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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 3D COL contraction. Fibroblasts were  pretreated with SN50, an inhibitor for NF-κB nuclear translocation, or SM, a peptide control, for 30 min before stimulation by  3D COL. The incubation was continued for 24 h. Cells released  from 3D COL by collagenase digestion were subcultured into 3D  COL for the measurement of gel contraction in the presence of  SN50 or other additives. The surface diameters (A) of 3D COLs  were measured from the photographs (B) taken at 4, 20, 48, and  72 h after subculture. The results represent 10 independent experiments. (C) Western analysis of proteins extracted from fibroblasts cultured in 3D COL for 20, 48, and 72 h in the presence of  either SM or SN50. The samples were run on 6% SDS–polyacrylamide gel. The blot was detected by an anti-α2 antibody and visualized with alkaline phosphatase/NBT/BCIP method as described  in the Materials and Methods. The integrin α2 subunit is marked  by an arrow. The results represent two independent experiments.
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Figure 8: NF-κB mediates 3D COL contraction. Fibroblasts were pretreated with SN50, an inhibitor for NF-κB nuclear translocation, or SM, a peptide control, for 30 min before stimulation by 3D COL. The incubation was continued for 24 h. Cells released from 3D COL by collagenase digestion were subcultured into 3D COL for the measurement of gel contraction in the presence of SN50 or other additives. The surface diameters (A) of 3D COLs were measured from the photographs (B) taken at 4, 20, 48, and 72 h after subculture. The results represent 10 independent experiments. (C) Western analysis of proteins extracted from fibroblasts cultured in 3D COL for 20, 48, and 72 h in the presence of either SM or SN50. The samples were run on 6% SDS–polyacrylamide gel. The blot was detected by an anti-α2 antibody and visualized with alkaline phosphatase/NBT/BCIP method as described in the Materials and Methods. The integrin α2 subunit is marked by an arrow. The results represent two independent experiments.

Mentions: It has been reported by various laboratories that integrin α2β1 mediates reorganization and contraction of collagen gels by human cells including fibroblasts (Schiro et al., 1991), cutaneous squamous carcinoma cells (Fujii et al., 1995), retinal pigment epithelial cells (Kupper and Ferguson, 1993), and transformed osteosarcoma cells (Riikonen et al., 1995). It was proposed that stimulants such as EGF (Fujii et al., 1995) and TGF-β (Riikonen et al., 1995b) induce 3D COL contraction by increasing integrin α2β1 expression. The transfection of integrin α2 cDNA into a cell line RD cells that expresses β1 chain but possess a very low level of α2β1 integrin restores the ability of RD cells to contract collagen gels (Schiro et al., 1991). We hypothesized that since NF-κB mediated α2 gene expression, it may be in the regulatory pathway leading to α2-mediated collagen gel contraction. The effects of NF-κB on 3D COL contraction by fibroblasts were examined. Treatment of cells with SN50, but not SM, significantly slowed the contraction process during 72 h examined (Fig. 8, A and B). The expression of integrin α2 protein by fibroblasts cultured in 3D COL was similarly reduced by SN50 but not SM (Fig. 8 C). The amount of a nonspecific band of low molecular weight, serving as an internal control, was similar in all conditions (Fig. 8 C), confirming the specificity of the inhibtion. Therefore, it further indicates that NF-κB was involved in the integrin α2 gene expression stimulated by 3D COL and tissue reorganization possibly by maintaining cellular level of integrin α2.


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 3D COL contraction. Fibroblasts were  pretreated with SN50, an inhibitor for NF-κB nuclear translocation, or SM, a peptide control, for 30 min before stimulation by  3D COL. The incubation was continued for 24 h. Cells released  from 3D COL by collagenase digestion were subcultured into 3D  COL for the measurement of gel contraction in the presence of  SN50 or other additives. The surface diameters (A) of 3D COLs  were measured from the photographs (B) taken at 4, 20, 48, and  72 h after subculture. The results represent 10 independent experiments. (C) Western analysis of proteins extracted from fibroblasts cultured in 3D COL for 20, 48, and 72 h in the presence of  either SM or SN50. The samples were run on 6% SDS–polyacrylamide gel. The blot was detected by an anti-α2 antibody and visualized with alkaline phosphatase/NBT/BCIP method as described  in the Materials and Methods. The integrin α2 subunit is marked  by an arrow. The results represent two independent experiments.
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Related In: Results  -  Collection

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Figure 8: NF-κB mediates 3D COL contraction. Fibroblasts were pretreated with SN50, an inhibitor for NF-κB nuclear translocation, or SM, a peptide control, for 30 min before stimulation by 3D COL. The incubation was continued for 24 h. Cells released from 3D COL by collagenase digestion were subcultured into 3D COL for the measurement of gel contraction in the presence of SN50 or other additives. The surface diameters (A) of 3D COLs were measured from the photographs (B) taken at 4, 20, 48, and 72 h after subculture. The results represent 10 independent experiments. (C) Western analysis of proteins extracted from fibroblasts cultured in 3D COL for 20, 48, and 72 h in the presence of either SM or SN50. The samples were run on 6% SDS–polyacrylamide gel. The blot was detected by an anti-α2 antibody and visualized with alkaline phosphatase/NBT/BCIP method as described in the Materials and Methods. The integrin α2 subunit is marked by an arrow. The results represent two independent experiments.
Mentions: It has been reported by various laboratories that integrin α2β1 mediates reorganization and contraction of collagen gels by human cells including fibroblasts (Schiro et al., 1991), cutaneous squamous carcinoma cells (Fujii et al., 1995), retinal pigment epithelial cells (Kupper and Ferguson, 1993), and transformed osteosarcoma cells (Riikonen et al., 1995). It was proposed that stimulants such as EGF (Fujii et al., 1995) and TGF-β (Riikonen et al., 1995b) induce 3D COL contraction by increasing integrin α2β1 expression. The transfection of integrin α2 cDNA into a cell line RD cells that expresses β1 chain but possess a very low level of α2β1 integrin restores the ability of RD cells to contract collagen gels (Schiro et al., 1991). We hypothesized that since NF-κB mediated α2 gene expression, it may be in the regulatory pathway leading to α2-mediated collagen gel contraction. The effects of NF-κB on 3D COL contraction by fibroblasts were examined. Treatment of cells with SN50, but not SM, significantly slowed the contraction process during 72 h examined (Fig. 8, A and B). The expression of integrin α2 protein by fibroblasts cultured in 3D COL was similarly reduced by SN50 but not SM (Fig. 8 C). The amount of a nonspecific band of low molecular weight, serving as an internal control, was similar in all conditions (Fig. 8 C), confirming the specificity of the inhibtion. Therefore, it further indicates that NF-κB was involved in the integrin α2 gene expression stimulated by 3D COL and tissue reorganization possibly by maintaining cellular level of integrin α2.

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