RESUMO
Previous studies revealed that type II cGMP-dependent protein kinase G (PKG II) could inhibit the activation of epidermal growth factor receptor (EGFR) which is a widely investigated RTK. PDGFR belongs to family of receptor tyrosine kinases (RTKs) too. However, the effect of PKG II on PDGFR activation is not clear yet. This study investigated potential regulatory effect of PKG II on activation of PDGFRß and the downstream signaling transductions in gastric cancer. The results from CCK8 assay and Transwell assay indicated that PDGF-BB induced cell proliferation and migration. Activated PKG II reversed the above variations caused by PDGF-BB. Immunoprecipitation and Western blotting results showed that PKG II combined with PDGFRß and phosphorylated this receptor, and thereby inhibited PDGF-BB induced activation of PDGFRß, and MAPK/ERK and PI3K/Akt mediated signal transduction pathways. Based on the prediction by phosphorylation site software, Ser643 and Ser712 were mutated to alanine respectively which prevented phosphorylation at these sites. Mutation at Ser712 abolished the inhibitory function of PKG II on PDGFRß activation but mutation of Ser643 had no such an effect, indicating that Ser712 was PKG II-specific phosphorylating site of PDGFRß. In conclusion, PKG II inhibited PDGFRß activation in gastric cancer via phosphorylating Ser712 of this RTK.
Assuntos
Proteína Quinase Dependente de GMP Cíclico Tipo II/metabolismo , Proteína Quinase Dependente de GMP Cíclico Tipo II/fisiologia , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Humanos , Fosforilação , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Proto-Oncogênicas c-sis/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Neoplasias Gástricas/metabolismoRESUMO
NO/cGMP signaling is important for bone remodeling in response to mechanical and hormonal stimuli, but the downstream mediator(s) regulating skeletal homeostasis are incompletely defined. We generated transgenic mice expressing a partly-activated, mutant cGMP-dependent protein kinase type 2 (PKG2R242Q) under control of the osteoblast-specific Col1a1 promoter to characterize the role of PKG2 in post-natal bone formation. Primary osteoblasts from these mice showed a two- to three-fold increase in basal and total PKG2 activity; they proliferated faster and were resistant to apoptosis compared to cells from WT mice. Male Col1a1-Prkg2R242Q transgenic mice had increased osteoblast numbers, bone formation rates and Wnt/ß-catenin-related gene expression in bone and a higher trabecular bone mass compared to their WT littermates. Streptozotocin-induced type 1 diabetes suppressed bone formation and caused rapid bone loss in WT mice, but male transgenic mice were protected from these effects. Surprisingly, we found no significant difference in bone micro-architecture or Wnt/ß-catenin-related gene expression between female WT and transgenic mice; female mice of both genotypes showed higher systemic and osteoblastic NO/cGMP generation compared to their male counterparts, and a higher level of endogenous PKG2 activity may be responsible for masking effects of the PKG2R242Q transgene in females. Our data support sexual dimorphism in Wnt/ß-catenin signaling and PKG2 regulation of this crucial pathway in bone homeostasis. This work establishes PKG2 as a key regulator of osteoblast proliferation and post-natal bone formation.
Assuntos
Doenças Ósseas Metabólicas/genética , Osso e Ossos/patologia , Proteína Quinase Dependente de GMP Cíclico Tipo II/fisiologia , Osteogênese/genética , Animais , Densidade Óssea/genética , Doenças Ósseas Metabólicas/metabolismo , Osso e Ossos/metabolismo , Diferenciação Celular/genética , Proliferação de Células/genética , Células Cultivadas , Proteína Quinase Dependente de GMP Cíclico Tipo II/genética , Complicações do Diabetes/genética , Complicações do Diabetes/metabolismo , Complicações do Diabetes/patologia , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Tamanho do Órgão/genética , Osteoblastos/metabolismo , Osteoblastos/fisiologiaRESUMO
Activity-dependent trafficking of AMPA receptors to synapses regulates synaptic strength. Activation of the NMDA receptor induces several second messenger pathways that contribute to receptor trafficking-dependent plasticity, including the NO pathway, which elevates cGMP. In turn, cGMP activates the cGMP-dependent protein kinase type II (cGKII), which phosphorylates the AMPA receptor subunit GluA1 at serine 845, a critical step facilitating synaptic delivery in the mechanism of activity-dependent synaptic potentiation. Since cGKII is expressed in the striatum, amygdala, cerebral cortex, and hippocampus, it has been proposed that mice lacking cGKII may present phenotypic differences compared to their wild-type littermates in emotion-dependent tasks, learning and memory, and drug reward salience. Previous studies have shown that cGKII KO mice ingest higher amounts of ethanol as well as exhibit elevated anxiety levels compared to wild-type (WT) littermates. Here, we show that cGKII KO mice are significantly deficient in spatial learning while exhibiting facilitated motor coordination, demonstrating a clear dependence of memory-based tasks on cGKII. We also show diminished GluA1 phosphorylation in the postsynaptic density (PSD) of cGKII KO prefrontal cortex while in hippocampal PSD fractions, phosphorylation was not significantly altered. These data suggest that the role of cGKII may be more robust in particular brain regions, thereby impacting complex behaviors dependent on these regions differently.
