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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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3D collagen lattice induces NF-κB activation. (A) Normal human dermal fibroblasts were transfected with a CAT reporter plasmid that contains 4× κB consensus sequence before  stimulation by collagen gel for 18–24 h. CAT activity was assayed  and subjected to TLC. T, tissue culture; C, collagen gel; Vector,  plasmid without 4× κB; κB, plasmid with 4× κB; mκB, plasmid  with mutated 4× κB. (B) Gel mobility supershift assay was performed with nuclear extracts prepared from fibroblasts grown on  tissue culture plates (TC) or collagen gel (COL). The results  shown are representative of four independent experiments. S1,  anti-P50–NF-κB–DNA complex; S2, anti-P65–NF-κB–DNA complex. NF-κB, (p50-p65)–DNA complex.
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Figure 1: 3D collagen lattice induces NF-κB activation. (A) Normal human dermal fibroblasts were transfected with a CAT reporter plasmid that contains 4× κB consensus sequence before stimulation by collagen gel for 18–24 h. CAT activity was assayed and subjected to TLC. T, tissue culture; C, collagen gel; Vector, plasmid without 4× κB; κB, plasmid with 4× κB; mκB, plasmid with mutated 4× κB. (B) Gel mobility supershift assay was performed with nuclear extracts prepared from fibroblasts grown on tissue culture plates (TC) or collagen gel (COL). The results shown are representative of four independent experiments. S1, anti-P50–NF-κB–DNA complex; S2, anti-P65–NF-κB–DNA complex. NF-κB, (p50-p65)–DNA complex.

Mentions: Previously we observed that 3D COL induces integrin α2 mRNA expression by activating protein kinase C (PKC)-ζ (Xu and Clark, 1997). PKC-ζ has been shown to play an essential role in activating NF-κB by dominant-negative PKC-ζ blocking TNF-α–stimulated NF-κB activity and constitutively active PKC-ζ activating NF-κB in NIH 3T3 cells (Diaz-Meco et al., 1993, 1994; Dominguez et al., 1993; Folgueira et al., 1996). We also obtained evidence that the DNA binding activity of NF-κB, along with the activation of PKC-ζ and integrin α2 mRNA expression, is induced by 3D COL (Xu and Clark, 1997). Here we examined whether 3D COL can signal the induction of transactivating activity of NF-κB in human dermal fibroblasts. A κB-CAT reporter plasmid was constructed by inserting four repeats of NF-κB consensus element into an SV-40 promoter CAT reporter plasmid vector. Fibroblasts were transfected with the plasmid followed by subculture in 3D COL. As shown in Fig. 1 A, 3D COL induced transactivating activity of NF-κB, consistent with the observation made in DNA binding activity (Xu and Clark, 1997). A control plasmid containing the mutant NF-κB recognition sequences did not demonstrate the inducibility by 3D COL (Fig. 1 A). The composition of the NF-κB DNA-binding complex was examined next by gel mobility supershift assay. The p50 and p65(RelA) of NF-κB family were found present in the binding complex (Fig. 1 B). The composition of the DNA-binding complex remained unchanged during full time-course (24 h) of the induction examined (data not shown).


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)

3D collagen lattice induces NF-κB activation. (A) Normal human dermal fibroblasts were transfected with a CAT reporter plasmid that contains 4× κB consensus sequence before  stimulation by collagen gel for 18–24 h. CAT activity was assayed  and subjected to TLC. T, tissue culture; C, collagen gel; Vector,  plasmid without 4× κB; κB, plasmid with 4× κB; mκB, plasmid  with mutated 4× κB. (B) Gel mobility supershift assay was performed with nuclear extracts prepared from fibroblasts grown on  tissue culture plates (TC) or collagen gel (COL). The results  shown are representative of four independent experiments. S1,  anti-P50–NF-κB–DNA complex; S2, anti-P65–NF-κB–DNA complex. NF-κB, (p50-p65)–DNA complex.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2140166&req=5

Figure 1: 3D collagen lattice induces NF-κB activation. (A) Normal human dermal fibroblasts were transfected with a CAT reporter plasmid that contains 4× κB consensus sequence before stimulation by collagen gel for 18–24 h. CAT activity was assayed and subjected to TLC. T, tissue culture; C, collagen gel; Vector, plasmid without 4× κB; κB, plasmid with 4× κB; mκB, plasmid with mutated 4× κB. (B) Gel mobility supershift assay was performed with nuclear extracts prepared from fibroblasts grown on tissue culture plates (TC) or collagen gel (COL). The results shown are representative of four independent experiments. S1, anti-P50–NF-κB–DNA complex; S2, anti-P65–NF-κB–DNA complex. NF-κB, (p50-p65)–DNA complex.
Mentions: Previously we observed that 3D COL induces integrin α2 mRNA expression by activating protein kinase C (PKC)-ζ (Xu and Clark, 1997). PKC-ζ has been shown to play an essential role in activating NF-κB by dominant-negative PKC-ζ blocking TNF-α–stimulated NF-κB activity and constitutively active PKC-ζ activating NF-κB in NIH 3T3 cells (Diaz-Meco et al., 1993, 1994; Dominguez et al., 1993; Folgueira et al., 1996). We also obtained evidence that the DNA binding activity of NF-κB, along with the activation of PKC-ζ and integrin α2 mRNA expression, is induced by 3D COL (Xu and Clark, 1997). Here we examined whether 3D COL can signal the induction of transactivating activity of NF-κB in human dermal fibroblasts. A κB-CAT reporter plasmid was constructed by inserting four repeats of NF-κB consensus element into an SV-40 promoter CAT reporter plasmid vector. Fibroblasts were transfected with the plasmid followed by subculture in 3D COL. As shown in Fig. 1 A, 3D COL induced transactivating activity of NF-κB, consistent with the observation made in DNA binding activity (Xu and Clark, 1997). A control plasmid containing the mutant NF-κB recognition sequences did not demonstrate the inducibility by 3D COL (Fig. 1 A). The composition of the NF-κB DNA-binding complex was examined next by gel mobility supershift assay. The p50 and p65(RelA) of NF-κB family were found present in the binding complex (Fig. 1 B). The composition of the DNA-binding complex remained unchanged during full time-course (24 h) of the induction examined (data not shown).

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