oa Loss Of Calreticulin Function Decreases NFKB Activity By Stabilizing IKB Protein

Background: Transcription factor NFKB is activated by several processes including inflammation, endoplasmic-reticulum (ER) stress, increased Akt signaling and enhanced proteasomal degradation. Calreticulin is an ER Ca²⁺ binding chaperone, which regulates many cellular processes. Previously, we have shown that loss of calreticulin function results in the activation of ER stress that is accompanied by a significant increase in the proteasome activity. These changes increase the resistance of calreticulin deficient cells to apoptosis. A role for calreticulin has also been described in the regulation of immune response.

Objectives: To examine the role of calreticulin in the activation of NFKB signaling leading to enhanced resistance to apoptosis of these cells.

Methods:: Wild type and calreticulin deficient cells were used for measurement of transcriptional activity of NFKB. Cells were co-transfected with of NFKB reporter and-gal reporter plasmids followed by reporter gene assays. Western blot analysis was utilized to examine changes in protein expression.

Results: Our data illustrate a significant decrease in the basal transcriptional activity of NFKB upon loss of calreticulin function. Furthermore, treatment with lipopolysaccharide increased the transcriptional activity of NFKB in both the wild type and calreticulin deficient cells. However, the transcriptional activity of NFKB was still significantly lower in the calreticulin deficient cells as compared to the wild type cells. Our data also showed that the reduced NFKB activity in calreticulin deficient cells is not due to decreased p65 or p50 protein levels. To determine the mechanism of decreased NFKB activity we examined changes in IKB protein stability. Our data showed a significant increase in the IKB protein level due to decreased level of phosphorylated IKB protein. Furthermore, we illustrated that loss of calreticulin function resulted in increased protein phosphatase2A activity that was abolished by Okadaic acid treatment. Inhibition of IKB de-phosphorylation decreased its ubiquitination and proteasomal degradation.

Conclusion: Our data suggests that the reduced transcriptional activity of NFKB upon loss of calreticulin function is mediated via stabilization of IKB protein. To our knowledge, this is the first report on the role of calreticulin in the regulation of NFKB function.