Transcription Characteristics And Regulation Of Collagen Gene In High-collagen-producing Fibroblast Clones Derived From Patients With Systemic Scleroderma

PartⅠIsolation of dermal fibroblast clones from systemic scleroderma patients and normal controls and separation of clones into different subpopulations by amount of collagen producedObjective To isolate dermal fibroblast clones from patients of systemic scleroderma(SSc) and normal controls and separate the clones into different subpopulations by the amount of collagen produced.Methods Fibroblast cell lines from sclerotic skin of SSc patients and skin of normal controls were established and identified by immunohistology.By means of modified limiting dilution cloning,single cells were isolated and cultured.In situ hybridization was used to detect mRNA steady-state level ofα1(Ⅰ) procollagen gene(COL1A1) of fibroblast clones.Results Five fibroblast cell lines from SSc and four from normal controls were established,from which 68 fibroblast clones(4th-passage fibroblast clones) were obtained,consisting of 36 clones from SSc group and 32 clones from normal controls. The mean COL1A1 mRNA steady-state level of fibroblast clones from SSc group (6.636±2.480) was much higher than normal controls(3.693±2.007)(t=-3.690, P=0.001).All the clones were divided into 3 subpopulations(high,intermediate and low) by K-means cluster analysis of COL1A1 mRNA levels.The total constituent ratios of the high,intermediate and low COL1A1 mRNA levels subpopulations between SSc group and normal controls were significantly different(P=0.005).A larger proportion of high COL1A1 mRNA level subpopulation was observed in SSc group compared with normal controls.Based on the above observations,another member of our project examined the typeⅠprocollagen production of the fibroblast clones by western blot and ELISA and COL1A1/α1(Ⅲ) procollagen(COL3A 1) mRNA levels by real-time RT-PCR(Taqman). The results showed the heterogeneity of COL1A1/COL3A1 mRNA of the fibroblast clones from SSc patients was more significant than clones from normal controls.The amounts of typeⅠprocollagen produced by the fibroblast clones paralleled their mRNA levels,and the COL1A1 mRNA levels paralleled their COL3A1mRNA levels. All the fibroblast clones were divided into high,intermediate and low collagen-producing subpopulations.A larger proportion and a higher extent of increased collagen production of high collagen-producing subpopulation were observed in SSc group compared with normal controls.Conclusions The proportion and the extent of increased collagen production of high collagen-producing subpopulation were elevated in fibroblast clones from patients with SSc compared with normal controls.PartⅡTranscription characteristics of procollagen gene in subpopulations of SSc fibroblast clones with different amounts of collagen producedObjective To study the transcription characteristics of COL1A1 and COL3A1 gene in subpopulations of SSc fibroblast clones with different amounts of collagen produced.Methods Five recombinant plasmids containing various lengths of sequences of human COL1A1 and COL3A1 promoter were constructed.By means of cationic liposome-mediated transient transfection and dual-luciferase reporter assay system, the activity of different fragments of COL1A1 and COL3A1 promoter in fibroblast clones subpopulations from SSc and normal controls with different amounts of collagen produced was measured.By ChIP and qPCR,the binding activity between Sp1 and COL1A1 proximal promoter was examined.Results All of the recombinant plasmids targeting on COL1A1 and COL3A1 promoter were confirmed by agarose electrophoresis and DNA sequencing analysis of the inserted segments.In most of the examined clones from different subpopulations,maximal activity was involved COL1A1 promoter region encompassing -174bp～+42bp and COL3A1 promoter region encompassing -96bp～+16bp,which paralleled the amounts of their typeⅠ/typeⅢprocollagen production.ChIP and qPCR showed that the binding activity between Sp1 and COL1A1-174bp～+42bp was elevated in high collagen-producing subpopulation, compared with low collagen-producing subpopulation(P＜0.05),and the difference was more significant in SSc group compared with normal controls(F=24.569, P=0.000).Conclusions The expression of COL1A1 and COL3A1 gene in high collagen-producing fibroblast clones from patients with SSc may be upregulated at the transcriptional level,in which Spl may be involved.PartⅢTranscription regulation of cytokines and Radix Salviae Miltiorrhizae on procollagen gene in subpopulations of SSc fibroblast clones with different amounts of collagen producedObjective To study the transcription regulation effect of cytokines and Radix Salviae Miltiorrhizae on COL1A1 and COL3A1 gene in subpopulations of SSc fibroblast clones with different amounts of collagen produced.Methods By transient transfection and dual-luciferase reporter assay system,the activities of COL1A1-174bp～+42bp and COL3A1-96bp～+16bp in subpopulations of SSc fibroblast clones with different amounts of collagen produced under cytokines, Radix Salviae Miltiorrhizae and its active monomers were detected.Results TGF-β1(5ng/mL) and CTGF(20ng/mL) upregulated the activities of COL1A1-174bp～+42bp and COL3A1-96bp～+16bp in fibroblast clones from patients with SSc and normal controls,and CTGF preferentially upregulated the activity of COL3A1-96bp～+16bp in low collagen-producing subpopulations of SSc fibroblast clones.No inhibiting effects of IFN-γ(100ng/mL) on COL1A1-174bp～+42bp or COL3A1-96b～+16bp activities were observed.The water soluble extracts of Radix Salviae Miltiorrhizae(1mg/mL) and Yanshinone IIA(5μg/mL) inhibited the activities of COL1A1-174bp～+42bp and COL3A1-96bp～+16bp in fibroblast clones from SSc patients and normal controls,preferentially downregulating the activities of COL1A1-174bp～+42bp and COL3A1-96bp～+16bp in high collagen-producing subpopulations of SSc fibroblast clones.Protocatechuic aldehyde(5μg/mL) and Salvianolic acid B(5μg/mL) respectively inhibited the activities of COL1A1-174bp～+42bp and COL3A1-96bp～+16bp in fibroblast clones.There were no significant effects of Danshensu(20μg/mL) on COL1A1-174bp～+42bp and COL3A1-96bp～+16bp activities in fibroblast clones.Conelusions Cytokines may constitute a complex network of transcriptional regulation involved in activation of procollagen gene in SSc fibroblasts.Radix Salviae Miltiorrhizae and some of its active monomers played different roles in inhibiting COL1A1 and COL3A1 gene transcription in SSc fibroblasts.