Signaling Mechanism Of Small GTPase In Bone Marrow Mesenchymal Stem Cells Motility And The Regulation Of Cdc42 In Skin Development And Barrier Formation

Part one Signaling Mechanism of Rho GTPases in Bone Marrow Mesenchymal Stem Cells MotilityBone Marrowmesenchymal stem cells(BMSCs)hold great promise for wound healing and tissue egeneration.During tissue repair,endogenous BMSCs or exogenously delivered MSCs migrate to the sites of injury and participate in the repair process.The mechanisms responsible for enhanced wound healing in the skin include the possible trans-differentiation of MSC to form cells of epidermal and dermal lineages,along with restorative paracrine effects of MSC synthesised growth factors.Cell migration is usually initiated in response to extracellular cues,which can be diffusible factors,signals on neighbouring cells,and/or signals from the extracellular matrix.Activins are members of the transforming growth factorβ(TGF-β)superfamily of dimeric proteins,consisting of βA and βB subunits which are connected by disulfide linkages.Activins are known to bind to heteromeric receptor complexes consisting of a type Ⅰ(ActRIA and ActRIB)and a type Ⅱ receptor(ActRⅡand ActRⅡB),which are all characterized by the presence of a cytoplasmic serine/threonine kinase domain.Activin is considered to play an important regulatory role in tissue repair and re-epithelialization process.Our previous studies have found that In vitro ACT B can induce the migration of BMSCs and promote the wound healing in vivo.However,at present,the intracellular mechanisms linking this event is far from understanding.TheGTPases are molecular switches which use a simple biochemical strategy to control complex cellular processes.It shuttles between the active GTP-bound form and the inactive GDP-bound form and works as a switch in the cell.The Rho subfamily of small GTPases are recognized to play a critical role in the regulation of actomyosin cytoskeletal organization.The consequences of activating Rho,Rac and Cdc42 in Swiss 3T3 fibroblasts have been well characterized.Rho regulates the assembly of actin stress fiber.Rac regulates the polymerization of actin at the cell periphery to produce lamellipodia and membrane ruffles,while Cdc42 triggers filopodia formation.In addition,all three GTPases regulate the formation of cell matrix adhesion sites called focal adhesions,which are intimately associated with the actin structures.Furthermore,the GTPases appear to be linked to one another in a cascade;activation of Cdc42,for example,leads to rapid localized activation of Rac.It has been suggested,therefore,that these GTPases may be important regulators of cell movement in response to extracellular signals.In this study,we addressed the signaling pathway downstream of ActivinB in BMSCs and identified a novel cascade of effectors that mediate reorganization of the actin cytoskeleton by this growth factor.Better understanding of the molecular mechanisms of BMSC migration will help to optimize therapeutic strategy to target BMSCs at injured tissues,and develop new therapies in regenerative medicine.Methods:The effect of ActivinB on the actin cytoskeleton of BMSCs was analyzed by direct fluorescence microscopy using rhodamine-phalloidin,and cell morphology was monitored by light microscopy.Control cells were serum-starved for 24 h and then stimulated with 10 ng/ml Activinb for 30 min up to maximum6h.Representative experiment showing the amount of active GTP-bound RhoA,Rac1 and Cdc42,determined by a GST pull-down assay,from non-stimulated(control)or ActivinB stimulated(10 ng/ml)BMSCs for 15 min,30min or 120min.RhoA,Racl and Cdc42 levels were determined by immunoblotting with antibodies specific for the respective GTPases following the pulldown or in the total cell extracts.BMSCs were infected with Lentivirus for empty vector,RhoA(N19)and RhoA(L63).At 24 h after infection,the cells were treated with activin B(10 ng/ml)for30 min,2h and 4h before being harvested for Western blot analyses,as described above.The cells were stained with rhodamine-phalloidin for F-actin and with Hoechst 33258 for nuclei,and the virus-infected cells were identified by GFP fluorescence.Analysis of Activition of RhoA in ActivinB-induced BMSCs migration by transwell migration assay and cell scratching assay in vitro.BMSCs were serum-starved for 12h and treated with10 ng/mL activin B for 15min,30min and 2h,harvested for Western blot analyses for LIMK2,Cofilin and Smad.Wild-type BMSCs were pretreated with Y27632(10μM)for 2h,followed by treated with ActivinB for 2h.Cells were analyzed for F-actin by using rhodamine-phalloidin,and nuclei were identified by Hoeschst staining.Wild-type BMSCs were pretreated with Y27632(10μM)for 2h,followed by treated with ActivinB for 15min,30min and 2h,harvested for Western blot analyses for LIMK2 and Cofilin.Results:1.Starved cells exhibited typical morphology,and their actin cytoskeleton was restricted to cortical actin while the main cell body became devoid of stress fibers.Treatment with ActivinB resulted in cell flattening and scattering,which was supported by changes in the organization of the actin cytoskeleton.Newly formed stress fibers were prominent in the treated cells,and their abundance and length clearly increased with prolonged ActivinB treatment.The actin reorganization was evident as early as 2h post-ActivinB treatment and persisted as long as6 h,at which point the cell shape changed became even more dramatic and cells appeared elongated or spindle shaped with a parallel arrangement of actin bundles.2.The amount of GTP-bound RhoA increased 15 min after ActivinB stimulation,reached maximal level at 30min(95%CI(1.45,1.90),(1.91,2.82)),and decreased to baseline after2h.Rac and Cdc42,in contrast to RhoA,exhibited no significance activation(F=0.297,P=0.754;F=0.129,P=0.882).3.In control cells transfected with GFP and stimulated with ActivinB for 2 h,reorganization of the actin cytoskeleton occurred normally and was not disturbed by the transient transfection process.In contrast,cells expressing dominant-negative RhoA could not form new actin stress fibers in response to ActivinB,whereas the surrounding non-transfected cells showed robust actin reorganization.In Trans well assay,the cell number of BMSCs transfeced with RhoA(N19)group in the lower chamber is significantly fewer than the empty-vector transfected group.In contrast,the number of RhoA(L63)group is higher than the control group(P<0.05).In vitro cell sctraching assays shows that RhoA(N19)could inhibit the ActivinB induced BMSCs migration.In scratching assay,the area of BMSCs transfeced with RhoA(N19)group is significantly larger than the empty-vector transfected group.In contrast,the area of RhoA(L63)group is smaller than the control group(P<0.05).4.The levels of phosphorylated LIMK2 increased at 15 min,30min and 2h post stimulation with ActivinB(95%CI(1.45,2.30),(2.44,2.91),(2.01,2.78)),and this was followed by an increase in phosphorylation of cofilin(95%CI(1.74,1.86),(2.61,2.75),(2.14,2.84)).The phosphorylated levels of both proteins persisted for as long as2 h.The levels of phosphorylated LIMK did not change(95%CI(0.66,1.25),(0.51,1.93),(0.91,1.13)).5.BMSCs treated with this inhibitor were unable to develop new actin filaments at 2 h indicating that ROCK1 function is necessary for actin reorganization by ActivinB.In cells pretreated with Y-27632 there was no change in the phosphorylation state of LIMK2(95%CI(0.86,1.24),(0.89,1.12),(0.88,1.29))or cofilin(95%CI(0.86,1.24),(0.89,1.12),(0.88,1.29)),indicating that ROCK1 mediates the ActivinB-induced actin reorganization in BMSCs through phosphorylation of LIMK2.Conclusion:ActivnB induced BMSCs rapid reorganization viaRhoA-ROCK-LIMK-Cofilin signaling cascade.Part two TheRegulation of Cdc42 in Skin Developmentand Barrier FormationThe skin is the largest organ in the body,divided into two main structural compartments,the epidermis and the dermis.Epidermis is a continually renewing epithelium,usually subdivided into several layers or strata,starting with the basal layer just above the dermis and proceeding upward through the spinous and granular layers to the top layer,the stratum corneum.Its main function is to protect the skin from potentially hazardous environmental threats,providing physical,chemical,biochemical barriers.The physical barrier mainly consists of the stratum corneum,although the cell-cell junctions and associated cytoskeletal proteins in the lower layers provide further important components.The chemical and biochemical barrier consists of lipids,acids,hydrolytic enzymes,antimicrobial peptides,and macrophages.The immunologic barrier is composed of humoral and cellular constituents of the immune system.The epidermal barrier is indispensable to the maintenance of organisms;manyexamples in experimental animals and humans have shown that severe defects in epidermal barrier formation lead to an early demise.CDC42 is a ubiquitously expressed small GTPase belonging to the Rho family It exists in an active GTP-bound and an inactive GDP-bound form.Signaling by integrins,growth factor and cytokine receptors,and cadherins can activate CDC42 by stimulating the exchange of GDP to GTP,catalyzed by guanine-nucleotide exchange factors(GEFs).Only in its active form can CDC42 interact with different effector molecules which in turn regulate the actin cytoskeleton,microtubule network,cell polarity,proliferation,apoptosis,endocytosis,and secretion.Since many of these effectors are expressed tissue specifically in different amounts,the consequences of CDC42 activation are dependent on the cell type.Mice with a constitutive knockout of CDC42 die around implantation,indicating the importance of CDC42 functionin vivo(Chen et al.2000),but preventing the analysis of CDC42 at later time points of development.The recent development of CDC42conditional knockout mice has provided new tools to study the function of CDC42 in development.To analyze the function of Cdc42 in skin and to test whether Cdc42 is important in skin development and barrier formation,we generated mice with a keratinocyte-restricted deletion of the Cdc42 gene(Cdc42loxp/loxp-K14-Cre)and found that the animals died within Id after birth with wrinkled skin.Dehydration assay and transepidermal water loss measurements revealed that in these mice the epidermal barrier was severely affected.However,there is no clues for the regulation mechanism in this process.To investigate the genetic mechanisms of CDC42 in skin development and barrier formation,the global gene expression profiles of skins of KO mice and WT mice were identified using the RNA-seq.To illustrate the dynamic change during the skin development,we choose 2 stages:Embryonic 17.5 days and first day after birth.The bioinformatics software were employed to analyze the different expressed genes of skin in KO and WT mice in different development stage.Our work would helpful for fully understanding of regulation networks of CDC42 in skin development and barrier formation.Methods:1.Generation of mutant mice,the skin tissue gathered and RNA extraction:Using Cre/loxP system,the mice analyzed in this study were generated by crossing Cdc42-loxp/loxp mice with K14-cre transgenic mice.The genotypes are identified by polymerase chain reaction(PCR)technology.The skin was obtained from the upper-right back of mice at E17.5 and E19.5(the first day of birth).Tissues were isolated and snap frozen in liquidnitrogen until use.Total RNA was extracted from the skins of wild type and KO mice at E17.5(N=4)and E19.5(the first day of birth)(N=4),using TRIZOL followingthe manufacturer’s instructions(Invitrogen).In total,eight samples were included in this study.2.RNA-Seq(Quantification)analysis:cDNALibrary was constructed.RNA-Seq(Quantification)analysiswere carried outusingIllumina HiSeqTM2000.3.Bioinformatics analysis:Clean reads were mapped to reference sequences(UCSC mm9)usingtophat.The gene expression level is calculated by using RPKM method with cuffdiff."The absolute value of log2Ratio≥1 and q≤0.001"is the threshold to judge the significance of gene expression difference.GO enrichment analysis is performed with Gene Ontology Consortium(AmiGO 2 version:2.1.4)based on significantly enriched terms of molecular function,cellular component and biological process respectively.4.Validation of Expression of common espressed genes of the skin tissues inE17.5 and E19.5(P1)using RT-PCR and Realtime PCRResults:1.The bioinformation analysis of differential expressed genes at E17.5:Among the 50 genes with>2-fold expression,12(24%)were upregulated and 38(76%)were downregulated in KO skin.The upregulated differential expressed genes in the cluster of biological process were found to be mainly related to keratinazation,keratinocyte differentiation,epidermal cell differentiation and epidermis development.Cellular component GO terms of down-regulated differentially expressed genes were related to intermedia filaments.The downregulated differential expressed genes in the cluster of biological process were found to be mainly related to extracellular matrix.2.The bioinformation analysis of differential expressed genes at E19.5:Among the 153 genes with>2-fold expression,111(72.5%)were upregulated and 42(27.5%)were downregulated in KO skin at E19.5d.The up-regulated differentially expressed genes in the cluster of biological process were found to be mainly related to keratinazation,keratinocyte differentiation,epidermal cell differentiation and epidermis development.Meanwhile,the inflammatory response,defense response and response to external stimulus et al.were also enrichment in the biological process GO term.The down-regulated differentially expressed genes enriched in the Cellular component GO terms related to keratin intermedia filament.3.The bioinformation analysis of common differential expressed genes in E17.5 and E19.5:Common up-regulated genes is enriched in biological process GO term of keratinazation and celluar component GO term of conified envelop.The commom down-regulated genes is enriched in the cellular component GO term of keratin intermedia filament.4.The common differentially expressed gene:SPRR1B,KRT71,KRT25,KRT27,KRT6B and KRTAP3-1 were validated by real time PCR.The result is cosistent of the sequencing results.Conclusion:Cdc42 would regulate the epidermal development and barrier formation by keratinazation,keratin intermedia filament and extracullular matrix.