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CARD9 Accelerates Rac1‐p38MAPK Axis in Pancreatic‐β Cells Under the Duress of Chronic Hyperglycemia
Author(s) -
Gamage Suhadinie,
Kowluru Anjaneyulu
Publication year - 2021
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2021.35.s1.05202
Subject(s) - rac1 , microbiology and biotechnology , transfection , signal transduction , downregulation and upregulation , chemistry , biology , cell culture , biochemistry , gene , genetics
Background‐ Caspase recruitment domain containing protein 9 (CARD9) is a scaffolding protein and a major signal transducer in innate and adaptive immune responses. Published evidence, albeit limited, suggests novel roles for CARD9‐RhoGDIβ‐Rac1 signaling module in the protection of host from infection via promoting optimal macrophage function. Recent evidence in the pancreatic β‐cell suggests novel roles for sustained activation of Rac1‐p38MAPK signaling module in the onset of cell dysregulation following prolonged exposure to high glucose (HG; glucotoxicity). However, putative roles of CARD9‐RhoGDI‐β module in the activation of HG‐induced CARD9‐RhoGDI‐β in stress kinase (p38MAPK and JNK1/2) activation under metabolic stress have not been verified in the beta cell. The current set of investigations were aimed at assessing potential regulatory roles of CARD9‐Rac1 in HG‐induced stress kinase (p38MAPK and JNK1/2) in insulin‐secreting INS‐1 832/13 cells exposed to glucotoxic conditions. Methods and Materials‐ Western blotting was used to demonstrate protein expression. CARD9 expression was suppressed using Horizon/ Dharmacon siRNA and transfection reagents. Membrane and cytosolic fractions were isolated using Membrane Extraction Kit from ThermoFisher Scientific. The degree of activation of Rac1 was determined by pull‐down assay from Cytoskeleton Inc. Co‐immunoprecipitation studies were preformed using reagents and protocols from ThermoFisher Scientific. Results ‐ CARD9 expression was significantly increased (1.6‐fold) in INS‐1 832/13 cells following exposure to glucotoxic conditions (20mM; 24 hrs). Such effects of glucose were not due to its osmotic effects since mannitol (20mM; 24 hrs; osmotic control) failed to induce CARD9 expression. Transfection of INS‐1 832/13 cells with siRNA‐CARD9 resulted in 70% suppression of its expression. Under these conditions, a significant inhibition (~ 50%) of HG‐induced activation of Rac1 was seen in CARD9‐depleted cells. Furthermore, a significant inhibition (~40%) of HG‐induced activation of p38MAPK, but not JNK1/2, was observed following siRNA‐mediated knockdown of CARD9. Lastly, co‐immunoprecipitation studies revealed a significant increase (~40%) in interaction between CARD9 and RhoGDI‐β, and a significant decrease (~50%) in the interaction between RhoGDI‐β and Rac1 in INS‐1 832/13 cells under glucotoxic conditions, thus suggesting potential alterations in the cross‐talk between these signaling proteins under in the beta‐cell under metabolic stress conditions. Conclusions‐ Based on these findings we conclude that CARD9 mediates activation of Rac1‐p38MAPK signaling pathway in pancreatic beta cells exposed to chronic glucotoxic conditions. Our data also suggest significant alterations in association between CARD9‐RhoGDIβ‐Rac1 that might be conducive to release of Rac1 from RhoGDI‐β leading to its activation under these conditions. Studies are underway to further assess the regulatory roles of CARD9 in the onset of metabolic dysfunction of the beta cell under metabolic stress.