RIPK3

RIPK3

A gene on chromosome 14q11.2 that encodes a serine/threonine/ tyrosine kinase essential for cellular necroptosis in response to the TNF-alpha family of death-inducing cytokines. RIPK3 interacts with and phosphorylates RIPK1 to form a necrosis-inducing complex, binding to and enhancing the activity of GLUL, GLUD1 and PYGL (metabolic enzymes involved in the tricarboxylic acid cycle and oxidative phosphorylation), thereby increasing reactive oxygen species production.
Mentioned in ?
References in periodicals archive ?
In particular, a kinase complex consisted of the RIPK1 and RIPK3 is a central step in the programmed necrotic cell death [12].
In order to verify our hypothesis, we sought to examine the influence of PA on the expression of RIPK1 and RIPK3 in NCMs and in H9c2 cells in this study.
Membranes were subsequently blocked with 5% skim milk in Tris-buffered saline and Tween 20 (TBST) solution for 2h and were incubated with primary antibodies at 4[degrees]C overnight: RIPK3 (Cell Signaling, number 14401), RIPK1 (Cell Signaling, number 3493), GRP78 (Cell Signaling, number 3183), Phospho-mTOR (Ser2481) (Cell Signaling, number 2974), mTOR (Cell Signaling, number 2983), Calreticulin (Cell Signaling, number 12238), AKT (Cell Signaling, number 4685), and Phospho-AKT (Ser473) (Cell Signaling, number 4058).
Images were collected using an Eclipse TE2000-U fluorescence microscope system (Nikon, Japan) and analyzed with ImageJ software (NIH, USA) to semiquantitatively determine the expression of RIPK1 and RIPK3.
After treatment with PA or OA for 24 h, the gene expression of both RIPK1 and RIPK3 in primary rat NCMs was upregulated only in the PA stimulation group compared with controls (without any treatment and with OA treatment), while Nec-1, a specific inhibitor of RIPK1, significantly decreased the PA-induced gene expression of RIPK1 and RIPK3 (Figure 2(a)).
The expression of RIPK1/RIPK3 protein obtained by immunofluorescence staining also indicated that growth-arrested H9c2 cells presented a slight RIPK1 and RIPK3 staining, while treatment with PA for 24 h significantly increased cytoplasmic RIPK1/RIPK3 staining (Figures 2(c) and 2(d)).
To further investigate the role of necroptosis in PA-induced cardiomyocyte hypertrophy, we also used a gene silencing approach to specifically knockdown RIPK1 and RIPK3 expression.
When the ER stress in H9c2 cells was suppressed, we observed that the ER stress markers were downregulated (Figure 3(b)) and the gene expression of RIPK1 and RIPK3 was also decreased compared to PA stimulation group (Figure 3(c)).
We have detected an increased expression of RIPK1 and RIPK3 in PA-treated cardiomyocytes, implying activation of necroptosis [15, 22, 29,30].
Moreover, Nec-1 pretreatment suppressed expression of both RIPK1 and RIPK3, demonstrating that the kinase complex which consisted of RIPK1 and RIPK3 might be essential in PA-induced cardiomyocyte necroptosis.
c) Immunofluorescence for RIPK1 and RIPK3 in H9c2 cells.