BNIP3 Regulation of Intracellular Calcium

Necrotizing enterocolitis is the most common gastrointestinal emergency affecting low‐birth weight premature infants, with an incidence between 3% and 10% in infants less than 1500 grams, and mortality ranging from 15% to 30%. The pathogenesis of NEC is not clearly defined, but is likely a multifactorial disease wherein intestinal ischemia and hypoxia play fundamental roles. The hypoxia‐inducible death gene Bnip3 has been recently identified as a key regulator of NEC, yet the molecular mechanisms surrounding Bnip3‐induced enterocyte necrosis are not fully defined. Calcium‐mediated opening of the mitochondrial permeability transition pore (mPTP) has been identified as an important precursor event to necrotic cell death. Therefore, we tested the hypothesis that Bnip3 initiates calcium‐dependent PTP opening in cultured cells. We demonstrate that expression of Bnip3 increases mitochondrial calcium content, determined by Rhod2 staining, and that this effect is attenuated by expression of the inhibitory alternative splice variant Bnip3ΔEx3. Consistent with these findings, Bnip3 expression opens the mPTP, which was also prevented by Bnip3ΔEx3. Using organelle‐targeted calcium indicators, we found that Bnip3ΔEx3 did not inhibit Bnip3‐induced calcium release from the endoplasmic reticulum. However, Bnip3ΔEx3 expression re‐directed calcium release and promoted nuclear accumulation of calcium. These novel findings indicate that Bnip3ΔEx3 may protect cells from necrosis by promoting calcium‐dependent signaling pathways that converge on the nucleus, and provide a novel mechanism that may serve to protect enterocytes from hypoxic injury during NEC. Support or Funding Information Athabasca University Children's Hospital Research Institute of Manitoba Natural Sciences and Engineering Research Council of Canada