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Darren D Browning - One of the best experts on this subject based on the ideXlab platform.
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abstract 1262 type 2 cgmp dependent Protein Kinase has a tumor suppressive role in the colon epithelium
Cancer Research, 2018Co-Authors: Bianca N Islam, Subbaramiah Sridhar, Justin Ashby, Ravindra Kolhe, Sarah K Sharman, Rui Wang, Darren D BrowningAbstract:The cGMP level plays a central role in regulating homeostasis in the colon epithelium and is emerging as a potential target for colon cancer prevention. The signaling components downstream of cGMP, and the tumor suppressive mechanism remain poorly understood. The present study has examined the expression of cGMP-dependent Protein Kinase isoforms (PKG1, PKG2) in normal intestinal mucosa and in colon cancer from human specimens. Immunohistochemical analysis detected PKG1 in supportive cells in the lamina propria and in smooth-muscle tissue, but not in the epithelium of normal intestine and colon. In contrast, PKG2 was detected exclusively in the epithelium. In colon cancer, PKG1 was restricted to the stroma where it colocalized with vimentin, whereas PKG2 was only detected in the tumor epithelium where it colocalized with cytokeratin. This pattern of PKG isoform expression was similar in the mouse intestine and colon, where only PKG2 was detected in purified colonic crypts. PKG2 knockout (KO) mice were used interrogate possible anti-carcinogenic roles in the colon epithelium. When subjected to the AOM/DSS model of colon carcinogenesis, the PKG2 KO animals produced significantly more polyps than wild type controls (1.75-fold, p=0.0037). While the polyps from PKG2 KO animals were slightly smaller than those from wild type, the trend was not significant (p=0.13). Wild type and PKG2 KO mice responded equally to DSS treatment, but the PKG2-deficient animals exhibited crypt hyperplasia and increased apoptosis in luminal epithelium relative to wild type animals. Taken together these findings suggest that PKG2 has a tumor suppressive role in the colon epithelium, and that reducing the proliferative compartment may be part of the mechanism. Citation Format: Bianca N. Islam, Sarah K. Sharman, Yali Hou, Justin Ashby, Rui Wang, Subbaramiah Sridhar, Ravindra Kolhe, Darren D. Browning. Type-2 cGMP-dependent Protein Kinase has a tumor-suppressive role in the colon epithelium [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1262.
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Abstract 1918: Type 2 cGMP-dependent Protein Kinase activates antineoplastic signaling in the colon
Cancer Research, 2015Co-Authors: Allison Bridges, Bianca N Islam, Subbaramiah Sridhar, Sarah K Sharman, Rui Wang, Darren D BrowningAbstract:Signaling through cGMP has emerged as a potentially important suppressor of tumorigenesis in the colon but the mechanisms are poorly defined. Studies of guanylyl-cyclase (GCC) knockout mice indicate that cGMP signaling inhibits proliferation in the colon epithelium and that AKT may have an important role. Type-2 cGMP-dependent Protein Kinase (PKG2) is a central effector of cGMP in the colon epithelium and likely mediates the suppressive signaling. An important growth-inhibitory effect of AKT is the inactivation of forkhead transcription factors (FoxO). FoxO Proteins are tumor suppressors that regulate growth-inhibitory and pro-apoptotic target gene expression but these Proteins have not previously been examined in the intestinal tract. The present study sought to determine whether PKG2 can activate FoxO in colon cancer cells and in the colon epithelium. Activation of PKG2 in LS174T colon cancer cells inhibited cell proliferation but did not significantly affect apoptosis. Suggestive of a mechanism, it was found that PKG2 inhibited the activity of AKT but not ERK in these cells. Activation of PKG2 also significantly increased luciferase reporter activity driven by FoxO-responsive elements in LS174T cells. In support of FoxO activation, PKG2 also increased the expression of several FoxO target genes, including catalase, GADD45, and p27kip. Treatment of colon explants with 8Br-cGMP also activated FoxO target gene expression at both RNA and Protein levels, and this was associated with reduced redox stress. The regulation of FoxO by cGMP in vivo most likely requires PKG2, because target gene expression was reduced in the colon mucosa of Prkg2-/- mice compared to wild type siblings. Since FoxO has not previously been studied in the colon, we first examined expression using IHC. These data showed that FoxO3a is the most prominent isoform, and was concentrated almost exclusively at the lumenal border. While FoxO1 showed negligible staining, FoxO4 was observed deeper in the crypt. In order to determine whether cGMP could activate FoxO in vivo, the PDE-5 inhibitor vardenafil (Levitra™) was used. Mice treated with vardenafil showed a dramatic mobilization of FoxO3a to the nucleus of lumenal epithelial cells. Analysis of the colon mucosa from treated animals showed increased levels of FoxO target genes and reduced redox stress. This was likely to be mediated by FoxO3a since vardenafil did not significantly affect the localization of FoxO1 or FoxO4. Taken together these data define a novel signaling pathway in the colon epithelium, where PKG2 leads to activation of FoxO. The established tumor suppressor role of FoxO Proteins highlights the potential therapeutic role for cGMP signaling in colon cancer prevention. Citation Format: Allison Bridges, Bianca Islam, Sarah Sharman, Rui Wang, Subbaramiah Sridhar, Darren Douglas Browning. Type 2 cGMP-dependent Protein Kinase activates antineoplastic signaling in the colon. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1918. doi:10.1158/1538-7445.AM2015-1918
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Type 2 cGMP-dependent Protein Kinase regulates proliferation and differentiation in the colonic mucosa
American Journal of Physiology-Gastrointestinal and Liver Physiology, 2012Co-Authors: Rui Wang, Franz Hofmann, In-kiu Kwon, Muthusamy Thangaraju, Nagendra Singh, Kebin Liu, Philippe Jay, Vadivel Ganapathy, Darren D BrowningAbstract:Signaling through cGMP has emerged as an important regulator of tissue homeostasis in the gastrointestinal tract, but the mechanism is not known. Type 2 cGMP-dependent Protein Kinase (PKG2) is a ma...
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An anti-tumor role for cGMP-dependent Protein Kinase
Cancer Letters, 2006Co-Authors: Yali Hou, Vadivel Ganapathy, Naren Gupta, Patricia V. Schoenlein, Elsie Wong, Robert G. Martindale, Darren D BrowningAbstract:This study compared Type-1 cGMP-dependent Protein Kinase (PKG) expression in normal and tumor tissues and examined PKG function in tumor growth. Studies with a cDNA array revealed that PKG expression was reduced in many tumors compared to respective normal tissue. This decrease in PKG expression was confirmed using quantitative RT-PCR and western blotting of matched colon specimens from normal epithelium and tumor tissue, and also in colon derived cell lines where luciferase reporter analysis revealed that the decreased expression occurred at the transcriptional level. Using SW620 colon carcinoma cells engineered for inducible expression of PKG1β, it was found that exogenous PKG1β lead to decreased tumor growth and invasiveness in nude mouse xenografts.
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Nitric oxide activation of p38 mitogen-activated Protein Kinase in 293T fibroblasts requires cGMP-dependent Protein Kinase.
Journal of Biological Chemistry, 2000Co-Authors: Darren D Browning, Marisa P. Mcshane, Caroline MartyAbstract:Abstract An increase in cellular levels of cyclic nucleotides activates serine/threonine-dependent Kinases that lead to diverse physiological effects. Recently we reported the activation of the p38 mitogen-activated Protein Kinase (MAPK) pathway in neutrophils by a cGMP-dependent mechanism. In this study we demonstrated that exogenously supplied nitric oxide leads to activation of p38 MAPK in 293T fibroblasts. Phosphorylation of p38 corresponded with an increase in ATF-2-dependent gene expression. The effect of nitric oxide was mimicked by addition of 8-bromo-cGMP, indicating that activation of soluble guanylyl cyclase was involved. The importance of cGMP-dependent Protein Kinase in the activation of p38 MAPK by nitric oxide in 293T cells was assessed in a transfection based assay. Overexpression of cGMP-dependent Protein Kinase-1α caused phosphorylation of p38 in these cells and potentiated the effectiveness of cGMP. Overexpression of a catalytically inactive mutant form of this enzyme (T516A) blocked the ability of both nitric oxide and 8-bromo-cGMP to activate p38 as measured by both p38 phosphorylation and ATF-2 driven gene expression. Together, these data demonstrate that nitric oxide stimulates a novel pathway leading to activation of p38 MAPK that requires activation of cGMP-dependent Protein Kinase.
Franz Hofmann - One of the best experts on this subject based on the ideXlab platform.
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Type 2 cGMP-dependent Protein Kinase regulates proliferation and differentiation in the colonic mucosa
American Journal of Physiology-Gastrointestinal and Liver Physiology, 2012Co-Authors: Rui Wang, Franz Hofmann, In-kiu Kwon, Muthusamy Thangaraju, Nagendra Singh, Kebin Liu, Philippe Jay, Vadivel Ganapathy, Darren D BrowningAbstract:Signaling through cGMP has emerged as an important regulator of tissue homeostasis in the gastrointestinal tract, but the mechanism is not known. Type 2 cGMP-dependent Protein Kinase (PKG2) is a ma...
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Type 2 cGMP-dependent Protein Kinase regulates proliferation and differentiation in the colonic mucosa
AJP - Gastrointestinal and Liver Physiology, 2012Co-Authors: Rui Wang, Franz Hofmann, In-kiu Kwon, Muthusamy Thangaraju, Nagendra Singh, Kebin Liu, Philippe Jay, Vadivel Ganapathy, Darren BrowningAbstract:Signaling through cGMP has emerged as an important regulator of tissue homeostasis in the gastrointestinal tract, but the mechanism is not known. Type 2 cGMP-dependent Protein Kinase (PKG2) is a major cGMP effector in the gut epithelium, and the present studies have tested its importance in the regulation of proliferation and differentiation in the mouse colon and in colon cancer cell lines. Tissue homeostasis was examined in the proximal colon of Prkg2(-/-) mice using histological markers of proliferation and differentiation. The effect of ectopic PKG2 on proliferation and differentiation was tested in vitro using inducible colon cancer cell lines. PCR and luciferase reporter assays were used to determine the importance of Sox9 downstream of PKG2. The colons of Prkg2(-/-) mice exhibited crypt hyperplasia, increased epithelial apoptosis, and reduced numbers of differentiated goblet and enteroendocrine cells. Ectopic PKG2 was able to inhibit proliferation and induce Muc2 and CDX2 expression in colon cancer cells, but did not significantly affect cell death. PKG2 reduced Sox9 levels and signaling, suggesting possible involvement of this pathway downstream of cGMP in the colon. The work presented here demonstrates a novel antiproliferative and prodifferentiation role for PKG2 in the colon. These homeostatic functions of PKG2 were reproducible in colon cancer cells lines where downregulation of Sox9 is a possible mechanism. The similarities in phenotype between PKG2 and GCC knockout mice positions PKG2 as a likely mediator of the homeostatic effects of cGMP signaling in the colon.
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the cgmp dependent Protein Kinase ii is an inhibitory modulator of the hyperpolarization activated hcn2 channel
PLOS ONE, 2011Co-Authors: Verena Hammelmann, Franz Hofmann, Xiangang Zong, Stylianos Michalakis, Martin BielAbstract:Opening of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels is facilitated by direct binding of cyclic nucleotides to a cyclic nucleotide-binding domain (CNBD) in the C-terminus. Here, we show for the first time that in the HCN2 channel cGMP can also exert an inhibitory effect on gating via cGMP-dependent Protein Kinase II (cGKII)-mediated phosphorylation. Using coimmunoprecipitation and immunohistochemistry we demonstrate that cGKII and HCN2 interact and colocalize with each other upon heterologous expression as well as in native mouse brain. We identify the proximal C-terminus of HCN2 as binding region of cGKII and show that cGKII phosphorylates HCN2 at a specific serine residue (S641) in the C-terminal end of the CNBD. The cGKII shifts the voltage-dependence of HCN2 activation to 2–5 mV more negative voltages and, hence, counteracts the stimulatory effect of cGMP on gating. The inhibitory cGMP effect can be either abolished by mutation of the phosphorylation site in HCN2 or by impairing the catalytic domain of cGKII. By contrast, the inhibitory effect is preserved in a HCN2 mutant carrying a CNBD deficient for cGMP binding. Our data suggest that bidirectional regulation of HCN2 gating by cGMP contributes to cellular fine-tuning of HCN channel activity.
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cGMP-dependent Protein Kinase II and aldosterone secretion.
FEBS Journal, 2009Co-Authors: Beate Spießberger, Dominik Bernhard, Stefan Herrmann, Susanne Feil, Claudia Werner, Peter B. Luppa, Franz HofmannAbstract:ACTH-stimulated aldosterone secretion can be inhibited by atrio-natriuretic peptide/cGMP. The mechanism behind this modulation has been reported to involve cGMP-dependent activation of phosphodiesterase 2 (PDE2) and hydrolysis of cAMP. Recently it was reported that activation of cGMP-dependent Protein Kinase II (cGKII) stimulated aldosterone secretion in rat zona glomerulosa cells. The zona glomerulosa of the murine adrenal cortex expresses cGKII and PDE2. We used mice with a homozygous inactivation of the cGKII gene to investigate in vivo the potential role of this Kinase in aldosterone secretion. Basal plasma renin and aldosterone levels were similar in wild-type and cGKII−/− mice. In vivo injection of atrio-natriuretic peptide decreased ACTH-stimulated aldosterone secretion in wild-type mice, but had no effect in cGKII-deficient mice. These results support the view that cGKII modulates aldosterone secretion in the murine adrenal cortex.
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Alternative splicing of cGMP-dependent Protein Kinase I and nitrate tolerance.
Circulation Research, 2003Co-Authors: Robert Feil, Franz HofmannAbstract:To the Editor: A major point of the recent study by Gerzanich et al1 is that angiotensin II–induced “alternative splicing of cGKI [cGMP-dependent Protein Kinase I] represents a novel mechanism for reducing sensitivity to NO/cGMP” (page 805). This conclusion was based on the authors’ finding of “a large increase in cGKIβ mRNA and a decrease in cGKIα mRNA” (page 805) in basilar arteries of angiotensin II–hypertensive versus control rats (their Figure 5). The cGKIα …
Thomas M. Lincoln - One of the best experts on this subject based on the ideXlab platform.
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Post-transcriptional regulation of cGMP-dependent Protein Kinase mRNA in vascular smooth muscle cells.
The FASEB Journal, 2006Co-Authors: Hassan Sellak, Thomas M. LincolnAbstract:It is well established that type-I cGMP-dependent Protein Kinase (PKG-I) mediates many functions in vascular smooth muscle cells (VSMC). Despite its important role in the vasculature, PKG expressio...
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Upstream stimulatory factors (USF-1/USF-2) regulate human cGMP-dependent Protein Kinase I gene expression in vascular smooth muscle cells.
Journal of Biological Chemistry, 2005Co-Authors: Hassan Sellak, Chung-sik Choi, Natasha C. Browner, Thomas M. LincolnAbstract:Cyclic GMP-dependent Protein Kinase I plays a pivotal role in regulating smooth muscle cell relaxation, growth, and differentiation. Expression of the enzyme varies greatly in smooth muscle and in other tissues and cell types, yet little is known regarding the mechanisms regulating cGMP-dependent Protein Kinase gene expression. The present work was undertaken to characterize the mechanisms controlling Kinase gene expression in vascular smooth muscle cells. A 2-kb human cGMP-dependent Protein Kinase I 5'-noncoding promoter sequence was characterized by serial deletion, and functional studies demonstrated that a 591-bp 5'-promoter construct possessed the highest activity compared with all other constructs generated from the larger promoter. Analysis of the sequence between -472 and -591 bp from the transcriptional start site revealed the existence of two E-like boxes known to bind upstream stimulatory factors. Electrophoretic mobility shift assays and functional studies using luciferase reporter gene assays identified upstream stimulatory factors as the transcription factors bound to the E-boxes in the 591-bp promoter. Site-directed mutagenesis of the E-boxes abolished the binding of upstream stimulatory factor Proteins and decreased the activity of the cGMP-dependent Protein Kinase I 591-bp promoter, thus confirming the involvement of these transcription factors in mediating gene expression. Cotransfection experiments demonstrated that overexpression of upstream stimulatory factors 1 and 2 increased cGMP-dependent Protein Kinase I promoter activity. Collectively, these data suggest that the human proximal cGMP-dependent Protein Kinase I promoter is regulated by tandem E-boxes that bind upstream stimulatory factors.
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invited review cgmp dependent Protein Kinase signaling mechanisms in smooth muscle from the regulation of tone to gene expression
Journal of Applied Physiology, 2001Co-Authors: Thomas M. Lincoln, Hassan SellakAbstract:cGMP is a second messenger that produces its effects by interacting with intracellular receptor Proteins. In smooth muscle cells, one of the major receptors for cGMP is the serine/threonine Protein Kinase, cGMP-dependent Protein Kinase (PKG). PKG has been shown to catalyze the phosphorylation of a number of physiologically relevant Proteins whose function it is to regulate the contractile activity of the smooth muscle cell. These include Proteins that regulate free intracellular calcium levels, the cytoskeleton, and the phosphorylation state of the regulatory light chain of smooth muscle myosin. Other studies have shown that vascular smooth muscle cells (VSMCs) that are cultured in vitro may cease to express PKG and will, coincidentally, acquire a noncontractile, synthetic phenotype. The restoration of PKG expression to the synthetic phenotype VSMC results in the cells acquiring a more contractile phenotype. These more recent studies suggest that PKG controls VSMC gene expression that, in turn, regulates phenotypic modulation of the cells. Therefore, the regulation of PKG gene expression appears to be linked to phenotypic modulation of VSMC. Because several vascular disorders are related to the accumulation of synthetic, fibroproliferative VSMC in the vessel wall, it is likely that changes in the activity of the nitric oxide/cGMP/PKG pathway is involved the development of these diseases.
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Regulation of Myosin Phosphatase by a Specific Interaction with cGMP- Dependent Protein Kinase Iα
Science, 1999Co-Authors: Howard K Surks, Thomas M. Lincoln, Naoki Mochizuki, Yasuyo Kasai, Serban P. Georgescu, K. Mary Tang, Masaaki Ito, Michael E. MendelsohnAbstract:Contraction and relaxation of smooth muscle are regulated by myosin light-chain Kinase and myosin phosphatase through phosphorylation and dephosphorylation of myosin light chains. Cyclic guanosine monophosphate (cGMP)–dependent Protein Kinase Iα (cGKIα) mediates physiologic relaxation of vascular smooth muscle in response to nitric oxide and cGMP. It is shown here that cGKIα is targeted to the smooth muscle cell contractile apparatus by a leucine zipper interaction with the myosin-binding subunit (MBS) of myosin phosphatase. Uncoupling of the cGKIα-MBS interaction prevents cGMP-dependent dephosphorylation of myosin light chain, demonstrating that this interaction is essential to the regulation of vascular smooth muscle cell tone.
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Regulation of Osteoclastic Acid Secretion by cGMP-dependent Protein Kinase
Biochemical and Biophysical Research Communications, 1994Co-Authors: C. Vaneppsfung, Thomas M. Lincoln, J.p. Williams, T.l. Cornwell, J.m. Mcdonald, W. Radding, Harry C. BlairAbstract:Nitric oxide (NO) down-regulates osteoclastic activity. The mechanism is unknown, although, in some cells NO acts by stimulating guanylate cyclase which activates cGMP-dependent Proteins. We demonstrated cGMP-dependent Protein Kinase in osteoclasts by immunofluorescence microscopy. Specificity was confirmed by Western blot analysis showing a single 78 kDa band, the size of the Type I isoform, in isolated avian osteoclasts. Osteoclast function centers on HCl secretion at a specialized membrane organelle. We found that purified cGMP-dependent Protein Kinase inhibits ATP-dependent acid transport in reconstituted osteoclast membrane vesicles >90%, while cAMP-dependent Kinase catalytic subunit, calmodulin Kinase II, or cGMP alone were ineffective. This novel, direct modulation of acid transport by cGMP-dependent Kinase and the occurrence of the enzyme in osteoclasts suggest that a mechanism of NO-regulation of bone turnover is via cGMP and cGMP-dependent Protein Kinase inhibition of HCl transport.
Hassan Sellak - One of the best experts on this subject based on the ideXlab platform.
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Post-transcriptional regulation of cGMP-dependent Protein Kinase mRNA in vascular smooth muscle cells.
The FASEB Journal, 2006Co-Authors: Hassan Sellak, Thomas M. LincolnAbstract:It is well established that type-I cGMP-dependent Protein Kinase (PKG-I) mediates many functions in vascular smooth muscle cells (VSMC). Despite its important role in the vasculature, PKG expressio...
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Upstream stimulatory factors (USF-1/USF-2) regulate human cGMP-dependent Protein Kinase I gene expression in vascular smooth muscle cells.
Journal of Biological Chemistry, 2005Co-Authors: Hassan Sellak, Chung-sik Choi, Natasha C. Browner, Thomas M. LincolnAbstract:Cyclic GMP-dependent Protein Kinase I plays a pivotal role in regulating smooth muscle cell relaxation, growth, and differentiation. Expression of the enzyme varies greatly in smooth muscle and in other tissues and cell types, yet little is known regarding the mechanisms regulating cGMP-dependent Protein Kinase gene expression. The present work was undertaken to characterize the mechanisms controlling Kinase gene expression in vascular smooth muscle cells. A 2-kb human cGMP-dependent Protein Kinase I 5'-noncoding promoter sequence was characterized by serial deletion, and functional studies demonstrated that a 591-bp 5'-promoter construct possessed the highest activity compared with all other constructs generated from the larger promoter. Analysis of the sequence between -472 and -591 bp from the transcriptional start site revealed the existence of two E-like boxes known to bind upstream stimulatory factors. Electrophoretic mobility shift assays and functional studies using luciferase reporter gene assays identified upstream stimulatory factors as the transcription factors bound to the E-boxes in the 591-bp promoter. Site-directed mutagenesis of the E-boxes abolished the binding of upstream stimulatory factor Proteins and decreased the activity of the cGMP-dependent Protein Kinase I 591-bp promoter, thus confirming the involvement of these transcription factors in mediating gene expression. Cotransfection experiments demonstrated that overexpression of upstream stimulatory factors 1 and 2 increased cGMP-dependent Protein Kinase I promoter activity. Collectively, these data suggest that the human proximal cGMP-dependent Protein Kinase I promoter is regulated by tandem E-boxes that bind upstream stimulatory factors.
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invited review cgmp dependent Protein Kinase signaling mechanisms in smooth muscle from the regulation of tone to gene expression
Journal of Applied Physiology, 2001Co-Authors: Thomas M. Lincoln, Hassan SellakAbstract:cGMP is a second messenger that produces its effects by interacting with intracellular receptor Proteins. In smooth muscle cells, one of the major receptors for cGMP is the serine/threonine Protein Kinase, cGMP-dependent Protein Kinase (PKG). PKG has been shown to catalyze the phosphorylation of a number of physiologically relevant Proteins whose function it is to regulate the contractile activity of the smooth muscle cell. These include Proteins that regulate free intracellular calcium levels, the cytoskeleton, and the phosphorylation state of the regulatory light chain of smooth muscle myosin. Other studies have shown that vascular smooth muscle cells (VSMCs) that are cultured in vitro may cease to express PKG and will, coincidentally, acquire a noncontractile, synthetic phenotype. The restoration of PKG expression to the synthetic phenotype VSMC results in the cells acquiring a more contractile phenotype. These more recent studies suggest that PKG controls VSMC gene expression that, in turn, regulates phenotypic modulation of the cells. Therefore, the regulation of PKG gene expression appears to be linked to phenotypic modulation of VSMC. Because several vascular disorders are related to the accumulation of synthetic, fibroproliferative VSMC in the vessel wall, it is likely that changes in the activity of the nitric oxide/cGMP/PKG pathway is involved the development of these diseases.
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Reorganization of myofilament Proteins and decreased cGMP-dependent Protein Kinase in the human uterus during pregnancy.
Journal of Clinical Endocrinology & Metabolism, 2001Co-Authors: Trudy L. Cornwell, Hassan Sellak, Rodney T. Miller, R. Ann WordAbstract:Excessive or premature contractions of uterine smooth muscle may contribute to preterm labor. Contractile stimuli induce myosin and actin filament interactions through calcium-dependent myosin phosphorylation. The mechanisms that maintain myometrial quiescence until term are not well established, but may include control of calcium levels by nitric oxide and cGMP signaling and thin filament (caldesmon and calponin) regulation. Previously, we reported that myometrial tissues from pregnant rats are not responsive to cGMP due to decreases in cGMP-dependent Protein Kinase. Considering the well documented differences in the endocrinology of parturition among species, this study was conducted to test the hypothesis that the levels and subcellular distribution of caldesmon, calponin, and cGMP-dependent Protein Kinase are regulated with the hormonal milieu of human pregnancy. Whereas cGMP-dependent Protein Kinase was significantly reduced in the human uterus during pregnancy, caldesmon expression was significantly increased, and both caldesmon and calponin were redistributed to a readily extractable subcellular pool. These data suggest that cGMP-dependent Protein Kinase does not mediate gestational quiescence. Redistribution of thin filament-associated Proteins, however, may alter uterine smooth muscle tone or the cytoskeletal framework of myocytes to maintain gestation despite the substantial distention that accompanies all intrauterine pregnancies.
Rui Wang - One of the best experts on this subject based on the ideXlab platform.
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abstract 1262 type 2 cgmp dependent Protein Kinase has a tumor suppressive role in the colon epithelium
Cancer Research, 2018Co-Authors: Bianca N Islam, Subbaramiah Sridhar, Justin Ashby, Ravindra Kolhe, Sarah K Sharman, Rui Wang, Darren D BrowningAbstract:The cGMP level plays a central role in regulating homeostasis in the colon epithelium and is emerging as a potential target for colon cancer prevention. The signaling components downstream of cGMP, and the tumor suppressive mechanism remain poorly understood. The present study has examined the expression of cGMP-dependent Protein Kinase isoforms (PKG1, PKG2) in normal intestinal mucosa and in colon cancer from human specimens. Immunohistochemical analysis detected PKG1 in supportive cells in the lamina propria and in smooth-muscle tissue, but not in the epithelium of normal intestine and colon. In contrast, PKG2 was detected exclusively in the epithelium. In colon cancer, PKG1 was restricted to the stroma where it colocalized with vimentin, whereas PKG2 was only detected in the tumor epithelium where it colocalized with cytokeratin. This pattern of PKG isoform expression was similar in the mouse intestine and colon, where only PKG2 was detected in purified colonic crypts. PKG2 knockout (KO) mice were used interrogate possible anti-carcinogenic roles in the colon epithelium. When subjected to the AOM/DSS model of colon carcinogenesis, the PKG2 KO animals produced significantly more polyps than wild type controls (1.75-fold, p=0.0037). While the polyps from PKG2 KO animals were slightly smaller than those from wild type, the trend was not significant (p=0.13). Wild type and PKG2 KO mice responded equally to DSS treatment, but the PKG2-deficient animals exhibited crypt hyperplasia and increased apoptosis in luminal epithelium relative to wild type animals. Taken together these findings suggest that PKG2 has a tumor suppressive role in the colon epithelium, and that reducing the proliferative compartment may be part of the mechanism. Citation Format: Bianca N. Islam, Sarah K. Sharman, Yali Hou, Justin Ashby, Rui Wang, Subbaramiah Sridhar, Ravindra Kolhe, Darren D. Browning. Type-2 cGMP-dependent Protein Kinase has a tumor-suppressive role in the colon epithelium [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1262.
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Abstract 1918: Type 2 cGMP-dependent Protein Kinase activates antineoplastic signaling in the colon
Cancer Research, 2015Co-Authors: Allison Bridges, Bianca N Islam, Subbaramiah Sridhar, Sarah K Sharman, Rui Wang, Darren D BrowningAbstract:Signaling through cGMP has emerged as a potentially important suppressor of tumorigenesis in the colon but the mechanisms are poorly defined. Studies of guanylyl-cyclase (GCC) knockout mice indicate that cGMP signaling inhibits proliferation in the colon epithelium and that AKT may have an important role. Type-2 cGMP-dependent Protein Kinase (PKG2) is a central effector of cGMP in the colon epithelium and likely mediates the suppressive signaling. An important growth-inhibitory effect of AKT is the inactivation of forkhead transcription factors (FoxO). FoxO Proteins are tumor suppressors that regulate growth-inhibitory and pro-apoptotic target gene expression but these Proteins have not previously been examined in the intestinal tract. The present study sought to determine whether PKG2 can activate FoxO in colon cancer cells and in the colon epithelium. Activation of PKG2 in LS174T colon cancer cells inhibited cell proliferation but did not significantly affect apoptosis. Suggestive of a mechanism, it was found that PKG2 inhibited the activity of AKT but not ERK in these cells. Activation of PKG2 also significantly increased luciferase reporter activity driven by FoxO-responsive elements in LS174T cells. In support of FoxO activation, PKG2 also increased the expression of several FoxO target genes, including catalase, GADD45, and p27kip. Treatment of colon explants with 8Br-cGMP also activated FoxO target gene expression at both RNA and Protein levels, and this was associated with reduced redox stress. The regulation of FoxO by cGMP in vivo most likely requires PKG2, because target gene expression was reduced in the colon mucosa of Prkg2-/- mice compared to wild type siblings. Since FoxO has not previously been studied in the colon, we first examined expression using IHC. These data showed that FoxO3a is the most prominent isoform, and was concentrated almost exclusively at the lumenal border. While FoxO1 showed negligible staining, FoxO4 was observed deeper in the crypt. In order to determine whether cGMP could activate FoxO in vivo, the PDE-5 inhibitor vardenafil (Levitra™) was used. Mice treated with vardenafil showed a dramatic mobilization of FoxO3a to the nucleus of lumenal epithelial cells. Analysis of the colon mucosa from treated animals showed increased levels of FoxO target genes and reduced redox stress. This was likely to be mediated by FoxO3a since vardenafil did not significantly affect the localization of FoxO1 or FoxO4. Taken together these data define a novel signaling pathway in the colon epithelium, where PKG2 leads to activation of FoxO. The established tumor suppressor role of FoxO Proteins highlights the potential therapeutic role for cGMP signaling in colon cancer prevention. Citation Format: Allison Bridges, Bianca Islam, Sarah Sharman, Rui Wang, Subbaramiah Sridhar, Darren Douglas Browning. Type 2 cGMP-dependent Protein Kinase activates antineoplastic signaling in the colon. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1918. doi:10.1158/1538-7445.AM2015-1918
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cGMP-dependent Protein Kinase contributes to hydrogen sulfide-stimulated vasorelaxation.
PLoS ONE, 2012Co-Authors: Mariarosaria Bucci, Andreas Papapetropoulos, Valentina Vellecco, Zongmin Zhou, Altaany Zaid, Panagiotis Giannogonas, Anna Cantalupo, Sandeep Dhayade, Katia Karalis, Rui WangAbstract:A growing body of evidence suggests that hydrogen sulfide (H2S) is a signaling molecule in mammalian cells. In the cardiovascular system, H2S enhances vasodilation and angiogenesis. H2S-induced vasodilation is hypothesized to occur through ATP-sensitive potassium channels (KATP); however, we recently demonstrated that it also increases cGMP levels in tissues. Herein, we studied the involvement of cGMP-dependent Protein Kinase-I in H2S-induced vasorelaxation. The effect of H2S on vessel tone was studied in phenylephrine-contracted aortic rings with or without endothelium. cGMP levels were determined in cultured cells or isolated vessel by enzyme immunoassay. Pretreatment of aortic rings with sildenafil attenuated NaHS-induced relaxation, confirming previous findings that H2S is a phosphodiesterase inhibitor. In addition, vascular tissue levels of cGMP in cystathionine gamma lyase knockouts were lower than those in wild-type control mice. Treatment of aortic rings with NaHS, a fast releasing H2S donor, enhanced phosphorylation of vasodilator-stimulated phosphoProtein in a time-dependent manner, suggesting that cGMP-dependent Protein Kinase (PKG) is activated after exposure to H2S. Incubation of aortic rings with a PKG-I inhibitor (DT-2) attenuated NaHS-stimulated relaxation. Interestingly, vasodilatory responses to a slowly releasing H2S donor (GYY 4137) were unaffected by DT-2, suggesting that this donor dilates mouse aorta through PKG-independent pathways. Dilatory responses to NaHS and L-cysteine (a substrate for H2S production) were reduced in vessels of PKG-I knockout mice (PKG-I−/−). Moreover, glibenclamide inhibited NaHS-induced vasorelaxation in vessels from wild-type animals, but not PKG-I−/−, suggesting that there is a cross-talk between KATP and PKG. Our results confirm the role of cGMP in the vascular responses to NaHS and demonstrate that genetic deletion of PKG-I attenuates NaHS and L-cysteine-stimulated vasodilation.
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Type 2 cGMP-dependent Protein Kinase regulates proliferation and differentiation in the colonic mucosa
American Journal of Physiology-Gastrointestinal and Liver Physiology, 2012Co-Authors: Rui Wang, Franz Hofmann, In-kiu Kwon, Muthusamy Thangaraju, Nagendra Singh, Kebin Liu, Philippe Jay, Vadivel Ganapathy, Darren D BrowningAbstract:Signaling through cGMP has emerged as an important regulator of tissue homeostasis in the gastrointestinal tract, but the mechanism is not known. Type 2 cGMP-dependent Protein Kinase (PKG2) is a ma...
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Type 2 cGMP-dependent Protein Kinase regulates proliferation and differentiation in the colonic mucosa
AJP - Gastrointestinal and Liver Physiology, 2012Co-Authors: Rui Wang, Franz Hofmann, In-kiu Kwon, Muthusamy Thangaraju, Nagendra Singh, Kebin Liu, Philippe Jay, Vadivel Ganapathy, Darren BrowningAbstract:Signaling through cGMP has emerged as an important regulator of tissue homeostasis in the gastrointestinal tract, but the mechanism is not known. Type 2 cGMP-dependent Protein Kinase (PKG2) is a major cGMP effector in the gut epithelium, and the present studies have tested its importance in the regulation of proliferation and differentiation in the mouse colon and in colon cancer cell lines. Tissue homeostasis was examined in the proximal colon of Prkg2(-/-) mice using histological markers of proliferation and differentiation. The effect of ectopic PKG2 on proliferation and differentiation was tested in vitro using inducible colon cancer cell lines. PCR and luciferase reporter assays were used to determine the importance of Sox9 downstream of PKG2. The colons of Prkg2(-/-) mice exhibited crypt hyperplasia, increased epithelial apoptosis, and reduced numbers of differentiated goblet and enteroendocrine cells. Ectopic PKG2 was able to inhibit proliferation and induce Muc2 and CDX2 expression in colon cancer cells, but did not significantly affect cell death. PKG2 reduced Sox9 levels and signaling, suggesting possible involvement of this pathway downstream of cGMP in the colon. The work presented here demonstrates a novel antiproliferative and prodifferentiation role for PKG2 in the colon. These homeostatic functions of PKG2 were reproducible in colon cancer cells lines where downregulation of Sox9 is a possible mechanism. The similarities in phenotype between PKG2 and GCC knockout mice positions PKG2 as a likely mediator of the homeostatic effects of cGMP signaling in the colon.