Laminin Reduces Expression of the Human α6 Integrin Subunit Gene by Altering the Level of the Transcription Factors Sp1 and Sp3

Abstract

Unlike FN, which functions as an activator of alpha5 gene transcription, LM repressed transcription, directed by the alpha6 gene promoter, by altering the nuclear levels of Sp1 and Sp3. Reappearance of LM in the basement membrane after repair of the corneal damage is therefore expected to substantially contribute to the final steps of this process by influencing the degree to which genes that encode protein products requested for cell adhesion and migration, such as integrin genes, are expressed during corneal wound healing.

Suppression of α5 gene expression is closely related to the tumorigenic properties of uveal melanoma cell lines

“…The plasmids ‐954α5‐CAT, ‐132α5‐CAT, and ‐92α5‐CAT have been previously described (Birkenmeier et al., 1991; Gaudreault et al., 2007). The oligonucleotides bearing the DNA‐binding sites for Sp1 (Dynan and Tjian, 1983), NFI (De Vries et al., 1987), and AP‐1 (Corbi et al., 2000; Gingras et al., 2009) were chemically synthesized using a Biosearch 8700 apparatus (Millipore, distributed by Fisher, Ottawa, ON, Canada).…”

“…Western blots were conducted as described (Larouche et al., 2000) using 30‐μg nuclear proteins with the following primary antibodies (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA, except for CLT‐9001 which came from Jackson Immuno Research Laboratories): rabbit polyclonal antibodies against Sp1 (sc‐59, 1:5000), Sp3 (sc‐644, 1:4000), NFI (sc‐5567, 1:1200), c‐jun (sc‐45, 1:3000), JunD (sc‐74, 1:2000), FosB (sc‐7203, 1:900), c‐Fos (sc‐52, 1:300), Fra‐1 (sc‐605, 1:1000), or Fra‐2 (sc‐604, 1:1200), or mouse monoclonal antibodies against JunB (sc‐46 1:3000) and actin (CLT 9001; 1:35000) as well as a peroxidase‐conjugated AffiniPure Goat secondary antibody against either mouse or rabbit IgG (1:1000 dilution). The labelling was revealed using a Detection kit (Amersham, distributed by GE Healthcare, Baie d’Urfé, Canada) as described (Gaudreault et al., 2007; Gingras et al., 2003). For detection of the α5 integrin subunit in T97α5 + (1) and T97α5 + (2) cells, a monoclonal antibody (final concentration of 10 μg/ml) directed against the human integrin subunits α5 (P1D6; Chemicon) was used as the primary antibody.…”

Rescue of the Transcription Factors Sp1 and NFI in Human Skin Keratinocytes through a Feeder-Layer-Dependent Suppression of the Proteasome Activity

“…The labeling was revealed using a detection kit (Amersham, Baie d'Urfé, Canada) as described previously. 91,92 Nuclear extracts and EMSA Except when indicated otherwise, all crude nuclear extracts were consistently prepared from all cell types when they reached 80 ± 5% confluence as described previously. 93 The protein concentration of each extract was determined by the Bradford procedure and precisely validated through Coomassie blue staining on SDS-PAGE.…”

“…33,91 The resulting DNA was analyzed by PCR using a pair of primers (ITGA5-U: 5′-CTTAGGGGTGGGGGACGC-3′ and ITGA5-L: 5′-CGCCCGCTCTTCCCTGTC-3′) spanning the α5 gene promoter. As a negative control, each ChIP sample was also subjected to PCR using primers (p21-U: 5′-AATTCCTCTGAAAGCTGACTGCC-3′ and p21-L: 5′-AGGTTTACCTGGGGTCTTTAGA-3′) specific to a region located ∼ 2 kb upstream from the human p21 promoter.…”

Leukocyte β7 Integrin Targeted by Krüppel-like Factors

“…Consequently, there has been interest in the characterization of the promoter elements of various integrins and the molecules regulating expression. Factors of the specificity protein family of transcription factors have been directly shown to bind to several integrin promoters (38)(39)(40)(41)(42). Notably, the promoter for b 7 has been identified, but there has been a paucity of defined factors that control expression of this lymphocyte-specific integrin (43,44).…”