KRIT1 loss of function causes a ROS-dependent upregulation of c-Jun

Abstract

Loss-of-function mutations in the KRIT1 gene (CCM1) have been associated with the pathogenesis of cerebral cavernous malformations (CCM), a major cerebrovascular disease. However, KRIT1 functions and CCM pathogenetic mechanisms remain incompletely understood. Indeed, recent experiments in animal models have clearly demonstrated that the homozygous loss of KRIT1 is not sufficient to induce CCM lesions, suggesting that additional factors are necessary to cause CCM disease. Previously, we found that KRIT1 is involved in the maintenance of the intracellular reactive oxygen species (ROS) homeostasis to prevent ROS-induced cellular dysfunctions, including a reduced ability to maintain a quiescent state. Here, we show that KRIT1 loss of function leads to enhanced expression and phosphorylation of the redox-sensitive transcription factor c-Jun, as well as induction of its downstream target COX-2, in both cellular models and human CCM tissues. Furthermore, we demonstrate that c-Jun upregulation can be reversed by either KRIT1 re-expression or ROS scavenging, whereas KRIT1 overexpression prevents forced upregulation of c-Jun induced by oxidative stimuli. Taken together with the reported role of c-Jun in vascular dysfunctions triggered by oxidative stress, our findings shed new light on the molecular mechanisms underlying KRIT1 function and CCM pathogenesis.

Keywords: COX-2; Cellular antioxidant defense mechanisms; Cerebral cavernous malformations; Free radicals; KRIT1; Reactive oxygen species; c-Jun.

Graphical abstract

Fig. 1

KRIT1 regulates c-Jun expression — KRIT1 knockout and re-expression approach. KRIT1^−/− (K^−/−) and wild-type (K^+/+) MEFs and KRIT1^−/− MEFs re-expressing KRIT1 (K9/6) were grown to confluence under standard conditions and analyzed by (A) RT-qPCR, (B, C) Western blotting, and (D) immunofluorescence as described under Material and methods. (A) RT-qPCR analysis of c-Jun mRNA expression levels. The amount of each target mRNA expressed in a sample was analyzed in triplicate using appropriate TaqMan gene expression assays (Roche) and normalized to the amounts of internal normalization control transcripts (18S rRNA). Results are expressed as relative mRNA level units referred to the average value obtained for the K^−/− samples and represent the mean (±SD) of n ≥ 3 independent RT-qPCR experiments. ***P≤0.001 versus K^−/− cells. Notice that c-Jun mRNA levels are significantly higher in K^−/− compared to K^+/+ and K9/6 MEFs. (B) Representative Western blot analysis of the relative c-Jun, phospho-c-Jun, and KRIT1 expression levels. Tubulin (α-Tub) was used as loading control. Notice that both c-Jun and phospho-c-Jun levels are significantly higher in K^−/− compared to K^+/+ and K9/6 MEFs. An inverse correlation between c-Jun/phospho-c-Jun and KRIT1 protein levels is also evident. (C) Histograms showing quantitative results of Western blot analysis of the relative c-Jun, phospho-c-Jun, and KRIT1 expression levels. Optical density values are expressed as relative protein level units referred to the average value obtained for the K^−/− samples and represent the mean (±SD) of n ≥ 3 independent Western blotting experiments. **P ≤ 0.01 and ***P ≤ 0.001 versus K^−/− cells. Notice that differences in phospho-c-Jun levels are correlated with differences in total c-Jun levels. (D) Confocal microscopy analysis of phospho-c-Jun levels and subcellular localization in K^−/− and K9/6 MEF cells. Phospho-c-Jun and nuclei were visualized with anti-phospho-c-Jun mAb coupled to Alexa Fluor 488 secondary antibody and DAPI dye, respectively. Notice that phospho-c-Jun is correctly localized to the nucleus in cells lacking KRIT1 (K^−/−) and shows enhanced levels compared to K9/6. Scale bar, 15 μm.

Fig. 2

KRIT1 regulates c-Jun expression — KRIT1 downregulation (siRNA) approach. (A–E) HeLa cells and (F) HUVECs were mock-transfected or transfected with either a KRIT1-specific siRNA (siK655 or siK469) or a negative control siRNA (siNC). 48 h posttransfection, cells were lysed and analyzed by (A, B) RT-qPCR and (C, D, F) Western blotting to assess c-Jun and KRIT1 mRNA and protein levels, respectively. 18S rRNA and tubulin (α-Tub) were used as endogenous controls for RT-qPCR normalization and Western blotting loading, respectively. Notice that the siRNA-mediated knockdown of KRIT1 results in the upregulation of c-Jun and phospho-c-Jun expression levels. Histograms show quantitative results of (A, B) RT-qPCR and (E, F) Western blot analysis of the relative c-Jun and KRIT1 mRNA and protein expression levels, respectively. mRNA levels (A, B) and optical density values of Western blot bands (E, F) are expressed as relative level units referred to the average value obtained for the mock- (E) or siNC-transfected (F) cells and represent the mean (±SD) of n = 3 independent experiments. ***P ≤ 0.001 versus mock-transfected cells.

Fig. 3

KRIT1 regulates c-Jun expression — KRIT1 overexpression approach. HeLa cells were mock-transfected or transiently transfected with a GFP-tagged KRIT1A construct. 48 h posttransfection, cells were lysed and analyzed by Western blotting with anti-c-Jun (c-Jun) and anti-GFP (GFP-KRIT1 and GFP) antibodies. Tubulin (α-Tub) was used as loading control. Notice that KRIT1 overexpression in HeLa cells results in the downregulation of c-Jun protein levels. Histograms show quantitative results of Western blotting analysis of the relative c-Jun and KRIT1 expression levels. Band optical density values are expressed as relative protein level units referred to the average value obtained for the mock-transfected cells and represent the mean (±SD) of n=3 independent Western blotting experiments. ***P≤0.001 versus mock-transfected cells.

Fig. 4

ROS scavenging overcomes the upregulation of c-Jun expression/phosphorylation caused by KRIT1 loss. KRIT1^−/− (K^−/−), wild-type (K^+/+), and KRIT1^−/− re-expressing KRIT1 (K9/6) MEFs grown to confluence were either mock-treated or treated with the ROS scavenging agent NAC (20 mM in complete medium) for 120 min at 37 °C. The cells were then lysed and analyzed by Western blotting with either (A) c-Jun (c-Jun) or (B) phospho-c-Jun (P-c-Jun) antibodies. Vinculin was used as loading control. Notice that c-Jun expression and phosphorylation levels in KRIT1^−/− cells treated with the ROS scavenger NAC (K^−/− NAC) are significantly reduced compared with untreated KRIT1^−/− cells and close to the levels of untreated wild-type cells (K^+/+). (C, D) Histograms showing quantitative results of Western blot analysis of c-Jun and phospho-c-Jun expression levels. Band optical density values are expressed as relative protein level units referred to the average value obtained for untreated K^−/− cells and represent the mean (±SD) of n = 3 independent Western blotting experiments. **P≤0.01 versus untreated K^−/− cells.

Fig. 5

KRIT1 overexpression prevents forced upregulation of c-Jun induced by oxidative stimuli. (A, D) KRIT1^−/− (K^−/−) and wild-type (K^+/+) MEFs and KRIT1^−/− re-expressing KRIT1 (K9/6) MEFs grown to confluence were either mock-treated or treated with H₂O₂ (0.1 mM in complete medium) for 60 min at 37 °C. The cells were then lysed and analyzed by Western blotting with anti-c-Jun (c-Jun) and anti-KRIT1 (KRIT1) antibodies. Tubulin (α-Tub) was used as loading control. Notice that KRIT1 overexpression prevents forced upregulation of c-Jun induced by oxidative stimuli. (B, C, E) Histograms show quantitative results of Western blot analysis of the relative c-Jun (B), KRIT1 (C), and P-c-Jun (E) expression levels. Band optical density values are expressed as relative protein level units referred to the average value obtained for untreated K^−/− cells and represent the mean (±SD) of n=3 independent Western blot experiments. ***P≤0.001 versus untreated K^−/− cells.

Fig. 6

c-Jun expression and phosphorylation are enhanced in CCM lesions from KRIT1 loss-of-function mutation carriers. Histological sections (4 µm) of paraffin-embedded CCM surgical specimens, deriving from a KRIT1 loss-of-function mutation carrier, were processed by a two-step immunohistochemical staining technique (DAKO EnVision+ System, HRP) with c-Jun and phospho-c-Jun antibodies to assess (A, B) c-Jun and (C, D) phospho-c-Jun expression in perilesional and CCM lesion vessels. Notice that many endothelial cells lining the lumen (l) of CCM vessels (B, D) were positive for c-Jun (B) and phospho-c-Jun (D), whereas neither c-Jun- (A) nor phospho-c-Jun- (C) positive staining was detected in perilesional normal vessels (A, C). Original magnification: 20×.

Fig. 7

KRIT1 loss of function induces a ROS-dependent activation of JNK. KRIT1^−/− (K^−/−) and KRIT1^−/− re-expressing KRIT1 (K9/6) MEFs grown to confluence were (A) left untreated or (B) either mock-treated or treated with the ROS-scavenging agent NAC (20 mM in complete medium) for 120 min at 37 °C. The cells were then lysed and analyzed by Western blotting as described under Material and methods. The phosphorylated JNK and total JNK were probed using anti-phospho-JNK (Thr-183/Tyr-185) antibody and anti-JNK antibody and compared to the relative P-c-Jun and KRIT1 expression levels. Total JNK and tubulin (α-Tub) served as loading controls. (A) Notice that JNK phosphorylation is significantly higher in K^−/− compared to K9/6 MEFs and correlated with P-c-Jun levels. An inverse correlation between P-JNK and KRIT1 protein levels is also evident. (B) Notice that P-JNK levels in K^−/− MEFs treated with the ROS scavenger NAC (K^−/− NAC+) are significantly reduced compared with untreated K^−/− cells (K^−/− NAC−) and close to the levels of untreated K9/6 MEFs (K9/6 NAC−). A direct correlation between P-JNK and P-c-Jun and an inverse correlation between P-JNK and KRIT1 levels are also evident.

Fig. 8

KRIT1 loss of function causes the upregulation of COX-2. (A, B) KRIT1^−/− (K^−/−), wild-type (K^+/+), and KRIT1^-/- re-expressing KRIT1 (K9/6) MEFs were grown to confluence under standard conditions and analyzed by (A) RT-qPCR and (B) Western blotting. (A) RT-qPCR analysis. COX-2 mRNA expression levels were analyzed in triplicate using appropriate TaqMan gene expression assays (Roche) and normalized to the amounts of internal normalization control transcripts (18S rRNA). Results are expressed as relative mRNA level units referred to the average value obtained for the K^−/− samples and represent the mean (±SD) of n=3 independent RT-qPCR experiments. ***P≤0.001 versus K^−/− cells. (B) Representative Western blot analysis. COX-2 levels in cell lysates were analyzed by Western blotting with an anti-COX-2 mAb and compared to the relative P-c-Jun and KRIT1 levels. Tubulin (α-Tub) was used as loading control. Notice that KRIT1 loss of function reproduced in K^−/− cells caused a significant upregulation of COX-2 expression at both mRNA and protein levels, which was completely rescued by the re-expression of KRIT1 (K9/6). It is also evident that COX-2 protein levels are directly and inversely correlated with P-c-Jun and KRIT1 protein levels, respectively. (C) Immunohistochemical analysis of paraffin-embedded human cerebral cavernous malformations with anti-COX-2 antibodies. Cavernous malformation tissue was collected from a CCM1 mutation carrier with familial disease at the time of surgical resection under an approved institutional review board protocol. Notice that many endothelial cells lining the lumen (l) of CCM vessels were positive for COX-2.

Fig. 9

Schematic model representing the inverse relationship between KRIT1 and c-Jun expression levels and its putative functional significance. See text for details.