UPARAP: A Functional Biomarker For The Phagocytic Subpopulation Of Fibroblasts

BACKGROUND AND OBJECTIVES:Fibroblasts consist of subpopulations with unique phenotypes and functions,which show obvious heterogeneity.Its main function is to maintain the balance between collagen synthesis and degradation,which plays an important role in organ fibrosis and wound healing.Collagen may be degraded via an extracellular pathway or an intracellular route.Intracellular collagen degradation through fibroblast phagocytosis is essential for physiological remodeling of the extracellular matrix and wound healing,but deregulation of collagen phagocytosis can lead to tissue fibrosis.It is also found that extracellular matrix gradation during malignant progression is related with collagen phagocytosis.Some reports show that 80%of human gingival fibroblasts are phagocytic subpopulation in vitro.Up to date,it is difficult to separate CPSF and nCPSF because their molecular characteristics have not been found.For this reason,it may be inaccurate to draw a conclusion from the experimental data on fibroblasts that consist of various subpopulations.To address this question,we attempted to establish method to separate the CPSF and nCPSF by utilizing different capability in collagen phagocytosis.To find out molecular biomarkers of CPSF and nCPSF,we used the indirect immunofluorescence assay to detect the protein expression in CPSF or nCPSF,which encoded by the differentially expressed genes screened using microarray analysis and confirmed through real-time PCR.And then,the function of the molecular markers was investigated in the process of collagen phagocytosis.Furthermore,as a targeting molecular,we hope to set up a better approach to sort CPSF and nCPSF,which will lay solid foundation for exploring the biological charateristics of CPSF and CPSF.METHODS:1.The fibroblasts come from gingival tissues of healthy volunteers, and were cultured in vitro.FITC-LB was coated with rat tail collagen typeâ… or rat IgG.After fibroblasts being incubated with COL-FITC-LB or IgG-FITC-LB at a 4:1 bead:cell ratio,it was performed respectively to examine the percentage of fibroblasts internalizing COL-FITC-LB or IgG-FITC-LB at 0.5,1,2,4,6,12, 24h.2.After fibroblasts being incubated with COL-FITC-LB in 6h,CPSF and nCPSF were separated by FCM.And then they were cultured in vitro and were observed under inverted microscope.After CPSF or nCPSF being incubated with COL-FITC-LB 4h,the percentage of internalizing cells was analyzed at passaging.3.CPSF and nCPSF were separated from the mixture of equal amount fibroblasts from 3 volunteers by FCM.Total RNA was respectively extracted from CPSF and nCPSF.Human Genome U133A 2.0 Array was used for screening differentially expressed genes.4.According to the relationship with collagen phagocytosis,the genes related with collagen phagocytosis were chosen from differential gene expression profile between CPSF and nCPSF, and were verified through real-time PCR.5.CPSF and nCPSF were from other 4 volunteers,and their mRNA and protein expression levels of uPARAP and integrinα₂ were determined using semiquantitive RT-PCR and indirect immuno-fluorescence assay.This was used to clarify the function of uPARAP and integrinα₂ in collagen phagocytosis,and determine which molecular can serve as a target biomolecular to set up a better approach to sort CPSF and nCPSF.RESULTS:1.Fibroblast Phagocytosis was observed at 0.5h after incubation with COL-FITC-LB or IgG-FITC-LB.Fibroblast Phagocytosis of IgG-FITC-LB reached the maximum at 1h,kept at the peak.The Fibroblasts internalizing COL-FITC-LB reached the maximum at 4h,and kept at stable.The results showed that the percentage of cell phagocytosing COL-FITC-LB was 10 fold of phagocytosing IgG-FITC-LB in 6h(p＜0.05).2.After sorting from fibroblasts,CPSF were cultured in vitro,and adhered to wall and died off after 24h.But majority nCPSF adhere to wall in vitro,nCPSF could grow and proliferate normally. There was an interesting phenomenon of nCPSF.At the first passage,the percentage of cells internalizing COL-FITC-LB was 12.17%among nCPSF.At the second,the percentage increased to 34.76%.At the third,the percentage is up to 60.65%,close to normal fibroblasts that were not sorted.3.17 differentially expressed genes,including 12 up-regulated and 5 down-regulated genes,were screened out from 18400 transcripts, representing 14500 distinct genes in CPSF and nCPSF.4.According the relationship with collagen phgaocytosis,3 genes of 12 up-regulated genes,including uPARAP,CYBB and HOOK1, were confirmed using real-time PCR.The results showed that uPARAP mRNA expression level was 2788 times in CPSF more than nCPSF.CYBB mRNA expression was only 0.85 times in CPSF more than nCPSF.HOOK1 mRNA expression was 1.96 times in CPSF more than nCPSF.5.mRNA expression levels of uPARAP and integrinα₂ were determined in CPSF and nCPSF from other 4 volunteers.The results revealed that uPARAP mRNA was highly expressed in CPSF,and was very low in nCPSF.But integrinα₂ mRNA expression was not significant difference between CPSF and nCPSF.6.Protein expression of uPARAP and integrinα₂ were displayed in CPSF and nCPSF from other 4 volunteers.The results showed that uPARAP was highly expressed in CPSF,and was very low in nCPSF.But integrinα₂ expression was not significant between CPSF and nCPSF.CONCLUSIONS:1.A novel method has been successfully established to separate CPSF and nCPSF using COL-FITC-LB phagocytosis of fibroblast and FCM sorting,which is different from the classical cells sorting method.This provides a new approach to explore the physiological and pathological roles of CPSF and nCPSF.2.uPARAP was the different expression gene in CPSF and nCPSF, uPARAP served as a functional biomarker of CPSF,was confirmed from gene and protein expression.It could be used to identify and sort CPSE This provides a novel molecular target for investigation of collagen degradation.3.In the present,we confirmed that uPARAP is a main collagen receptor of fibroblast and a candidate biomarker for treatment of collagen diseases.