Endoplasmic Reticulum to Mitochondria Ca2+ Signaling Inhibits Factor VIII Secretion and Mediates Oxidative Stress and Apoptosis Upon Factor VIII Expression
Authors: Malhotra Jyoti , Xiong Mon , Miao Hongzhi , Pipe Steven , Kaufman Randal J. 
Published: October 2019

Abstract
Abstract 2212
Factor VIII (FVIII) is the clotting factor that is deficient in hemophilia A, an × chromosome-linked bleeding disorder. Newly synthesized FVIII is subject to misfolding in the endoplasmic reticulum (ER) lumen, activates the unfolded protein response (UPR), causes oxidative stress and induces apoptosis in vitro and in vivo in mice (PNAS, 105:1825-30, 2008). We have recently demonstrated that FVIII expression causes mitochondrial inner membrane depolarization. Although accumulation of misfolded protein within the ER is a central event in cell death, the relationship between protein misfolding in the ER, ER Ca2+ release, oxidative stress and mitochondrial dysfunction has not been explored. To elucidate the relationship between protein misfolding in the ER and mitochondrial function, we disrupted Ca2+ signaling in cells that were induced to express FVIII bothin vitro and in vivo in mice. Intracellular Ca2+ chelation, IP3 receptor antagonist, or Bcl2 overexpression (which stabilizes the pool of ER Ca2+), all prevented mitochondrial membrane depolarization upon FVIII expression. The requirement for Ca2+ uptake into the mitochondrial matrix was studied by interfering with cyclophilin D (CypD), a cis-trans prolyl isomerase located in the mitochondrial matrix that is required for Ca2+ uptake into mitochondria. Cyclosporin A (CsA) inhibits cis-trans prolyl isomerases and prevents Ca2+ uptake into the mitochondria upon ER stress. CsA addition to the media of CHO cells that were induced to express FVIII reduced ER stress, mitochondrial dysfunction, oxidative stress, and apoptosis. In addition, CsA increased the secretion of FVIII from the CHO cells. For in vivo studies, CsA was administered by intraperitoneal injection (10 mg/kg-1/day) into mice for three days prior to tail vein injection of FVIII DNA expression vectors for liver delivery. In addition, FVIII expression was analyzed in CypD-/- mice. Hepatocyte responses and FVIII secretion were studied at 24 hr after DNA injection. Either CsA treatment or CypD deletion prevented UPR activation, oxidative stress and apoptosis associated with wild-type or B domain-deleted (BDD) FVIII expression. Furthermore, either CsA treatment or CypD deletion increased secretion of BDD into the plasma, where these interventions had no effect upon expression of 226/N6 FVIII, a molecule that is efficiently secreted from the cell (Blood,103:3412-9). These studies indicate that misfolded FVIII accumulation within the ER induces Ca2+ leak and subsequent uptake into mitochondria that leads to membrane depolarization and oxidative stress. Cyclophilin D may therefore be a critical mediator of the oxidative stress and apoptotic response that occurs upon protein misfolding in the ER. These findings may provide an avenue to novel therapies to treat and/or prevent hemophilia A.

References:
Malhotra JD, Miao H, Zhang K, Wolfson A, Pennathur S, Pipe SW, Kaufman RJ. (2008) Antioxidants reduce endoplasmic reticulum stress and improve protein secretion. Proc Natl Acad Sci 105: 18525-30
Malhotra JD, Kaufman RJ. (2007). Endoplasmic reticulum stress and oxidative stress: a vicious cycle and double edged sword? Antioxid Redox Signal 9(12):2277-93
Miao HZ, Srichainan N, Palmer L, Kucab P, Cunningham MA, Kaufman RJ and Pipe SW(2004). Bioengineering of coagulation factor VIII for improved secretion Blood, 103:3412-9

Disclosures:
No relevant conflicts of interest to declare.