Functional role of Meox2 during the epithelial cytostatic response to TGF-β

Expression profile of Meox2 in response to TGF-β1. (A) Semi-quantitative RT-PCR analysis of Meox2 and Gapdh from NMuMG cells during a time course experiment with 5ng/ml TGF-β1. Normalized Meox2 mRNA expression values are listed. (B) Quantitative real time RT-PCR of MEOX2 and GAPDH from HaCaT cells stimulated with 5ng/ml TGF-β1 for the indicated time periods. (C) Semi-quantitative RT-PCR analysis of Meox2 and Gapdh mRNA expression in NMuMG cells infected with adenoviruses (MOI 100) expressing the indicated proteins and stimulated with 5ng/ml TGF-β1 for 2h. Normalized Meox2 mRNA expression values are listed. Immunoblots with anti-Flag or anti-HA antibodies of duplicate cell cultures treated under identical conditions demonstrate expression of the infected proteins (Smads, anti-FLAG and ALK5, anti-HA). (D) Quantitative real time RT-PCR of MEOX2 and GAPDH from HaCaT cells infected with adenoviruses (MOI 100) expressing the indicated proteins and stimulated with 5ng/ml TGF-β1 for 16h.

Meox2 mediates HaCaT cytostasis in response to TGF-β1. (A) Quantitative real time RT-PCR analysis of MEOX2 and GAPDH mRNA levels in HaCaT cells transiently transfected with control (siLuc) and specific (siMEOX2) siRNAs prior to stimulation with 5ng/ml TGF-β1 for the indicated time periods. (B) Thymidine incorporation assay in HaCaT cells transiently transfected with siRNAs as in panel A, and stimulated with 5ng/ml TGF-β1 for 18h. The control conditions have been normalized to 100%. (C) Thymidine incorporation assay in HaCaT cells transiently infected with the indicated adenoviral MOI and stimulated with 5ng/ml TGF-β1 for 20h. (D) Cell proliferation assay in HaCaT cells infected as in panel C and stimulated with TGF-β1 for 72h.

Meox2 induces expression of the cell cycle inhibitor p21 in epithelial cells. (A–C) Quantitative real time RT-PCR analysis of P21 and GAPDH mRNA levels in HaCaT (A), MCF-7 (B) and NMuMG (C) cells transiently infected with Ad-LacZ or Ad-Meox2 (MOI 100) for 48h. (D–F) Immunoblots with anti-HA, anti-p21 or anti-β-tubulin (control) antibodies of duplicate cell cultures treated under identical conditions demonstrate expression of ectopic Meox2 and endogenous p21 and β-tubulin proteins, the latter serving as protein loading control. (G) Immunoblot analysis of endogenous p21 protein expression in HaCaT cells infected with increasing MOI (10, 20, 40, 80, 100; triangle) of Meox2 or LacZ (MOI 100, -) adenoviruses for 48h. Anti-HA immunoblots show the expression of ectopic Meox2.

Meox2 is required for optimal p21 levels after TGF-β stimulation. (A) Quantitative real time RT-PCR analysis of MEOX2 and GAPDH mRNA expression in HaCaT cells transfected with the indicated siRNAs and stimulated with 5ng/ml TGF-β1 for 0 or 16h. (B) Quantitative real time RT-PCR analysis of p21 and GAPDH mRNA expression in the same HaCaT cells of panel A. (C) Immunoblot analysis of endogenous p21 and β-tubulin protein expression in HaCaT cells transfected and stimulated under identical conditions as in panel A. (D) Semi-quantitative RT-PCR analysis of endogenous MEOX2, ID2, PAI-1 and GAPDH levels in HaCaT cells transfected and stimulated under identical conditions as in panel A.

Regulation of other genes of the TGF-β-dependent cytostatic program by Meox2. Quantitative real time RT-PCR analysis of (A) p21, (B) p15, (C) c-Myc and (D) Id2 mRNA normalized over GAPDH mRNA expression in HaCaT cells infected with the indicated adenoviruses (MOI 100) and stimulated with 5ng/ml TGF-β1 for 0, 2 and 8h. (E) Immunoblots with anti-HA or anti-β-tubulin (control) antibodies of duplicate cell cultures treated under identical conditions demonstrate expression of ectopic Meox2. (F) Semi-quantitative RT-PCR analysis of p21, p15, c-MYC, ID2 and GAPDH mRNA expression in MCF-7 cells infected with adenoviruses (MOI 100) expressing the indicated proteins for 48h. Normalized mRNA expression values are listed and fold-differences between control and Meox2 expression are tabulated on the right hand side. (G) Immunoblot analysis of p21 and Id2 protein expression in MCF-7 cells infected with the indicated adenoviruses (MOI 100) for 48h. Anti-HA immunoblots show the expression of the infected Meox2 and β-tubulin serves as loading control.

Meox2 induces the p21 promoter–enhancer via its distal region. (A) Transactivation of the −2400/+8p21 promoter in NMuMG and HaCaT cells transiently infected with Ad-Meox2 or Ad-LacZ (MOI 100) for 24h. Normalized relative luciferase activity is shown in arbitrary units. (B) Luciferase p21 promoter assays of the indicated promoter deletion constructs in HepG2 and HEK-293 cells transfected with mock vector (pcDNA3) or specific Meox2-expressing vectors (wild type, Meox2, and homeodomain deletion mutant, Meox2ΔHD). Values express average normalized relative luciferase activity and standard deviations from triplicate determinations. Similar data were obtained from both cell lines. All basal promoter activities (Mock) are shown as 1, relative to which the promoter activities obtained after Meox2 transfections are calculated. The differences in basal promoter activity between the various deletion constructs are shown in parenthesis under Mock. (C) Immunoblot analysis of transfected wild type Meox2 and mutant lacking its homeodomain (ΔHD) in cell lysates used for the luciferase assay of panel B, using the anti-HA antibody. (D) Diagram of the human p21 promoter–enhancer indicating the distal and proximal regions (double-headed arrows) with the corresponding p53, FoxO3a-Smad and Sp1 binding elements, and the transcriptional initiation (+1) site. Vertical arrowheads indicate the putative Meox2-binding sites and the PCR fragments amplified during ChIP analysis are shown as grey rectangles. (E) ChIP analysis of the human p21 promoter–enhancer in transfected HEK-293 cells with the two indicated p21 genomic fragments and wild type (WT) Meox2 expression vector. The homeodomain deletion mutant Meox2(ΔHD) serves as negative control. Q-PCR assays measure genomic sequences corresponding to a distal and a proximal p21 genomic fragment as illustrated in panel D (grey rectangles).

Meox2 and Smad proteins interact and cooperate in inducing endogenous p21 levels. (A) Co-immunoprecipitation assays in HaCaT cells infected with the indicated adenoviruses (MOI 200) and stimulated with 5ng/ml TGF-β1 for 2h. Anti-HA (Meox2) immunoprecipitation (IP) was followed by immunoblot (IB) with antibodies against Flag (Smads) and HA (Meox2). Immunoblots of the corresponding total extracts with the two antibodies are shown below. (B) Immunoblot analysis of endogenous p21 expression in HaCaT cells infected with the indicated adenoviral MOI for 32h and stimulated with 5ng/ml TGF-β1 for another 16h. Anti-HA immunoblot shows the expression of the infected Meox2 and anti-Flag immunoblot shows the expression of Smad3 and Smad4. (C) Immunoblot analysis of endogenous p21 protein expression in MDA-MB-468 cells infected with the indicated adenoviruses (MOI 100) for 48h. Anti-HA immunoblot shows the expression of the exogenous Meox2 and β-tubulin serves as protein loading control.

Meox2 partially blocks the EMT response to TGF-β1. (A, B) Immunofluorescence microscopy of endogenous E-cadherin in HaCaT (A) or NMuMG (B) cells infected with the indicated adenoviruses (MOI 100) and treated with 5ng/ml TGF-β1 for 48h. Bars represent 10μm. (C, D) Immunoblot analysis of endogenous E-cadherin, ectopic Meox2 (anti-HA) and endogenous β-tubulin serving as control in duplicate cultures infected and treated exactly as in panels A and B.