Transforming growth factor-beta1 up-regulation of human alpha(1)(I) collagen is mediated by Sp1 and Smad2 transacting factors - PubMed (original) (raw)

Transforming growth factor-beta1 up-regulation of human alpha(1)(I) collagen is mediated by Sp1 and Smad2 transacting factors

Polina Sysa et al. DNA Cell Biol. 2009 Sep.

Abstract

Hepatic fibrosis results from excessive deposition of type I collagen. The roles of Smads in mediating the effect of transforming growth factor-beta1 (TGFbeta1) on activation of the alpha(1)(I) collagen promoter were determined. Smads bind in association with Sp1 to the CC(GG)-rich TGFbeta1 responsive element of the promoter that lacks the classical Smad recognition element, and enhance binding of Sp1. In transfection experiments, TGFbeta1 activated a proximal promoter, but not promoters mutated at sites that prevented Sp1 binding. Sp1 alone or the combination of Smad2 and Smad4 activated the promoter in transfected human LX-2 stellate cells. Sp1 or Smad2 knockdowns with siRNAs prevented the effect of TGFbeta1 in enhancing the promoter. In conclusion, this study shows that Smads bind in association with Sp1 to the CC(GG)-rich TGFbeta1 responsive element of the human alpha(1)(I) collagen promoter that lacks the classical Smad recognition element, thus enhancing the binding of Sp1 and in this manner activating the collagen promoter.

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Figures

FIG. 1.

FIG. 1.

Mutational analysis of the TGFβ1 responsive element of the α1(I) collagen promoter. EMSA was performed with 1 pg of the labeled Sp1.1 oligonucleotide and 8 μg of nuclear extracts form LX-2 human stellate cells. The arrowheads indicate the protein–DNA complexes formed. The boldface letters indicate the mutated sequences in each mutant. NS, nonspecific band; F, the free probe; TGFβ1, transforming growth factor β1; EMSA, electrophoretic mobility shift assay.

  1. WT: 5′-CTTCCCTCCTCCTCCCCCTCTCC-3′
  2. M1: 5′-CTTTTCTCCTCCTCCCCCTCTCC-3′
  3. M2: 5′-CTTCCTACCTCCTCCCCCTCTCC-3′
  4. M3: 5′-CTTCCCTTTTCCTCCCCCTCTCC-3′
  5. M4: 5′-CTTCCCTCCATCTCCCCCTCTCC-3′
  6. M5: 5′-CTTCCCTCCTTTACCCCCTCTCC-3′
  7. M6: 5′-CTTCCCTCCTCCTTTCCCTCTCC-3′
  8. M7: 5′-CTTCCCTCCTCCTCCTTCTCTCC-3′
  9. M8: 5′-CTTCCCTCCTCCTCCCCTACTCC-3′
  10. M9: 5′-CTTCCCTCCTCCTCCCCCTTACC-3′

FIG. 2.

FIG. 2.

EMSA showing binding of nuclear extracts form LX-2 stellate cells to the wild-type (wt) Sp1.1 oligonucleotide in the presence of excess competing unlabeled oligonucleotide. The complexes formed are indicated by the arrowheads. NS indicates nonspecific band; F indicates the position of the free probe.

FIG. 3.

FIG. 3.

EMSA with supershift showing binding of nuclear extracts form LX-2 stellate cells to the Sp1.1 oligonucleotide. The EMSAs were performed with 10 μg of nuclear extract and the labeled oligonucleotide. For supershifts the antibodies (Ab) to Smad2/3, Smad4, and Sp1 (1:200 dilution) or preimmune serum (PI) were added to the reaction mixture. The arrowheads indicate the protein–DNA complexes formed. The arrows indicate the location of the supershifted complexes. NS indicates a nonspecific band; F indicates the position of the free probe.

FIG. 4.

FIG. 4.

EMSA with supershift showing binding of nuclear extracts form LX-2 stellate cells to the Sp1.1 oligonucleotide. The EMSAs were performed with 10 μg of nuclear extract and the labeled oligonucleotide. For supershifts the antibody (Ab) to cKrox (1:200 dilution) or preimmune serum (PI) was added to the reaction mixture. The arrowheads indicate the protein–DNA complexes formed. The arrows indicate the location of the supershifted complexes. NS indicates a nonspecific band; F indicates the position of the free probe.

FIG. 5.

FIG. 5.

Autoradiography and Western blot showing the presence of Smad2, Smad3, Smad4, pSmad2, pSmad3, Sp1, and cKrox in nuclear extract from TGFβ1-treated LX-2 cells UV crosslinked to the labeled Sp1.1 oligonucleotide. The cells were exposed to TGFβ1 (10 ng/mL) for 24 h. The blots were exposed to film to determine the UV binding of the proteins and incubated with antibodies to the Smads, Sp1, and cKrox. The location of the bound proteins and their molecular mass (kDa) obtained from protein standards is shown.

FIG. 6.

FIG. 6.

Chromatin immunoprecipitation (ChIP) assay of Sp1 and Smad binding. One- (1×) and two-step (2×) cross-linked DNA–protein complexes were immunoprecipitated with antibodies to Sp1, Smad 2/3, and Smad4. Negative control was performed with purified rabbit IgG. The covalent linkage was reversed, and the precipitated double-stranded DNA was amplified to the region −199 to −93 (106 bp) of the α1(I) collagen promoter.

FIG. 7.

FIG. 7.

Effect of Smad expression vectors (pRK5F-Smad2, pRK5F-Smad3, and pRK-DPC4–Flag [Smad4]) on the activity of α1(I) collagen promoters in transfected LX-2 cells in (A) the absence and (B) the presence of the Sp1 expression vector (pPacSp1). Luciferase activity is expressed as the percentages of the control empty vectors, pRK5 (for Smads), pPadh (for Sp1), or their combination. The data are expressed as means ± SE of six determinations. *p < 0.05 versus control; **p < 0.01 versus control; ***p < 0.001 versus control.

FIG. 8.

FIG. 8.

Effect of Smad expression vectors (pRK5F-Smad2, pRK5F-Smad3, and pRK-DPC4–Flag [Smad4]) on the activity of α1(I) collagen promoters in transfected Drosophila Schneider cells in the absence and the presence of the Sp1 expression vector (pPacSp1). Luciferase activity is expressed as the percentages of the control empty vectors, pRK5 (for Smads), pPadh (for Sp1), or their combination. The data are expressed as means ± SE of six determinations. *p < 0.05 versus control; **p < 0.01 versus control; ***p < 0.001 versus control.

FIG. 9.

FIG. 9.

Effects of Sp1 and Smad siRNAs on protein expression of Sp1 and Smads determined by Western blot in the presence of TGFβ1 (10 ng/mL). C, negative control siRNA. KD, knock down. The values are expressed as means ± SE of relative densitometry of six measurements per group *p < 0.05 versus control; **p < 0.01 versus control.

FIG. 10.

FIG. 10.

Effects of selective inhibition of Sp1, Smad2, Smad3, and Smad4 by siRNAs on the activity of the cotransfected pGL3-2.3k α1 wt collagen promoter in LX-2 cells treated or not treated with TGFβ1 (10 ng/mL). C, negative control siRNA. KD, knock down. The data are expressed as means ± SE of six determinations. *p < 0.05 versus respective group not treated with TGFβ1; **p < 0.01 versus respective group not treated with TGFβ1; X_p_ < 0.05 versus control not treated with TGFβ1; XX_p_ < 0.01 versus control not treated with TGFβ1; XXX_p_ < 0.01versus control not treated with TGFβ1.

FIG. 11.

FIG. 11.

Effect of TGFβ1 on the α1(I) collagen promoter in _Smad3_−/− (Smad3 KO) mouse embryonic fibroblasts (MEF). The pGL3-2.3k α1 wt promoter and the p3TP-lux reporter, a TGFβ1 inducible reporter containing three TGFβ1-binding elements, were transfected into wt and Smad3−/− MEF. The cells were exposed or not exposed to TGFβ1 (10 ng/mL) for 24 h. Luciferase activity is expressed as a percentage of control. Data are presented as means ± SE of six determinations. *p < 0.05 versus control without TGFβ1; **p < 0.001 versus control without TGFβ1.

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