Dataset Information

Hyperglycemic Conditions Promote Rac1-Mediated Serine536 Phosphorylation of p65 Subunit of NFκB (RelA) in Pancreatic Beta Cells.

ABSTRACT:

Background/aims

We recently reported increased phosphorylation (at S536) of the p65 subunit of NFκB (Rel A) in pancreatic beta (INS-1 832/13) cells following exposure to hyperglycemic (HG) conditions. We also demonstrated that HG-induced S536 phosphorylation of p65 is downstream to the regulatory effects of CARD9 since deletion of CARD9 expression significantly attenuated HG-induced S536 phosphorylation of p65 in beta cells. The overall objective of the current investigation is to identify putative mechanisms underlying HG-induced phosphorylation of p65 in islet beta cells following exposure to HG conditions.

Methods

INS-1 832/13 cells were incubated in low glucose (LG; 2.5 mM) or high glucose (HG; 20 mM) containing media for 24 hours in the absence or presence of small molecule inhibitors of G protein prenylation and activation. Non-nuclear and nuclear fractions were isolated from INS-1 832/13 cells using a commercially available (NE-PER) kit. Degree of S536 phosphorylation of the p65 subunit was quantified by western blotting and densitometry.

Results

HG-induced p65 phosphorylation was significantly attenuated by inhibitors of protein prenylation (e.g., simvastatin and L-788,123). Pharmacological inhibition of Tiam1-Rac1 (e.g., NSC23766) and Vav2-Rac1 (e.g., Ehop-016) signaling pathways exerted minimal effects on HG-induced p65 phosphorylation. However, EHT-1864, a small molecule compound, which binds to Rac1 thereby preventing GDP/GTP exchange, markedly suppressed HG-induced p65 phosphorylation, suggesting that Rac1 activation is requisite for HG-mediated p65 phosphorylation. Lastly, EHT-1864 significantly inhibited nuclear association of STAT3, but not total p65, in INS-1 832/13 cells exposed to HG conditions.

Conclusion

Activation of Rac1, a step downstream to HG-induced activation of CARD9, might represent a requisite signaling step in the cascade of events leading to HG-induced S536 phosphorylation of p65 and nuclear association of STAT3 in pancreatic beta cells. Data from these investigations further affirm the role(s) of Rac1 as a mediator of metabolic stress- induced dysfunction of the islet beta cell.

SUBMITTER: Kowluru A

PROVIDER: S-EPMC9644397 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature

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Publications

Hyperglycemic Conditions Promote Rac1-Mediated Serine536 Phosphorylation of p65 Subunit of NFκB (RelA) in Pancreatic Beta Cells.

Kowluru Anjaneyulu A Gamage Suhadinie S Hali Mirabela M Gleason Noah N

Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology 20220801 4

<h4>Background/aims</h4>We recently reported increased phosphorylation (at S536) of the p65 subunit of NFκB (Rel A) in pancreatic beta (INS-1 832/13) cells following exposure to hyperglycemic (HG) conditions. We also demonstrated that HG-induced S536 phosphorylation of p65 is downstream to the regulatory effects of CARD9 since deletion of CARD9 expression significantly attenuated HG-induced S536 phosphorylation of p65 in beta cells. The overall objective of the current investigation is to identi ...[more]

PMID: 35981264

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Project description:BackgroundIntrahepatic and extrahepatic metastases contribute to the high recurrence rate and mortality of hepatocellular carcinoma (HCC). Constitutive activation of nuclear factor-κB (NF-κB) is a crucial feature of HCC. NF-κB p65 (p50-p65) is the most common dimeric form. Ser536 acts as an essential phosphorylation site of RelA/p65. However, the effect of RelA/p65 Ser536 phosphorylation on progression and metastases during intermediate and advanced HCC has not been reported.MethodsPhosphorylation of RelA/p65 (p-p65 Ser536) and NF-κB p65 were detected by using immunohistochemical staining in HCC tissue samples. The biological effects of RelA/p65 Ser536 phosphorylation were evaluated by using xenograft and metastasis models. NF-κB p65 nuclear translocation was detected by using Western blotting. The binding of NF-κB p65 to the BCL2, SNAIL, and MMP9 promoters was detected by using chromatin immunoprecipitation. The biological effects on proliferation, migration, invasion, and epithelial-mesenchymal transition were assessed by using tetrazolium-based colorimetry, colony formation, EdU incorporation, flow cytometry, cell wound healing, and transwell assay.ResultsNF-κB p65 is highly expressed, while p-p65 Ser536 is not well expressed in intermediate and advanced HCC tissues. In vivo experiments demonstrated that a phosphorylation-mimetic mutant of RelA/p65 Ser536 (p65/S536D) prevents tumor progression and metastasis. In vitro experiments showed that p65/S536D inhibits proliferation, migration, and invasion. Mechanistically, RelA/p65 Ser536 phosphorylation inhibits NF-κB p65 nuclear translocation and reduces NF-κB p65 binding to the BCL2, SNAIL, and MMP9 promoters.ConclusionsRelA/p65 Ser536 phosphorylation was detrimental to NF-κB p65 entry into the nucleus and inhibited HCC progression and metastasis by reducing BCL2, SNAIL, and MMP9. The phosphorylation site of RelA/p65 Ser536 has excellent potential to be a promising target for NF-κB-targeted therapy in HCC.

Dataset Information

Hyperglycemic Conditions Promote Rac1-Mediated Serine536 Phosphorylation of p65 Subunit of NFκB (RelA) in Pancreatic Beta Cells.

Background/aims

Methods

Results

Conclusion

Publications

Hyperglycemic Conditions Promote Rac1-Mediated Serine536 Phosphorylation of p65 Subunit of NFκB (RelA) in Pancreatic Beta Cells.

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