The Role Of Furin In The Initiation Of Parturition And Its Mechanism

The global preterm prevalence is up to 10%,which is the leading cause of infant death.Survivors experience a series of complications in their adulthood.Therefore,it is urgent to figure out the regulatory factors of preterm birth aiming to control its occurrence and development.Considering human pregnancy and delivery as a complex and species-specific process.The mechanisms of parturition have not been clarified,there is still no effective clinical means to prevent preterm birth.Human myometrial tissues were obtained from pregnant women undergoing elective cesarean section at term.Globle genome anlysis showed that the expression of Furin,SULF1 and FN14 in human myometrial tissues at term labor(TL)was significantly up-regulated from that in term non-labor(TNL).As Furin has been reported acting as the proprotein convertase,it participates in various disease-related processes,such as cancer,atherosclerosis,infections.Therefore,exploring the role of Furin in uterine activation will help explain the molecular mechanism of normal human parturition and preterm birth.We focus on the regulation mechanism of Furin on uterine muscle activation.First,the abundance of Furin was detected in human pregnant related tissue samples((including myometrium,chorion and amniotic membrane)at the initiation of human parturition.As followed,we used primary cultured myometrial cells to explore the effect of Furin on the cell contractile force.We also constructed normal pregnancy and infectious/non-infectious induced preterm birth models in mice to determine whether Furin was involved in the initiation of parturition in vivo.The above study will provide new ideas for finding therapeutic targets for clinical prevention and treatment of preterm birth.Main Results:1.The myometrial tissues?chorionic and amniotic membranes were collected from pregnant women undergoing elective cesarean section in TL or in TNL.Western blotting,RT-PCR and IHC were used to determine the expression of Furin.The expression of Furin was only significantly up-regulated in myometrial tissues of TL group compared with TNL group.2.The human primary myometrial cells were cultured to study the roles of Furin in the regulation of UAPs and cell contractility.The expression of OTR,FP,CX43,COX2,p-P65 and p-MLC20 significantly decreased in the myometrial cells transfected with Furin siRNA,accompanying with the attenuated cell contractility.The cell contractility was significantly weakened in the myometrial cells treated with Furin inhibitor CMK(20ug/ml),while significantly enhanced in the myometrial cells transfected with Furin overexpression lentivirus.3.The myometrial tissues were collected from mice during pregnancy(on GD15,GD17 and GD19)and at term labor.And the myometrial tissues were also collected from pregnant mice administrated with Furin inhibitor D6R(1.5mg/kg)on GD17.5 and GD18.5.The expression of Furin were significantly up-regulated in GD19 and term labor groups compared with GD15 and GD17 groups.Administration of Furin inhibitor CMK(1mg/kg)or(D6R(1mg/kg or 1.5mg/kg)on GD17.5 and GD18.5 was significantly postponed the delivery time.The expression of Furin in the myometrial tissues did not change,but COX2,CX43 and FP were significantly decreased in the group treated with D6 R.More evidence indicated that D6 R affected the structure of myometrium at labor,as abnormal accumulation of glycogen,expansion of endoplasmic reticulum,abnormal vacuoles in smooth muscle,and the presence of autophagic vacuoles,as well as pyknosis of the nucleus and accompanied with the breakage of partial myofilament.4.However,administration of D6R(1mg/kg)or CMK(1mg/kg)on GD15 did not affect the time of preterm birth induced by RU38486.The expression of Furin did not change in the group treated with RU38486 compared with control group.Similarly,the time of preterm birth induced by LPS was also not affected with administration of Furin inhibitor D6R(1mg/kg)on GD15.5.5.As one of the classic Furin substrate,the expression of full length TWEAK was significantly increased in the myometrial cells treated with D6R(20ug/ml).The human primary myometrial cells were transfected with Furin OVE lentivirus,Furin siRNA or treated with D6R(20ug/ml).The content of TWEAK and human 27 factors in culture supernatant were determined by ELISA.The content of TWEAK was increased when cells transfected with Furin OVE,and decreased when cells transfected with Furin siRNA or treated with D6R(20ug/ml),but the human 27 factors have no change.The myometrial tissues and serum were collected from pregnant mice which were treated with Furin inhibitor D6R(1.5mg/kg)on GD17.5 and GD18.5.The content of TWEAK was decreased in serum of the pregnant mice treated with D6R(1.5mg/kg).The expression of FN14 was specifically upregulated in myometrial tissues of TL group compared with TNL group.The expression of OTR,FP,CX43,COX2,p-P65 and p-MLC20 were significantly decreased in the myometrial cells transfected with FN14 siRNA.The cell contractility was significantly weakened in the myometrial cells transfected with FN14 siRNA or treated with L524-0366(20uM),while significantly enhanced in the myometrial cells treated with TWEAK(100ng/ml).The protein expression of FN14 was significantly up-regulated in GD19 and term labor groups compared with GD17 group.The length of mice normal pregnancy can be significantly delayed by L524-0366(1mg/kg).And the expression of p-P65 was significantly up-regulated in myometrial cells treated with TWEAK.Conclusions:1.Furin specifically expressed in the human myometrium at term labor.It promotes the expression of UAPs in myometrial cells.In vivo,Furin inhibitors significantly postponed the delivery time of normal parturition,but did not affect the preterm birth induced by RU38486 or LPS.It indicated that Furin may directly affect the cell contractility of uterine smooth muscle by stimulating the expression of UAPs and further initiate the parturition.2.The TNFR superfamily member FN14 is also specifically upregulated in the myometrial tissues of term labor.Furin can cleave its ligand TWEAK,The TWEAK-FN14 pathway promotes the phosphorylation of transcription factor P65 and increases the expression of UAPs,thereby enhances the cell contractility.We further confirmed that blockage of FN14 significantly delayed the normal delivery of mice.It indicated that TWEAK-FN14 may participate in the initiation of parturition by activating the NF?B pathway.