Participation of transcription factors in the α2(I) collagen gene's down-regulation by PKC inhibitors in normal fibroblasts

<p><b>Copyright information:</b></p><p>Taken from "α2(I) collagen gene regulation by protein kinase C signaling in human dermal fibroblasts"</p><p>Nucleic Acids Research 2005;33(4):1337-1351.</p><p>Published online 1 Mar 2005</p><p>PMCID:PMC552962.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> () To determine the amounts of Sp1 or Sp3 in cell lysates, fibroblasts were serum-starved for 24 h and treated with PKC inhibitors for the period indicated. Immunoblotting was performed using anti-Sp1 or Sp3 antibody. The same membrane was then stripped and reprobed with anti-β-actin antibody as a loading control. () Dermal fibroblasts were serum-starved for 24 h and treated with PKC inhibitors for 24 h. Cell lysates were immunoprecipitated with anti-Sp1 antibody to examine the phosphorylation levels of Sp1 as described in ‘Materials and Methods’. One experiment representative of three independent experiments is shown. Phosphorylated Sp1 levels quantitated by scanning densitometry and corrected for the total level of Sp1 in the same samples are shown relative to the level in untreated cells (1.0). ( and ) For immunoblot analysis, the transient transfection of the Sp1 or Sp3 expression vector in normal dermal fibroblasts was performed as described in ‘Materials and Methods’. Twenty-four hours after the transfection, cells were treated with the indicated dose of PKC inhibitors for 72 h. Conditioned media and cell lysates (normalized for protein concentrations as measured with the Bio-Rad reagent) were subjected to immunoblotting with anti-type I collagen antibody and antibody for HA or β-actin, respectively. Type I procollagen protein levels quantitated by scanning densitometry and corrected for the levels of β-actin in the same samples are shown relative to the level in untreated cells (1.0). () To determine the amounts of Ets1 or Fli1 in cell lysates, human dermal fibroblasts were serum-starved for 24 h and treated with rottlerin for the period indicated. Immunoblotting was performed using anti-Ets1 or Fli1 antibody. The same membrane was then stripped and reprobed with anti-β-actin antibody as a loading control. () Cell lysates were prepared from normal fibroblasts in the presence or absence of rottlerin for 12 h and incubated with the COL-EBS oligo or the COL-EBS-M oligo as described under ‘Materials and Methods’. Proteins bound to these nucleotides were isolated with streptavidin-agarose beads, and Sp1, Ets1 or Fli1 was detected by immunoblotting. The levels of Ets1 (open bars) and Fli1 (closed bars) bound to COL-EBS oligo quantitated by scanning densitometry are shown relative to the level in untreated cells (1.0). The mean ± SE from four independent experiments is presented. * < 0.05 as compared with the value in untreated cells. EBS, Ets-binding site. () Ets1 or Fli1 phosphorylation on threonine (top panel) or serine (middle panel) was determined in fibroblasts treated with rottlerin for the indicated periods of time. Ets1 or Fli1 levels were determined using monoclonal anti-Ets1 or Fli1 antibody on the same filter (bottom panel)