KRIT1 loss-of-function induces a chronic Nrf2-mediated adaptive homeostasis that sensitizes cells to oxidative stress: Implication for Cerebral Cavernous Malformation disease

Abstract

KRIT1 (CCM1) is a disease gene responsible for Cerebral Cavernous Malformations (CCM), a major cerebrovascular disease of proven genetic origin affecting 0.3-0.5% of the population. Previously, we demonstrated that KRIT1 loss-of-function is associated with altered redox homeostasis and abnormal activation of the redox-sensitive transcription factor c-Jun, which collectively result in pro-oxidative, pro-inflammatory and pro-angiogenic effects, suggesting a novel pathogenic mechanism for CCM disease and raising the possibility that KRIT1 loss-of-function exerts pleiotropic effects on multiple redox-sensitive mechanisms. To address this possibility, we investigated major redox-sensitive pathways and enzymatic systems that play critical roles in fundamental cytoprotective mechanisms of adaptive responses to oxidative stress, including the master Nrf2 antioxidant defense pathway and its downstream target Glyoxalase 1 (Glo1), a pivotal stress-responsive defense enzyme involved in cellular protection against glycative and oxidative stress through the metabolism of methylglyoxal (MG). This is a potent post-translational protein modifier that may either contribute to increased oxidative molecular damage and cellular susceptibility to apoptosis, or enhance the activity of major apoptosis-protective proteins, including heat shock proteins (Hsps), promoting cell survival. Experimental outcomes showed that KRIT1 loss-of-function induces a redox-sensitive sustained upregulation of Nrf2 and Glo1, and a drop in intracellular levels of MG-modified Hsp70 and Hsp27 proteins, leading to a chronic adaptive redox homeostasis that counteracts intrinsic oxidative stress but increases susceptibility to oxidative DNA damage and apoptosis, sensitizing cells to further oxidative challenges. While supporting and extending the pleiotropic functions of KRIT1, these findings shed new light on the mechanistic relationship between KRIT1 loss-of-function and enhanced cell predisposition to oxidative damage, thus providing valuable new insights into CCM pathogenesis and novel options for the development of preventive and therapeutic strategies.

Keywords: Adaptive redox homeostasis; Antioxidant defense; Argpyrimidine-modified heat-shock proteins; CCM1/KRIT1; Cerebral Cavernous Malformations; Cerebrovascular disease; Glyoxalase 1 (Glo1); Heme oxygenase-1 (HO-1); Nuclear factor erythroid 2-related factor 2 (Nrf2); Oxidative DNA damage and apoptosis; Oxidative stress; Redox signaling; c-Jun.

Graphical abstract

Fig. 1

KRIT1 modulates the expression of the anti-glycation enzyme Glyoxalase 1 (Glo1) and the formation of MG-derived argpyrimidine protein adducts. Wild type (K^+/+), KRIT1^-/- (K^-/-), and KRIT1^-/- re-expressing KRIT1 (K9/6) MEF cells grown to confluence under standard conditions were lysed and analyzed by Western blotting (a,d), qRT-PCR (b), and spectrophotometric enzymatic assay (c) as described in Materials and Methods. a) Representative Western blot and quantitative histogram of the relative KRIT1 and Glo1 protein expression levels. α-tubulin was used as internal loading control for WB normalization. The WB bands of Glo1 were quantified by densitometric analysis, and normalized optical density values were expressed as relative protein level units referred to average value obtained for K9/6 samples. b) Glo1 mRNA expression levels were analyzed in triplicate by qRT-PCR and normalized to the amount of an internal control transcript (GAPDH). Results are expressed as relative mRNA level units referred to the average value obtained for K9/6 cells, and represent the mean (± SD) of n ≥ 3 independent qRT-PCR experiments. c) Glo1 enzyme activity was measured in cytosolic extracts according to a spectrophotometric method monitoring the increase in absorbance at 240 nm due to the formation of S-D- lactoylglutathione. Glo1 activity is expressed in milliunits per mg of protein, where one milliunit is the amount of enzyme that catalyzes the formation of 1 nmol of S-D-lactoylglutathione per min under assay conditions. Results represent the mean (± SD) of n ≥ 3 independent experiments performed in triplicate. d) Representative WB and quantitative histogram of argpyrimidine (AP) adducts as detected using a specific mAb. α-tubulin was used as internal loading control for WB normalization. Western blots are representative of three separate experiments. **p ≤ 0.01 versus K9/6 cells, ***p ≤ 0.001 versus K9/6 cells. Notice that KRIT1 loss-of-function leads to a significant increase in Glo1 expression and activity, and a decrease in the intracellular levels of major AP adducts of 70 and 27 kDa.

Fig. 2

The upregulation of Glyoxalase 1 (Glo1) and the downregulation of argpyrimidine adducts occur also in human brain microvascular endothelial cells upon KRIT1 knockdown. Human brain microvascular endothelial cells (hBMEC) grown under standard conditions were mock transfected (CTR) or transfected with either KRIT1-targeting siRNA (siKRIT1) or a scrambled control (siCTR). Cells were then either lysed and analyzed by Western blotting (a,e), qRT-PCR (b), and spectrophotometric enzymatic assay (c), or treated with H₂DCF-DA for measurement of cellular levels of general reactive oxidative species by image-based cytometry (d), as described in Materials and Methods. a) Representative WB and quantitative histogram of the relative KRIT1 and Glo1 protein expression levels in si-CTR and si-KRIT1 cells. β-actin was used as internal loading control for WB normalization. The WB bands of Glo1 were quantified by densitometric analysis, and normalized optical density values were expressed as relative protein level units referred to the average value obtained for si-CTR samples. b) Glo1 mRNA expression levels were analyzed in triplicate by qRT-PCR and normalized to the amount of an internal control transcript (human β-actin). Results are expressed as relative mRNA level units referred to the average value obtained for control cells (CTR), and represent the mean (± SD) of n ≥ 3 independent qRT-PCR experiments. c) Glo1 enzyme activity was measured in cytosolic extracts of si-CTR and si-KRIT1 cells according to a spectrophotometric method monitoring the increase in absorbance at 240 nm due to the formation of S-D-lactoylglutathione. Glo1 activity is expressed in milliunits per mg of protein, where one milliunit is the amount of enzyme that catalyzes the formation of 1 nmol of S-D-lactoylglutathione per min under assay conditions. Results represent the mean (± SD) of n ≥ 3 independent experiments performed in triplicate. d) Measurement of cellular levels of general reactive oxidative species. si-CTR and si-KRIT1 endothelial cells were left untreated or treated with H₂DCF-DA, and DCF fluorescence intensity was analyzed by a Tali® Image-Based Cytometer. The representative cytometer profile shows the increase in DCF fluorescence intensity in si-KRIT1 cells as compared to the control (si-CTR). e) Representative WB and quantitative histogram of argpyrimidine (AP) adducts in si-CTR and si-KRIT1 endothelial cells as detected using a specific mAb. β-actin was used as internal loading control for WB normalization. Western blots are representative of three separate experiments. **p ≤ 0.01 compared to control (CTR or si-CTR) cells. Notice that KRIT1 knockdown in human brain microvascular endothelial cells leads to a significant increase in Glo1 expression and activity, and a decrease in the intracellular levels of major AP adducts of 70 and 27 kDa.

Fig. 3

The upregulation of Glyoxalase 1 (Glo1) and the decrease of argpyrimidine levels caused by KRIT1 loss are redox-dependent. Wild type (K^+/+), KRIT1^-/- (K^-/-), and KRIT1^-/- re-expressing KRIT1 (K9/6) MEF cells grown to confluence under standard conditions were either mock-pretreated or pretreated with the ROS scavenger Tiron (Tir). Cells were then either treated with H₂DCF-DA for measurement of cellular levels of general reactive oxidative species by image-based cytometry (a), or lysed and analyzed by qRT-PCR (b), Western blotting (c,e), and spectrophotometric enzymatic assay (d) as described in Materials and Methods. a) Measurement of cellular levels of general reactive oxidative species. Cells mock-pretreated or pretreated with Tiron (Tir) were left untreated or treated with H₂DCF-DA, and DCF fluorescence intensity was analyzed by a Tali® Image-Based Cytometer. Fluorescence readings were expressed as relative units of DCF fluorescence intensity referred to the average value obtained for K9/6 cells. b-d) Glo1 mRNA, protein and specific activity levels in cells mock-pretreated (-) or pretreated (+) with Tiron (Tir) were analyzed and expressed as described in Fig. 1a-c. e-f) Representative WB (e) and quantitative histogram (f) of intracellular levels of argpyrimidine (AP) adducts in cells mock-pretreated (-) or pretreated (+) with Tiron (Tir). WB analysis and densitometric quantification of WB bands were performed as described in Fig. 1d. Results are representative of n ≥ 3 independent experiments, and histograms represent quantifications expressed as means ± SD. **p ≤ 0.01 versus K9/6 cells; ***p ≤ 0.001 versus K9/6 cells. Notice that the normalization of intracellular ROS levels induced by cell pre-treatment with the antioxidant Tiron was accompanied by the rescue of Glo1 upregulation to near-physiological levels, as well as by increased levels of AP adducts.

Fig. 4

KRIT1 loss-dependent upregulation of Glo1 is part of a cell adaptive response to oxidative stress involving the master redox-sensitive transcriptional regulator Nrf2. Wild type (K^+/+), KRIT1^-/- (K^-/-), and KRIT1^-/- re-expressing KRIT1 (K9/6) MEF cells grown to confluence under standard conditions were left untreated (a) or either mock-pretreated (-) or pretreated (+) with the ROS scavenger Tiron (b,c). Nuclear and cytoplasmic fractions (a,b) or total cell extracts (c) were then obtained and analyzed by Western blotting for the indicated proteins as described in Section 2. Nuclear levels of p-c-Jun were used as a control of redox-dependent effect of KRIT1 loss-of-function . Lamin-β1 and α-tubulin were used as internal loading controls for WB normalization of nuclear and total/cytoplasmic proteins, respectively. (a,b,c) The histograms below their respective Western blots represent the mean (± SD) of the densitometric quantification of three independent experiments. *p < 0.05 versus K9/6 cells, **p ≤ 0.01 versus K9/6 cells, ***p ≤ 0.001 versus K9/6 cells. Notice that the upregulation of c-Jun and p-c-Jun nuclear levels induced by KRIT1 loss-of-function is paralleled by a marked nuclear accumulation of Nrf2 (a,b) and the upregulation of its downstream effector HO-1 (c), both of which are significantly reverted by cell treatment with the ROS scavenger Tiron (b,c).

Fig. 5

Defective autophagy and JNK activation associated with KRIT1 loss-of-function contribute to the sustained upregulation of Nrf2 and its downstream effectors HO-1 and Glo1. Wild type (K^+/+), KRIT1^-/- (K^-/-), and KRIT1^-/- re-expressing KRIT1 (K9/6) MEF cells grown to confluence under standard conditions were either mock-pretreated (-) or pretreated (+) with the JNK inhibitor SP600125 (a-d), or the autophagy inducer Rapamycin (e-h). Nuclear and cytoplasmic fractions or total cell extracts were then obtained and analyzed by Western blotting (a-c, e-g) and spectrophotometric enzymatic assay (d,h) for the indicated proteins, as described in Materials and Methods and Fig. 1a-c. Nuclear levels of p-c-Jun were used as a control of redox-dependent effect of KRIT1 loss-of-function. Lamin-β1 and α-tubulin were used as internal loading controls for WB normalization of nuclear and total/cytoplasmic proteins, respectively. Histograms represent the mean (± SD) of n ≥ 3 independent experiments performed in triplicate. **p ≤ 0.01 and ***p ≤ 0.001 versus K9/6 cells; °p ≤ 0.05 and °°p ≤ 0.01 versus mock-pretreated (-) cells. Notice that the redox-sensitive nuclear accumulation of Nrf2 and upregulation of its downstream effectors HO-1 and Glo1 induced by KRIT1 loss-of-function were significantly reverted by cell treatment with either the JNK inhibitor SP600125 (a-d) or the autophagy inducer Rapamycin (e-h).

Fig. 6

Nuclear accumulation of Nrf2 in endothelial cells lining human CCM lesions. Nrf2 immunohistochemical (IHC) staining in histological sections of human CCM surgical specimens deriving from a KRIT1 loss-of-function mutation carrier. a,b) Hematoxylin/eosin (H&E) (a) and Nrf2 IHC (b) staining of a representative CCM surgical sample containing normal vessels in a perilesional area (left side, box c), which served as an internal negative control, and a large CCM lesion lined by a thin endothelium (right side and box d). c,d) Magnifications of the two representative areas indicated in panel (b). Notice that endothelial cells lining the lumen of normal vessels are negative for Nrf2 nuclear staining (panel c and magnified inset), while a significant positive Nrf2 nuclear staining is evident in endothelial cells lining the lumen of CCM lesions (black arrows, panel d and magnified inset). Scale bars: a,b 200 µm; c,d 50 µm, insets 30 µm.

Fig. 7

KRIT1 loss-of-function is associated with a redox-sensitive downregulation of AP-modified heat shock proteins Hsp27 and Hsp70, and a redox-sensitive increase in cell susceptibility to oxidative DNA damage and apoptosis. a) Identification of the 70 kDa and 27 kDa AP-modified and redox-sensitive proteins downregulated in K^-/- versus K^+/+ and K9/6 MEF cells (Figs. 1d and 3e). AP-modified proteins were purified from the lysate of K9/6 cells by immunoaffinity chromatography with an anti-AP mAb. Chromatography fractions containing the eluted 70 kDa and 27 kDa AP-modified proteins were identified by SDS-PAGE and staining with Coomassie Blue. The isolated 70 kDa and 27 kDa AP-modified proteins were then digested and resolved as individual peptides by HPLC. The respective internal peptides were identified as mouse heat-shock protein 70 (Hsp70) and 27 (Hsp27) upon comparison with standard sequencing databases in the public domain (BLAST). b,c) Wild type (K^+/+), KRIT1^-/- (K^-/-), and KRIT1^-/- re-expressing KRIT1 (K9/6) MEF cells were either mock-treated or treated with the ROS scavenger Tiron, and oxidative DNA damage (b) and apoptotic cell death (c) were evaluated by ELISA-based quantification of abasic sites in DNA and TUNEL assay, respectively. Histograms represent the mean (± SD) of n ≥ 3 independent experiments performed in triplicate. **p ≤ 0.01 and ***p ≤ 0.001 versus K9/6 cells; °p ≤ 0.05 and °°p ≤ 0.01 versus untreated cells. Notice that cell treatment with Tiron rescued the enhanced background levels of DNA abasic sites (b) and apoptosis (c) induced by KRIT1 loss-of-function (K^-/- cells) near to levels observed in K^+/+ cells.

Fig. 8

The increased susceptibility to apoptotic cell death induced by KRIT1 loss-of-function occurs through the intrinsic, mitochondria-mediated pathway. Wild type (K^+/+), KRIT1^-/- (K^-/-), and KRIT1^-/- re-expressing KRIT1 (K9/6) MEF cells were grown to confluence under standard conditions. Total cell extracts (a) or mitochondria and cytosolic fractions (b) were then obtained, and proteins involved in the intrinsic, mitochondria-dependent apoptotic pathway, including the anti-apoptotic Bcl-2 and pro-apoptotic Bax proteins (a), and Cytochrome c (Cyt c) and Caspase-3 (Casp-3) (b), were analyzed by Western blotting as described in Materials and Methods. Cox IV and α-tubulin were used as internal loading controls for WB normalization of mitochondria and total/cytoplasmic proteins, respectively. Pro-Casp-3, intact protein; Casp-3, active fragment. A representative immunoblot is shown for each experiment. Histograms alongside their respective Western blots represent the mean (± SD) of the densitometric quantification of three independent experiments. **p ≤ 0.01 and ***p ≤ 0.001 versus K9/6 cells. Notice that the basal levels of Bcl-2 and Bax proteins in K^-/- cells were significantly down-regulated and up-regulated, respectively, as compared with K^+/+ and K9/6 cells (a). In addition, a significant increase in Cyt c release into the cytosol and activation of Casp-3 was also evident in K^-/- cells (b).

Fig. 9

Schematic models representing adaptive redox responses and cellular states associated with KRIT1 loss-of-function. a) Redox-sensitive pathways modulated by KRIT1 functions. KRIT1 loss-of-function causes a persistent activation of the major redox-sensitive transcription factors c-Jun and Nrf2 and consequent upregulation of downstream targets, including cycloxygenase-2 (COX-2), heme oxygenase-1 (HO-1) and glyoxalase 1 (GLO1). While the c-Jun/COX-2 axis promotes pro-oxidant and pro-inflammatory effects, the Nrf2/HO-1 and Nrf2/GLO1 pathways mediate adaptive antioxidant responses that counteract these effects by limiting ROS and MG intracellular accumulation, thus contributing to reduce a vicious cycle of oxidative stress and providing an adaptive defense for long-term cell survival. However, this sustained adaptive redox homeostasis occurs at the expense of other cytoprotective mechanisms, including the MG-dependent formation of cytoprotective AP-Hsp27 protein adducts, leading to enhanced cell susceptibility to oxidative DNA damage and apoptosis, and sensitizing cells to additional stressful insults. b) Spectrum of cellular states associated with KRIT1 loss-of-function. Cellular stress and defense responses associated with KRIT1 dysfunctions can be viewed as distinct but overlapping components of a spectrum of cellular states that ranges from basal homeostatic state, to adaptive stress response, insufficient defense, and defense failure, each of which can be defined in terms of the maintenance of molecular and cellular functions within an acceptable dynamic range. In turn, differences in expression and functional levels of stress-responsive proteins and adaptive defense mechanisms acting within the range of each cellular state may be influenced by genetic variation, resulting in inter-individual differences in adaptive stress responses and susceptibility to disease onset and progression. See text for details.