Transcriptional activation of the type I collagen genes COL1A1 and COL1A2 in fibroblasts by interleukin-4: analysis of the functional collagen promoter sequences
Authors: Annett Skupin
Cordula Büttner
Ernst Peter Rieber
Journal: Journal of Cellular Physiology (2004)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: Pneumonitis; Lung fibrosis; Radiation therapy; Chest tumors; Extracellular matrix proteins
Affiliations: Institute of Immunology, Medical Faculty, Technical University Dresden, Dresden, Germany. 

Abstract:  Pneumonitis followed by lung fibrosis is a frequent complication of radiation therapy of chest tumors. A hallmark of these fibrotic lesions is the excessive production and accumulation of extracellular matrix proteins such as type I collagen. In addition to TGF-beta1, IL-4 has been recognized as a potent inducer of collagen gene synthesis in fibroblasts. In this study, we analyzed the regulation of the alpha1(I) procollagen (COL1A1) promoter and the alpha2(I) procollagen (COL1A2) promoter by IL-4 in normal human lung fibroblasts. We provide evidence that the IL-4-induced transcriptional activator STAT6 binds to various sequences within the COL1A1 and COL1A2 promoter. The regulatory function of these regions was tested by reporter gene analysis using 5' deletions of the COL1A1 and COL1A2 promoter fused to the luciferase gene. Interleukin-4 treatment of human fibroblasts transiently transfected with COL1A1 promoter deletion constructs resulted in luciferase activity exceeding that of untreated fibroblasts by 25%, while luciferase activity driven by the COL1A2 promoter was enhanced by about 70% upon IL-4 treatment. A combined action of SP1, NFkappaB, and STAT6 essentially contributes to the IL-4 mediated COL1A2 gene activation. An AP2 site adjacent to the reverse orientated STAT6 consensus motif TTC N(3/4) GCT is located within 205 bases from the transcription start site and seems to support the moderate IL-4-induced COL1A1 gene activation. Interferon-gamma downregulation of transcription is mainly seen with the COL1A1 promoter. 

Retracted: No

PubPeer comments:
 *Primers for the amplification of the COL1A1, COL1A2, and u03b2u2010actin gene were tested versus BLAST algorithm to ensure that these primers, when combined, would only amplify the respective cDNAs:* *human COL1A1 sense 5u2032u2010GGGTGACCGTGGTGAGAu20103u2032, antisense 5u2032u2010CCAGGAGAGCCAGAGGTCCu20103u2032 spanning nucleotides of exon 42 and 46 (nt 3,218u20133,511) to yield a PCR product of 194 bp;* 1) Putting these sequences into NCBI Primer Blast against the Homo sapiens reference mRNA sequences does not return any COL1A1 transcripts, only COL2A1 with 4 mismatches in F and R primers. 2) Only the F Primer is present in COL1A1 sequence 3) A PCR reaction amplifying from 3218-3511 should anyway be 293 bp long, not 194. 4) I have tried Revcomp, reversing the R primer sequence in case of error, no matches. 5) The nearest sequence match is CCAGGGGGACCAGGAGGTCC. It has 2 insertion changes and some mismatches, and would generate a 473 bp product. Is this the result of sequence revision, transcription error or something else? Could the authors look at this issue, please? The COL1A2 primers look fine (294bp amplified as stated), but the Beta Actin primers listed do not amplify sequence from the ACTB gene. ,