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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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Effect of 5′ deletion on the expression of integrin α2-CAT fusion gene induced by 3D COL. (A) The 5′ flanking region  of integrin α2 gene fused to the CAT structural gene. These constructs were deletion mutants derived from a pα2961-CAT (Zutter  et al., 1994). (B) Fibroblasts were cotransfected with the 5′ deletion mutant-CAT and RSV–β-galactosidase fusion genes followed by subculture in 3D COL as described in the Materials and  Methods. (C) Fibroblasts were cotransfected with pα2549-CAT  and RSV–β-galactosidase fusion genes followed by subculture on  tissue culture plastic plates (TC), collagen-coated surface (mCOL),  and 3D COL. Cells were harvested 1 d after subculture and cell  extracts were assayed for CAT activity. The results represent at  least five independent experiments. RSV–β-galactosidase activity  was used as a control. pCMV, cytomegalovirus promoter-CAT  construct.
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Figure 4: Effect of 5′ deletion on the expression of integrin α2-CAT fusion gene induced by 3D COL. (A) The 5′ flanking region of integrin α2 gene fused to the CAT structural gene. These constructs were deletion mutants derived from a pα2961-CAT (Zutter et al., 1994). (B) Fibroblasts were cotransfected with the 5′ deletion mutant-CAT and RSV–β-galactosidase fusion genes followed by subculture in 3D COL as described in the Materials and Methods. (C) Fibroblasts were cotransfected with pα2549-CAT and RSV–β-galactosidase fusion genes followed by subculture on tissue culture plastic plates (TC), collagen-coated surface (mCOL), and 3D COL. Cells were harvested 1 d after subculture and cell extracts were assayed for CAT activity. The results represent at least five independent experiments. RSV–β-galactosidase activity was used as a control. pCMV, cytomegalovirus promoter-CAT construct.

Mentions: Since NF-κB is a transcription factor, the requirement of its activity in 3D COL stimulation of integrin α2 mRNA expression indicates that 3D COL may control the transcription of integrin α2 gene. To assess this possibility, we examined whether 3D COL regulates the promoter activity of integrin α2 gene. The promoter region of the integrin α2 gene was cloned and a series of deletion mutants of 5′ flanking sequence were inserted into a CAT reporter vector (Fig. 4 A) (Zutter et al., 1994, 1995b). Fibroblasts were transiently transfected with these CAT reporter constructs followed by incubation in 3D COL or on two-dimensional surfaces. As shown in Fig. 4 B, the reporter gene directed by sequences upstream of −92 bp of integrin α2 promoter (pα2122CAT, pα2351CAT, pα2549CAT, and pα2776CAT) showed inhibited basal expression when compared to the activity of pα292CAT (Fig. 4 B), indicating the presence of potential silencer element(s). Upon stimuilation by 3D COL, the upstream sequences up to −351 bp of integrin α2 promoter (pα292CAT, pα2122CAT, and pα2351CAT) did not show a positive response. In contrast, the plasmids containing either −549 or −776 bp of upstream sequences (pα2549CAT and pα2776CAT) demonstrated 3D COL inducibility. Therefore a region located between −92 and −122 bp probably has negative regulatory sequences for basal promoter activity, whereas the sequences located between −549 and −351 bp of integrin α2 promoter appear to modulate positive response to 3D COL. For the convenience of this report, we designate this region α2549–351. To understand whether the positive response to 3D COL is elicited by collagen signals resident in collagen molecules of any form or a special collagen structure, we cultured fibroblasts transfected with pα2549CAT on tissue plastic plates, on monolayer collagen-coated plates, and in 3D COL. Cells grown on collagen-coated plates failed to induce reporter gene activity when compared to cells grown on plastic surface (Fig. 4 C), supporting our previous observations of α2 mRNA (Xu and Clark, 1997). Therefore signals from collagen in a particular 3D structure seemed responsible for the positive response of α2 promoter modulated by α2549–351.


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)

Effect of 5′ deletion on the expression of integrin α2-CAT fusion gene induced by 3D COL. (A) The 5′ flanking region  of integrin α2 gene fused to the CAT structural gene. These constructs were deletion mutants derived from a pα2961-CAT (Zutter  et al., 1994). (B) Fibroblasts were cotransfected with the 5′ deletion mutant-CAT and RSV–β-galactosidase fusion genes followed by subculture in 3D COL as described in the Materials and  Methods. (C) Fibroblasts were cotransfected with pα2549-CAT  and RSV–β-galactosidase fusion genes followed by subculture on  tissue culture plastic plates (TC), collagen-coated surface (mCOL),  and 3D COL. Cells were harvested 1 d after subculture and cell  extracts were assayed for CAT activity. The results represent at  least five independent experiments. RSV–β-galactosidase activity  was used as a control. pCMV, cytomegalovirus promoter-CAT  construct.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2140166&req=5

Figure 4: Effect of 5′ deletion on the expression of integrin α2-CAT fusion gene induced by 3D COL. (A) The 5′ flanking region of integrin α2 gene fused to the CAT structural gene. These constructs were deletion mutants derived from a pα2961-CAT (Zutter et al., 1994). (B) Fibroblasts were cotransfected with the 5′ deletion mutant-CAT and RSV–β-galactosidase fusion genes followed by subculture in 3D COL as described in the Materials and Methods. (C) Fibroblasts were cotransfected with pα2549-CAT and RSV–β-galactosidase fusion genes followed by subculture on tissue culture plastic plates (TC), collagen-coated surface (mCOL), and 3D COL. Cells were harvested 1 d after subculture and cell extracts were assayed for CAT activity. The results represent at least five independent experiments. RSV–β-galactosidase activity was used as a control. pCMV, cytomegalovirus promoter-CAT construct.
Mentions: Since NF-κB is a transcription factor, the requirement of its activity in 3D COL stimulation of integrin α2 mRNA expression indicates that 3D COL may control the transcription of integrin α2 gene. To assess this possibility, we examined whether 3D COL regulates the promoter activity of integrin α2 gene. The promoter region of the integrin α2 gene was cloned and a series of deletion mutants of 5′ flanking sequence were inserted into a CAT reporter vector (Fig. 4 A) (Zutter et al., 1994, 1995b). Fibroblasts were transiently transfected with these CAT reporter constructs followed by incubation in 3D COL or on two-dimensional surfaces. As shown in Fig. 4 B, the reporter gene directed by sequences upstream of −92 bp of integrin α2 promoter (pα2122CAT, pα2351CAT, pα2549CAT, and pα2776CAT) showed inhibited basal expression when compared to the activity of pα292CAT (Fig. 4 B), indicating the presence of potential silencer element(s). Upon stimuilation by 3D COL, the upstream sequences up to −351 bp of integrin α2 promoter (pα292CAT, pα2122CAT, and pα2351CAT) did not show a positive response. In contrast, the plasmids containing either −549 or −776 bp of upstream sequences (pα2549CAT and pα2776CAT) demonstrated 3D COL inducibility. Therefore a region located between −92 and −122 bp probably has negative regulatory sequences for basal promoter activity, whereas the sequences located between −549 and −351 bp of integrin α2 promoter appear to modulate positive response to 3D COL. For the convenience of this report, we designate this region α2549–351. To understand whether the positive response to 3D COL is elicited by collagen signals resident in collagen molecules of any form or a special collagen structure, we cultured fibroblasts transfected with pα2549CAT on tissue plastic plates, on monolayer collagen-coated plates, and in 3D COL. Cells grown on collagen-coated plates failed to induce reporter gene activity when compared to cells grown on plastic surface (Fig. 4 C), supporting our previous observations of α2 mRNA (Xu and Clark, 1997). Therefore signals from collagen in a particular 3D structure seemed responsible for the positive response of α2 promoter modulated by α2549–351.

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