Figure 1.
HTRA1 inhibits TGF-β-mediated transcription.
(A) The effect of HTRA1 on TGF-β transcription was examined by co-transfecting HeLa cells with a luciferase reporter plasmid driven by tandem Smad responsive elements and with either control plasmid or 0, 25, 50, 100 or 150 ng of a plasmid that overexpresses HTRA1. After 24 hours, cells were either left untreated or treated with 10 ng/ml TGF-β for 16 hours to induce the Smad-responsive reporter gene. Relative luciferase activity was determined by calculating the ratio of firefly and Renilla luciferase activities. (B) The effect of exogenous treatment of HTRA1 on TGF-β-mediated transcription. HeLa cells were transfected with a Smad-responsive reporter and then either left untreated or treated with the indicated concentrations of purified HTRA1 or an inactive mutant, S328A. Relative luciferase activity was determined by calculating the ratio of firefly and Renilla luciferase activities. (C) TGF-β-induced expression of PAI-1 mRNA. HeLa cells were transfected with either a control plasmid or an HTRA1 plasmid, and then treated with 10 ng/ml of TGF-β for the indicated times. (D) Expression of PAI-1 mRNA in HeLa cells transfected with either a nonspecific control siRNA or with HTRA1 siRNA, then treated with 10 ng/ml of TGF-β for the indicated times. *P<0.05, **P<0.01, ***P<0.001, compared to TGF-β treated control (A, B), or empty plasmid and non-specific siRNA controls (C, D). Student’s t-test, n = 3.
Figure 2.
HTRA1 cleaves TGF-β receptors.
(A) Cleavage of TGF-β receptors by HTRA1 in vitro. Equal amounts of purified TGF-β receptors were incubated with increasing amount of purified HTRA1 with or without CPII, an HTRA1 agonist. The inactive mutant, S328A, was also incubated with the receptors in the presence or absence of CPII. Protein digestions were analyzed by western blot. UD = undigested proteins. (B) HTRA1 cleavage of TGF-β receptors from the cell surface. HeLa cells were transfected with either a control or HTRA1 plasmid in addition to plasmids encoding either TβRIII, TβRII or TβRI. Membrane proteins were isolated and analyzed by western blot for cleavage of membrane bound receptors. Cadherin was used as a loading control. Overexpression of His-tagged HTRA1 was confirmed with an anti-His antibody. (C) mRNA levels of the TGF-β receptors after HeLa cells were transfected with either a control or HTRA1 plasmid.
Figure 3.
Regulation of TGF-β signaling by HTRA1.
(A)(B) Binding of TGF-β to the cell surface as measured by flow cytometry. Cells were transfected with either nonspecific control siRNA or HTRA1 siRNA and then incubated with either biotinylated TGF-β or a biotinylated negative control protein to measure the background. Solid gray = background, dashed line = control siRNA, black line = HTRA1 siRNA. (B) Bar chart indicates the mean FITC values after subtraction of the background level. **P<0.01, Student’s t-test, n = 4. (C) Immunoblot of total and phosphorylated Smad2 (465/467) in A549 cells transfected with either control or HTRA1 siRNA, and then treated with 1 ng/ml TGF-β for the indicated times. β-actin was used as a loading control. (D) The effect of HTRA1 knockdown on the expression of TGF-β-regulated genes. A549 cells were transfected with either nonspecific control siRNA or HTRA1 siRNA and then treated with 1 ng/ml TGF-β for the indicated times. *P<0.05, **P<0.01, ***P<0.001, Student’s t-test, n = 3.
Figure 4.
The in vivo effects of loss of HTRA1.
(A) Schematic of the HTRA1 knockout strategy. Primers were designed with one forward primer (For) that amplifies an amplicon for the wild type and heterozygous alleles with a corresponding reverse primer (Rev), and a second reverse primer designed to only amplify the knockout. The knockout reverse primer (KO Rev) only amplifies following CRE-LOX recombination. Solid triangles = loxP sites; EV = EcoRI. E2 = Exon2; E3 = Exon3. (B) Genotype results of mouse embryonic fibroblasts showing confirmation of the generation of HTRA1+/− and HTRA1−/− mice. (C) Expression of TGF-β-regulated genes in embryonic fibroblasts from wild type (WT) and HTRA1 knockout (KO) mice. Cells were treated with 1 ng/ml TGF-β for the indicated times. *P<0.05, **P<0.01, ***P<0.001, Student’s t-test, n = 9 for each cell type. (D) Microcomputed tomography and parameters of trabecular bone in the distal femurs in wild type (WT), HTRA1 heterozygous (HET) and HTRA1 knockout (KO) mice (all males, 3 months old). (E) Microcomputed tomography and parameters of trabecular bone in the fourth vertebrae in wild type (WT), HTRA1 heterozygous (HET) and HTRA1 knockout (KO) mice. For both (D) and (E), *P<0.05, **P<0.01, ***P<0.001, ANOVA and Fisher’s protected least significant difference test, n = 13.