Figure 1.
Catalytic function of TG2 is not essential for activation of NF-κB.
A- Western blot analysis for TG2 expression in MCF10A cells stably transfected with either a lentiviral construct alone (Vec), or a lentiviral containing a TG2-wt or a TG2-C277S construct (upper frame). The membrane was stripped and reprobed with anti-β actin antibody to ensure even protein loading in each lane (middle frame). Electrophoretic mobility shift assay (EMSA) was performed using 32P end-labeled 45-mer double-stranded NF-κB oligonucleotide probe (5′-TTG TTA CAA GGG ACT TTC CGC TGG GGA CTT TCC AGG GAG GCG TGG-3′) using nuclear extracts prepared from vector, TG2-wt or TG-C227S-transfected MCF10A cells (lower panel). B- Localization of p65/RelA (green) protein in nuclei (blue) of vector alone or after their stimulation with TNFα (10 ng/ml for 30 minutes) and TG2-C2277S-transfected MCF10A cells, as determined by an immunofluorescence assay. TG2 was stained with PE-conjugated secondary antibody (red). Magnification- ×100 original, ×500 magnified view. C- Cross-linking activity of endogenous TG2 was determined by BPA conjugation using total cell extracts prepared from MCF10A cells transfected with vector alone or with TG2-WT or TG2-C277S. The BPA conjugation was determined in the presence (+CaCl2) or absence (+EDTA) of 5 mM Ca2+, as described in the Materials and Methods section. Fifteen micrograms of protein from each reaction mixture was subjected to Western blot analysis to determine TG2 and β actin levels (lower panel). Results shown are from a representative experiment, repeated at least twice with similar results.
Figure 2.
TG2 expression correlates with constitutive NF-κB activation.
A- Western blot analyses of nuclear and cytosolic fractions prepared from vector, TG2-WT, and TG2-C277S-expressing MCF10A cells. Membranes were probed with either anti-TG2 or anti-p65/RelA antibodies, stripped, and reprobed with anti-IκBα, GAPDH or anti-histone antibodies to determine even protein loading and purity of cytosolic and nuclear fractions, respectively. The low IκBα protein levels in cytosolic fractions were related to TG2 expression, as suggested by the even β actin band in all the three cytosolic fractions (data not shown). B- Detection of p65/RelA in the nuclear fraction of TG2-C277S transfected MCF10A cells and C- in MCF-7/RT cells after transfection with either the control or TG2-specific shRNA. Membranes were reprobed with anti-IκBα, GAPDH or anti-histone antibodies to ensure the even loading and purity of cytosolic and nuclear fractions. D- Quantitative RT-PCR array showing relative changes in the expression of NF-κB target genes in TG2-C277S-transfected MCF10A cells and MCF10A-vector cells after stimulation with TNFα (10 ng/mL) for the indicated times. The Y-axis denotes the fold-expression, and the x-axis denotes the genes. Expression of GAPDH, β actin, and 18S ribosomal RNA was used to normalize the variable template loading. E- RT-PCR analysis for basal and TNFα-induced expression of selected NF-κB target genes in TG2-C277S and control vector-transfected MCF10A cells. F- Immunoblot analysis for basal expression of indicated NF-κB target genes in TG2-C277S and vector-transfected MCF10A cells. Results shown are from a representative experiment repeated at least 2 or 3 times with similar results.
Figure 3.
TG2-induced expression of transcription repressor genes is mediated though NF-κB.
A- Quantitative RT-PCR array showing relative changes in the expression of NF-κB related genes in TG2-C277S and vector-transfected MCF10A cells before and after transfection with control or p65/RelA - siRNA. B- Immunoblot analysis of NF-κB target genes in cells transfected with TG2-C277S or the vector alone before and after transfection with control or p65/RelA- siRNA. Membranes were reprobed with anti-TG2 and β-actin antibodies to ensure, if any, the effect of p65/RelA downregulation of TG2 expression and even protein loading in each lane. C- Quantitative RT-PCR array showing relative changes in the expression of NF-κB responsive genes- in vector-control andTG2-C277S MCF10A cells in response to TG2 downregulation. The y-axis denotes the fold- expression and the x-axis denotes the genes. The expression of GAPDH, β-actin, and 18S ribosomal RNA was used to normalize variable template loadings. D- Immunoblot analysis was performed to validate the effect of TG2 downregulation on NF-κB target genes in TG2-C277S MCF10A cells. The membrane was reprobed with anti-β-actin to ensure equal protein loading. Results shown are from representative experiments repeated at least 3 times with similar results.
Figure 4.
TG2-induced NF-κB activation is independent of IKK activity.
A- Quantitative RT-PCR array showing relative changes in the expression of NF-κB target genes in response to TNFα (10 ng/mL, 2 hours) treatment and the effect of IKK inhibition (parthenolide, 5 µM) on TNFα induced gene expression in MCF10Avec cells. B- Quantitative RT-PCR analysis of NF-κB responsive genes in TG2-C277S MCF10A cells incubated with TNFα (10 ng/mL, 2 hours) in the presence or absence of parthenolide. C- RT-PCR analysis was performed to validate the expression of Zeb1, Zeb2 and Snail1 in MCF10A-Vec cells incubated with TNFα (10 ng/ml, 2 hours) and TG2-C277S MCF10A cells in the presence or absence of parthenolide (5 µM). Results shown are from a representative experiments repeated at least twice with similar results.
Figure 5.
TG2 mediate IκBα degradation via non-proteasomal pathway and results in HIF-1α expression.
A- Total cell extracts prepared from MCF10A-Vec, MCF10A TG2-C277S, drug-sensitive (MCF-7/WT) and drug resistant (MCF-7/RT) MCF-7 cell were immunoprecipitated with anti-p65/RelA or anti-IκBα antibody and immunoblotted with anti-TG2, anti- p65/RelA, or anti- IκBα antibody. Total cell extracts (15 µg protein) were also directly subjected to immunoblotting to determine TG2 and p65/RelA levels. B- MCF10A-Vec and MCF10A TG2-C277S cells were incubated with medium alone (Cont.) or medium containing the indicated proteasomal inhibitor [Bortezomib (20 nM), N-acetyl-leucyl-leucyl-norleucinal (ALLN) (50 µg/mL), and MG-132 (1 µM)] for 2 hours. At the end of incubation, cells extracts were prepared and subjected to immunoblot analysis, using anti-TG2, anti-IκBα and anti-p21 antibody (p21 served as a positive control) and β-actin to ensure equal protein loading. C- RT-PCR analysis showing the basal transcript level of HIF-1α in MCF10A-Vec, MCF10A TG2-C277S, MCF7-WT and MCF7/RT cells. Results shown are from a representative experiment repeated at least 2 times with similar results. D- Immunoblot analysis showing the basal HIF-1α and TG2 protein expression in MCF10A-Vec, MCF10A TG2-C277S, MCF7-WT and MCF7/RT cells under normoxic conditions.
Figure 6.
Chromatin immunoprecipitation (ChIP) showing direct binding of p65/RelA in complex with TG2-C277S protein to the HIF-1α promoter.
A- ChIP assay was performed on formaldehyde fixed MCF10A-Vec, MCF10A TG2-C277S, MCF7-WT, and MCF7/RT cell extracts using p65/RelA (upper panel), TG2 (lower panel), or the control anti-mouse IgG antibodies. PCR reaction was performed on immunoprecipitates, using a specific set of primers (shown in the Materials and Methods section) to amplify HIF-1α promoter sequence containing the NF-κB binding site (−197/−188 bp). Prior to immunoprecipitation, an aliquot from each sample was saved to determine the ‘input’ representing the PCR amplification of 1% of the genomic DNA without immunoprecipitation (PCR control). The sonicated DNA fraction from each sample was also subjected to PCR reaction (data not shown. B- Immunoblot analysis of Zeb1, Snail, and Twist protein expression in HIF-1α downregulated MCF10A TG2-C277S and MCF-7/RT cells. Membranes were stripped and reprobed with anti- HIF-1α antibody to determine the extent of HIF-1α inhibition by gene-specific or control siRNA and with anti β-actin antibodies to ensure even protein loading in each lane.
Figure 7.
Schematic representation of TG2-regulated signaling.
Association of TG2 with IκBα results in its rapid degradation via non-proteasomal pathway and liberates the p65/RelA:p50 NF-κB complex. TG2 also associates in complex with the p65/RealA subunit of NF-κB and translocates to the nucleus where it binds to the cognate NF-κB binding site on the HIF-1α promoter and results in its transcription regulation. Increased expression of HIF-1α under normoxic conditions results in increased expression of various downstream target genes such as transcription repressors Zeb1, Zeb2, Snail1 and Twist, which induce EMT and promote cell survival, invasiveness, and cellular plasticity. Overall, these TG2-regulated changes contribute to increased chemoresistance and metastatic potential.