Pigment epithelium‑derived factor facilitates NLRP3 inflammasome activation through downregulating cytidine monophosphate kinase 2: A potential treatment strategy for missed abortion

Abstract

A number of conditions may underlie the occurrence of missed abortion (MA), including inflammation. Pigment epithelium‑derived factor (PEDF) is a novel mediator of the inflammation‑related nucleotide‑binding oligomerization domain‑like receptor protein 3 (NLRP3) inflammasome, which is associated with several human diseases. However, the association between MA and NLRP3 inflammasome, and whether PEDF is reduced in MA, remain unknown. In the present study, the decidua and chorion tissues of patients who had suffered a MA were examined, and a lipopolysaccharide (LPS)‑induced human chorionic trophoblast HTR8/SVneo cell model was established to mimic MA in vitro. The results revealed that cytidine monophosphate kinase 2 (CMPK2) expression and NLRP3 inflammasome activation, downstream pro‑IL‑18 and pro‑IL‑1β expression, and IL‑18 and IL‑1β release, were all significantly increased in MA tissues or LPS‑induced HTR8/SVneo cells. PEDF reversed the increase in CMPK2 expression and activation of the NLRP3 inflammasome axis and, thus, downregulated the production of mitochondrial reactive oxygen species and mitochondrial DNA release, resulting in reduced lactate dehydrogenase release, and a resultant decrease in cell viability. Recovery of CMPK2 expression abolished all the effects of PEDF, indicating that CMPK2 may be an effector downstream of PEDF. PEDF reduced CMPK2 protein levels but did not affect the mRNA levels, and treatment with the proteasomal inhibitor MG132 significantly reversed this reduction in CMPK2 protein levels. Furthermore, a ubiquitination assay of immunoprecipitation demonstrated that CMPK2 was polyubiquitinated in the presence of LPS, PEDF and MG132. These results indicated that the NLRP3 inflammasome is implicated in the pathogenesis of MA, and PEDF may reduce MA through ubiquitin‑dependent proteasomal degradation of CMPK2 to inhibit NLRP3 activation, which may serve as a novel strategy for preventing or reducing the risk of MA.

Figure 1

Activation of the NLRP3 inflammasome was observed in the decidua and chorion tissues of MA patients. (A) Immunohistochemistry staining of NLRP3 in the decidua and chorion tissues from patients who underwent IA or MA. Scale bar, 100 μm. (B) Ratio of moderate and strong expression to negative and weak expression of NLRP3 in decidua and chorion tissues; n=20. (C) Western blot of an IP of the NLRP3 inflammasome in the decidua and chorion tissues and the cell lysate input controls. Quantitative analysis of (D) PEDF and CMPK2, and (E) NLRP3, ASC and Casp1 protein expression levels; n=20. ^*P<0.05, ^**P<0.01 vs. indicated or relative IA group; ^#P<0.05 vs. relative decidua tissue of MA patients. MA, missed abortion; IA, induced abortion; NLRP3, nucleotide-binding oligomerization domain-like receptor protein 3; IP, immunoprecipitation; IB, immunoblotting; PEDF, pigment epithelium-derived factor; ASC, apoptosis-associated speck-like protein containing a caspase recruitment domain; CMPK2, cytidine monophosphate kinase 2; Casp1, caspase1.

Figure 2

NLRP3 inflammasome increases downstream IL-18 and IL-1β levels in decidua and chorion tissues of MA patients. (A) The relative mRNA expression levels of pro-IL-18 and pro-IL-1β in the IA and MA groups were determined; n=20. (B) Western blot and densitometry analysis of pro-IL-18 and pro-IL-1β protein expression in IA and MA tissues; n=20. (C) The serum concentrations of IL-18 and IL-1β in IA and MA tissues were analyzed using ELISA; n=20. ^*P<0.05, ^**P<0.01 vs. relative IA group or the indicated group; ^#P<0.05, ^##P<0.01 vs. relative decidua tissue of MA patients. NLRP3, nucleotide-binding oligomerization domain-like receptor protein 3; MA, missed abortion; IA, induced abortion; IL, interleukin.

Figure 3

PEDF inhibits the activation of the NLRP3 inflammasome and reduces downstream increases in IL-18 and IL-1β through downregulation of CMPK2 in the HTR8/SVneo human chorionic trophoblast cell line. (A) The NLRP3 inflammasome in HTR8/SVneo cells was immunoprecipitated using an NLRP3 antibody, and the cell lysate input controls were tested by western blotting. The HTR8/SVneo cells were treated with 10 μg/ml LPS for 6 h. Densitometry analysis of (B) PEDF and CMPK2, (C) NLRP3, ASC and Casp1, and (D) pro-IL-18 and pro-IL-1β protein expression in HTR8/SVneo cells; n=5. (E) Relative mRNA levels of pro-IL-18 and pre-IL-1β in HTR8/SVneo cells; n=5. (F) The protein concentration of IL-18 and IL-1β in the cultured supernatants was analyzed by ELISA. n=5. ^**P<0.01 vs. relative normal group; ^#P<0.05, ^##P<0.01 vs. relative LPS group; ^&P<0.05, ^&&P<0.01 vs. relative LPS + PEDF group. PEDF, pigment epithelium-derived factor; NLRP3, nucleotide-binding oligomerization domain-like receptor protein 3; LPS, lipopolysaccharide; ASC, apoptosis-associated speck-like protein containing a caspase recruitment domain; CMPK2, cytidine monophosphate kinase 2; Casp1, caspase 1; CMPK2 group, LPS + PEDF-LVs + CMPK2-LV; LV, lentivirus; IP, immunoprecipitation; IB, immunoblotting.

Figure 4

PEDF alleviates LPS-induced mitochondrial and cellular damage by decreasing the expression of CMPK2 in HTR8/SVneo cells. (A) Fluorescence microscopy for Mito-SOX Red-labelled ROS in cytosol of HTR8/SVneo cells. Scale bar, 100 μm. HTR8/SVneo cells, cells overexpressing PEDF, or cells overexpressing CMPK2, were treated with 10 μg/ml LPS for 6 h. (B) Quantification of fluorescence density of mtROS in HTR8/SVneo cells. n=5. (C) Cytosolic mtDNA copy numbers were measured using by quantitative PCR; n=5. HTR8/SVneo (D) cell death and (E) cell viability were determined by assessing LDH release or using a Cell Counting Kit-8 assay, respectively. n=5. ^**P<0.01 vs. relative normal group; ^##P<0.01 vs. relative LPS group; ^&&P<0.01 vs. relative LPS + PEDF group. PEDF, pigment epithelium-derived factor; LPS, lipopolysaccharide; CMPK2, cytidine monophosphate kinase 2; mt, mitochondrial; ROS, reactive oxygen species; LDH, lactate dehydrogenase.

Figure 5

PEDF induces degradation of the CMPK2 protein via a ubiquitin-dependent proteasomal mechanism. (A) Relative mRNA expression levels of CMPK2 in normal, LPS-induced, PEDF-overexpressing cells, and LPS + PEDF overexpression HTR8/SVneo cells; n=5. (B) Western blot and densitometry analysis of CMPK2 in HTR8/SVneo cells treated with LPS, leupeptin general protease inhibitor, NH4Cl lysosomal inhibitor, MG132 proteasome-specific inhibitor or DMSO for 6 h following overexpression of PEDF; n=5. (C) Cells were pre-treated with PEDF or MG132 and subsequently treated with LPS, and CMPK2 was immunoprecipitated; n=5. CMPK2 ubiquitination was determined using an anti-ubiquitin antibody. ^*P<0.05, ^**P<0.01. PEDF, pigment epithelium-derived factor; LPS, lipopolysaccharide; CMPK2, cytidine monophosphate kinase 2; IP, immunoprecipitation; IB, immunoblotting.