Receptor Of Activated Protein Kinase C 1 Research Articles

RACK1 Acts as a Potential Tumor Promoter in Colorectal Cancer.

Background The receptor of activated protein kinase C 1 (RACK1) promotes the progression and invasion of several cancers. However, the role of RACK1 in the pathogenesis of colorectal cancer (CRC) has not been clearly defined. Herein, we aimed to investigate the biological role of RACK1 in CRC. Materials and Methods The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) dataset were searched, and the expression of RACK1 in CRC tissues and adjacent normal tissues was evaluated. Immunohistochemical staining was performed to detect the expression of RACK1 in human CRC, adenoma, and normal tissues. Western blotting was used to detect the expression of RACK1 in human CRC cell lines. Functional assays, such as BrdU, colony formation, and wound healing and transwell invasion assays, were used to explore the biological role of RACK1 in CRC. Results RACK1 was upregulated in CRC tissues compared with its expression in adjacent normal tissues in TCGA and the GEO dataset (P < 0.05). Moreover, RACK1 was significantly overexpressed in CRC and adenoma tissues compared with its expression in normal tissues (P < 0.05). Loss-of-function experiments showed that RACK1 promoted cell proliferation, migration, and invasion in vitro. Conclusions Our data indicated that RACK1, as an oncogene, markedly promoted the progression of CRC, which suggested that RACK1 is a potential therapeutic target for CRC management.

Downregulation of tumor suppressor RACK1 by Helicobacter pylori infection promotes gastric carcinogenesis through the integrin β-1/NF-κB signaling pathway

Receptor of activated protein kinase C 1 (RACK1) is downregulated in gastric cancer and is involved in modulating NF-κB signaling pathway activity. However, the underlying molecular mechanisms regulating RACK1 expression are unclear. In this study, we demonstrated that downregulated expression of RACK1 was observed in gastric cancer tissue compared to adjacent normal tissue and was correlated with poor prognosis in patients. Helicobacter pylori (H. pylori) infection downregulated RACK1 expression in concert with canonical NF-κB signaling pathway activation in vivo and in vitro. RACK1 overexpression suppressed NF-κB signaling pathway activation as well as the release of downstream proinflammatory cytokines. In addition, RACK1 downregulation increased integrin β-1 expression, while integrin β-1 silencing decreased NF-κB signaling activation. Moreover, H. pylori infection downregulated RACK1 but upregulated integrin β-1 expression at the precancerous lesion stages in human subjects. Our data indicate that H. pylori infection promotes the upregulation of integrin β-1 expression via downregulation of RACK1 expression, which subsequently leads to the elevated activation of the NF-κB signaling pathway, an essential step in H. pylori-induced carcinogenesis.

Overexpression of RACK1 Promotes Metastasis by Enhancing Epithelial-Mesenchymal Transition and Predicts Poor Prognosis in Human Glioma.

Emerging studies show that dysregulation of the receptor of activated protein kinase C1 (RACK1) plays a crucial role in tumorigenesis and progression of various cancers. However, the biological function and underlying mechanism of RACK1 in glioma remains poorly defined. Here, we found that RACK1 was significantly up-regulated in glioma tissues compared with normal brain tissues, being closely related to clinical stage of glioma both in mRNA and protein levels. Moreover, Kaplan-Meier analysis demonstrated that patients with high RACK1 expression had a poor prognosis (p = 0.0062, HR = 1.898, 95% CI: 1.225–3.203). In vitro functional assays indicated that silencing of RACK1 could dramatically promote apoptosis and inhibit cell proliferation, migration, and invasion of glioma cells. More importantly, knockdown of RACK1 led to a vast accumulation of cells in G0/G1 phase and their reduced proportions at the S phase by suppressing the expression of G1/S transition key regulators Cyclin D1 and CDK6. Additionally, this forced down-regulation of RACK1 significantly suppressed migration and invasion via inhibiting the epithelial-mesenchymal transition (EMT) markers, such as MMP2, MMP9, ZEB1, N-Cadherin, and Integrin-β1. Collectively, our study revealed that RACK1 might act as a valuable prognostic biomarker and potential therapeutic target for glioma.

A Varp-Binding Protein, RACK1, Regulates Dendrite Outgrowth through Stabilization of Varp Protein in Mouse Melanocytes

Varp (VPS9-ankyrin repeat protein) in melanocytes is thought to function as a key player in the pigmentation of mammals. Varp regulates two different melanocyte functions: (i) transport of melanogenic enzymes to melanosomes by functioning as a Rab32/38 effector and (ii) promotion of dendrite outgrowth by functioning as a Rab21-guanine nucleotide exchange factor. The Varp protein level has recently been shown to be negatively regulated by proteasomal degradation through interaction of the ankyrin repeat 2 (ANKR2) domain of Varp with Rab40C. However, the molecular mechanisms by which Varp escapes from Rab40C and retains its own expression level remain completely unknown. Here, we identified RACK1 (receptor of activated protein kinase C 1) as a Varp-ANKR2 binding partner and investigated its involvement in Varp stabilization in mouse melanocytes. The results showed that knockdown of endogenous RACK1 in melanocytes caused dramatic reduction of the Varp protein level and inhibition of dendrite outgrowth, and intriguingly, overexpression of RACK1 inhibited the interaction between Varp and Rab40C and counteracted the negative effect of Rab40C on dendrite outgrowth. These findings indicated that RACK1 competes with Rab40C for binding to the ANKR2 domain of Varp and regulates dendrite outgrowth through stabilization of Varp in mouse melanocytes.

The Association of Receptor of Activated Protein Kinase C 1(RACK1) with Infectious Bursal Disease Virus Viral Protein VP5 and Voltage-dependent Anion Channel 2 (VDAC2) Inhibits Apoptosis and Enhances Viral Replication

Infectious bursal disease (IBD) is an acute, highly contagious, and immunosuppressive avian disease caused by IBD virus (IBDV). Our previous report indicates that IBDV VP5 induces apoptosis via interaction with voltage-dependent anion channel 2 (VDAC2). However, the underlying molecular mechanism is still unclear. We report here that receptor of activated protein kinase C 1 (RACK1) interacts with both VDAC2 and VP5 and that they could form a complex. We found that overexpression of RACK1 inhibited IBDV-induced apoptosis in DF-1 cells and that knockdown of RACK1 by small interfering RNA induced apoptosis associated with activation of caspases 9 and 3 and suppressed IBDV growth. These results indicate that RACK1 plays an antiapoptotic role during IBDV infection via interaction with VDAC2 and VP5, suggesting that VP5 sequesters RACK1 and VDAC2 in the apoptosis-inducing process.

Hepatitis B virus core protein sensitizes hepatocytes to tumor necrosis factor-induced apoptosis by suppression of the phosphorylation of mitogen-activated protein kinase kinase 7.

Hepatitis B, which caused by hepatitis B virus (HBV) infection, remains a major health threat worldwide. Hepatic injury and regeneration from chronic inflammation are the main driving factors of liver fibrosis and cirrhosis in chronic hepatitis B. Proinflammatory tumor necrosis factor alpha (TNF-α) has been implicated as a major inducer of liver cell death during viral hepatitis. Here, we report that in hepatoma cell lines and in primary mouse and human hepatocytes, expression of hepatitis B virus core (HBc) protein made cells susceptible to TNF-α-induced apoptosis. We found by tandem affinity purification and mass spectrometry that receptor of activated protein kinase C 1 (RACK1) interacted with HBc. RACK1 was recently reported as a scaffold protein that facilitates the phosphorylation of mitogen-activated protein kinase kinase 7 (MKK7) by its upstream activators. Our study showed that HBc abrogated the interaction between MKK7 and RACK1 by competitively binding to RACK1, thereby downregulating TNF-α-induced phosphorylation of MKK7 and the activation of c-Jun N-terminal kinase (JNK). In line with this finding, specific knockdown of MKK7 increased the sensitivity of hepatocytes to TNF-α-induced apoptosis, while overexpression of RACK1 counteracted the proapoptotic activity of HBc. Capsid particle formation was not obligatory for HBc proapoptotic activity, as analyzed using an assembly-defective HBc mutant. In conclusion, the expression of HBc sensitized hepatocytes to TNF-α-induced apoptosis by disrupting the interaction between MKK7 and RACK1. Our study is thus the first indication of the pathogenic effects of HBc in liver injury during hepatitis B. Our study revealed a previously unappreciated role of HBc in TNF-α-mediated apoptosis. The proapoptotic activity of HBc is important for understanding hepatitis B pathogenesis. In particular, HBV variants associated with severe hepatitis may upregulate apoptosis of hepatocytes through enhanced HBc expression. Our study also found that MKK7 is centrally involved in TNF-α-induced hepatocyte apoptosis and revealed a multifaceted role for JNK signaling in this process.

Essential Function for PDLIM2 in Cell Polarization in Three-Dimensional Cultures by Feedback Regulation of the β1-Integrin–RhoA Signaling Axis

PDLIM2 is a cytoskeletal and nuclear PDZ-LIM domain protein that regulates the stability of Nuclear Factor kappa-B (NFκB) and other transcription factors, and is required for polarized cell migration. PDLIM2 expression is suppressed by methylation in different cancers, but is strongly expressed in invasive breast cancer cells that have undergone an Epithelial Mesenchymal Transition (EMT). PDLIM2 is also expressed in non-transformed breast myoepithelial MCF10A cells and here we asked whether it is important for maintaining the polarized, epithelial phenotype of these cells. Suppression of PDLIM2 in MCF10A cells was sufficient to disrupt cell polarization and acini formation with increased proliferation and reduced apoptosis in the luminal space compared to control acini with hollow lumina. Spheroids with suppressed PDLIM2 exhibited increased expression of cell-cell and cell-matrix adhesion proteins including beta 1 (β1) integrin. Interestingly, levels of the Insulin-like growth factor 1 receptor (IGF-1 R) and Receptor of activated protein kinase C 1 (RACK1), which scaffolds IGF-1R to β1 integrin, were also increased, indicating a transformed phenotype. Focal Adhesion Kinase (FAK) and cofilin phosphorylation, and RhoA Guanosine Triphosphatase (GTPase) activity were all enhanced in these spheroids compared to control acini. Importantly, inhibition of either FAK or Rho Kinase (ROCK) was sufficient to rescue the polarity defect. We conclude that PDLIM2 expression is essential for feedback regulation of the β1-integrin-RhoA signalling axis and integration of cellular microenvironment signals with gene expression to control the polarity of breast epithelial acini structures. This is a mechanism by which PDLIM2 could mediate tumour suppression in breast epithelium.

Compound danshen tablet ameliorated aβ25-35-induced spatial memory impairment in mice via rescuing imbalance between cytokines and neurotrophins

BackgroundCompound Danshen Tablet (CDT), a Traditional Chinese Medicine, has recently been reported to improve spatial cognition in a rat model of Alzheimer’s disease. However, in vivo neuroprotective mechanism of the CDT in models of spatial memory impairment is not yet evaluated. The present study is aimed to elucidate the cellular mechanism of CDT on Aβ25-35-induced cognitive impairment in mice.MethodsMice were randomly divided into 5 groups: the control group (sham operated), the Aβ25-35 treated group, the positive drug group, and large and small dosage of the CDT groups, respectively. CDT was administered at a dose of 0.81 g/kg and 0.405 g/kg for 3 weeks. The mice in the positive drug group were treated with 0.4 mg/kg of Huperzine A, whereas the mice of the control and Aβ25-35 treated groups were administrated orally with equivalent saline. After 7 days of preventive treatment, mice were subjected to lateral ventricle injection of Aβ25-35 to establish the mice model of Alzheimer’s disease. Spatial memory impairment was evaluated by Morris water maze test. Choline acetyltransferase (ChAT) contents in hippocampus and cortex were quantified by ELISA. The levels of cytokines, receptor of activated protein kinase C1 (RACK1) and brain-derived neurotrophic factor (BDNF) in hippocampus were measured by RT-PCR and ELISA.ResultsThe results showed that Aβ25-35 caused spatial memory impairment as demonstrated by performance in the Morris water maze test. CDT was able to confer a significant improvement in spatial memory, and protect mice from Aβ25-35-induced neurotoxicity. Additionally, CDT also inhibited the increase of TNF-α and IL-6 level, and increased the expression of choline acetyltransferase (ChAT), receptor of activated protein kinase C1 (RACK1) and brain-derived neurotrophic factor (BDNF) in brain as compared to model mice.ConclusionThese findings strongly implicate that CDT may be a useful treatment against learning and memory deficits in mice by rescuing imbalance between cytokines and neurotrophins.

Cancer-derived immunoglobulin G promotes tumor cell growth and proliferation through inducing production of reactive oxygen species

Cancer cells have been found to express immunoglobulin G (IgG), but the exact functions and underlying mechanisms of cancer-derived IgG remain elusive. In this study, we first confirmed that downregulation of IgG restrained the growth and proliferation of cancer cells in vitro and in vivo. To elucidate its mechanism, we carried out a co-immunoprecipitation assay in HeLa cells and identified 27 potential IgG-interacting proteins. Among them, receptor of activated protein kinase C 1 (RACK1), ras-related nuclear protein (RAN) and peroxiredoxin 1 (PRDX1) are closely related to cell growth and oxidative stress, which prompted us to investigate the mechanism of action of IgG in the above phenomena. Upon confirmation of the interactions between IgG and the three proteins, further experiments revealed that downregulation of cancer-derived IgG lowered levels of intracellular reactive oxygen species (ROS) by enhancing cellular total antioxidant capacity. In addition, a few ROS scavengers, including catalase (CAT), dimethylsulfoxide (DMSO), n-acetylcysteine (NAC) and superoxide dismutase (SOD), further inhibited the growth of IgG-deficient cancer cells through suppressing mitogen-activated protein kinase/extracellular-regulated kinase (MAPK/ERK) signaling pathway induced by a low level of intracellular ROS, whereas exogenous hydrogen peroxide (H2O2) at low concentration promoted their survival via increasing intracellular ROS levels. Similar results were obtained in an animal model and human tissues. Taken together, our results demonstrate that cancer-derived IgG can enhance the growth and proliferation of cancer cells via inducing the production of ROS at low level. These findings provide new clues for understanding tumor proliferation and designing cancer therapy.

Endothelin-1 induces hypoxia inducible factor 1α expression in pulmonary artery smooth muscle cells

Endothelin-1 (ET-1) dose-dependently increased HIF1α expression in pulmonary artery smooth muscle cells (PASMCs). Inhibition of protein synthesis did not affect ET-1-induced HIF1α expression. The maximum effect of ET-1 was similar to that caused by proteasome inhibitor MG132. Further study indicates that ET-1 also dose-dependently stimulated calcineurin activation, specific calcineurin inhibitor cyclosporine A (CsA), abolished ET-1-induced HIF1α elevation, and reversed ET-1-induced RACK1 (receptor of activated protein kinase C 1) de-phosphorylation. Endothelin receptor A was found to specifically mediate the effects of ET-1. To examine whether RACK1 is particularly involved in proteasome-dependent HIF1α degradation, RACK1 was silenced by siRNA transfection. Cells lacking RACK1 exhibited significant elevation of HIF1α protein level. Taken together, our study suggests that ET-1 suppressed proteasome-dependent HIF1α degradation by calcineurin-dependent RACK1 de-phosphorylation.

RACK1 promotes breast carcinoma proliferation and invasion/metastasis in vitro and in vivo

A yeast two-hybrid system was utilized to identify novel PI3K p110alpha-interacting proteins, of which receptor of activated protein kinase C1 (RACK1) was chosen for successive detailed analyses. Our aim was to investigate the function(s) of RACK1 and its involvement in mechanisms of breast carcinoma proliferation and invasion/metastasis. Experiments in breast carcinoma cell lines stably transfected with RACK1, as well as nude mouse models, showed that RACK1 promotes breast carcinoma proliferation and invasion/metastasis in vitro and in vivo. Conversely, knockdown of RACK1 by siRNA in vitro inhibited proliferation, migration, and invasion. In cell lines stably transfected with RACK1, p-AKT, cyclin D1, cyclin D3, and CD147 expression, as well as MMP2 activity, were elevated. RACK1-induced migration could be inhibited by the addition of Rho-kinase inhibitor. In 160 breast carcinoma cases, survival analyses established that RACK1 is an independent prognostic factor for poor outcome (P < 0.001). In conclusion, RACK1 is an independent prognosis-related factor and promotes breast carcinoma proliferation and invasion/metastasis in vitro and in vivo.