TG2 modulates the expression of MMP-2 and other metastasis-related proteins. A, a metastasis-focused Superarray was used to compare the expression levels of mRNAs for 120 metastasis-related genes. SKOV3 cells stably transfected with pcDNA3.1 were compared with cells transfected with AS-TG2. The image shows differences of hybridization between AS-TG2 cells and control cells. The arrow points to the probe on the array corresponding to MMP-2. Similar results were obtained from AS-TG2 and pcDNA3.1 xenograft-derived cultures (not shown). B, the table shows the differential expression levels of tumor metastasis related genes in pcDNA3.1- and AS-TG2-transfected cells.

Down-regulation of TG2 diminishes MMP-2 mRNA expression and MMP-2 transactivation. A, semiquantitative reverse transcription-PCR for MMP-2 and GAPDH in SKOV3 cells stably transfected with AS-TG2 and pcDNA3.1. B, real-time PCR quantifies MMP-2 mRNA expression relative to GAPDH in AS-TG2 and pcDNA3.1 cells. Data are the means of duplicate measurements ± S.D.; p < 0.001. Error bars are too small to be seen. Experiments were independently repeated three times with comparable results. C, a gene reporter assay of MMP-2 promoter activity in AS-TG2 and pcDNA3.1 SKOV3 cells, conducted as described under “Materials and Methods.” Cells were co-transfected with an MMP-2 promoter luciferase reporter and Renilla plasmids. To control for variations in transfection efficiency, values for luminescence were normalized to Renilla activity. Data are shown as means of triplicate measurements ± S.D. (p = 0.03).

Down-regulation of TG2 diminishes MMP-2 expression, and activity, in cancer cells. A: left, a Western blot prepared using lysates of control and AS-TG2 SKOV3 cells was probed with antibodies to MMP-2, TG2, and GAPDH. Right, a Western blot prepared after fractionating equal volumes of media conditioned by control and AS-TG2 SKOV3 cells was probed with an antibody to MMP-2. B: a Western blot prepared using lysates of IGROV-1 ovarian cancer cells transfected with siRNA to TG2 (si TG2) or a scrambled siRNA (si C) was probed with antibodies to MMP-2, TG2, and GAPDH. C: left, zymography on media conditioned by AS-TG2 or pcDNA3.1 SKOV3 cells for 24 h, conducted as described under “Materials and Methods.” Gelatinolytic MMP-2 activity produces white bands of hydrolyzed gelatin. Right, zymography using media conditioned by pcDNA3.1 and AS-TG2 SKOV3 cells for 1, 3, 6, and 24 h. D: left, a Western blot was prepared from lysates of MDA-MB 436 breast cancer cells transfected with siRNA targeting TG2 (si TG2) or scrambled siRNA (si C) was probed with antibodies to MMP-2, TG2, and GAPDH. Right, zymography using conditioned media from MDA-MB 436 cells transfected with siRNA targeting TG2 or scrambled siRNA. E: a Western blot prepared using lysates of PC-3 prostate cancer cells transfected with siRNA targeting TG2 (si TG2) or scrambled siRNA (si C) was probed with antibodies to MMP-2, TG2, and GAPDH. All results shown are representative of observations made in at least two independent experiments. Densitometry quantifies the levels of expression for MMP-2 and TG2 relative to GAPDH.

TG2 regulates the activity of MMP-2. A, in vitro assay of MMP-2 activity based on cleavage of a fluorogenic peptide substrate in a cell-free system, as described under “Materials and Methods.” The bar graph shows the effects of a negative control (DMSO), a positive control (1 mm aminophenylmercuric acetate (AMPA)), and 1 mm and 5 mm recombinant TG2 on MMP-2 activity. Results are the mean MMP-2 activity in relative fluorescence units ±S.D. for each condition, assayed in triplicate. B, a Western blot probed with antibodies to MMP-2 and GAPDH quantifies MMP-2 expression in SKOV3 cells cultured in the absence or presence of the TG2 inhibitor, KCC009 (0, 250, and 500 μm) for 24 h. Densitometry quantifies the expression of MMP-2 relative to GAPDH. C, zymography on conditioned media from SKOV3 cells cultured in the absence or presence of 250 or 500 μm KCC009.

PP2A-α is a target of TG2. A, AS-TG2 and pcDNA3.1 cells were incubated in the absence or presence of 100 nm okadaic acid (OA) for 3 h in serum-free media. A Western blot prepared from nuclear extracts of the cells was probed with antibodies to pSer¹³³CREB and poly(ADP-ribose) polymerase (PARP). B, SKOV3 cells were transfected with control siRNA or siRNAi targeting PP2A-α. A Western blot prepared from lysates of the cells was probed with antibodies to PP2A-α and GAPDH. C, Western blot analysis for pSer¹³³CREB in nuclear extracts of SKOV3 cells transfected with PP2A-α targeted siRNA or control siRNA. Poly(ADP-ribose) polymerase was used as loading control. D, a Western blot prepared from lysates of AS-TG2 or pcDNA3.1 SKOV3 cells was probed with antibodies to PP2A-α, TG2, and GAPDH. E, a Western blot prepared from lysates of OV90 cells transduced with human TG2 or a control vector was probed with antibodies to PP2A-α, TG2, and GAPDH. F, a Western blot prepared from lysates of A2780 cells transduced with human TG2 or a control vector was probed with antibodies to PP2A-α, TG2, and GAPDH. TG2 overexpression correlates with reduced PP2A-α. G, Western blots prepared from TG2 (left) or PP2A-α (right) immunoprecipitates of SKOV3 cells were probed with anti-TG2 and anti-PP2A-α. Immunoprecipitations with IgG served as controls for each experiment. H, SKOV3 cells were incubated in the absence or presence of KCC009 for 24 h. A Western blot prepared from lysates of the cells was probed with antibodies to PP2A-α and GAPDH. Densitometry quantifies PP2A-α expression. I, AS-TG2- and pcDNA3.1-transfected cells were treated with 20 μg/ml cycloheximide for up to 6 h. A Western blot prepared from cell lysates was probed with antibodies to PP2A-α and GAPDH, a loading control. Densitometry quantifies PP2A-α expression. J, semiquantitative reverse transcription-PCR for PP2A-α and GAPDH in SKOV3 cells stably transfected with AS-TG2 and pcDNA3.1. K, proposed model for regulation of MMP-2 by TG2 in ovarian cancer cells.

TG2 regulates MMP-2 transcription by activating CREB. A, schematic of a CREB binding site at −205 bp in the human MMP-2 promoter and the positions of primers used to amplify this region in the ChIP assay (below). B, Western blot assay of MMP-2 (left panel) and total CREB (right panel) and GAPDH (control) in SKOV3 cells transfected with siRNA targeting CREB or control siRNA. C, ChIP demonstrates that CREB binds the MMP-2 promoter region. Immunoprecipitated chromatin from SKOV3 cells was used for PCR amplification. A 120-bp PCR product was detected by gel agarose electrophoresis from chromatin immunoprecipitated with an antibody against CREB (lane 4) using primers flanking the CREB-binding region of the MMP-2 promoter (−275 to −156 bp). Positive controls consist of 10% of the total chromatin in the absence of immunoprecipitation (lane 2, input) and chromatin immunoprecipitated with CREB antibody and amplified with primers for NR4A2 promoter, known to be transcriptionally regulated by CREB (lane 3). Negative controls consist of chromatin immunoprecipitated with IgG and amplified with MMP-2 promoter primers (lane 6) and chromatin immunoprecipitated with CREB antibody and amplified with primers to a region of the MMP-2 promoter upstream of the predicted CREB binding site (lane 5). D, MMP-2 promoter activity measured by a reporter assay using SKOV3 cells stably transfected with a MMP-2 promoter luciferase construct or a MMP-2 promoter luciferase construct with a deleted CREB-binding sequence. To control for variations in transfection efficiency, values for luminescence were normalized to Renilla activity. Data are the means of duplicate measurements ± S.D. (p = 0.0002).

TG2 regulates CREB phosphorylation and activity. A, Western blots were prepared from nuclear (left) and whole cell extracts (middle and right) isolated from pcDNA3.1 and AS-TG2 SKOV3 cells. The blots were probed with an antibody to pSer¹³³CREB (left and middle) and total CREB (right). Poly(ADP-ribose) polymerase (PARP) was used as an indicator of the purity of the nuclear fraction and served as a loading control (left). GAPDH was used as a loading control for the whole cell extracts (middle and right). B, SKOV3 cells were incubated in the absence or presence of KCC009 (250 or 500 μm) for 24 h. Western blots prepared from lysates of the cells were probed with antibodies to pSer¹³³ CREB and GAPDH (left panel) and total CREB and GAPDH (right panel). C, CREB reporter activity was assayed in AS-TG2 or pcDNA3.1 SKOV3 cells co-transfected with a 3XCRE reporter construct and Renilla plasmids. To control for variations of transfection efficiencies, luminescence values are normalized to Renilla activity. Data are the means of duplicate measurements ± S.D.; p = 0.008. D, CREB reporter activity was assayed in SKOV3 cells treated with KCC009. Data are the means of duplicate measurements ± S.D.; p < 0.004. E, ChIP shows that CREB binding to the MMP-2 promoter region is decreased in cells in which TG2 has been knocked down. Immunoprecipitated chromatin from AS-TG2 and pcDNA3.1 cells was used for PCR amplification. Results of ChIP assay correspond to PCR products, as follows: DNA ladder; chromatin from pcDNA3.1 cells not subjected to immunoprecipitation (IP) (input, lane 1, positive control); chromatin from AS-TG2 cells not subjected to IP (input, lane 2, positive control), chromatin from pcDNA3.1 cells immunoprecipitated with CREB antibody and amplified with MMP-2 specific primers (lane 3); chromatin from AS-TG2 cells immunoprecipitated with CREB antibody and amplified with MMP-2 specific primers (lane 4); and negative controls consisting of chromatin from pcDNA3.1 (lane 5) and AS-TG2 cells (lane 6) immunoprecipitated with IgG and amplified with MMP-2-specific primers. Densitometry (below the gel) quantifies the results. F, real-time PCR quantifies amplification of the MMP-2 promoter bound to the CREB antibody. Data represent means of duplicate measurements ± S.D.; p = 0.03. Experiments were independently repeated three times with comparable results.