Control). Colonies were counted after 3 weeks of growth in medium containing 0.8 mg/ml of neomycin. The cells were plated in triplicates and experiments were repeated twice. Results are presented from one representative experiment and significant differences were found for Prkg2 transfections compared with empty vector transfections (P<0.05) with paired t-test for every cell line, but not for ΔPrkg2 and empty vector in U-Cl2:6MG. The U-87MG cell line yielded too few colonies after transfection and was not included.

Figure 2

(a) Western blot of cGKII (only showing the specific 86 kDa band) and Sox9 protein expression in human glioma cell lines (U-2987MG, U-343MG, U-87MG and U-1242MG) and clones transfected with Prkg2 from U-2987MG (U-2987-P3 and U-2987-P6), U-343MG (U-343-P7 and U-343-P8), U-87MG (U-87-P1) and U-1242MG (U-1242-P1) human glioma cell lines (after four passages in culture). (b) Real-time PCR showing relative mRNA expression levels of PRKG2 as well as SOX9 in glioma cell lines compared with a normal human adult brain RNA. The expression levels are also compared with total RNA from normal human fetal brain and two of the Prkg2-transfected cell lines U-2987-P6 and U-87-P1. (c) Western blot showing phosphorylated Sox9 (Ser181) and both phosphorylated VASP residues (Ser 239 and Ser157) in untransfected and Prkg2-transfected glioma cell clones after 1 h of treatment with 8-pCPT-cGMP (250 μM) compared with β-actin loading. (d) Immunostaining of pSox9 (Ser181) in the glioma cells (U-2987MG and U-2987-P6) with or without 4 h of cyclic guanosine monophosphate (cGMP) analog (250 μM) treatment.

Figure 3

(a) Growth of human glioma cell lines stably transfected with Prkg2 (U-2987-P6, U-87-P1 and U-1242-P1) or empty pcDNA vector (denoted U-2987MG, U-87MG and U-1242MG) when treated with 250 μM of cyclic guanosine monophosphate (cGMP) analog over 7 days. M, million cells; Statistical significance: **P<0.01 and *P<0.05 using Student’s unpaired t-test (error bars indicate standard deviation from triplicates). (b) Flow cytometry of U-2987-MG glioma cells stably transfected with Prkg2 (U-2987-P6) or pcDNA (denoted U-2987MG) and treated with 250 μM of cGMP analog for 72 h.

Figure 4

(a) Inhibition of Akt phosphorylation (Thr308) in Prkg2-transfected U-2987MG cell line (U-2987-P6) when stimulated with cyclic guanosine monophosphate (cGMP) analog. (b) Phalloidin staining (red) of U-2987MG and U-2987-P6 glioma cells unstimulated or when stimulated with a cGMP analog for indicated time periods. Yellow arrowheads show the start of arborization and loss of adhesion of the cGKII-positive cells. Blue, DAPI; red, phalloidin coupled to rhodamine; cGMP, 8-pCPT-cGMP (250 μM); R-cGMPS, Rp-pCPT-cGMPS (10 μM).

Figure 5

(a) Real-time PCR showing relative expression of the markers of immature cells (CD133, NES), astrocytic (GFAP), oligodendrocytic (PLP) or neuronal (TuJ1, MAP2) lineage for U-2987MG and U-2987-P6 (Prkg2-transfected) glioma cells with or without 72 h of cGMP analog treatment (added every day). Relative expression levels of PDGFRα and SOX9 were also studied. The expression level in U-2987MG cells for each gene was set to 1. Significant different levels: *P<0.05, using Student’s t-test after triplicate measures (after 72 h cGMP analog treatment in U-2987-P6). (b) Western blot showing Sox9 protein levels in U-2987MG and U-2987-P6 after different cGMP analog treatment periods (4, 24 and 72 h) compared with levels of β-actin. For the 72-h treated cells, cGMP analog was added daily. For 72+24 h, cGMP was added daily for the first 72 h, then the cGMP analog was removed for the media for an additional 24 h. (c) Real-time PCR showing relative expression of SOX9 and PRKG2 between the glioma cell lines U-2987MG and U-87MG (and the corresponding Prkg2-transfected clones) when cultured in 10% serum compared with when the cell lines are cultured in neurobasal media (containing EGF and FGF). As compared to controls of total RNA from normal brain (set to 1). (d) Immunostaining of SOX9, TuJ1 and GFAP in glioma cells when differentiating for 72 h in neurobasal media (without growth factors) with or without the cGMP analog (added daily). Relative percentage of TuJ1-expressing cells (after triplicate cell counts) within parentheses under the different conditions are shown.

Figure 6

(a) Western blot of U-2987MG and U-2987-P6 after siRNA inhibition of VASP (siVASP) and Sox9 (siSox9) with or without stimulation with 250 μM of 8-pCPT-cGMP for 8 h. (b) Cell proliferation of glioma cell lines (U-2987MG, U-2987-P6 and U-1242MG) transfected with control siRNA-A (siControl) or siRNA directed against Sox9 (siSox9). Fluorescence (measurement of cellular DNA content) proportional to cell number after 1, 3, 5 and 7 days of culturing after siRNA transfection is presented (from quadruplicate CyQUANT measurements). Statistical significance: ***P<0.001, using Student’s unpaired t-test (error bars indicate standard deviation from quadruplicates for siSox9 and siSox9 (+cGMP) compared with siControl for both U-2987MG and U-2987-P6 and also for siControl+cGMP compared with siControl for U-2987-P6).