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A three-dimensional collagen lattice activates NF-kappaB in human fibroblasts: role in integrin alpha2 gene expression and tissue remodeling.

Xu J, Zutter MM, Santoro SA, Clark RA - J. Cell Biol. (1998)

Bottom Line: Clark. 1997.The inhibition of NF-kappaB activity by SN50, a peptide inhibitor targeted at nuclear translocation of NF-kappaB, significantly reduced the induction of integrin alpha2 mRNA and protein by the collagen lattice.Therefore, an indirect regulatory mechanism by NF-kappaB in integrin alpha2 gene expression induced by three-dimensional collagen lattice is suggested.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, School of Medicine, State University of New York, Stony Brook, New York 11794-8165, USA. JXu@epo.som.sunysb.edu

ABSTRACT
Normal adult human dermal fibroblasts grown in a three-dimensional collagen lattice increase mRNA level of collagen receptor integrin subunit alpha2 (Xu, J., and R.A.F. Clark. 1996. J. Cell Biol. 132:239- 249.) and DNA binding activity of a nuclear transcription factor, NF-kappaB (Xu, J., and R.A.F. Clark. 1997. J. Cell Biol. 136:473-483.). Here we present evidence that the collagen lattice induced the nuclear translocation of p50, one member of NF-kappaB family, and the degradation of an NF-kappaB inhibitor protein, IkappaB-alpha. The inhibition of NF-kappaB activity by SN50, a peptide inhibitor targeted at nuclear translocation of NF-kappaB, significantly reduced the induction of integrin alpha2 mRNA and protein by the collagen lattice. A region located between -549 and -351 bp in the promoter of integrin alpha2 gene conferred the inducibility by three-dimensional collagen lattice. The presence of either SN50 or IkappaB-alpha32, 36, a stable mutant of IkappaB-alpha, abrogated this inducibility, indicating that the activation of integrin alpha2 gene expression was possibly mediated by NF-kappaB through this region. Although there were three DNA-protein binding complexes forming in this region that are sensitive to the inhibition of NF-kappaB nuclear translocation, NF-kappaB was not directly present in the binding complexes. Therefore, an indirect regulatory mechanism by NF-kappaB in integrin alpha2 gene expression induced by three-dimensional collagen lattice is suggested. The involvement of NF-kappaB in reorganization and contraction of three-dimensional collagen lattice, a process that requires the presence of abundant integrin alpha2beta1, was also examined. The inhibition of NF-kappaB activity by SN50 greatly blocked the contraction, suggesting its critical role in not only the induction of integrin alpha2 gene expression by three-dimensional collagen lattice, but also alpha2beta1-mediated tissue-remodeling process.

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NF-κB mediates DNA–protein binding complex formation on integrin α2 promoter induced by 3D COL. (A) Three  DNA fragments were generated by PCR using integrin α2 promoter as template and used for gel mobility shift assay. F1, DNA  fragment spanning from −590 to −514 bp; F2, DAN fragment  spanning from −516 to −413 bp; F3, DNA fragment spanning  from −415 to −342 bp. (B) Nuclear extracts were prepared from  fibroblasts grown in 3D COL for the time periods as indicated  and from cells grown in 3D COL for 4 h with or without SN50.  Gel mobility shift assay was performed using labeled F2, an upstream sequence of integrin α2 (−516 to −413 bp). Unlabeled F2  was used as DNA competitor. The results represent two independent experiments. F2, specific F2–protein complexes; NS, nonspecific binding.
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Figure 7: NF-κB mediates DNA–protein binding complex formation on integrin α2 promoter induced by 3D COL. (A) Three DNA fragments were generated by PCR using integrin α2 promoter as template and used for gel mobility shift assay. F1, DNA fragment spanning from −590 to −514 bp; F2, DAN fragment spanning from −516 to −413 bp; F3, DNA fragment spanning from −415 to −342 bp. (B) Nuclear extracts were prepared from fibroblasts grown in 3D COL for the time periods as indicated and from cells grown in 3D COL for 4 h with or without SN50. Gel mobility shift assay was performed using labeled F2, an upstream sequence of integrin α2 (−516 to −413 bp). Unlabeled F2 was used as DNA competitor. The results represent two independent experiments. F2, specific F2–protein complexes; NS, nonspecific binding.

Mentions: Analysis of DNA sequence in this region revealed that a site located between −457 and −447 bp (GGGACGCACC) shares sequence homology but for one base (underlined) to the NF-κB consensus sequence GGGRNNYYCC (R indicates purine; N is any base; Y is pyrimidine) present in a set of inducible genes expressed by human monocytic and endothelial cells (Parry and Mackman, 1994). The oligonulceotides synthesized based on this sequence, however, mediated neither DNA–protein complex formation as judged by gel mobility shift assay nor transactivation when inserted into an SV-40 promoter-CAT reporter vector as judged by CAT analysis of transfected cells cultured in 3D COL (data not shown). The observation raised a possibility that α2 promoter context is required for the detection of NF-κB binding activity to the region. Nuclear protein binding of the entire α2549–351 region was thus examined. Three DNA fragments were generated by PCR: F1, −590 and −514 bp; F2, −516 and −413 bp; and F3, −415 and −342 bp (Fig. 7 A). Gel mobility shift assay showed the formation of three specific F2– protein complexes, which were moderately increased by 3D COL (Fig. 7 B) whereas F1 and F3 did not demonstrate the specific nuclear protein binding (data not shown). Three approaches were taken to determine whether NF-κB was directly involved in the F2-protein complexes. First, the DNA binding activity of 3D COL–induced nuclear proteins to F2 region of α2 promoter was reduced to approximately basal level by the incubation of cells with SN50, the p50 nuclear translocation inhibitor (Fig. 7 B), suggesting NF-κB activity is required for the binding complex formation. However, the competition with unlabeled NF-κB consensus sequence did not affect the DNA complex formation on F2 (data not shown), suggesting the absence of direct contact of NF-κB with this DNA fragment. Third, supershift assays with antibodies against various proteins of NF-κB family did not yield band shifts (data not shown), supporting the failure of NF-κB to bind this promoter region. Therefore, NF-κB appears to play a critical role in integrin α2 promoter activation by 3D COL through α2549–351 without direct binding to the region.


A three-dimensional collagen lattice activates NF-kappaB in human fibroblasts: role in integrin alpha2 gene expression and tissue remodeling.

Xu J, Zutter MM, Santoro SA, Clark RA - J. Cell Biol. (1998)

NF-κB mediates DNA–protein binding complex formation on integrin α2 promoter induced by 3D COL. (A) Three  DNA fragments were generated by PCR using integrin α2 promoter as template and used for gel mobility shift assay. F1, DNA  fragment spanning from −590 to −514 bp; F2, DAN fragment  spanning from −516 to −413 bp; F3, DNA fragment spanning  from −415 to −342 bp. (B) Nuclear extracts were prepared from  fibroblasts grown in 3D COL for the time periods as indicated  and from cells grown in 3D COL for 4 h with or without SN50.  Gel mobility shift assay was performed using labeled F2, an upstream sequence of integrin α2 (−516 to −413 bp). Unlabeled F2  was used as DNA competitor. The results represent two independent experiments. F2, specific F2–protein complexes; NS, nonspecific binding.
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Related In: Results  -  Collection

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Figure 7: NF-κB mediates DNA–protein binding complex formation on integrin α2 promoter induced by 3D COL. (A) Three DNA fragments were generated by PCR using integrin α2 promoter as template and used for gel mobility shift assay. F1, DNA fragment spanning from −590 to −514 bp; F2, DAN fragment spanning from −516 to −413 bp; F3, DNA fragment spanning from −415 to −342 bp. (B) Nuclear extracts were prepared from fibroblasts grown in 3D COL for the time periods as indicated and from cells grown in 3D COL for 4 h with or without SN50. Gel mobility shift assay was performed using labeled F2, an upstream sequence of integrin α2 (−516 to −413 bp). Unlabeled F2 was used as DNA competitor. The results represent two independent experiments. F2, specific F2–protein complexes; NS, nonspecific binding.
Mentions: Analysis of DNA sequence in this region revealed that a site located between −457 and −447 bp (GGGACGCACC) shares sequence homology but for one base (underlined) to the NF-κB consensus sequence GGGRNNYYCC (R indicates purine; N is any base; Y is pyrimidine) present in a set of inducible genes expressed by human monocytic and endothelial cells (Parry and Mackman, 1994). The oligonulceotides synthesized based on this sequence, however, mediated neither DNA–protein complex formation as judged by gel mobility shift assay nor transactivation when inserted into an SV-40 promoter-CAT reporter vector as judged by CAT analysis of transfected cells cultured in 3D COL (data not shown). The observation raised a possibility that α2 promoter context is required for the detection of NF-κB binding activity to the region. Nuclear protein binding of the entire α2549–351 region was thus examined. Three DNA fragments were generated by PCR: F1, −590 and −514 bp; F2, −516 and −413 bp; and F3, −415 and −342 bp (Fig. 7 A). Gel mobility shift assay showed the formation of three specific F2– protein complexes, which were moderately increased by 3D COL (Fig. 7 B) whereas F1 and F3 did not demonstrate the specific nuclear protein binding (data not shown). Three approaches were taken to determine whether NF-κB was directly involved in the F2-protein complexes. First, the DNA binding activity of 3D COL–induced nuclear proteins to F2 region of α2 promoter was reduced to approximately basal level by the incubation of cells with SN50, the p50 nuclear translocation inhibitor (Fig. 7 B), suggesting NF-κB activity is required for the binding complex formation. However, the competition with unlabeled NF-κB consensus sequence did not affect the DNA complex formation on F2 (data not shown), suggesting the absence of direct contact of NF-κB with this DNA fragment. Third, supershift assays with antibodies against various proteins of NF-κB family did not yield band shifts (data not shown), supporting the failure of NF-κB to bind this promoter region. Therefore, NF-κB appears to play a critical role in integrin α2 promoter activation by 3D COL through α2549–351 without direct binding to the region.

Bottom Line: Clark. 1997.The inhibition of NF-kappaB activity by SN50, a peptide inhibitor targeted at nuclear translocation of NF-kappaB, significantly reduced the induction of integrin alpha2 mRNA and protein by the collagen lattice.Therefore, an indirect regulatory mechanism by NF-kappaB in integrin alpha2 gene expression induced by three-dimensional collagen lattice is suggested.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, School of Medicine, State University of New York, Stony Brook, New York 11794-8165, USA. JXu@epo.som.sunysb.edu

ABSTRACT
Normal adult human dermal fibroblasts grown in a three-dimensional collagen lattice increase mRNA level of collagen receptor integrin subunit alpha2 (Xu, J., and R.A.F. Clark. 1996. J. Cell Biol. 132:239- 249.) and DNA binding activity of a nuclear transcription factor, NF-kappaB (Xu, J., and R.A.F. Clark. 1997. J. Cell Biol. 136:473-483.). Here we present evidence that the collagen lattice induced the nuclear translocation of p50, one member of NF-kappaB family, and the degradation of an NF-kappaB inhibitor protein, IkappaB-alpha. The inhibition of NF-kappaB activity by SN50, a peptide inhibitor targeted at nuclear translocation of NF-kappaB, significantly reduced the induction of integrin alpha2 mRNA and protein by the collagen lattice. A region located between -549 and -351 bp in the promoter of integrin alpha2 gene conferred the inducibility by three-dimensional collagen lattice. The presence of either SN50 or IkappaB-alpha32, 36, a stable mutant of IkappaB-alpha, abrogated this inducibility, indicating that the activation of integrin alpha2 gene expression was possibly mediated by NF-kappaB through this region. Although there were three DNA-protein binding complexes forming in this region that are sensitive to the inhibition of NF-kappaB nuclear translocation, NF-kappaB was not directly present in the binding complexes. Therefore, an indirect regulatory mechanism by NF-kappaB in integrin alpha2 gene expression induced by three-dimensional collagen lattice is suggested. The involvement of NF-kappaB in reorganization and contraction of three-dimensional collagen lattice, a process that requires the presence of abundant integrin alpha2beta1, was also examined. The inhibition of NF-kappaB activity by SN50 greatly blocked the contraction, suggesting its critical role in not only the induction of integrin alpha2 gene expression by three-dimensional collagen lattice, but also alpha2beta1-mediated tissue-remodeling process.

Show MeSH
Related in: MedlinePlus