Y Yamaguchi1, S Crane, L Zhou, S M Ochoa, V Falanga. 1. Boston University School of Medicine, Roger Williams Medical Center, Department of Dermatology and Skin Surgery, 50 Maude Street, Providence, RI 02908, USA.
Abstract
BACKGROUND: Several extracellular matrix genes, most notably alpha1(I) and alpha1(III) procollagen, are reported to be co-ordinately expressed in cultures of dermal fibroblasts. However, it remains unclear whether the expression of these genes is truly co-ordinate or whether it may be the result of averaging the phenotypic expression of different fibroblast subpopulations present within each culture. Objectives To determine by Northern analysis the correlation between alpha1(I) and alpha1(III) procollagen mRNA levels in clonal populations of human dermal fibroblasts. METHODS: As previously described, clonal cultures were derived from parent strains of human dermal fibroblasts by a microscopically controlled dilution technique and by stimulation of single cells with low oxygen tension in the early phases of clonal growth. RESULTS: In agreement with previous reports, we found that baseline steady-state levels of alpha1(I) procollagen mRNA were co-ordinately regulated with the alpha1(III) procollagen mRNA in 26 parent strains (r = 0. 9003; P < 0.0001). However, this close correlation between the expression of these two procollagen chains was absent in a total of 40 unselected clonal strains derived from four of the parent cultures (r = 0.5745; P < 0.0001). Moreover, this intrachain heterogeneity in alpha1(I) and alpha1(III) procollagen mRNA levels in clonal cultures was statistically significant from that measured in parent strains (P = 0.0016). CONCLUSIONS: alpha1(I) and alpha1(III) procollagen mRNA levels in clonal cultures do not show the tight co-ordinate regulation observed in non-clonal cultures, suggesting that these two genes operate under different sets of regulatory controls. This clonal heterogeneity may provide additional flexibility to the process of tissue repair and fibroblast clonal expansion.
BACKGROUND: Several extracellular matrix genes, most notably alpha1(I) and alpha1(III) procollagen, are reported to be co-ordinately expressed in cultures of dermal fibroblasts. However, it remains unclear whether the expression of these genes is truly co-ordinate or whether it may be the result of averaging the phenotypic expression of different fibroblast subpopulations present within each culture. Objectives To determine by Northern analysis the correlation between alpha1(I) and alpha1(III) procollagen mRNA levels in clonal populations of human dermal fibroblasts. METHODS: As previously described, clonal cultures were derived from parent strains of human dermal fibroblasts by a microscopically controlled dilution technique and by stimulation of single cells with low oxygen tension in the early phases of clonal growth. RESULTS: In agreement with previous reports, we found that baseline steady-state levels of alpha1(I) procollagen mRNA were co-ordinately regulated with the alpha1(III) procollagen mRNA in 26 parent strains (r = 0. 9003; P < 0.0001). However, this close correlation between the expression of these two procollagen chains was absent in a total of 40 unselected clonal strains derived from four of the parent cultures (r = 0.5745; P < 0.0001). Moreover, this intrachain heterogeneity in alpha1(I) and alpha1(III) procollagen mRNA levels in clonal cultures was statistically significant from that measured in parent strains (P = 0.0016). CONCLUSIONS: alpha1(I) and alpha1(III) procollagen mRNA levels in clonal cultures do not show the tight co-ordinate regulation observed in non-clonal cultures, suggesting that these two genes operate under different sets of regulatory controls. This clonal heterogeneity may provide additional flexibility to the process of tissue repair and fibroblast clonal expansion.