TRANSCRIPTIONAL ACTIVATION OF THE ALPHA-1(I) PROCOLLAGEN GENE IN SYSTEMIC-SCLEROSIS DERMAL FIBROBLASTS - ROLE OF INTRONIC SEQUENCES

Objective, To investigate the transcriptional regulation of the alpha 1(I) procollagen gene (COL1A1) in cultured dermal fibroblasts from pat ients with diffuse systemic sclerosis (SSc) of recent onset and to eva luate the role that intronic sequences may play in the upregulated exp ression of COL1A1 in SSc dermal fibroblasts, Methods, Dermal fibroblas ts from 6 patients with diffuse SSc of recent onset and from 3 healthy individuals were studied, The steady-state levels of alpha 1(I) proco llagen messenger RNA were evaluated by Northern hybridization analysis , and the transcriptional regulation of COL1A1 was examined by transie nt transfection experiments,vith deletion constructs containing portio ns of COL1A1 promoter (with 5' end points at -5.3 kb, -2.3 kb, and -80 4 bp and 3' end point at +42 bp) ligated to the chloramphenicol acetyl transferase (CAT) reporter gene, To examine the role of intronic seque nces, constructs containing, in addition to the COL1A1 promoter, a por tion of the first intron (+380 bp to +1,440 bp) cloned in front of the CAT gene were transfected, The efficiency of transfections was normal ized relative to the net amount of CAT plasmid actually transfected in to recipient cells, determined by a modified Southern hybridization pr ocedure, Results, Maximal CAT activity was observed with constructs ex tending from -804 bp to +42 bp in both normal and SSc fibroblasts, How ever, the activity driven by this construct was 80-110% higher in SSc fibroblasts, The CAT activity driven by a construct with a 5' end poin t at -5.3 kb was only 15-20% higher in SSc cells, and the CAT activity driven by a construct with a 5' end point -2.3 kb was 35-45% higher i n SSc fibroblasts, The CAT activity driven by the -804-bp promoter con struct was increased up to 4-fold in SSc fibroblasts in comparison wit h normal cells when the intronic segment spanning +380 bp to +1,440 bp was included in the transfected construct, Conclusion, The results di rectly demonstrate the transcriptional activation of COL1A1 in dermal fibroblasts from SSc patients, The data also indicate that first-intro n sequences of COL1A1 are required for maximal transcriptional activit y of the collagen gene and may play an important role in the up-regula tion of its expression in SSc fibroblasts.