Regulatory Mechanism Of Hippo Pathway By Cytoskeletal Dynamics And Its Biological Significance On Nuclues Pulpsosus Cell Morphology And Viability

Part 1 Dysregulation of Hippo pathway is involved in intervertebral disc degeneration and cell senescenceObjectiveTo explore the mechanism of Hippo signaling pathway in intervertebral disc degeneration and cell senescenceMethodsRats(10 weeks)were anesthetized using an intraperitoneal injection of 10%chloral hydrate(0.3 ml/100 g).The coccygeal 5-6,6-7,7-8,and 8-9 discs were identified and percutaneously punctured using a 21 G needle.Analysis was performed at 4 weeks(P4w)and 12 weeks(P12w)post-operatively.A natural IDD group and a control group(healthy rats with no lesions)were also included and analyzed at 4 weeks(4w),14weeks(14w)as P4w control,22 weeks(22w)as P12w control and 50 weeks(50w).disc height index,Magnetic resonance imaging(MRI)examination and Pfirrmann grade evaluation and Hematoxylin and eosin staining were performed to observe IDD.Real-time PCR and Western blotting were tested to detect core molecules of Hippo signaling pathway.Because in vitro subcultivation(replication senescence)can lead to senescence of Nucleus pulposus cells(NPCs),the control passage 3(P3)cells were used to induce the premature senescence of cells(P10)according to our preliminary work.The cells were plated at a density of 1×10~6 cells per well into 6-well plates.A cholecystokinin(CCK)-8 assay and cell cycle analysis were performed and Senescence?-galactosidase(SA-?-Gal)activity was measured according to the manufacturer's instructions.P3 and P10groups cells were seeded in a six-well plate 24 h at medium density(1?10~6)before transfection,each group has three holes.After 24 h,cells were transfected with shYAP or sh-Control duplexes according to the manufacturer's instructions.Extracting genes and proteins to identify YAP interference efficiency and comparison of key molecular of Hippo signaling pathways among groups(P3 sh-Control,P3 shYAP,P10 sh-Control and P10 shYAP groups),SA-?-Gal activity was measured to detect cell senescence and Immunofluorescence staining was used to observe the intracellular distribution of Yes-associated protein(YAP)and p-YAP.ResultsChanges in Disc height index(DHI)in the natural IDD groups.We observe statistically significant differences in DHI among the natural IDD groups(4w,14w and 50w)(0.155±0.005,0.109±0.001,0.072±0.002,P<0.05).The IDD values at 14w and 50w were lower than those at 4w,indicating that discs continuously and slowly degenerate with age.Indeed,older discs displayed more serious IDD.MRI of natural group tail discs showed normal signals,and the gray scales and Pfirrmann grades increased gradually over time.To identify IDD patterns,H&E staining was performed to examine the morphology of the nucleus pulposus cells(NP)and annulus fibrosus(AF).Compared with the other groups,the 4w group contained more vacuolated notochordal cells and less small chondrocyte-like cells with even cellular distribution.In the 50w group,the level of vacuolated notochordal cells decreased gradually over time,and NPCs clusters and serpentine AF were found,indicating disc degeneration.Compared with the control group,significant disc morphological changes,including small NP areas and an increased number of chondrocyte-like cells,were observed after puncturing.Disrupted borders between AF,NP,and AF serpentine fibers were also found.Real time PCR and Western blot in NP of P4w group also indicated that Hippo signal was inhibited,YAP expression was increased,and p-YAP expression was decreased.Compared with the P3 sh-Control group,the shYAP group markedly accelerates the premature senescence of NPCs by activating level of p53 and p21 which mediate cell replicative senescence,the parameters showed a significant difference(69.3±5.13 and8.13±1.80,P<0.05).ConclusionOur results demonstrate that the activity of YAP decreased gradually with age in natural IDD.Hippo signaling was suppressed in the early stages of disc injury,demonstrating the potential for endogenous repair,but this repair was weak and did not prevent the progression of disc degeneration.Moreover,YAP was inhibited by NPCs contact via the Hippo pathway in vitro.Indeed,YAP was primarily localized to the cytoplasm and its response to growth inhibition at a high cell density was restrained.Dephosphorylated YAP levels were increased and mainly localized in the cytoplasm,with NPCs developing toward senescence.Part 2 Cytoskeleton protein-mediated Hippo signaling pathway regulates disc degenerationObjectiveTo explore the mechanism of Hippo signaling pathway and cytoskeleton protein in regulating intervertebral disc degeneration,cell density and extracellular matrix.MethodsThere are four groups in the research,including a natural IDD group 4 weeks(4w),14weeks(14w),50 weeks(50w)and P4w group post-operatively.we investigated the characteristics of Hippo pathway and three major cytoskeletal elements-F-actin,?-tubulin and vimentin by Immunofluorescence staining,Real-time Polymerase Chain Reaction(Real-time PCR)and Western blot.NPCs from the third passage cultured in Dulbecco's modified Eagle's medium(DMEM)/F12 with 10%fetal bovine serum(FBS)were plated at a low density(8×10~4),medium density(1×10~6),and high density(2×10~6)per well into 6-well plates containing different protein(Fibronectin 5?g/cm2,Laminin 2?g/cm2 and Polylysine0.1mg/ml)coated coverslips(24 mm in diameter and coated overnight).24 hours after plating.The distribution and expression of YAP protein and cytoskeletal protein F-actin,?-tubulin and Vimentin were observed by immunofluorescence.The expression of core molecules of Hippo signaling pathway and cytoskeletal proteins were detected by Real-time PCR and Western blotting.ResultsF-actin mildly decreased with age in natural IVD,whether in AF(Outer annulus fibrosus(IAF)and Inner annulus fibrosus(OAF))or NP.F-actin is partly punctate in nucleus pulposus cells,but extends into the processes of the OAF and IAF cells in P4w group.What is more,F-actin displays an extensive network traversing the cytoplasm of both NP and AF cells and actin filaments also extend into the processes of the NPCs.?-tubulin was observed throughout the cytoplasm in cells from NP,IAF and OAF tissue in P4w.And as age increased,the?-tubulin in NP gradually decreased,and unlike the expression of F-actin,there was no increase in the P4w group.Quantitative mRNA analysis demonstrated appreciably more vimentin mRNA and protein in 4w group and P4w group,although vimentin gene expression in the NP decreased with age.YAP decreased with age in natural IVD and activated in puncture group.Although there are differences in cell density,F-actin filaments displayed a flattened morphology when cultured as a monolayer seeded onto polylysine-coated and laminin-coated coverslip in three densities.F-actin filaments were predominately localized beneath the cell membrane;F-actin is mainly located in the nucleus and mostly punctate distribution in medium density on the laminin-coated coverslip.?-tubulin was mainly distributed throughout the cytoplasm,with evidence of intense peri-nuclear staining in NPCs with fibronectin-coated.The effect of cell densities on the cells is only a morphological change,yet has no role on the subcellular distribution of?-tubulin.In all groups,the vimentin network appeared as a fibrous meshwork radiating from the cell nucleus towards the cell periphery.In our study,YAP gene and protein expression was the highest in medium density,p-YAP and Connective tissue growth factor(CTGF)corresponding changes.Meanwhile,YAP is mainly localized in the nucleus.ConclusionHippo signaling was suppressed in the early stages of disc injury,demonstrating the potential for endogenous repair,but this repair was weak and did not prevent the progression of disc degeneration.Meanwhile,the Hippo pathway and cytoskeleton were also shown to be regulated by cell density and ECM.The main functions of?-tubulin and vimentin filaments are to provide the cell with its shape and its elastic properties in resisting mechanical forces.YAP was primarily localized to the cytoplasm and its response to growth inhibition at a low cell density was restrained,suggesting the specificity of NPCs and the complex regulation mechanism of Hippo pathway.Part3 AMOT130 linking F-actin to YAP is involved in intervertebral disc degenerationObjectiveTo explore the mechanism of Hippo signaling pathway and F-actin and its role in intervertebral disc degenerationMethodsNPCs were passaged three times(P3)in Dulbecco's modified Eagle's medium(DMEM)/F12 with 10%FBS,then plated into 6-well plates at a density of 1×10~6cells/well for 24 h.Then the cells were treated with F-actin inhibitor latrunculin B(Lat B)for 1 h.Stock solutions of Lat B was prepared at a final concentration of 2.5 mM in dimethyl sulfoxide(DMSO),with working solutions diluted 1:1000(2.5?M)in serum-free DMEM/F12.Control samples were treated with DMSO in serum-free DMEM/F12.Passage 2 NPCs were seeded in a 6-well plate before transfection.After 24 h,cells were transfected with Sh-LATS A,Sh-LATS B,or Sh-Control duplexes for 12 h according to the manufacturer's instructions.Passage 3 NPCs were seeded in 6-well plates for 24h at a density of 1×10~6 cells/well(Sh-Control,Sh-LATS1/2,Sh-LATS1/2+Lat B groups).Cell senescence was observed by SA-?-gal staining.Ki67,YAP and F-actin were detected by immunofluorescence,and Hippo signal molecules were detected by Real time PCR and Western blot.The interaction between F-actin and AMOT130 was confirmed by CO-IP assay.What is more,AMOT130 and F-actin co-localized in NPCs were detected by laser scanning confocal microscopy.ResultsIn non-senescent NPCs(P3,DMSO group),F-actin filaments were predominately localized beneath the cell membrane;however,large numbers of F-actin stress fibers were observed throughout the cytoplasm and extending into the cellular protrusions.These fibers disappeared in response to Lat B treatment,reverting to a more punctate distribution,consistent with a depolymerization of F-actin.The Hippo pathway was suppressed in the DMSO group,with YAP primarily localized in the nucleus,indicative of greater target gene transcription and cell growth.By contrast,when F-actin was disrupted,cells became smaller,with YAP protein primarily sequestered in the cytoplasm.The percentage of Ki67-positive cells in the DMSO group was 40%,compared to only 10%after Lat B treatment(P<0.05).To determine whether YAP localization was affected by LATS in the absence of direct cell–cell contact,cells were treated with either vehicle control(Sh-Control)or Sh-LATS1/2 to silence LATS gene expression.The results of Real time PCR and Western blotting showed that the interfering vector had a good effect,and the level of LATS1/2gene and protein decreased significantly.What is more,Unlike Lat B,which disrupted the peripheral F-actin band and excluded YAP from the nucleus,treatment with Sh-LATS1/2 led to greater nuclear accumulation of YAP.However,this effect was not seen in cells treated with Sh-LATS 1/2+Lat B,with cells exhibiting a primarily punctate F-actin distribution,which suggests that F-actin integrity is essential for LATS-mediated regulation of YAP.Although the expression of YAP and its downstream target gene CTGF increased in NPCs treated with Sh-LATS1/2,Ki67 and SA-?-Gal staining showed no increase in cell proliferation(43.51%and 45.84%)and senescence positive ratio(17.25%and 18.83%)compared to control group.Sh-LATS1/2 treatment decreased p-YAP(YAP activation),relative to controls,while p-YAP more decreased in response to Sh-LATS1/2+Lat-B.To investigate the mechanism behind the decreased NPCs proliferation and increased senescence following the F-actin inhibition,and also observe whether F-actin and AMOT130 were interrelated,we examined for Angiomotin130(AMOT130)that interact with F-actin using Co-Immunoprecipitation(Co-IP)and co-localization.In the present study,the interaction between F-actin and AMOT130 was confirmed by CO-IP assay.What is more,laser scanning confocal microscopy showed that AMOT130and F-actin were co-localized in NPCs.Conclusion In summary,defects in the ability of YAP to localize to the cytoplasm in the context of F-actin disruption suggest a LATS1/2 dependent mechanism for YAP localization.In contrast,knockdown of LATS1/2 have a minor influence on YAP to regulate either cell shape or proliferation.Taken together,our results demonstrate that YAP activity can be inhibited through two distinct mechanisms:(i)the recruitment of YAP to the tight junction by direct AMOT130-YAP interactions,(ii)and YAP phosphorylation by the canonical Hippo signaling,which in turn promotes YAP cytoplasmic retention and inactivation by 14-3-3.