Depletion of cyclophilins B and C leads to dysregulation of endoplasmic reticulum redox homeostasis

Abstract

Protein folding within the endoplasmic reticulum is assisted by molecular chaperones and folding catalysts that include members of the protein-disulfide isomerase and peptidyl-prolyl isomerase families. In this report, we examined the contributions of the cyclophilin subset of peptidyl-prolyl isomerases to protein folding and identified cyclophilin C as an endoplasmic reticulum (ER) cyclophilin in addition to cyclophilin B. Using albumin and transferrin as models of cis-proline-containing proteins in human hepatoma cells, we found that combined knockdown of cyclophilins B and C delayed transferrin secretion but surprisingly resulted in more efficient oxidative folding and secretion of albumin. Examination of the oxidation status of ER protein-disulfide isomerase family members revealed a shift to a more oxidized state. This was accompanied by a >5-fold elevation in the ratio of oxidized to total glutathione. This "hyperoxidation" phenotype could be duplicated by incubating cells with the cyclophilin inhibitor cyclosporine A, a treatment that triggered efficient ER depletion of cyclophilins B and C by inducing their secretion to the medium. To identify the pathway responsible for ER hyperoxidation, we individually depleted several enzymes that are known or suspected to deliver oxidizing equivalents to the ER: Ero1αβ, VKOR, PRDX4, or QSOX1. Remarkably, none of these enzymes contributed to the elevated oxidized to total glutathione ratio induced by cyclosporine A treatment. These findings establish cyclophilin C as an ER cyclophilin, demonstrate the novel involvement of cyclophilins B and C in ER redox homeostasis, and suggest the existence of an additional ER oxidative pathway that is modulated by ER cyclophilins.

Keywords: Cyclosporine A; Disulfide; Endoplasmic Reticulum (ER); Oxidase; Peptidyl-Prolyl Isomerase; Protein Folding; Protein-disulfide Isomerases; Redox Regulation.

FIGURE 1.

CypB and CypC expression profile and localization in HepG2 cells. A, immunoblotting was used to detect CypB and CypC in HepG2 cells. The specificity of the anti-PPIs antibodies for CypB and CypC was established by comparison of the band profiles of either CypB or CypC specific knockdowns (KD) versus a mock control. B, the N-glycosylation status of CypC was tested by PNGase F (PNG) digestion of cell lysates. Prion protein (PrP) and CypB were used as glycosylated and unglycosylated protein controls, respectively. C, to test if unglycosylated CypC is cytosolically localized, cells were treated for 30 min with the indicated concentrations of digitonin to selectively permeabilize the cell membrane and release cytosolic proteins. The cells were then lysed and analyzed by immunoblotting. CypA and CypB were used as cytosolic and ER-residing protein controls, respectively. D, glycosylated CypC possesses an endoglycosidase H-sensitive glycan. Cell lysates were digested with Endo H and PNGase F and analyzed by immunoblotting. MHC class I was used as a control glycoprotein that traverses the entire secretory pathway and GAPDH served as a loading control. E, UPR activation was tested in cells treated with CsA (5 or 20 μg/ml), FK506 (20 μg/ml), or thapsigargin (Tg; 3 μm) using an Xbp1 splicing assay. mRNA from the treated cells was isolated and tested for the splicing status of Xbp1 mRNA, unspliced (Xbp1^u) or spliced Xbp1 (Xbp1^s) indicate negative or positive UPR activation, respectively. F, the efficiency of CypB and CypC retention within the ER was assessed under different conditions. Cells were treated with CsA (5 or 20 μg/ml), FK506 (20 μg/ml), or Tg (3 μm) overnight. Cell lysate and media samples were tested by immunoblot for the relative levels of CypB and CypC as well as the ER luminal protein PDI. G, BFA blocks CsA-induced secretion of CypB and CypC. HepG2 cells were treated with 5 μg/ml of CsA and/or 0.5 μg/ml of BFA overnight and the cell lysates were tested for CypB and CypC retention. Dimethyl sulfoxide (DMSO) was used as a vehicle control.

FIGURE 2.

Single knockdown of either CypB or CypC results in compensatory up-regulation of the remaining ER-residing cyclophilin in HepG2 cells. A, CypB or CypC knockdown (KD) was performed using specific siRNA and compared with the mock knockdown performed with negative control siRNA. Knockdown efficiency was assessed by immunoblotting. CypA served as a knockdown specificity control, the ER Hsp70 BiP was used as a marker of UPR induction and GAPDH was a loading control. B, band intensities from the CypB and CypC immunoblots were quantified by densitometry, normalized to GAPDH levels, and expressed as a percentage change in expression as a function of the indicated knockdown condition (n = 3, ±S.D.). *, indicates significant difference from control (p < 0.05). C, UPR activation was tested on day 6 in HepG2 cells depleted for either CypB or CypC using the Xbp1 splicing assay. HepG2 cells treated with 3 mm DTT for 2 h were used as a positive control for UPR induction.

FIGURE 3.

Combined knockdown of CypB and CypC improves oxidative folding and secretion of albumin in HepG2 cells. A, combined CypB and CypC knockdown (CypB/C) was performed using specific siRNAs and compared with the mock knockdown (KD) performed using negative control siRNA. The efficiency of the knockdown was assessed by immunoblotting. GAPDH was used as a loading control and BiP served as an indicator of UPR activation. B, UPR activation was additionally tested on day 6 in double knockdown cells using the Xbp1 splicing assay. HepG2 cells treated with 3 mm DTT for 2 h were used as a positive control. C, kinetics of albumin disulfide formation and secretion. Cells depleted of both CypB and CypC were radiolabeled with [³⁵S]Met for 10 min and then chased with unlabeled Met for the indicated times. NEM (20 mm) was added to alkylate free thiols and then albumin was immunoisolated from cell lysates and media. The samples were analyzed by SDS-PAGE gel under reducing (+DTT) or non-reducing (−DTT) conditions. The mobilities of reduced (R), partially oxidized (PO), and oxidized (O) forms of albumin are indicated. D, kinetics of albumin oxidation presented as the amount of fully oxidized protein in cells and media as a percentage of total radiolabeled albumin at each chase time (n = 3, ±S.D.). E, secretion kinetics of albumin presented as a percentage of total albumin signal at each chase time (n = 3, ±S.D.). F, the kinetics of transferrin oxidation were determined as described for albumin above and are presented as the amount of fully oxidized protein as a percentage of all forms of transferrin at each chase time (n = 3, ±S.D.). G, secretion kinetics of transferrin presented as a percentage of total transferrin signal at each chase time (n = 3, ±S.D.). Data are representative of two independent experiments.

FIGURE 4.

CsA treatment or double knockdown of CypB and CypC results in increased oxidation of PDIs and an elevated GSSG:GS ratio in HepG2 cells. A, single and double knockdown (KD) efficiencies of CypB and CypC were assessed by immunoblotting with GAPDH serving as a loading control. B, oxidation state of PDIs under single or double depletion of CypB and CypC. Before lysis, cells were incubated for 10 min with 20 mm NEM in ice-cold PBS to alkylate free protein thiols. Cells were then lysed and treated with TCEP to reduce disulfide bonds followed by treatment with the bulkier alkylating agent AMS to modify newly exposed thiol groups. The lysates were analyzed by SDS-PAGE and immunoblotted for ERp72, ERp57, and P5. The upward mobility shifts indicated by arrows represent an increase in the proportion of PDI members with oxidized active sites. As controls, before the NEM incubation, cells were treated for 5 min with either 5 mm DTT or 5 mm diamide (diam.) to reduce or oxidize PDIs, respectively. C, assessment of P5 oxidation state upon treatment with different PPI inhibitors. HepG2 cells were treated overnight with CsA, FK506, rapamycin (Rapa) or the UPR inducer Tm at the indicated concentrations. D, UPR activation was assessed by Xbp1 splicing assay under the same conditions as in panel C. E, assessment of the oxidation state of ERp72 and ERp57 upon overnight treatment with CsA (5 μg/ml), FK506 (20 μg/ml), or Tm (5 μg/ml). F, evaluation of the oxidation state of cellular glutathione. Cells depleted of CypB and CypC as well as mock knockdown cells were either left untreated or treated overnight with CsA (5 μg/ml). The cells were then lysed and the ratio of GSSG to total GS (GSSG + GSH) was determined (n = 3, ±S.D.). *, indicates significant difference between mock and CypB/C combined knockdown (p < 0.05)

FIGURE 5.

Known ER oxidative enzymes do not contribute to the hyperoxidation arising from CsA treatment. A, knockdown (KD) efficiency of ER oxidative enzymes. Knockdowns in HepG2 cells included double knockdown of Ero1α and Ero1β or single knockdown of PRDX4, secreted QSOX1, or VKOR. The efficiency of the knockdowns was assessed by immunoblotting with GAPDH or secreted albumin (in the case of QSOX1) serving as loading control. B, the indicated enzymes were depleted and then cells were incubated overnight in the absence or presence of CsA (5 μg/ml). The ratio of GSSG to total GS (GSSG + GSH) in cell lysates was assessed (n = 3, ±S.D.).