8-Br-cGMP suppresses tumor progression through EGFR/PLC γ1 pathway in epithelial ovarian cancer.

BACKGROUND: Cyclic guanosine monophosphate (cGMP)-dependent protein kinase I (PKG-I), a serine/threonine kinase, is important in tumor development. The present study determines that the cGMP/PKG I pathway is essential for promoting cell proliferation and survival in human ovarian cancer cells, whereas cGMP analog has been shown to lead to growth inhibition and apoptosis of various cancer cells. The role of cGMP/PKG I pathway in epithelial ovarian cancer (EOC), therefore, remains controversial. We investigated the effect of cGMP/PKG I pathway and the underlying mechanism in EOC. METHODS AND RESULTS: The results showed that exogenous 8-Bromoguanosine-3', 5'-cyclic monophosphate (8-Br-cGMP) (cGMP analog) could antagonize the effects by EGF, including suppressing proliferation, invasion and migration of EOC cells. In vivo, 8-Br-cGMP hampered the growth of the xenograft tumor. Additionally, the expressions of epidermal growth factor receptor (EGFR), matrix metallopeptidase 9 (MMP9), proliferating cell nuclear antigen and Ki67 in xenograft tumor were decreased after 8-Br-cGMP intervention. Further research demonstrated that 8-Br-cGMP decreased the phosphorylation of EGFR (Y992) and downstream proteins phospholipase Cγ1 (PLC γ1) (Y783), calmodulin kinase II (T286) and inhibited cytoplasmic Ca2+ release as well as PKC transferring to cell membrane. It's worth noting that the inhibition was 8-Br-cGMP dose-dependent and 8-Br-cGMP showed similar inhibitory effect on EOC cells compared with U-73122, a specific inhibitor of PLC γ1. CONCLUSIONS: The activation of endogenous PKG I by addition of exogenous 8-Br-cGMP could inhibit EOC development probably via EGFR/PLCγ1 signaling pathway. 8-Br-cGMP/PKG I provide a new insight and strategy for EOC treatment.

VEGFB-VEGFR1 ameliorates Ang II-induced cardiomyocyte hypertrophy through Ca2+ -mediated PKG I pathway.

In response to assorted stimuli, the heart will develop into cardiomyocyte hypertrophy, but sustained cardiomyocyte hypertrophy will finally lead to heart failure. This research is aimed to examine the effect of VEGFB on cardiomyocyte hypertrophy by using the cardiomyocyte-derived cell line H9C2 of cultured rates. It turns out that VEGFB can positively prevent the Ang II-induced rising in the size of cardiomyocyte as well as reduce Ang II-induced mRNA and protein levels of ß-MHC (ß-myosin heavy chain), BNP (brain natriuretic peptide), and ANP (atrial natriuretic peptide). Moreover, VEGFB can regulate the decline of the Ang II-induced rising in Ca2+ . After VEGFR1 knockdown, these effects of VEGFB were partially reversed. Moreover, VEGFB attenuated the suppression of PKG I, p-VASP, and RGS2 caused by Ang II; whereas VEGFR1 knockdown partially abolished the indicated effect of VEGFB. In a word, the effect of VEGFB on relevant downstream targets and the pathways of PKG I by VEGFR1 may explain its efficacy on cardiomyocyte hypertrophy. Thus, it can be suggested that it is feasible to apply VEGFB-VEGFR1 for reducing the symptoms of cardiomyocyte hypertrophy.

Murine cardiac growth, TRPC channels, and cGMP kinase I.

Signaling via cGMP-dependent protein kinase I (cGKI) and canonical transient receptor potential (TRPC) channels appears to be involved in the regulation of cardiac hypertrophy. Recent evidence suggests that TRPC channels are targets for cGKI, and phosphorylation of these channels may mediate the antihypertrophic effects of cGMP signaling. We tested this concept by investigating the role of cGMP/cGKI signaling on angiotensin II (A II)-induced cardiac hypertrophy using a control group (Ctr), trpc6(-/-), trpc3(-/-), trpc3(-/-)/6(-/-), ßRM mice, and trpc3(-/-)/6(-/-) × ßRM mice. ßRM mice express cGKIß only in the smooth muscle on a cGKI(-/-) background. The control group was composed of littermate mice that contained at least one wild type gene of the respective genotype. A II was infused by minipumps (7 days; 2 mg/kg/day) in Ctr, trpc6(-/-), trpc3(-/-), trpc3(-/-)/6(-/-), ßRM, and trpc3(-/-)/6(-/-) × ßRM mice. Hypertrophy was assessed by measuring heart weight per tibia length (HW/TL) and fibrosis by staining of heart slices. A II-induced increase in HW/TL and fibrosis was absent in trpc3 (-/-) mice, whereas an increase in HW/TL and fibrosis was evident in Ctr and trpc6(-/-), minimal or absent in trpc3(-/-), moderate in ßRM, and dramatic in trpc3(-/-)/6(-/-) ßRM mice. These results suggest that TRPC3 may be necessary for A II-induced cardiac hypertrophy. On the other hand, hypertrophy and fibrosis were massively increased in ßRM mice on a TRPC3/6 × cGKI(-/-)KO background, indicating an "additive" coupling between both signaling pathways.

Triiodo-L-thyronine (T3) downregulates Npr1 gene (coding for natriuretic peptide receptor-A) transcription in H9c2 cells: involvement of ß-AR-ROS signaling.

INTRODUCTION: Natriuretic peptide receptor-A (NPR-A) signaling system is considered as an intrinsic productive mechanism of the heart that opposes abnormal cardiac remodeling and hypertrophic growth. NPR-A is coded by Npr1 gene, and its expression is downregulated in the hypertrophied heart. AIM: We sought to examine the levels of Npr1 gene transcription in triiodo-L-thyronine (T3) treated hypertrophied cardiomyocyte (H9c2) cells, in vitro, and also the involvement of ß-adrenergic receptor (ß-AR) - Reactive oxygen species (ROS) signaling system in the down-regulation of Npr1 transcription also studied. MAIN METHODS: Anti-hypertrophic Npr1 gene transcription was monitored in control and T3-treated (dose and time dependent) H9c2 cells, using a real time PCR method. Further, cell size, intracellular cGMP, ROS, hypertrophy markers (ANP, BNP, α-sk, α-MHC and ß-MHC), ß-AR, and protein kinase cGMP-dependent 1 (PKG-I) genes expression were also determined. The intracellular cGMP and ROS levels were determined by ELISA and DCF dye method, respectively. In addition, to neutralize T3 mediated ROS generation, H9c2 cells were treated with T3 in the presence and absence of antioxidants [curcumin (CU) or N-acetyl-L-cysteine (NAC)]. RESULTS: A dose dependent (10 pM, 100 pM, 1 nM and 10 nM) and time dependent (12 h, 24 h and 48 h) down-regulation of Npr1 gene transcription (20, 39, 60, and 74% respectively; 18, 55, and 85%, respectively) were observed in T3-treated H9c2 cells as compared with control cells. Immunofluorescence analysis also revealed that a marked down regulation of NPR- A protein in T3-treated cells as compared with control cells. Further, a parallel downregulation of cGMP and PKG-I (2.4 fold) were noticed in the T3-treated cells. In contrast, a time dependent increased expression of ß-AR (60, 72, and 80% respectively) and ROS (26, 48, and 74%, respectively) levels were noticed in T3-treated H9c2 cells as compared with control cells. Interestingly, antioxidants, CU or NAC co-treated T3 cells displayed a significant reduction in ROS (69 and 81%, respectively) generation and to increased Npr1 gene transcription (81 and 88%, respectively) as compared with T3 alone treated cells. CONCLUSION: Our result suggest that down regulation of Npr1 gene transcription is critically involved in T3- induced hypertrophic growth in H9c2 cells, and identifies the cross-talk between T3-ß-AR-ROS and NPR-A signaling.

Increasing cGMP-dependent protein kinase I activity attenuates cisplatin-induced kidney injury through protection of mitochondria function.

Cisplatin is widely used to treat malignancies. However, its major limitation is the development of dose-dependent nephrotoxicity. The precise mechanisms of cisplatin-induced kidney damage remain unclear, and the renoprotective agents during cisplatin treatment are still lacking. Here, we demonstrated that the expression and activity of cGMP-dependent protein kinase-I (PKG-I) were reduced in cisplatin-treated renal tubular cells in vitro as well as in the kidney tissues from cisplatin-treated mice in vivo. Increasing PKG activity by both pharmacological and genetic approaches attenuated cisplatin-induced kidney cell apoptosis in vitro. This was accompanied by decreased Bax/Bcl2 ratio, caspase 3 activity, and cytochrome c release. Cisplatin-induced mitochondria membrane potential loss in the tubular cells was also prevented by increased PKG activity. All of these data suggest a protective effect of PKG on mitochondria function in renal tubular cells. Importantly, increasing PKG activity pharmacologically or genetically diminished cisplatin-induced tubular damage and preserved renal function during cisplatin treatment in vivo. Mitochondria structural and functional damage in the kidney from cisplatin-treated mice was inhibited by increased PKG activity. In addition, increasing PKG activity enhanced ciaplatin-induced cell death in several cancer cell lines. Taken together, these results suggest that increasing PKG activity may be a novel option for renoprotection during cisplatin-based chemotherapy.

Examining a role for PKG Iα oxidation in the pathogenesis of cardiovascular dysfunction during diet-induced obesity.

BACKGROUND: Protein kinase G (PKG) Iα is the end-effector kinase that mediates nitric oxide (NO)-dependent and oxidant-dependent vasorelaxation to maintain blood pressure during health. A hallmark of cardiovascular disease is attenuated NO production, which in part is caused by NO Synthase (NOS) uncoupling, which in turn increases oxidative stress because of superoxide generation. NOS uncoupling promotes PKG Iα oxidation to the interprotein disulfide state, likely mediated by superoxide-derived hydrogen peroxide, and because the NO-cyclic guanosine monophosphate (cGMP) pathway otherwise negatively regulates oxidation of the kinase to its active disulfide dimeric state. Diet-induced obesity is associated with NOS uncoupling, which may in part contribute to the associated cardiovascular dysfunction due to exacerbated PKG Iα disulfide oxidation to the disulfide state. This is a rational hypothesis because PKG Iα oxidation is known to significantly contribute to heart failure that arises from chronic myocardial oxidative stress. METHODS AND RESULTS: Bovine arterial endothelial cells (BAECs) or smooth muscle cells (SMCs) were exposed to drugs that uncouple NOS. These included 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) which promotes its S-glutathiolation, 4-diamino-6-hydroxy-pyrimidine (DAHP) which inhibits guanosine-5'-triphosphate-cyclohydrolase 2 to prevent BH4 synthesis or methotrexate (MTX) which inhibits the regeneration of BH4 from BH2 by dihydrofolate reductase. While all the drugs mentioned above induced robust PKG Iα disulfide dimerization in cells, exposure of BAECs to NOS inhibitor L-NMMA did not. Increased PKG Iα disulfide formation occurred in hearts and aortae from mice treated in vivo with DAHP (10mM in a drinking water for 3 weeks). Redox-dead C42S PKG Iα knock-in (KI) mice developed less pronounced cardiac posterior wall hypertrophy and did not develop cardiac dysfunction, assessed by echocardiography, compared to the wild-type (WT) mice after chronic DAHP treatment. WT or KI mice were then subjected to a diet-induced obesity protocol by feeding them with a high fat Western-type diet (RM 60% AFE) for 27 weeks, which increased body mass, adiposity, plasma leptin, resistin and glucagon levels comparably in each genotype. Obesity-induced hypertension, assessed by radiotelemetry, was mild and transient in the WT, while the basally hypertensive KI mice were resistant to further increases in blood pressure following high fat feeding. Although the obesogenic diet caused mild cardiac dysfunction in the WT but not the KI mice, gross changes in myocardial structure monitored by echocardiography were not apparent in either genotype. The level of cyclic guanosine monophosphate (cGMP) was decreased in the aortae of WT and KI mice following high fat feeding. PKG Iα oxidation was not evident in the hearts of WT mice fed a high fat diet. CONCLUSIONS: Despite robust evidence for PKG Iα oxidation during NOS uncoupling in cell models, it is unlikely that PKG Iα oxidation occurs to a significant extent in vivo during diet-induced obesity and so is unlikely to mediate the associated cardiovascular dysfunction.